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Unformatted text preview: 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 20 C H A P T E R 2 Pe ar so About 4.5 billion years ago the planet Earth coalesced from clumps of debris floating through space after the Big Bang. For another billion years Earth's surface was a harsh place; stantly remodeling the face of the planet. Yet it was during this tumultuous period that life on Earth began. Some researchers believe that organic molecules arose from a pri- r sa So bu le lu Chemistry, Biochemistry, and Cell Physiology tio tio n n tri Le No ar tF n or in Di Re g s O n with the capacity for catalysis and self-replication. At some point around 4 billion years ago, these purely chemical processes produced the earliest life-form, the progenote. The progenote was likely a chemoautolithotroph, capable of surviving without oxygen and living on inorganic sources of energy and carbon. The closest living relatives to the progenote are likely the archaea. These modern prokaryotes can survive in the harshest environments that now exist on Earth, such as sulfuric hot springs and deep-sea vents. The progenote was the ancestor to all organisms on the planet and, as a result, it is likely that many of the ubiquitous biological features arose in the progenote. The dependence on water, the role of nucleic acids, the use of only 20 amino asteroid bombardment and volcanic eruptions were con- s mordial soup of methane, ammonia, and water, energized by atmospheric electrical discharges. Others believe that the first organic molecules arose from chemical reactions of products of deep-sea volcanoes. Regardless of the origins of the first small organic molecules, the pathway to living organisms required the formation of larger macromolecules 20 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 21 Pe ar so A species of domain Archaea found in deep-sea vents. acids in proteins, and the basic pathways of intermediary metabolism are shared attributes of all living organisms. Within the first billion years, the progenote gave rise to Connective tissue. three distinct types of organisms: eubacteria, archaea, and eukaryotes. Each lineage diversified independently over the and archaea, remained single-cell organisms with little inotes experienced evolutionary changes that resulted in the next 3 billion years. The two prokaryote lineages, eubacteria tracellular organization. In contrast, the ancestral eukary- production of membranous, subcellular compartments, when the earliest eukaryotes found a way to package their thereby increasing intracellular organization. This began DNA into a membrane-bound compartment: the nucleus. Later, around 3 billion years ago, a eukaryote engulfed a bacterium that resembled a modern purple bacterium. Although the purple bacterium was probably ingested as food, it developed a symbiotic relationship with its host, replicating with the host cell. Over time, the bacterial endosymbiont lost its Le No ar tF n or in Di Re g s O tri r bu tio sa n n The transition from single-cell organisms to multicellu- lar organisms occurred independently in the ancestors of plants, fungi, and animals. Each lineage found different solutions to the challenge of building multicellular tissues. The strategy used by fungi and plants relies on a cell wall for resistance to osmotic swelling and intercellular connections. Animal cells, in contrast, found other solutions to these physical challenges. Na /K ATPase appeared early in animal evolution, enabling animal cells to regulate cell volume, ionic balance, and osmotic balance. Collagen, one of the vital proteins used to construct tissues, also arose very early in metazoan evolution. Once these physical associations were established, more elaborate pathways for intercellular communication became possible and necessary. Even plants and fungi use chemical messengers to communicate, but animals possess much more complicated mechanisms for cell-to-cell signaling. We cannot understand the basis of animal diversity without an awareness of the evolutionary origins of animals. On the one hand, many cellular processes are similar across broad taxa, so what we learn from studies on model species of fungi and plants tells us a lot about how these features work in animals. On the other hand, each lineage evolved novel ways of using similar machinery to face the chemical and physical stresses imposed by the environment. By understanding how different taxa solved similar problems, we can better understand the constraints on animal cell function and evolution. Modern animal physiology builds upon studies of organisms in diverse taxa to understand the cellular origins of diversity in animals.2 21 capacity to exist outside the cell, and the host cell became re- liant on the metabolic contributions of the endosymbiont, the ancestor of mitochondria. By 2 billion years ago the diverse groups of protists were established. The protists include organisms like the euglena (with features of both animals and plants), the trypanosomes (the single-cell flagellate parasites of blood that cause malaria), and amoebas (ciliated cells that are the namesake of amoeboid movement). These protists were once called protozoans because they were considered to be primitive animals, but we now recognize the protists to be a group of over 50 different phyla that emerged prior to the origins of the three main eukaryote kingdoms: plants, fungi, and animals. The term metazoan, which arose to distinguish multicellular animals from the single-cell protozoans, is now used synonymously with "animal," although some taxonomists separate sponges, the most primitive animals, from true metazoans (eumetazoans). So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 22 22 PART ONE The Cellular Basis of Animal Physiology Overview Physiology is the study of how animals work and how they solve the challenges of surviving in the natural environment. Though we often think of animal physiology as a study of organs, systems, and whole animals, it is important to recognize that reasons for many of these features can be traced back to underlying rules of chemistry, biochemistry, and cell biology. Many of the properties of organs and systems emerge from regulation of cellular processes, such as energy production, membrane transport, cellular anatomy, and gene expression (Figure 2.1). While the physiology of an animal is much more than the sum of these molecular and cellular processes, an awareness of how cells work is vital to understanding complex physiological processes. Pe ar so entropy, states that the universe is becoming more chaotic. Both laws describe the constraints that exist when energy is transferred between systems. With any spontaneous transfer of energy, some energy is diverted in a way that increases the entropy of a system, another form of energy. Although each chemical reaction conforms to these principles, living organisms are able to delay the inevitable increase in chaos, or entropy. The survival of living organisms depends upon an ability to obstruct the natural processes that lead to chemical breakdown. Chemistry In the purely chemical world, chemical reactions proceed according to the rules of thermodynamics. The first law of thermodynamics, also called the law of conservation of energy, states that energy can be converted from one form to another but the total amount of energy in the universe is constant. The second law, also called the law of ATP Le No ar tF n or in Di Re g s O tri ATP n Energy Energy is the ability to do work. In our world, gasoline is an important form of chemical energy. We know that if the fuel tank of a car is full of gasoline, we have the potential to use this fuel to move the car from place to place. Burning the gasoline causes the pistons in the engine to move, turning the drive-shaft and ultimately the wheels. This familiar analogy illustrates many important principles that govern energy transfers or energetics. The gasoline in the tank has potential energy trapped within its chemical structure. When gasoline is ignited, the resulting explosion releases heat and carbon dioxide, moving the piston in its r bu tio ATP sa Energy production n So le lu t Membrane transport io n s Cellular anatomy Gene expression Figure 2.1 Cells and tissues Many cellular processes underlie physiological systems. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 23 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 23 Pe ar so cylinder. This type of energy is kinetic energy, the energy of movement. The standard SI (Systme International) unit of energy is the joule, although the imperial unit, the calorie, persists in the scientific literature. A joule is defined many ways, depending on the circumstances. In electrical terms, a joule (J) is the amount of energy used when 1 watt of power (W) is expended for the period of 1 second (1 J 1 W sec). Conversely, a watt is defined as a joule per second. You are probably most familiar with the units of energy from your household electrical bill, with energy consumption expressed in kilowatt hours (1 kW h 3.6 106 J). In more biological terms, a piece of toast with butter has about 300 kJ of energy, which is enough energy to allow you to run for about 6 minutes or light a 100-watt bulb for about 1 hour. All energy is kinetic energy, potential energy, or a combination of both. However, in the context of biological systems it is more useful to classify types of energy by other categories. Thermal energy is a form of kinetic energy that is reflected in the movement of particles, and serves to increase temperature. Chemical energy is a form of potential energy that is held within the bonds of chemical structures. Food webs transfer energy Most biological processes are essentially transfers of energy from one form to another. When you see and smell a rose, the perception is essentially a cascade of chemical and electrical energy transfers between the sensory system and the brain. We are more familiar with the concept of energy transfers in the context of food webs. Plants capture the energy of photons and use it to create sugars. Herbivorous animals eat the plants, and carnivores eat the herbivores. At each level, some potential energy in the diet is assimilated to form animal tissues. Some potential energy is converted to heat, which is either lost to the environment or retained within the animal. Dietary potential energy is also transferred to kinetic energy, when animals use nutrients to fuel locomotion. A portion of the potential energy in the diet is locked in chemical structures that can't be liberated by the animal, and is excreted in waste products. Light is the ultimate source of dietary energy for most animals; it also provides the energy that allows animals to use vision and perceive color. The chemical energy transferred between trophic levels is stored within molecules in the bonds between atoms. Chemical reactions liberate energy from one bond in order to produce other bonds. We discuss the nature of chemical bonds, and the role of energy in bond formation, a bit later in this chapter. However, other forms of energy are also critical components of biological function. Radiant energy is energy that is released from an object and transmitted to another object by waves or particles. The sun is the most obvious source of radiant energy, emitting light that serves as an energy source for photosynthetic organisms. Other forms of radiant energy occur in animals, such as the infrared radiation given off from warm-bodied objects. Radiant energy is important in the thermal biology of animals, and is discussed in more detail in Chapter 13: Thermal Physiology. Le No ar tF n or in Di Re g s O tri r bu tio sa n n Mechanical energy is a combination of potential and kinetic energy that can be used to move objects from place to place. A flying bird uses its wings to produce the mechanical energy necessary for flight. A kangaroo uses its legs to store mechanical energy in the form of elastic storage energy. Recoil of the springs helps the kangaroo hop. Many forms of mechanical energy have important roles in animal locomotion, which is discussed in more detail in Chapter 12: Locomotion. Electrical energy is a combination of potential and kinetic energy that results from the movement of charged particles down a charge gradient. So le lu t io n Energy is stored in electrochemical gradients s Molecules within a system tend to disperse or diffuse randomly within the available space. Imagine starting a dozen spinning tops in the center of a box. The tops collide frequently at first, eventually dispersing randomly throughout the box. Two aspects of diffusion govern the properties of many biological processes. First, diffusion is 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 24 24 PART ONE The Cellular Basis of Animal Physiology certain to lead to a random distribution of molecules, but the rate of diffusion can be slow. Many physiological systems function to reduce the reliance on slow rates of diffusion. Second, the tendency of molecules to diffuse is a source of energy that cells can use to drive other processes. Living organisms can invest energy to delay the inevitable tendency toward randomness. In the previous example, you could prevent the spinning tops from randomly distributing by moving each top back to the center position. Your efforts to reverse the random distribution reflect an energetic investment on your part. Similarly, biological systems can invest energy to move molecules out of a random distribution. The resulting diffusion gradient is a form of energy storage that the cell can use for other purposes. Of particular importance are the gradients established across biological membranes. Transmembrane gradients created by cells differ in terms of the nature of the molecules (Figure 2.2). A chemical gradient arises when one type of molecule occurs at a higher concentration on one side of a membrane. The magnitude of the chemical gradient is expressed as a ratio of the concentrations of the specific molecule on either side of the membrane. For example, you might say that a given molecule is 10-fold more concentrated outside the cell. The second type of gradient, an electrical gradient, arises if the distribution of charged molecules is unequal on either side of an electrical barrier in a circuit. The electrical gradient across the barrier is dependent on the distributions of all the charged molecules combined. The strength of the electrical gradient Pe ar so Le No ar tF n or in Di Re g s O tri r bu tio + + Plasma membrane (bilayer) n is expressed in the electrical unit of volts. In cells, membranes are the electrical barrier and the electrical gradient is called the membrane potential. The nature of the molecule determines whether the potential energy of the gradient is primarily electrical or chemical. If a molecule is uncharged, then it can only form a chemical gradient. A charged molecule can form a chemical gradient and influence the electrical gradient. For instance, if the concentration of Na is greater outside the cell than inside, there is both an electrical gradient (more positive charges outside the cell) and a chemical gradient (more Na ions outside the cell). Consequently, these gradients are often discussed as electrochemical gradients. Thermal energy is the movement of molecules It is impossible to ignore the importance of thermal energy, or heat energy, in discussing chemical or biological processes. When a system gains thermal energy, there is an increase in the movement of molecules within that system. This type of movement has a profound effect on molecular reactivity and the rate of chemical reactions. Most chemical reactions involve changes in thermal energy. Exothermic reactions release heat and endothermic reactions absorb heat. To understand the reasons for these changes in thermal energy, let's consider a simple reaction in which a single substrate, A, becomes a single product, Z: sa + n + Positive charge Negative charge + So le lu t AZ + + + + + + + + + (a) Chemical gradient (b) Electrical gradient Figure 2.2 Storage of potential energy in electrochemical gradients Animals can use the energy stored as (a) chemical gradients or (b) electrical gradients, or membrane potential. At any given time, each molecule of A is vibrating in solution, experiencing subtle changes in its structure. A single molecule of A at times moves quickly (lots of kinetic energy) and at other times moves slowly (less kinetic energy). Occasionally, a molecule of A has so much kinetic energy that it is able to assume a specific structure that is vulnerable to a more significant change. This structure, intermediate between A and Z, is called the transition state. The energy required for a molecule to reach the transition state is the activation energy, or EA. Once a molecule reaches the transition state, it is equally likely to revert to io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 25 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 25 Pe ar so H Number of S molecules the substrate, S, or convert to the product, P. The progression of the reaction from S to P, expressed in terms of energy content, is shown in Figure 2.3. Since the energy content, or enthalpy (H), of S is greater than the enthalpy of P, the chemical reaction leads to a change in enthalpy ( H), calculated as Hproducts Hsubstrates In this reaction (Figure 2.3), P has a lower enthalpy, making H negative. S is converted to P, and the difference in enthalpy, or H, is released to the environment, primarily as heat. Thus, an exothermic reaction is defined in thermodynamic terms as a reaction with a negative H. An endothermic reaction has a positive H. Low (a) Le No ar tF n or in Di Re g s O EA Enthalpy of S molecules High Transition state n All chemical reactions are reversible under the right conditions. The reaction of S to P is favored only because the activation energy barrier is lower for S than it is for P. Because thermal energy was released when S was converted to P, thermal energy must be absorbed if P is to be converted to S. The reverse reaction, with its positive H, is an endothermic reaction. If both S and P are present, at any point in time both forward and reverse reactions occur simultaneously. The net reaction is the difference between the forward rate and the reverse rate. Because heat is released in one direction and absorbed in the other, the balance between forward and reverse directions depends on temperature. At high temperatures, endothermic reactions become more feasible. Thus, temperature influences chemical reactions in two ways. Increasing temperature allows more molecules to reach activation energy, and increases the likelihood of endothermic reactions. Chemical Bonds Most biologically available energy is stored in the form of chemical bonds. Covalent bonds hold individual atoms together to form a molecule. These strong bonds involve the sharing of electrons between two atoms. Noncovalent bonds organize molecules into three-dimensional structures. In general, noncovalent bonds are called weak bonds or sometimes weak interactions to further distinguish them from strong bonds. Enthalpy (kcal/mol) tri S r bu tio P sa Activation energy (EA ) Covalent bonds involve shared electrons Each element has a characteristic arrangement of electrons that influences the types of bonds it can form. Specifically, for the six common biological elements, each atom has at least one unpaired electron in its outer electron shell. Atoms with unpaired electrons can readily form covalent bonds with other atoms with unpaired electrons. These atoms share electrons so readily that they are rarely present in elemental form. Atoms with more than one unpaired electron can form multiple covalent bonds. For instance, molecular oxygen has two oxygen atoms joined by a double covalent bond. Many atoms are covalently bonded to more than one other atom. Methane, for example, is composed of four hydrogen atoms covalently bound to a single carbon atom. Each type of Difference in E content of substrate and product (H ) n So le lu t io n Chemical reaction (b) s Figure 2.3 Chemical reactions, substrates, products, and thermal energy (a) A collection of substrate molecules, S, possesses an average energy level and an average arrangement of electrons around its nucleus. But at any given time, some S molecules are energy-rich and others energy-poor. (b) Occasionally a molecule of S might absorb enough energy from the surroundings (perhaps from a molecular collision) that it achieves the transition state (S*). At this point it could revert to S, or change into a novel product P. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 26 26 PART ONE The Cellular Basis of Animal Physiology covalent bond has a characteristic bond energy, the energy required to either form or break the bond. The greater the bond energy, the stronger the bond. Multiple bonds possess more bond energy than single bonds. Large molecules are built from a collection of individual atoms attached by covalent bonds. Functional groups are combinations of atoms and bonds that recur in biological molecules (Figure 2.4). Weak bonds control macromolecular structure Weak bonds arise between atoms with asymmetrical distributions of electrons either within the atom or between atoms. Four types of weak bonds can be distinguished based on how they form mo- Pe ar so Functional groups O C OH Carboxyl H N H Amino OH Hydroxyl Le No ar tF n or in Di Re g s O Covalent bonds S S Disulfide C O Ester O n tri O P OH C O C H C H O P OH OH Phosphoryl H Methyl r bu tio O P O OH S Thioester O C Ether C O Peptide Phosphodiester sa lecular interactions: van der Waals forces, hydrogen bonds, ionic bonds, and hydrophobic bonds (Figure 2.5). The electrons in a bond between two atoms can be shared unequally. This asymmetry in electron distribution creates a polarity, or transient dipole, within the molecular structure. One region is slightly negative ( ), and the other is slightly positive ( ). When an atom with a transient dipole encounters another atom, the distribution of electrons in the second atom is altered. The weak interaction between the two dipoles is the van der Waals interaction. Van der Waals interactions are effective only over a very narrow range of atomic distances. When two atoms are far away, the dipole of one atom has no effect on the electron cloud of the other. As the atoms approach, the attraction between the atoms increases. When the atoms get too close, their electron shells repel each atom away from the other. The van der Waals radius is the distance at which the attractive force is at its greatest. Each atom has a characteristic van der Waals radius. Hydrogen bonds arise from the asymmetric sharing of electrons between two atoms. They are critical to the organization of water molecules. In a single water molecule, each hydrogen atom is covalently linked to the oxygen atom. However, the oxygen atom is just a bit better at attracting the van der Waals interaction n H N H So le lu t Hydrogen bond H Hydrophobic bond H H io n H C C C H C C C H H H H H H C H C C C C H H H C s H H H Ionic bond H N O H H H SH Sulfhydryl + H O C Figure 2.4 Important functional groups and bonds Although there are many types of bonds and functional groups, those illustrated here are particularly common in macromolecular structure. Figure 2.5 Weak bonds Four types of weak bonds are involved in building macromolecules: hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 27 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 27 Pe ar so electron of hydrogen. More precisely, hydrogen's electron spends a bit more time closer to the oxygen atom than the hydrogen atom. Consequently, the hydrogen is slightly positive ( ), and the oxygen atom slightly negative ( ). The attraction between the of hydrogen in one water molecule and the of oxygen in another water molecule constitutes a hydrogen bond (Figure 2.6). In some cases, a nucleus is so good at attracting electrons that when a bond breaks, an electron from one atom remains with the other to create ions. Electronegative ions, or anions, possess extra electrons, whereas electropositive ions, or cations, have lost electrons. Anions and cations can interact to form an ionic bond. Most of the molecules we think of as salts, acids, and bases rely on ionic bonds to join anions and cations. Van der Waals forces, hydrogen bonds, and ionic interactions form on the basis of mutual attraction between two charged or slightly charged atoms. However, hydrophobic bonds form between atoms because of a mutual aversion to water. Whole molecules or specific regions of large molecules can be hydrophobic ("water-fearing"). The bonds within hydrophobic molecules share electrons equally and therefore do not possess significant dipoles. With little internal charge, they cannot interact effectively with the more polar molecules such as water. Weak bonds are sensitive to temperature Bond energy reflects the amount of thermal energy required to break (or form) a bond. Weak bonds are more vulnerable to the effects of temperature because their bond energies are much lower than the bond energies of covalent bonds. Whereas covalent bonds have energies of formation of 200900 kcal/mol, weak bonds have energies of formation less than 5 kcal/mol. The three-dimensional macromolecular structures of proteins, membranes, and DNA, which primarily depend upon weak bonds, are also sensitive to temperature. As a result, rising temperature can cause macromolecules to unfold, or denature, when these weak bonds break. However, not all weak bonds are affected by temperature the same way. Hydrogen bonds, ionic bonds, and van der Waals forces each have positive energy of formation and tend to break when temperature increases. In contrast, hydrophobic bonds have negative energy of formation and are strengthened by thermal energy. Le No ar tF n or in Di Re g s O 2 n CO NCE P T CH E CK 1. What are the five main forms of energy used by animals? Provide biological and nonbiological examples of processes that represent conversion of energy from one form to another. tri + H O + H + H + + H + H O O r bu tio O + H O H O H + H + sa 2. What are the four types of weak bonds, and how do they differ from each other and from covalent bonds? 3. What is the difference between (a) thermal energy and temperature and (b) exergonic and exothermic? n H + Properties of Water So le lu t H + H + Most cells are composed primarily of water. Aquatic organisms also live in water, and even the cells of terrestrial organisms are bathed in the aquatic environment of their extracellular fluids. Many physiological processes arose to meet the challenges of the physical and chemical properties of water. io n s The properties of water are unique A solvent is the most abundant molecule in a liquid, whereas the other molecules within the liquid are solutes. Collectively, the solutes and solvents constitute the solution. In biological systems, the solvent is usually water. Water's unusual combination Figure 2.6 Water dipole and hydrogen bonds Oxygen atoms in water strongly attract the electron of the hydrogen atom. The result is a small charge difference ( ). The hydrogen atom is slightly positive ( ) and the oxygen atom is slightly negative ( ). These charges influence the way that water molecules interact. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 28 28 PART ONE The Cellular Basis of Animal Physiology of physicochemical properties, which can be attributed to its ability to form hydrogen bonds, have special significance in biological processes and constrain the direction of biological evolution. Liquid water is actually a network of interconnected water molecules. Each water molecule interacts strongly with other water molecules, creating internal cohesiveness. At the interface between air and water, the attraction between water molecules creates a force called surface tension. This prevents most water molecules from spontaneously escaping to the air. Many animals take advantage of surface tension to move over water (Figure 2.7). Their mass exerts a force on the water, but it is not great enough to disrupt the molecular interactions between water molecules. The organization of water molecules changes in relation to temperature. At high temperatures, the water molecules possess enough thermal energy to escape the restraining force of surface tension. At this point, the water "boils," and water molecules can escape as gaseous water (steam). In contrast, low temperatures stabilize water structure as a result of the formation of additional hydrogen bonds. Water solidifies, or freezes, when each water molecule forms four hydrogen bonds to create a stable lattice of water molecules. Changes in temperature also influence the density of water. Although frozen water molecules incorporate more hydrogen bonds, the geometry is such that the water molecules are held further apart than in liquid water. Consequently, ice is less Pe ar so Le No ar tF n or in Di Re g s O tri r bu tio sa n n dense than liquid water and tends to float. These physical properties of water have important effects on aquatic ecosystems. In temperate regions of Earth, a layer of ice forms on the surface of lakes in early winter. The ice layer insulates the lake water from the air conditions, creating a more stable environment for aquatic organisms. Temperature also alters the density of liquid water. Because the density of water is greatest at 4C, the deepest parts of large water bodies tend to be a constant 4C, whereas surface waters can be colder or warmer, depending on the latitude and season. Other physical properties of water have an important impact on biological processes. Water has a higher melting point (0C) and a higher boiling point (100C) than other solvents. In most habitable locations on Earth, then, water is a very stable liquid. Water's high heat of vaporization, the amount of energy required to cause liquid water to boil or evaporate, makes sweating an effective cooling strategy for mammals. A great deal of energy is absorbed when liquid water vaporizes. Water on the skin absorbs a lot of thermal energy from the body in the process of evaporation. Solutes influence the physical properties of water Figure 2.7 Surface tension The basilisk lizard is able to run across the surface of water. The surface tension of water can support the lizard because the force is distributed over the large surface area of the feet. Many solutes can dissolve in water because they can form hydrogen bonds with water molecules. Water-soluble molecules in solution are often surrounded by a coat of water molecules called the hydration shell. The hydration shell increases the functional size of the molecule, and influences how the solute interacts with other molecules in complex biological systems. In the tissues of most animals, the most common solutes are inorganic ions. K is the most abundant cation inside cells, and Na is the most abundant cation in the extracellular fluid. However, in some species, particularly marine animals, the most abundant solutes are organic ones such as urea, amino acids, and sugars. Each type of solute can exert specific, distinct effects on the chemical properties of other molecules within the solution. However, all solutes, regardless of their chemical nature, exhibit four basic properties, known as colligative properties. Solutes reduce the freezing point of the solution, and increase the boiling point, the vapor pressure, and the osmotic pressure of the solution. The colligative properties depend only on the concentration of solutes, not their size or charge. So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 29 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 29 Pe ar so In a solution with high concentrations of solutes, cooling to 0C will not induce freezing. The thermal energy of the system is low enough to form the extra hydrogen bonds, but the solutes block the formation of hydrogen bonds necessary to form the ice crystal. When solutes are present, the solution must be cooled below 0C before the extra hydrogen bonds can form. The freezing point of biological fluids, such as cytoplasm or blood, is always lower than freshwater, and sometimes even as low as seawater. The difference in the freezing point of body fluids and the aquatic environment has important ramifications for aquatic animals. Similar mechanisms are responsible for the effects of solutes on the vapor pressure and boiling point of water. A water molecule can escape liquid water only at the water-gas interface. When solute molecules are also present at the surface, they reduce the likelihood that a water molecule will escape. We discuss the fourth colligative property, osmotic pressure, after we discuss a related concept: diffusion. that forms around many molecules enlarges the functional size of the molecule, restricting its mobility. Other factors that influence how the solute interacts with the solvent, such as charge and solubility, also affect the rate of diffusion. Each solute has an experimentally determined diffusion coefficient (Ds), which is influenced by the structural properties of the solute. The rate of diffusion of a solute (dQs /dt) depends on the diffusion coefficient of the solute (Ds), the diffusion area (A), and the concentration gradient (dC/dX). The relationship between these parameters is defined by the Fick equation: dQs dt Ds A dC dX Solutes move through water by diffusion The direction of diffusion of molecules in a solution depends on the concentration gradient, but the rate of diffusion depends on many additional factors. Molecules move more rapidly when the gradients are steeper. The properties of the solute itself also influence the rate of diffusion. If solute molecules are relatively large, they have a more difficult time moving through the restrictive structure of water. Large molecules like proteins diffuse much more slowly than small molecules like K . The hydration shell Le No ar tF n or in Di Re g s O tri r bu tio NaCl n Small solutes, such as inorganic ions, are able to traverse the width of the cell, typically about 10 m, in a fraction of a second. The time required for a molecule to diffuse a given distance increases with the square of the distance. If a molecule takes 1 sec to diffuse 0.1 mm, it would take about 3 h to diffuse 1 cm. Many biological processes depend on diffusion, such that physiological and anatomical strategies have evolved to prevent these processes from becoming "diffusion limited." Solutes in biological systems impose osmotic pressure sa The semipermeable membranes of cells allow some molecules to cross while restricting the movement of others. Imagine a situation where two identical solutions of pure water exist on either side of a membrane that allows free movement of water molecules only (Figure 2.8). On each Semipermeable membrane n H2O So le lu t H2O Gravity io n s Osmotic pressure H2O H2O (a) (b) (c) (d) Figure 2.8 Osmotic pressure The two solutions differing in solute concentration are separated by a semipermeable membrane. The movement of water creates osmotic pressure. Movement will continue until the force of gravity is equal to the osmotic pressure. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 30 30 PART ONE The Cellular Basis of Animal Physiology side of the membrane is approximately 55.5 mol of water per liter. Water molecules freely cross the membrane in both directions. If you added NaCl to one side of the membrane, a concentration gradient would be created for Na and Cl . Since the membrane is permeable to water alone, only water molecules could move across to equalize the concentration gradients. There would be a net movement of water molecules from the side with pure water to the side with solutes. This would increase the volume on the side with solutes. Eventually, the net movement of water would stop when the force generated by the movement of water equaled the force of gravity, which prevents the water column from getting any higher. In cells, the movement of water is restricted not by gravity but by the flexibility of the cell membrane. In either case, the force associated with the movement of water is the osmotic pressure, the fourth colligative property of solutes. The ability of solutions to induce water to cross a membrane is expressed as the osmolarity, expressed in units of osmoles per liter (OsM). Osmolarity is analogous in many respects to molarity (M). Whereas molarity is a reflection of the concentration of specific molecules in a solution, osmolarity depends on the total concentration of particles in solution. The osmolarity of a solution of known molarity can be calculated on the basis of the number of particles derived from each molecule. If a solution has only one solute, and that solute does not dissociate, then molarity and osmolarity are equivalent. For instance, 1 mol/l (or 1 M) glucose solution has an osmolarity of 1 osmol/l (or 1 OsM). Some solutes dissociate into multiple particles. Each mole of NaCl produces 1 mol of Na and 1 mol of Cl . Thus, a 1 M NaCl solution has an osmolarity of 2 OsM. Knowledge of the concentration and valency of the solutes would allow you to estimate osmolarity, but in reality the osmolarity is somewhat less. Some of the salt does not dissociate, and some of the water molecules become associated with the hydration shell of the ions. The osmolarity and osmotic pressure of a solution are physical properties of a solution. However, in a biological setting the absolute osmolarity is often less important than the osmolarity of an extracellular fluid relative to the osmolarity of the intracellular fluid (Figure 2.9a). If a cell is placed in a solution with greater osmolarity, then the solution is considered hyperosmotic (relative to the cell). Similarly, if a cell is placed in pure water, the solu- tion is hyposmotic. When the osmolarity is the same on both sides of the cell membrane, the solution is isosmotic. Differences in osmolarity can alter cell volume Biologists usually make distinctions between osmolarity, which is related to the osmotic pressure, and tonicity, which is the effect of a solution on cell volume. Tonicity depends on differences in osmolarity, but also on the types of solutes and the permeability of the membrane to those solutes. To understand the distinction between osmolarity and tonicity, consider the following example (Figure 2.9b). A cell that is placed in an isosmotic salt solution neither shrinks nor swells (an isotonic solution). If more salt is added, the cell loses water and shrinks. Thus, this solution is both hyperosmotic and hypertonic. Imagine now that small amounts of urea, a permeant solute, are added to the isotonic salt solution. The urea would equilibrate across the cell membrane, and thus prevent the net movement of water in or out of the cell; this is an isotonic solution. Of course, if the cell were placed in a solution containing only urea, the movement of urea into the cell, combined with the high internal salt concentration, would draw water into the cell, causing it to swell or even burst; this is a hypotonic solution. Pe ar so Le No ar tF n or in Di Re g s O tri r bu tio sa n n pH and the Ionization of Water A small proportion of the H2O molecules in any solution dissociates into ions by breaking one of the covalent bonds between oxygen and hydrogen. In reality, water is in equilibrium with itself. H2O H2O H3O So le lu t For simplicity, the cation is treated as a proton (H ) rather than a hydronium ion (H3O ). The dissociation of water into ions is reversible. Both the forward reaction (water dissociation) and the reverse direction (water formation) occur simultaneously. Only a very small proportion of water molecules are dissociated at any given time, about 1 in 55,500,000 water molecules at room temperature (25C). Under these conditions (pure water at 25C), the concentration of protons arising from water dissociation is 10 7 M. For the sake of convenience, the concentration of protons is usually converted to the pH scale. The pH of a solution is io n OH s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 31 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 31 Na+ Cell Cl Na+ Cell Cl Pe ar so More salt added Hyperosmotic Solution (a) Osmolarity Isosmotic Solution More water added Salt added Isotonic Solution Urea added Figure 2.9 Osmolarity versus tonicity (a) A cell is in an isosmotic solution when the solution has an osmotic pressure equal to that of the cell cytoplasm. If salt is added to the solution it becomes a hyperosmotic solution. Water leaves the cell, causing the cell volume to shrink, until the osmotic pressures are again equal. If the salt concentration is reduced, as would be the case if more water was added, the solution becomes hyposmotic. Water flows into the cell, causing it to Le No ar tF n or in Di Re g s O Hyposmotic Solution (b) Tonicity n Urea Hypertonic Solution Isotonic Solution calculated as the negative logarithm of proton concentration (denoted as [H ]). Thus, the pH of pure water at 25C is pH 7 ( log 10 7). As we see later in this chapter, the negative logarithmic scale, designated by the prefix p, is also a convenient way to express low concentrations of other ions, such as pOH for [OH ] and pCa for [Ca2 ]. tri r bu tio sa swell. (b) The effects of solutes on cell volume depend on the ability of the solute to enter the cell. If NaCl is added to an isosmotic solution, the cell shrinks and the solution is considered hypertonic. If urea is added to the solution, there is little change in cell volume because urea can cross the cell membrane. Thus, adding urea to this solution makes it hyperosmotic, but it is also isotonic. n molecules dissociate, raising the pH to 7.28. In each of these situations, water remains neutral, but the pH at neutrality, or pN, varies inversely with temperature. In practice, pure water changes its pH at a rate of 0.014 units per degree Celsius. So le lu t Acids and bases alter the pH of water io n Neutrality is not always at pH 7 A solution is considered neutral when [H ] [OH ], or pH pOH. Pure water at 25C possesses 10 7 M concentrations of both H and OH : pH 7 and pOH 7. The temperature of a solution of pure water alters the proportion of water molecules with enough thermal energy to break the covalent OH bond. For instance, at 45C almost twice as many H2O molecules dissociate, lowering the pH to 6.72. At 5C, about half as many H2O Pure water is never anything but neutral. However, ionizable solutes can influence the pH of a solution. An acid releases one or more protons. Hydrochloric acid (HCl) is an acid because it dissociates into H and Cl . A base causes a reduction in the [H ] of the solution. When the base sodium hydroxide (NaOH) is dissolved into water, it rapidly dissociates into Na and OH . The extra OH arising from NaOH dissociation rapidly interacts with H to form H2O, reducing the [H ] and increasing pH. s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 32 32 PART ONE The Cellular Basis of Animal Physiology The degree to which acids and bases change the pH of a solution depends on the ease with which the molecule dissociates under physiological conditions. Inorganic acids such as HCl and H2SO4 are considered strong acids because they readily release their protons to the solution. Similarly, NaOH and KOH are strong bases because they readily dissociate to release OH . Many biological molecules are weak acids or weak bases, which are only partially ionized under physiological conditions. If an acid is defined as something that releases a proton, then we can discuss acids with the general formula of HA. Dissociation of the acid HA produces H and the anion, A . We can describe a reversible chemical reaction with the equation Pe ar so We define the relationship between the substrate (HA) and products (H and A ) as the mass action ratio, using the equation Mass action ratio 3H 4 3HA4 3A 4 To understand how these parameters change, consider an experiment where the acid HA is added to pure water. When first added to water, HA remains intact and [A ] is equal to zero; the mass action ratio is also close to zero. However, very quickly at least some of the acid dissociates. There is an increase in both [H ] and [A ], and as a result an increase in the mass action ratio. At some point the reaction slows, with [HA] reaching a minimum and [H ] and [A ] reaching a maximum. When this occurs, the reaction is at equilibrium. It is important to recognize that although there is no net change in the concentrations of reactants, both forward and reverse reactions continue, but at equal rates. When the reaction is at equilibrium, the mass action ratio attains a specific value, Keq, the equilibrium constant. Under most circumstances, the equilibrium constant is converted to its negative log ( log10 Keq), analogous to the way we converted [H ] to pH. Thus, the equilibrium equation can be rewritten after log transformation as Le No ar tF n or in Di Re g s O tri r bu tio 3A 4 HA H n A This simple equation is useful for understanding many different biochemical and physiological principles. For example, the pK value reflects the strength of acids and bases. A strong acid will give up its proton even when the concentration of protons in the surrounding area is very high (low pH). Thus, the pH must be very low to prevent a strong acid from dissociating. The pK value is low for a strong acid, less than 3 for hydrochloric acid and sulfuric acid. Similarly, strong bases, such as sodium hydroxide and ammonium hydroxide, have pK values greater than 11. The pK values for some common biological acids and bases are shown in Table 2.1. The equilibrium equation is a powerful tool for analyzing biological solutions. Once we know the values of three of the four parameters, we can calculate the one that is unknown. To determine pH, we can rearrange the equation into the form pH pK log 3HA4 3A 4 This rearrangement is known as the Henderson-Hasselbalch equation, named after the researchers who used the relationship to explain the behavior of CO2 (HA) and HCO3 (A ), which is important in respiratory physiology. sa Acid Table 2.1 Acids and bases. Carbonic acid Phosphoric acid H3PO4 H2PO4 n So le lu t Reaction H2CO3 HCO3 HCO3 CO3 H2PO4 HPO42 HPO42 PO43 NH4 NH3 H N CH N H+ pK H 3.8 10.2 3.1 6.9 Ammonium Acetic acid Glycine (amino) (carboxy) H CH3COOH CH3COO RNH3 RNH2 RCOOH RCOO H N RC HC io n H H H H H H N H s 12.4 9.3 4.8 2.3 9.6 pK pH log 3HA4 Histidine R C HC CH + H+ 6.0 Put another way, the pK is the pH at which half the acid is dissociated, [A ] [HA], [A ]/[HA] 1 and log [A ]/[HA] 0. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 33 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 33 Pe ar so of the glycine molecule, or its pKCOOH. Adding more base causes deprotonation of the amino group. At pH 9.6, exactly half the amino groups are protoChanges in pH can alter the dissociation of other nated. This pH value is the equilibrium constant molecules with ionizable groups. Let's look at the for the amino group of glycine, or its pKNH2. At still amino acid glycine to explore how pH affects its higher pH values, the carboxyl groups remain structure (Figure 2.10). Glycine has a carboxyl charged and the amino groups are fully deprotogroup that can be protonated (COOH) or deprotonated, giving the glycine molecule a net negative nated (COO ). It has an amino group that can be charge. Midway between the two pK values, the deprotonated (NH2) or protonated (NH3 ). The glycine molecule has no net charge, as the charges protonation state of the carboxyl and amino on the carboxyl group (COO ) balance the charges groups in a molecule of glycine depends on the pH on the amino group (NH3 ). Glycine and other molof the solution. ecules that have both negative and positive We can observe the effects of pH on glycine charges are called zwitterions. structure by performing a titration, where an acid The ionization state of molecules is very sensior base is added to a solution. We start our titrative to temperature. Let's return to the previous extion by dissolving glycine in an acidic solution. At ample where we titrated the ionizable groups of very low pH, where [H ] is high, both amino and glycine. As pH rose to equal pKCOOH, protons became carboxyl groups are protonated. The carboxyl so scarce that half of the carboxyl groups lost their group is uncharged (COOH) and the amino group proton. If we repeated the titration at lower temperhas a positive charge (NH3 ), giving glycine a net ature, the pK value would change because the lower positive charge. When we add base to the solution temperature increases the strength of the bond to increase pH, the protonation state of both of holding the proton to the carboxyl group. At any these groups begins to change. First, the carboxyl given pH the colder glycine would be more protogroups become deprotonated (COOH COO2 nated. Put another way, a higher pH would be reH ). At pH 2.3, exactly half of the carboxyl groups quired to lure half the protons off the carboxyl in the glycine molecule are ionized. This pH value group. Thus, the pK value increases as temperature is the equilibrium constant for the carboxyl group decreases. Each ionizable group has a characteristic sensitivity to temperature, expressed as pK/C. For example, 14 H+ H H the ionization of phosphoric acid 13 H H N N O is relatively insensitive to tem12 O = H C C H C C perature ( pK/C 0.005), 11 O O H whereas the ionization of the H 10 imidazole group of histidine is pKa = 9.6 9 more sensitive to temperature 8 + ( pK/C 0.017). + H 7 H H H H H The protonation state of 6 N N O O many molecules can have im5 H C C =H C C portant effects on molecular O OH 4 H H processes. Many of the effects of 3 temperature and pH on cells pKa = 2.3 2 can be traced to the effects on 1 the protonation state of critical 0 molecules. For example, many Added base proteins form structures that Figure 2.10 Changing pH and the ionization state of acids and depend on particular ionization bases The amino acid glycine occurs in several different ionization states that states of amino acids. Changes change with pH. At low pH (high [H ]) both the amino group and the carboxyl group are in pH or temperature can affect protonated. As pH increases, the carboxyl loses its proton first, becoming half ionized how these proteins fold and at pH 2.3, its pKa value. The amino group does not lose its proton until much higher pH function. By actively regulating values are reached. Near neutral pH, glycine is primarily neutral. Both pH and temperature affect the ionization of biological molecules Le No ar tF n or in Di Re g s O tri r bu tio sa n n So le lu t io n pH s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 34 34 PART ONE The Cellular Basis of Animal Physiology temperature and pH, animals diminish the debilitating effects of changes in protonation state. Buffers limit changes in pH A variety of mechanisms help cells regulate pH. The first level of defense is a buffer. A buffer is a chemical found in a solution that dampens the effect of added acid or base on the pH of the solution. Buffers are often described as if they were single molecules. In reality we should think of them as buffer systems, because they are mixtures of at least two forms of a molecule, typically protonated and deprotonated. If we add a buffer to the solution, the protons liberated from the acid can associate with the buffer. As a result, the addition of acid has less effect on pH than it does in the absence of buffer. Most buffer systems rely on weak acids, present in both the acid form (HA) and the anion form (A ). Furthermore, a buffer works only over a particular range of pH values. Acetic acid is a weak acid that can be used as a buffer. The effects of an acetic acid/acetate buffer are illustrated by the titration curve shown in Figure 2.11. If you started your titration at low pH, most of the acetic acid would be in the protonated form (HA). If you added small volumes of NaOH, the pH would increase proportionately. Below pH 3.75, acetic acid would remain mostly protonated (HA). If you add more base, the increase in pH would induce some acetic acid (HA) to become deprotonated (HA H A ). Since Pe ar so Le No ar tF n or in Di Re g s O tri r bu tio sa n H n 10 9 8 7 6 Buffering range pH 5 4 3 2 1 0 some of the protons are liberated from acetic acid, the added NaOH has a reduced effect on the pH of the solution. This buffering effect is evident over the pH range of about 3.75 to 5.75, where the titration curve is quite shallow. Once pH reaches 5.75, most of the acetic acid is in the deprotonated form (A ), which cannot act as a buffer. This pH range corresponds to the greatest buffering capacity of the solution, and is centered on the pK value for the buffer, about 4.75, where about half of the buffer is protonated (HA) and half deprotonated (A ). Animals use a variety of different molecules as buffers. The best buffers in animal cells have pK values that approach the pH of the compartment in which they are used. Phosphate (H2PO4 /HPO42 ) is an important buffer in the cytoplasm of most cells, with a pKA of 6.9. The amino acid histidine contributes to buffering in many animal cells because the pK value of its imidazole side chain is very close to intracellular pH. Histidine residues within large proteins help buffer the cytoplasm against changes in pH. Many species use amino acids with imidazole groups to produce dipeptides that serve as important intracellular buffers. The dipeptides carnosine (histidine and -alanine), anserine (1-methylhistidine and -alanine), and ophidine (3-methylhistidine and -alanine) are important buffers in the muscle of many species. In air-breathing animals, the most important extracellular buffer is bicarbonate/CO2, but it works by a different mechanism than a simple A /HA buffer pair. In a closed test tube bicarbonate/CO2 would have little buffering capacity at physiological pH because its pK is much too low (3.8). It works as a biological buffer because animals can expire CO2. As [H ] increases, bicarbonate is consumed and carbonic acid is produced (H2CO3), which in turn forms H2O and CO2. So le lu t HCO3 H2CO3 H2O Same amount of base has different effects on pH. When an animal expires CO2 as a gas, it is essentially eliminating a weak acid from the body, buffering against a change in pH. You will learn more about the interaction between CO2 and acidbase balance in Chapter 9: Respiratory Systems. io n CO2 s 2 Added base CO NCE P T CH E CK 4. What is the relationship between pK and pH? 5. How does temperature influence the pK of water? What might this mean for animals that experience changes in body temperature? Figure 2.11 Effects of buffers on changes in pH Buffers blunt the effects of added bases (or acid) on the pH of a solution. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 35 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 35 6. What change in pH has a greater effect on proton concentration: pH 6 to 7 or pH 7 to 8? 7. What properties of a particle influence its rate of diffusion across a membrane? What membrane properties influence this rate? Pe ar so Biochemistry that are metals, such as copper, iron, magnesium, zinc, and selenium. Organic cofactors, or coenzymes, are usually derived from vitamins; coenzyme A is derived from panthothenic acid, FAD from riboflavin, and NAD from niacin. Many of the life-threatening diseases we associate with vitamin deficiencies can be traced back to perturbations of metabolism due to loss of function of specific enzymes. Animals control the inner workings of cells through the use of enzymes, which interconvert macromolecules to create building blocks and control the flow of chemical energy. A metabolic pathway is a series of consecutive enzymatic reactions that catalyze the conversion of substrates to products, with multiple stable intermediates. Flow through the pathway is called metabolic flux. Metabolic pathways can be either synthetic (anabolic), degradative (catabolic), or a combination of both (amphibolic). Energy metabolism revolves around production of ATP and other energyrich molecules. Metabolism is the sum of all these metabolic pathways within the cell, tissue, or organism. Many metabolic pathways span multiple cellular compartments, allowing cells to create distinct microenvironments. For example, the mitochondria are specialized compartments with a major role in energy metabolism. In the following sections, we discuss the nature of enzymes and metabolic energy, and the metabolism of three of the four major classes of biological macromolecules: proteins, carbohydrates, and lipids. The fourth class of macromolecules, nucleic acids, is discussed later in this chapter when we consider genetics. Enzymes accelerate reactions by reducing the reaction activation energy The laws of thermodynamics that govern chemical reactions in test tubes also apply to chemical reactions in living cells (see Box 2.1, Mathematical Underpinnings: Thermodynamics). Enzymes do not determine whether or not a chemical reaction is thermodynamically possible. However, enzymes do have the ability to accelerate thermodynamically feasible reactions by factors of 108 to 1012. Previously we discussed how substrate molecules in an uncatalyzed reaction must obtain sufficient energy to meet the activation energy barrier (EA). Once the EA is met, the substrate can adopt the transition state and then spontaneously change into the product. Although enzymatic reaction uses the same substrate and yields the same product as an uncatalyzed reaction, it produces a different intermediate at the transition state. First, the enzyme (E) and substrate (S) bind to form the ES complex. After conversion to transition states (ES*, EP*), the final product (P) is formed and then is released by the enzyme. This is represented as shown: S E ES ES* EP* EP E Le No ar tF n or in Di Re g s O tri r bu tio sa n n Enzymes Enzymes are biological catalysts that convert a substrate to a product. Enzymes, like other types of catalysts, have three properties: (1) they are active at very low concentrations within the cell; (2) they increase the rate of reactions but they themselves are not altered in the process; (3) they do not change the nature of the products. Although some enzymes, called ribozymes, are made of RNA, most enzymes are composed of protein. Many enzymes possess nonprotein components, called cofactors. A cofactor that is covalently bonded into the enzyme is called a prosthetic group. Some enzymes use cofactors The energy required to reach this intermediate state is lower than in the uncatalyzed reaction (Figure 2.12). With a lower energy barrier, more of the substrate molecules possess enough energy to reach the transition state, and the reaction is accelerated. Like other chemical reactions, enzyme reactions are reversible, proceeding through the same set of reaction intermediates. An enzymatic reaction begins with the substrate binding at a specific location called the active site. Think of the active site as a pocket into which the substrate fits. The enzyme can bind the substrate only if it possesses the proper conformation. The three-dimensional folding of the enzyme, maintained by weak bonds, forms the active site. So le lu t io n P s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 36 36 PART ONE The Cellular Basis of Animal Physiology BOX 2.1 MATHEMATICAL UNDERPINNINGS Thermodynamics G. All reactions that occur spontaneously possess a negative G; free energy was released. Chemists evaluate these parameters under standard conditions. The standard free energy, or G is assessed at 25C, with each reactant, including H , present at a concentration of 1 M. The proton concentration used by chemists equates to pH 0, which is not relevant to biological systems. When we use the laws of thermodynamics to discuss biological systems, the parameters must be altered to reflect normal cellular conditions. When biochemists adjust G for standard conditions, including a pH of 7.0, the symbol G is used. It is important to distinguish between G and G when discussing chemical reactions. The value of G is a constant. It tells how much free energy is available when a reaction begins under standard conditions. The value of G, the actual free energy of a reaction in a cell, depends upon the concentrations of reactants. If a reaction is close to equilibrium, then G equals zero. For the reaction A BY Z Pe ar so All chemical reactions, whether they occur in test tubes or biological systems, are governed by the laws of thermodynamics. The first law of thermodynamics deals with conservation of energy. The energy within a substrate is either transferred to the product or released. The first law doesn't tell us if the reaction will go forward or backward, only that the energy transformations must be balanced. The second law of thermodynamics provides a way of predicting if a reaction is likely to occur. It says that spontaneous processes occur in the direction that will increase randomness, or entropy ( S). Throughout this chapter we discuss many examples of increases in entropy. When table salt dissolves in water or ice melts, the molecules that were once in a well-ordered crystal begin to disperse. Diffusion also illustrates the principle of spontaneous increases in entropy. Solutes at high concentration tend to disperse to regions of lower concentration. Collectively, these laws tell us that the total energy of the universe is constant but that it tends toward randomness. What does this mean for chemical reactions? As we discovered earlier, spontaneous chemical reactions liberate thermal energy. Some of this thermal energy is used within the system to increase randomness or entropy. The remainder of the thermal energy is called free energy ( G) because it is available for other purposes. The equation relating enthalpy ( H), entropy ( S), free energy ( G), and temperature (T) was first proposed by J. Willard Gibbs in 1878. Le No ar tF n or in Di Re g s O H n the relationship between G, G, and concentration is defined by the following equation where R is the gas constant: From this equation, we see two factors that influence biological systems. First, the change in energy associated with randomness is dependent upon temperature. This is because the potency of a fixed amount of thermal energy, or its ability to induce randomness, depends on temperature. Thermal energy is more effective at inducing entropy at low temperature. The second principle is more apparent if we rearrange the equation to isolate G. tri G H r bu tio T S T S sa or When the reaction is at equilibrium G 0 and the mass action ratio is equal to Keq, the equation is reduced to 0 n G The amount of free energy available in a reaction is the difference between the total energy change and the amount of energy associated with the change in randomness. This equation allows us to predict if a reaction will occur spontaneously. If a reaction is to occur spontaneously, the amount of energy potentially released by a reaction ( H) must be greater than the energy used to increase entropy (T S). Recall that exothermic reactions, those that release heat, have a negative H. Similarly, reactions that release free energy have a negative We can measure Keq directly by letting the reaction reach equilibrium. The value of G can be calculated from the equation above. Knowing Keq and G, we can calculate the amount of actual free energy G available for a reaction at any concentration of reactants. Remember that G represents the maximal amount of free energy theoretically available from a reaction, under a constant temperature and pressure that approximates conditions found in the cell. Cells use enzymes to mediate chemical reactions and transfer as much energy as possible to other useful forms. Some enzymes mediate reactions that store energy as chemical energy, such as ATP or NADH. Free energy can also be used to create electrochemical gradients. The ability to divert free energy into useful forms is central to the success of living organisms. So le lu t G G RT ln 3A 4 3B4 3Y4 3Z 4 G RT ln Keq G RT ln Keq io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 37 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 37 EA (uncatalyzed) Free energy ES S EP Pe ar so Figure 2.12 EA (catalyzed) [P] Change in free energy Slope = V = ( P Time P ) Time Time Enzymes and EA Enzymes are biological catalysts that accelerate reactions without changing the nature of the product. When the substrate (S) binds the enzyme (E), the enzyme-substrate complex (ES) is formed. The enzyme alters the substrate through a series of transition states, ultimately releasing the product (P). The rate is faster than the noncatalyzed rate because of the lower activation energy (EA). Once it binds the substrate, the enzyme induces a change in the molecular structure of the substrate, perhaps as subtle as a shift in the distribution of electrons across a particular bond or a twist in the substrate molecule. By inducing these subtle structural changes in the substrate, the enzyme makes the substrate more likely to spontaneously undergo more significant changes. Many enzymes require two or more substrates. These enzymes accelerate reactions by bringing destabilized reactants in close proximity. All together, these changes increase the probability that the substrate will undergo a major change in structure toward the formation of EP*. Le No ar tF n or in Di Re g s O tri r bu tio sa n n Figure 2.13 Time course of enzyme reaction Enzyme assays begin with the addition of substrate to the reaction. The enzyme rapidly converts the substrate (S) to product (P). The buildup of [P] eventually slows the reaction, as P competes with S for the active site. The initial velocity (V) is the fastest because P has not yet accumulated. Enzyme kinetics describe enzymatic properties Enzymes accelerate reaction rates, and make possible reactions that would not normally occur at a useful rate. However, cells must ensure that enzymatic reactions occur not at the fastest possible rate, but at the appropriate rate. Thus, enzyme activity is regulated within complex metabolic pathways. The conditions that influence the rate of enzymatic reactions are referred to as enzyme kinetics. The simplest way to influence an enzymatic reaction is to change the concentration of substrates (S) or products (P). We use the reaction S P to illustrate the importance of substrate concentration ([S]) in two experimental scenarios. The first scenario illustrates how the buildup of [P] influences the rate of the forward reaction (Figure 2.13). When the reaction begins, there is no product ([P] 0). As it proceeds, molecules of P accumulate and eventually compete with molecules of S for the same active site. Finally, the reaction approaches equilibrium, where the forward and reverse reaction rates are equal and the mass action ratio equals Keq. We can determine the initial velocity of the forward reaction (V) from the slope of the curve before P accumulates. The second scenario illustrates how the initial [S] influences the enzymatic rate (Figure 2.14). The experiment previously described is repeated many times using a wide range of starting [S]. Increasing [S] from a low concentration to a higher concentration causes a proportional increase in V. Under these conditions, a higher [S] increases the frequency with which molecules of S find the active site. However, after a point, increases in [S] no longer cause a proportional increase in V. The higher abundance of S molecules still increases the probability of a collision with E. However, if S encounters E in the midst of a reaction cycle, the enzyme is unable to bind S. Eventually, E is saturated with S molecules and further increases in [S] do not increase V beyond a maximal rate So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 38 38 PART ONE The Cellular Basis of Animal Physiology Vmax Vmax Initial velocity (mmoles/min) Pe ar so 1/2 Vmax 1 V 1/2Vmax Km [S] Km Substrate concentration (mM) Figure 2.14 The Michaelis-Menten rectangular hyperbola Each point on the curve represents the initial velocity (V) calculated as shown in Figure 2.16. The maximal velocity (Vmax) is the velocity at which the curve reaches an asymptote. The Km is the [S] required to reach a velocity that is one-half of the maximal velocity. (Vmax). When enzymes are at Vmax, each molecule of enzyme has a characteristic number of catalytic cycles per unit time, known as the turnover number or kcat. A high rate of enzymatic activity could be achieved by a cell, in principle, in either of two ways. Some enzymes work very fast, and show a high kcat. The cell does not need many molecules of the enzyme because each molecule works quickly. The fastest enzymes can undergo more than 40,000,000 catalytic cycles each second. Alternatively, cells can make many copies of an enzyme with a low kcat. The relative importance of each strategy--faster enzymes versus more enzymes-- depends on the nature of the reaction and the design of the enzyme. The relationship between [S] and V was first described mathematically by the biochemists Leonar Michaelis and Maud Menten as a rectangular hyperbola. The Michaelis-Menten equation is Le No ar tF n or in Di Re g s O tri r bu tio 3S4 3S4 Km n Figure 2.15 Homotropic enzymes and sigmoidal kinetics Not all enzymes obey MichaelisMenten kinetics. Homotropic enzymes show sigmoidal kinetics. The enzymes usually possess multiple active sites. When the enzyme binds one molecule of S, the changes in conformation increase the ability to bind a second molecule of S. The slope of the linear range of the curve indicates the degree of cooperativity. The slope of this region provides the Hill coefficient. sa n V VMAX The value for the Michaelis-Menten constant (Km) is the concentration of substrate [S] required to obtain an initial velocity (V) that is half the maximal velocity (Vmax). Km is an indicator of the affinity of an enzyme for a substrate. A low Km means that the enzyme has high affinity for the substrate, and little substrate is needed to drive the reaction at a high rate. Not all enzymes demonstrate hyperbolic Michaelis-Menten kinetics. For instance, homotropic enzymes show a sigmoidal relationship between V and [S] (Figure 2.15). Homotropic enzymes typically have multiple subunits that can each bind a substrate molecule. At low [S], each active site has a low affinity for S. The enzyme does not bind S very well and the reaction velocity is slow. Once one subunit binds one molecule of S, it undergoes a change in conformation that in turn alters the ability of other subunits to bind a substrate molecule. As a result, doubling of [S] more than doubles V, a phenomenon called cooperativity. The degree of cooperativity is described by the Hill coefficient, which is the slope of relationship at the point of inflection. Enzyme kinetics are assessed under carefully controlled experimental conditions that do not approximate normal cellular conditions. Interpreting the impact of enzyme kinetics in living cells is often difficult. The conditions necessary to evaluate Vmax require [P] to be zero, which never occurs in living cells. Thus, enzymes in cells almost never could proceed at Vmax. As with other chemical reactions, the rate and direction of the enzymatic re- So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 39 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 39 Pe ar so action depend on the difference between the mass action ratio, which is calculated from the actual [S] and [P], and the Keq value, which is the expected [S] and [P] when the reaction reaches equilibrium. In a near-equilibrium reaction, the mass action ratio is close to Keq; the forward and reverse directions continue at equal rates, with little net change in [S] or [P]. Most enzyme reactions are far from equilibrium in cells. If the mass action ratio is lower than Keq, then the reaction will proceed in the forward direction. When the mass action ratio is higher than Keq, the reaction will tend to favor the reverse direction. By altering the concentrations of substrates and products, cells can regulate enzyme activities and metabolic pathways. Vmax 0 0.1 0.2 0.3 [KCI] (M) 0.4 0.5 (a) The physicochemical environment alters enzyme kinetics Every enzyme has a characteristic optimal activity under a specific set of environmental conditions (Figure 2.16). Enzyme kinetics are influenced by environmental conditions, such as temperature, pH, salt concentration, and hydrostatic pressure. While these factors generally have little impact on your metabolism, such environmental factors can influence the metabolic biochemistry of other species. Some enzymes function optimally under conditions that resemble normal cellular conditions. For instance, mammalian enzymes often function optimally at normal body temperatures of 3740C. However, the optimal conditions for many enzymes bear little similarity to normal cellular conditions; the optimal temperature for some mammalian enzymes is well above normal body temperatures. Environmental conditions typically influence enzyme kinetics through effects on weak bonds. First, changes in weak bonds can alter the threedimensional structure of the enzyme. For instance, warm temperatures could break bonds that are necessary to form the active site. Second, environmental conditions can alter the ionization state of critical amino acids within the active site. For instance, the amino acid histidine is important in many active sites, and changes in pH can alter its protonation state and consequently substrate affinity (Km). Any environmentally induced change in Km, either an increase or a decrease, can be disruptive to a cell. Third, environmental conditions can alter the ability of the enzyme to undergo structural changes necessary for catalysis. Enzymes must be rigid enough to maintain the Le No ar tF n or in Di Re g s O Vmax 0 n 10 20 30 40 50 60 tri r bu tio sa (b) Figure 2.16 Effects of salt and temperature on enzyme kinetics Most enzymes function optimally under n physiologically-realistic conditions. (a) The activity of mammalian enzymes changes in response to the concentration of the salt KCl. Maximal activity occurs at concentrations that approximate those found within the cell (100150 mM K ). (b) Increasing temperature accelerates enzymes. Beyond an optimal temperature, the enzyme denatures and loses catalytic activity. So le lu t Temperature (C) io n proper conformation, but flexible enough to incur conformational changes during catalysis. Many of the studies assessing the effects of environmental conditions have focused on the effects of temperature on the enzyme lactate dehydrogenase (LDH). This enzyme has an important role in glucose metabolism, which we discuss in more detail later in this chapter. It catalyzes the following reversible reaction: Pyruvate NADH H lactate NAD s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 40 40 PART ONE The Cellular Basis of Animal Physiology Environmental conditions can change the Km value of LDH for pyruvate and NADH. Lowering temperature increases the affinity of the enzyme for its substrate pyruvate (Figure 2.17). When comparing the effects of temperature on Km in different species, several patterns emerge. First, in every species, the Km value decreases as temperature decreases. Second, at any temperature, each species shows a very different Km value. For example, when assayed at 15C, Antarctic fish LDH has a high Km, LDH from temperate fish has an intermediate Km, and desert lizard LDH has a low Km. Third, when the LDH from each species is assayed at its normal body temperature, the resulting Km values fall within a narrow range, from 0.1 to 0.3 mM. Evolutionary variation in LDH structure is responsible for the differences between species. These structural variations provide all the species with an enzyme that demonstrates similar kinetics under their natural conditions. This pattern, called conservation of Km, is common when comparing enzyme kinetics of different animals. Pe ar so Allosteric and covalent regulation control enzymatic rates Molecules that do not participate directly in catalysis can also alter enzyme kinetics. Competitive inhibitors are molecules that can bind to the active site, preventing substrate molecules from binding (Figure 2.18a). The effectiveness of a com0.6 0.5 Le No ar tF n or in Di Re g s O tri 30 Temperate fish Desert lizard Conserved range Polar fish n Km (mM) 0.4 0.3 0.2 0.1 0 0 Antarctic fish r bu tio 40 50 sa n petitive inhibitor depends on [S]. When [S] is low, the inhibitor outcompetes S for the active site, reducing the reaction rate. At a very high [S], the inhibition by the competitor is greatly reduced. Thus, a competitive inhibitor increases Km but doesn't affect Vmax. Allosteric regulators are molecules that alter enzyme kinetics by binding to the protein at locations far away from the active site. The allosteric regulator alters the three-dimensional structure of the enzyme, inducing complex changes in enzyme kinetics. For example, an allosteric activator could increase the affinity of the enzyme for the substrate, as depicted in Figure 2.18b. Allosteric effectors can activate or inhibit enzyme activity, changing either Km or Vmax. Enzymes often possess multiple sites for different allosteric regulators. Enzymes controlled by allosteric regulators are often larger and more complex than other enzymes. Typically, each metabolic pathway is regulated by one or more key allosteric enzymes. Enzymes can also be regulated by the covalent modification of critical amino acid residues within the protein. The most common type of covalent modification is protein phosphorylation, where a specific protein kinase transfers the phosphate group from ATP to an amino acid of the target enzyme. For instance, tyrosine kinase is a regulatory enzyme that phosphorylates target proteins at specific tyrosine residues. Another common class of protein kinases is specific for threonine and serine residues. Protein phosphorylation is reversible. Cells possess suites of protein phosphatases that cleave phosphate groups from phosphorylated amino acid residues. Phosphorylation might stimulate an enzyme, as depicted in Figure 2.18c, or inhibit it. So le lu t Enzymes convert nutrients to reducing energy io n 10 20 Temperature (C) Figure 2.17 Conservation of Km The Km of an enzyme often changes with temperature. For a number of unrelated species, the Km of LDH for pyruvate (KmPYR) increases with an increase in temperature; that is, at warmer temperatures LDH is less able to bind pyruvate. However, when you examine the kinetic values that would occur at the actual body temperatures for the animal, you find that the Km values are very similar across species. (Source: Data from Hochachka and Somero, 2002) Enzymes transfer energy from nutrients to molecules that function as energy stores. These energyrich molecules are a type of energy currency, acting as substrates and products for hundreds of different enzymes. Cells store chemical energy in two main forms: reducing energy and high-energy molecules. Many enzymatic reactions capture energy in the form of reducing equivalents: NAD and NADP. The enzymes that use reducing equivalents are called oxidoreductases and include enzymes s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 41 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 41 Vmax Substrate Inhibitor Inhibitor Pe ar so Inhibitor (a) Competitive inhibition Substrate bound with low affinity Active site Activator (b) Allosteric activation Active site + Inhibitor + Inhibitor V [S] Km Km (inhibited)(uninhibited) Le No ar tF n or in Di Re g s O Substrate bound with high affinity Allosteric site + Activator Allosteric regulator n Vmax + Activator Activator V [S] Km Km (+ Activator) (No activator) Phosphorylated Active site tri ATP Protein kinase V OH Pi Protein phosphatase Unphosphorylated Phosphorylated (c) Covalent activation r bu tio ADP O n sa So le lu t Vmax O P O O Vmax [S] Figure 2.18 Enzyme regulation (a) Competitive inhibitors are able to bind to the active site of enzymes, thereby preventing the real substrate from binding. At low [S], the inhibitor outcompetes the substrate. However, if [S] is increased to very high levels, the true substrate outcompetes the inhibitor, and thus these regulators have no effect on Vmax. (b) Allosteric enzymes are regulated by molecules that bind at sites distant from the active site. The resulting structural change in the enzyme alters its kinetic properties. In this figure, the allosteric regulator activates the enzyme by increasing the affinity for the substrate, shown in the graph as a decrease in the Km. (c) Many enzymes are controlled by phosphorylation-dephosphorylation. Protein kinases phosphorylate the target enzyme, transferring a phosphate group from ATP to specific hydroxy groups. Protein phosphatases remove the phosphate group. In this figure, the enzyme is activated by phosphorylation, greatly increasing the Vmax. io n Unphosphorylated s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 42 42 PART ONE The Cellular Basis of Animal Physiology with the common names dehydrogenase, reductase, and oxidase. When an enzymatic reaction transfers an electron to NAD (or NADP ), the reduced NADH (or NADPH) that is formed can be used to drive other reactions. In other words, energy can be stored by reducing a molecule and this energy can be recovered, in part, by oxidizing the reduced compound. Consider the nonenzymatic and enzymatic reactions for lactate oxidation. Without an enzyme, lactate is oxidized to form pyruvate with the following reaction: NH2 O O O O P O O O P O H OH ATP COO O CH2 H N C +NH2 N CH3 O CH2 O H H N N N P O N The negative standard free energy ( G) means that energy is liberated in this reaction, and without an enzyme the energy released would be lost as heat. Cells possess the enzyme LDH, introduced earlier in this chapter, which couples lactate oxidation to NADH reduction. The NAD reduction reaction has a positive G. NAD 2e G 2H NADH 62 kJ/mol H Pe ar so H OH Lactate pyruvate 2H G 36 kJ/mol 2e O P O By coupling lactate oxidation to NAD reduction, the enzymatic reaction captures free energy from lactate oxidation in the form of NADH. Lactate NAD NADH H G 26 kJ/mol Note that the enzymatic reaction for lactate oxidation has a positive G, which means the reverse direction of this reaction (lactate formation) is normally favored. The most important reducing equivalent in energy metabolism is NADH. The reducing energy within the cell, or redox status, is best expressed as [NADH]/[NAD ]. This ratio is high when a cell is rich in reducing energy, and low when cells are energy poor. NAD is a reactant in many enzymes of energy metabolism, but other enzymes are allosterically regulated by NAD. Whether acting through mass action effects or allosteric regulation, enzymes sensitive to [NADH]/[NAD ] allow metabolic pathways to respond to the energy state. Le No ar tF n or in Di Re g s O O n Phosphocreatine O P H N C H2N O NH3 H N H C H H C H H C H C C O Phosphoarginine H O tri pyruvate r bu tio Figure 2.19 n High-energy molecules Cells use several energy-rich molecules, such as ATP, phosphocreatine, phosphoarginine, and acetyl CoA, as energy currency. sa So le lu t O S C CH3 CoA Acetyl CoA less reactions. ATP synthesis requires energy, and ATP breakdown liberates energy. ADP3 HPO42 G H ATP4 30.5 kJ/mol H2O io n s ATP is a carrier of free energy Cells use many types of molecules to store energy (Figure 2.19), but ATP is the most versatile of these high-energy molecules and participates in count- ATP possesses two phosphodiester bonds (POP). Some enzymes break the bond between the second and third phosphate groups, forming ADP. In some cases the inorganic phosphate (Pi) is released as a product, but often the Pi is transferred to another molecule. Other enzymes target the bond between the first and second phosphate groups, forming AMP and pyrophosphate (PPi). Because these energy exchange reactions involve 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 43 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 43 Pe ar so Gp a breakdown of a phosphodiester bond, they are often called high-energy bonds. It is important to realize that the energy is not stored in the bond per se, but is released when ATP hydrolysis occurs--a reaction with large, negative free energy. The importance of utilizing a metabolite like ATP is, first, to avoid high concentrations of other metabolites; participation of ATP permits reactions that otherwise would be thermodynamically unfavorable. Second, ATP links major metabolic pathways that require cellular energy, such as endergonic pathways of biosynthesis, with those that generate energy, such as the exergonic process of carbohydrate catabolism. The relative abundance of ATP reflects the energy status of a cell. The absolute concentration of ATP is unimportant; what counts is the relative proportion of the adenylate pool (ATP ADP AMP) that exists in the energy-rich forms ATP and ADP. The ATP status of the cell is best expressed by the phosphorylation potential ( Gp), the free energy associated with ATP hydrolysis (ATP ADP Pi): dine is transferred to ADP to form ATP. In vertebrates, creatine phosphokinase (CPK) catalyzes this reaction. Phosphocreatine ADP ATP creatine Acetyl coenzyme A, or acetyl CoA, is another important high-energy store. Energy is released in reactions that hydrolyze its thioester bond (OS). As we see later in this chapter, many pathways of biosynthesis and energy metabolism intersect at acetyl CoA. Collectively, reducing energy and high-energy compounds provide the energetic support for many cellular processes. ATP is the most common form of energy currency, but the other nucleotides--GTP, TTP, and CTP--have the same energetic value, although only GTP is commonly used in energy metabolism. Phosphorylated guanidine derivatives are important energy stores in many animals. Vertebrates use phosphocreatine and invertebrates use phosphoarginine, phosphoglycocyamine, phosphotaurocyamine, or phospholombricine. Phosphoguanidine compounds, each with a PN bond, are useful energy stores because they do not participate in many reactions within the cell. Consequently, cells can accumulate very high concentrations of phosphoguanidines without affecting other pathways. The concentration of ATP, in contrast, is kept low and relatively constant. Major changes in ATP concentration would have kinetic consequences for countless enzymes that use ATP as a substrate or product. For instance, the ATP concentration in vertebrate muscle is typically about 5 mM, whereas phosphocreatine concentrations might be 1050 mM. Animal tissues use these high-energy compounds when the need for ATP temporarily outstrips the capacity to produce ATP. When ATP levels decline, the energy within phosphoguani- Le No ar tF n or in Di Re g s O G RT ln 3ADP4 3Pi 4 3ATP4 n Proteins Proteins play many important roles in cell structure and function. Almost all enzymes are proteins (though many have nonprotein components). Proteins form the internal skeleton of a cell (cytoskeleton) as well the extracellular matrix needed to organize cells into complex tissues. The diversity in protein structure is afforded by the use of 20 amino acids that can be strung together in countless combinations. The blueprint for all proteins in a cell is in the form of DNA, which is transcribed into RNA and translated to form the appropriate proteins at the right time. tri r bu tio sa Proteins are polymers of amino acids n Animals build proteins from combinations of 20 amino acids. As the name implies, amino acids share the general structure of an amino group (NH2) and a carboxylic acid group (COOH). They are called -amino acids because both the amino and carboxyl groups are located on the first, or , carbon. Amino acids are distinguished from one another by their side groups (R). The R groups of polar amino acids form hydrogen bonds with water. Some polar amino acids are uncharged at physiological pH values (serine, threonine, cysteine, tyrosine, asparagines, glutamine), while others possess R groups with side chains that can become charged. Acidic amino acids (aspartate, glutamate) are negatively charged at physiological pH when carboxyl groups become deprotonated (COOH COO H ). Basic amino acids (arginine, lysine) take on a positive charge when amino groups become protonated (NH2 H NH3 ). So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 44 44 PART ONE The Cellular Basis of Animal Physiology Many amino acids are nonpolar because their R groups are aliphatic chains (alanine, valine, leucine, isoleucine, methionine) or aromatic rings (phenylalanine, tryptophan) that do not readily interact with water. The collection of amino acids, with their unique properties of side chain length, shape, charge, and polarity, provides cells with the building blocks necessary to construct thousands of different proteins. Proteins are folded into threedimensional shapes Amino acids are polymerized into linear chains by covalent peptide bonds that link the amino group (NH3) of one amino acid to the carboxyl group (COOH) of another amino acid. H | R1-N-H | H O H O || | || H-O-C-R2 R1-N-C Pe ar so Two amino acids in a chain is a dipeptide. Polypeptides are longer chains of amino acids. At one end of the polymer, called the C terminus, the amino acid has an unbonded carboxyl group. At the other end, the N terminus, the amino acid has an unbonded amino group. The linear sequence of amino acids in a protein is called the primary structure. Once the primary structure is established, proteins are organized into more complex threedimensional conformations (Figure 2.20). First, the Le No ar tF n or in Di Re g s O R2 H2O n tri H N R H N R C H C O C C H r bu tio sa protein folds onto itself to assume its secondary structure. The information for proper folding is contained directly in the primary structure. The size, charge, and polarity of the side groups influence the interactions between amino acids in the chain. Secondary structures arise when side groups of amino acids interact to form a structure that is more stable than the simple linear conformation. The two most common protein secondary structural motifs are the -helix and the -sheet (Figure 2.21). In the -helix, the protein is twisted into a spiral with 3.6 amino acids per turn and side chains extending outward. The structure is stabilized in two ways. First, hydrogen bonds form between the C O of one amino acid and the NH of the amino acid four positions along the chain. Second, the -helix structure is stabilized when opposing side chains can interact. With the period of 3.6 amino acids, a side chain is exposed to the side chain of the amino acid three or four positions away. For example, if two aromatic amino acids are three positions apart, when the protein twists into an -helix the structure will be stabilized by the hydrophobic interactions between the side chains. Similarly, negatively charged amino acids are often found three residues away from positively charged amino acids. Their electrostatic interactions stabilize the protein. The other common type of secondary structure, the -sheet, forms when linear regions of a protein align side by side and form hydrogen bonds. In this conformation, the side chains extend above and below the face of the sheet. Once a protein forms its secondary structure, the different regions fold together to create its n So le lu t io n s Primary structure Secondary structure Tertiary structure Quaternary structure Figure 2.20 Protein structural levels The amino acid sequence of a protein is its primary structure. This polypeptide can then be folded and organized into three-dimensional conformations. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 45 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 45 Disulfide bond Ionic bond H C S S H C H C C O C O+ H H N H H C H H C H Pe ar so -Helix -Pleated sheet H Figure 2.21 Protein secondary structure: The -helix and -sheet The most common secondary structures in proteins are the -helix and -sheet. Weak bonds stabilize both types of secondary structures. The information that is used to fold the protein is contained within the primary sequence. tertiary structure (Figure 2.22). If the protein folds in a way that allows two adjacent cysteine residues to come into close proximity, their sulfhydryl groups (SH) can form a covalent bond (SS) called a disulfide bond or bridge. Multiple weak bonds link various amino acids and side chains to stabilize three-dimensional structure. Many proteins assume a globular structure when hydrophobic interactions form between regions scattered throughout the protein. By pulling together hydrophobic regions, a hydrophobic core is formed that stabilizes the structure of the protein. A protein achieves its quaternary structure when multiple subunits, or polypeptide chains, are brought together. Proteins with two subunits are called dimmers--a homodimer if the monomers are identical, otherwise a heterodimer. Proteins can be composed of even larger numbers of subunits, such as trimers (three subunits) and tetramers (four subunits). Le No ar tF n or in Di Re g s O H C H H n Folded protein Hydrophobic interaction H C H H H C H H C H Hydrogen bond H N H O=C tri r bu tio sa Figure 2.22 Weak bonds and protein tertiary structure Both covalent bonds and weak bonds contribute to protein three-dimensional structures. n Molecular chaperones help proteins fold Proteins can function properly only when they are folded into the correct conformation. Many proteins can use the information within the primary sequence to fold spontaneously, but others require the help of molecular chaperones. Each cell contains different types of chaperones to ensure that proteins are properly folded. They work by forcing the protein into a conformation that allows the appropriate weak bonds to form. Environmental conditions, such as temperature, can alter weak bonds and disrupt threedimensional protein structure. Increasing temperature weakens the hydrogen bonds that stabilize -helices and -sheets. High temperature can cause the protein to unfold, or denature. Once denatured, a protein can no longer perform its proper function and may even damage cells. Therefore, a partially denatured protein must be So le lu t C H C H H H io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 46 46 PART ONE The Cellular Basis of Animal Physiology HOCH2 6 5 4 HOCH2 O 1 2 4 6 5 O 1 2 OH OH 3 OH 3 HO OH HO OH OH -D-Glucose (Glc) Pe ar so HOCH2 HO O OH OH OH -D-Galactose (Gal) HOCH2 O OH OH HO H NH2 -D-Glucosamine (GlcN) -D-Glucose (Glc) refolded or destroyed before it can damage the cell. Molecular chaperones bind to denatured proteins, folding them into the proper configuration. During heat stress, cells increase the levels of molecular chaperones called heat shock proteins to cope with the increased number of denatured proteins. HOCH2 O OH OH OH CH2OH Carbohydrates Carbohydrates share a preponderance of hydroxyl (OH), or alcohol, groups, and for this reason they are often called polyols. For any animal, the diet is a vital source of the carbohydrates used to build and fuel cells. Glucose, the most common carbohydrate in animal diets, is central to cellular energy metabolism and biosynthesis because of its metabolic versatility. Cells can break glucose down for energy, or store it for later consumption, or use it to build other carbohydrates needed by the cell. -D-Fructose (Fru) Monosaccharides are small carbohydrates that have from three to seven carbons. The most comFigure 2.23 Common monosaccharides These mon monosaccharides are the six-carbon sugars structural models of monosaccharides show how side (hexoses) including glucose, fructose, and galacgroups extend above and below the plane of the ring tose (Figure 2.23). Glucose and galactose, as well structures. The and forms of glucose differ in the orientation of the hydroxy group on C-1. as mannose, can be modified by the addition of acidic groups, amino groups, and modified amino groups. These sugar derivatives serve many purposes in the cell, primarily as modifications of other macromolecules, HOCH2 HOCH2 including proteins, lipids, and nuO OH O HOCH2 cleic acids. 1 4 O O OH HO OH OH O Many of the sugars that animals O HOCH2 HO 1 1 OH OH obtain in the diet are disaccharides, OH OH two monosaccharides connected by OH a covalent bond (Figure 2.24). In orOH Lactose (Gal (1-4) Glc) Trehalose (Glc (1-1) Glc) der to use disaccharides, animals first break them down into monosaccharides. Animals can also proHOCH2 HOCH2 HOCH2 duce disaccharides such as lactose, O O OH O HOCH2 O HO an important component of milk in 1 2 1 4 OH OH OH HO O mammalian mammary secretions, O CH2OH HO and trehalose, an energy store and OH OH OH OH solute. Sucrose (Fru (2-1) Glc) Maltose (Glc (1-4) Glc) The addition of carbohydrates to other macromolecules is called Figure 2.24 Common disaccharides Both trehalose and maltose are made glycosylation. Glycosylated lipids from two glucose molecules but with bonds forming between different pairs of carbons. (glycolipids) and proteins (glycoSucrose and maltose are synthesized in plants; animals obtain them by eating the plants. Le No ar tF n or in Di Re g s O O OH OH H HO NH C O CH3 N-Acetyl--D-glucosamine (GlcNAc) n HOCH2 Animals use monosaccharides for energy and biosynthesis tri r bu tio sa n So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 47 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 47 proteins) are common in the plasma membrane of cells. A glycosylated macromolecule displays an altered molecular profile, changing how it interacts with other macromolecules and reducing its susceptibility to degradation. HOCH2 O HOCH2 O O OH OH OH O HOCH2 O OH OH O HOCH2 O OH OH O OH O Amylose Pe ar so Complex carbohydrates perform many functional and structural roles Complex carbohydrates, or polysaccharides, are larger polymers of carbohydrates that serve in energy storage and structure. Polysaccharides can be composed of long chains of a single type of monosaccharide or combinations of two alternating monosaccharides. Common polysaccharides important in metabolism and structure are shown in Figure 2.25. Starch is a general term for the glucose polysaccharides used by plants and animals for energy storage. Plant starch, a mixture of amylose and amylopectin, is an important dietary source of energy for many animals. Animal starch, or glycogen, is central to animal energy metabolism, acting as an internal energy store for most animals and a nutrient for animals that eat other animals. Amylose, amylopectin, and glycogen differ in the linkage between glucose molecules and the nature of the branching pattern. Cellulose, another plant-derived glucose polymer, is essentially indigestible in animals because of the nature of the bonds between glucose units. Cellulose, in most animals, provides dietary fiber. However, some animals, such as ruminants and termites, possess gastrointestinal symbionts that can degrade cellulose for energy. Polysaccharides are also critical structural components of animal cells. Arthropods build their exoskeletons with chitin, a polysaccharide of N-acetyl-glucosamine. Vertebrates secrete hyaluronate, a polymer of N-acetyl-glucosamine and glucuronic acid, into the extracellular space, where its gel-like properties act as a spacer between cells and tissues. Hyaluronate is a member of a class of compounds called glycosaminoglycans that include chondroitin sulfate and keratan sulfate. These compounds are important components of animal tissues, such as cartilage. In order to use glycogen as an energy store, animals control the balance between glycogen synthesis (glycogenesis) and glycogen breakdown (glycogenolysis). Glycogen phosphorylase initiates glycogenolysis, releasing glucose in the form of glucose 1-phosphate. When glucose is Amylopectin Le No ar tF n or in Di Re g s O Glycogen HOCH2 O OH n (a) Glucose polymers HOCH2 O O OH NH O C CH3 O O O OH NH C CH3 O HOCH2 O O NH C CH3 GlcNAc GlcNAc GlcNAc tri r bu tio n sa So le lu t Chitin HOCH2 COO O O COO O O HO OH O O NH OH OH C O OH CH3 HOCH2 O OH HO O NH H O GlcA Hyaluronate GlcNAc GlcNAc (b) Glucose and amino sugar polymers io n CH3 C O GlcNAc Figure 2.25 Polysaccharides (a) Plants and animals use polymers of glucose as energy stores. Amylose and amylopectin are the two polysaccharides that compose starch, an important dietary source of energy for animals. Animals produce glycogen, which resembles the plant polysaccharides but with much greater branching. (b) Animals build many polysaccharides from combinations of monosaccharides and amino sugars, such as N-acetylglucosamine (GlcNAc). Chitin is a polymer of N-acetylglucosamine, whereas hyaluronate is a polymer of N-acetyl-glucosamine and glucuronic acid (GlcA). s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 48 48 PART ONE The Cellular Basis of Animal Physiology Glycogen synthase (inactive) Protein kinase Protein phosphatase Pyruvate Glycogen synthase (active) Pyruvate CO2 Pe ar so Glycogen (n glucose) Pyruvate carboxylase Glycogen (n+1 glucose) GTP GDP Oxaloacetate CO2 GTP Glycogen phosphorylase (active) Glycogen phosphorylase phosphatase PEPCK Malate Glycogen phosphorylase kinase GDP Phosphoenolpyruvate Glycogen phosphorylase (inactive) Figure 2.26 Control of glycogen synthase and glycogen phosphorylase Under conditions in which glycogen breakdown is desirable, both glycogen synthase and glycogen phosphorylase are phosphorylated by protein kinases. Phosphorylation inhibits glycogen synthase but stimulates glycogen phosphorylase. Similarly, dephosphorylation of these two enzymes by protein phosphatases favors glycogen synthesis. abundant, glycogen synthase is activated and glucose 1-phosphate is used to increase the size of the glycogen particle. Protein kinases and protein phosphatases regulate both glycogen synthase and glycogen phosphorylase (Figure 2.26). Le No ar tF n or in Di Re g s O tri r bu tio sa n n Malate NAD+ NADH Oxaloacetate CO2 GTP GDP Phosphoenolpyruvate 2-Phosphoglycerate PEPCK Gluconeogenesis builds glucose from noncarbohydrate precursors Glucose is essential for energy metabolism and biosynthesis. When dietary glucose is inadequate or when glycogen stores are compromised, animals can produce glucose from noncarbohydrate precursors via gluconeogenesis. The gluconeogenic pathway (Figure 2.27) using mitochondrial pyruvate as a starting point has the following overall reaction: 2pyruvate 4ATP 2GTP 2NADH 4H2O 2H glucose 4ADP 2GDP 6Pi 2NAD So le lu t 3-Phosphoglycerate ATP NADH NAD+ ADP 1,3-Bisphosphoglycerate Glyceraldehyde 3-phosphate Dihydroxyacetone phosphate Fructose 1,6-bisphosphate io n FBPase Pi s Fructose 6-phosphate Pi Glucose 6-phosphate Figure 2.27 Gluconeogenesis Cells convert pyruvate to glucose and glycogen using the enzymes of gluconeogenesis. The exact route of phosphoenolpyruvate synthesis depends upon tissue and species. Some species use a mitochondrial PEPCK to produce phosphoenolpyruvate. Glucose 1-phosphate UTP Glucose Glycogen UDP 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 49 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 49 Pe ar so Gluconeogenesis begins in the mitochondria, where pyruvate carboxylase converts pyruvate to oxaloacetate, the substrate for PEP carboxykinase (PEPCK). In species with a mitochondrial PEPCK, PEP is transported to the cytoplasm; if PEPCK is cytoplasmic, the mitochondria convert oxaloacetate to malate, export it, and then resynthesize oxaloacetate within the cytoplasm. A series of reactions produces glucose 6-phosphate, which can be used to produce glycogen, or in some tissues converted to glucose by glucose 6-phosphatase. Because gluconeogenesis requires a great deal of energy, cells stimulate gluconeogenesis only when they have excess energy available. The metabolic indicators of energy status, such as acetyl CoA and adenylates (AMP, ADP, and ATP), regulate the gluconeogenic rate. The pathway is controlled mainly by availability of gluconeogenic substrates and allosteric regulation of pyruvate carboxylase and fructose 1,6-bisphosphatase (FBPase). Glucose Hexokinase ATP ADP Glucose 6-phosphate Phosphoglucose isomerase Glycogen Fructose 6-phosphate Phosphofructokinase ATP ADP Fructose bisphosphate Aldolase Glycolysis is a low-efficiency, high-velocity pathway Glycolysis is the pathway that breaks down glucose obtained from the blood and glucose 6-phosphate derived from processing of the glucose 1-phosphate liberated from stored glycogen. This pathway is a vital source of ATP because it can proceed in the absence of oxygen (anoxia) and can produce ATP very rapidly (albeit for brief periods). Although glycolysis is usually discussed from the perspective of glucose or glycogen breakdown, other carbohydrates derived from the diet are also processed into hexoses that can enter glycolysis. Disaccharides are first broken down into monosaccharides: trehalose into two glucose, lactose into glucose and galactose, sucrose into glucose and fructose. The glycolytic pathway (Figure 2.28) using glucose as initial substrate has the following overall reaction: Le No ar tF n or in Di Re g s O tri r bu tio sa n n Glyceraldehyde 3-phosphate Dihydroxyacetone phosphate Triosephosphate isomerase Glyceraldehyde 3-phosphate dehydrogenase 2 NAD+ 2 NADH 1,3-Bisphosphoglycerate (2) 2 ADP 2 ATP 3-Phosphoglycerate (2) Phosphoglycerokinase Phosphoglycerate mutase 2-Phosphoglycerate (2) Enolase Glucose 2ADP 2NAD 2ATP 2pyruvate 2NADH 2H When glucose is carried into the cell, the enzyme hexokinase rapidly phosphorylates it, using a molecule of ATP. Since glucose 6-phosphate is not readily transported across the cell membrane, phosphorylation of glucose traps glucose within the cell. The next steps in glycolysis are a series of enzymatic reactions that convert the glucose backbone to fructose, which is then hydrolyzed to form two trioses that are ultimately converted to pyruvate. Figure 2.28 Glycolysis Glycolysis is a series of cytoplasmic enzymes that breaks down glucose or glycogen to produce ATP. Because ATP is required by hexokinase, glycolysis from glycogen produces more ATP (three ATP per glucosyl) than it does from glucose (two ATP per glucose). The other important products of glycolysis are pyruvate and NADH. The three irreversible reactions are highlighted. So le lu t H2O Pyruvate kinase Phosphoenol pyruvate (2) 2 ADP 2 ATP Pyruvate (2) io n s Seven of the ten glycolytic reactions are freely reversible, and catalyzed by the enzymes shared with the gluconeogenic pathway. The three irreversible glycolytic reactions--hexokinase, phosphofructokinase (PFK), and pyruvate kinase (PK)--are important sites of regulation for the pathway, acting via mass action effects, allosteric regulation, and covalent modification. During periods of high energy demand, much of the ATP is broken down to ADP and 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 50 50 PART ONE The Cellular Basis of Animal Physiology AMP, affecting the mass action ratios for all three regulatory enzymes. Both ADP and AMP are powerful activators of PFK enzymatic activity, whereas ATP inhibits PFK, as well as PK. When cells do not need energy, glycolysis is inhibited at PFK and PK. With PFK inhibited, glucose 6-phosphate is diverted into glycogen synthesis. Thus, the fate of the glucose 6-phosphate--glycolysis or glycogen synthesis--is linked to energy status through regulation of PFK. This is an example of negative feedback regulation, where an increase in the concentration of products inhibits the pathway. In addition to the 2 mol of ATP per glucose, glycolysis produces 2 mol of pyruvate and NADH. Glycolysis can continue only if the cell can remove the pyruvate and NADH produced. The fate of these products depends on two factors: the metabolic demands of the cell and the availability of oxygen. Pe ar so Mitochondria oxidize glycolytic pyruvate and NADH under aerobic conditions When energy is required and oxygen abundant, pyrvuate produced in glycolysis enters the mitochondria for further oxidation. First, the enzyme Glucose NAD+ Le No ar tF n or in Di Re g s O Glycolysis Pyruvate n pyruvate dehydrogenase (PDH) produces acetyl CoA, which is further oxidized to produce CO2. The reducing energy (4 NADH and 1 FADH2) and nucleotides (1 GTP) allow mitochondria to produce the equivalent of 15 ATP from pyruvate. Since the cytoplasmic production of pyruvate produces only 1 ATP per pyruvate, considerably more energy is produced by glucose oxidation (glucose CO2) than by glycolysis (glucose pyruvate). Mitochondria also dispose of the cytoplasmic NADH produced in glycolysis. Although they cannot oxidize NADH directly, mitochondria use two redox shuttles to obtain the reducing energy of cytoplasmic NADH: the -glycerophosphate shuttle and the malate-aspartate shuttle (Figure 2.29). In the -glycerophosphate shuttle, cytoplasmic NADH is first oxidized by the enzyme -glycerophosphate dehydrogenase ( -GPDH), embedded within the mitochondrial inner membrane. Oxidation of glycolytic NADH in the -glycerophosphate shuttle generates two ATP. The malate-aspartate shuttle uses pairs of enzymes that are located in both the cytoplasm and the mitochondria. First, cytoplasmic malate dehydrogenase oxidizes NADH. This transfers the reducing energy of NADH to malate, which AspAT Oxaloacetate 6 NADH 1 G3PDH tri FADH2 NADH + H+ G3P r bu tio DHAP Aspartate G3PDH Cytosol Inner membrane FAD+ n sa So le lu t 2-Oxoglutarate 5 Inner membrane 2-Oxoglutarate 4 MDH NAD+ Malate Glutamate Aspartate Q Glutamate io n 2 Malate 3 NADH NAD+ Oxaloacetate Matrix (a) Glycerophosphate shuttle (b) Malate-aspartate shuttle s AspAT Figure 2.29 Redox shuttles (a) Glycolytic NADH can be oxidized by the combined actions of cytoplasmic and mitochondrial forms of glycerol 3-phosphate dehydrogenase. The complete -glycerophosphate shuttle leads to transfer of the reducing power of NADH to mitochondrial FADH2. (b) The malate-aspartate shuttle, shown as a series of reactions from 1 to 6, results in net transfer of NADH into the mitochondria with complete cycling of the reactants. The enzymes malate dehydrogenase (MDH) and aspartate aminotransferase (AspAT) are located in both the cytoplasm and mitochondria. This cycle also requires specific transporters capable of carrying metabolites across the mitochondrial membrane. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 51 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 51 Pe ar so enters the mitochondria and is oxidized by malate dehydrogenase. The remainder of the cycle ensures that the cytoplasm is provided with adequate oxaloacetate and the mitochondrial oxaloacetate is removed. The enzyme aspartate aminotransferase converts oxaloacetate to aspartate in the mitochondria. Aspartate is exported to the cytoplasm where another aspartate aminotransferase regenerates oxaloacetate. The other substrates and products in the aspartate aminotransferase reaction, glutamate and 2-oxoglutarate, are required in the transport of malate and aspartate. Terminal dehydrogenases oxidize NADH under anaerobic conditions Since mitochondria require oxygen to process pyruvate and NADH, the nature of the end products of glycolysis depends on the availability of oxygen. Environmental hypoxia arises when external oxygen levels fall below critical levels for prolonged periods. Intertidal bivalves may close their shells during tidal cycles, inducing hours of hypoxia, whereas parasites of the gastrointestinal tract live perpetually under hypoxia. Functional anoxia can arise when tissue oxygen demands outstrip oxygen delivery from the blood. For example, muscle can become hypoxic during intense exercise. Diving animals gradually deplete their onboard oxygen stores, causing short-term hypoxia in some tissues. In each of these situations, animals depend on glycolysis for energy and must be able to oxidize NADH to allow glycolysis to continue. One of the most common pathways for NAD regeneration is through the activity of LDH, an enzyme we introduced earlier in this chapter. Le No ar tF n or in Di Re g s O CO2 n demands of specific tissues. For example, a turtle can depress its metabolic rate at the onset of a dive. Second, animals can extend hypoxic survival by storing high levels of glycogen. For some bivalve molluscs, for example, almost half their dry weight is glycogen, providing many days of anoxia tolerance. Third, some anoxia-tolerant organisms alter the nature of glycolysis to produce an alternative end product that is less toxic than lactate. Some molluscs produce strombine, alanopine, or octopine. Some species of fish can convert lactate to ethanol, which is then excreted into the water. This additional reaction spares them the toxicity of lactate, but in the process they lose a valuable source of energy. Bivalve molluscs and some endoparasites also produce alternate end products, but gain extra energy along the way. Phosphoenolpyruvate (PEP) can be diverted from glycolysis to produce succinate (4 ATP/glucose) or propionate (6 ATP/glucose). The various alternative pathways and anaerobic end products are summarized in Figure 2.30. Glucose NADH Pyruvate X NADH NADH tri Pyruvate NADH This reaction regenerates NAD and disposes of pyruvate, permitting glycolysis to continue. For many species, lactate production is a good indication of glycolytic flux. Once produced in the LDH reaction, lactate can either be retained in the tissue or exported from the cell into extracellular fluid. Although lactate is slightly toxic, it can be tolerated for short periods. When the anoxia bout ends, lactate is metabolized, and is often used as a substrate to regenerate glucose and glycogen. The most hypoxia-tolerant and anoxia-tolerant animals use three general mechanisms to extend survival. One is to reduce their metabolic demands to extend the life of their energy stores by entering some form of dormancy or reducing the metabolic r bu tio H lactate sa Ethanol n NAD So le lu t Oxaloacetate NADH Malate Fumarate Succinate Propionate NADH Lactate Tauropine (x=taurine) Nopaline (proline) Alanopine (alanine) Strombine (glycine) Lysopine (lysine) Octopine (arginine) io n s Figure 2.30 Anaerobic end products of glycolysis Animals collectively have many different ways to oxidize NADH when oxygen is limiting. Many animals and tissues rely on lactate dehydrogenase, but other pathways occur in hypoxia-tolerant animals. Some anaerobic end products can lead to production of extra ATP. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 52 52 PART ONE The Cellular Basis of Animal Physiology Collectively, these variations of glycolysis allow animals to succeed in anoxic environments that are toxic to other species. Lipids Lipids are a class of hydrophobic organic molecules including fatty acids, triglycerides, phospholipids, steroids, and steroid derivatives. They have many roles in animal cells, acting as substrates for energy production, building blocks for membranes, and signaling molecules. Fatty acids are long aliphatic chains produced from acetyl CoA Fatty acids are long chains of carbon atoms (aliphatic) ending with a carboxyl group (Figure 2.31). They can vary in chain length from two carbons, as with acetate, to more than 30 carbons. The shortest fatty acids are often called volatile fatty acids, or VFAs, because they readily evaporate from solution. VFAs are produced by ruminants with the bacterial fermentation of cellulose. 2 4 6 8 10 12 14 16 18 Pe ar so O C 1 C HO 18:0 - Stearic acid (saturated) Le No ar tF n or in Di Re g s O C C C C C C C C C 3 n C C 5 C 7 C 9 C C C 11 13 15 17 O C HO C C C C tri C C C C C C 18:1 (9) - Oleic acid (monounsaturated) r bu tio C C C C C C C C C C C C sa n Medium chain fatty acids (MCFAs) and long chain fatty acids (LCFAs) are common in energy stores and as part of phospholipids that make up cell membranes. Fatty acids also differ in the number and position of double bonds between carbon atoms. Saturated fatty acids have no double bonds and are linear in structure. The introduction of a double bond into a linear fatty acid causes a bend in the chain, which has important consequences for membrane structure. Monounsaturated fatty acids have one double bond. Polyunsaturated fatty acids, or PUFAs, possess multiple double bonds. Fatty acid nomenclature considers both the chain length and the number of bonds. Palmitic acid is denoted as 16:0, meaning it is 16 carbons long and has no double bonds. There are two naming systems to denote fatty acids, which differ in how they identify the location of the first double bond. In the delta ( ) system, a number corresponds to location of the double bond relative to the carboxyl carbon; in the omega () system, the number refers to the distance from the methyl end of the fatty acid. Thus, the 18-carbon fatty acid oleic acid can be either 18:1 9 or 18:1 9. Linoleic acid is denoted as either 18:2 9,12 or 18:2 6. Animals can produce many fatty acids using the enzyme fatty acid synthase, which cyclically adds two-carbon units to the fatty acid. Though fatty acids grow by adding acetyl groups, malonyl CoA, a three-carbon activated fatty acid is the actual substrate for the enzyme fatty acid synthase. Malonyl CoA is produced by acetyl CoA carboxylase. Using the reducing energy of NADPH, fatty acid synthase repeatedly adds acetyl CoA groups to the fatty acid. After seven cycles, when the fatty acid has grown to 16 carbons, palmitate has been produced and is released by the enzyme. The overall reaction for palmitate synthesis is So le lu t Acetyl CoA 7malonyl CoA 7NADPH 7H palmitate 7NADP io n O C HO C C C C C C C C C C C C 18:2 (9,12) - Linoleic acid (polyunsaturated) Figure 2.31 Fatty acids Saturated fats such as stearic acid (18:0) are linear in structure. Addition of a double bond, as shown with the monounsaturated fatty acid oleic acid (18:1), introduces a bend in the structure. The second double bond shown in the polyunsaturated acid linoleic acid (18:2) causes further disruption of the linear structure. Though palmitate is the product of fatty acid synthase, accessory enzymes process much of it to produce other fatty acids. These enzymes elongate the carbon chain and introduce double bonds to produce the other important fatty acids, such as oleic acid. Many animals can produce all of the fatty acids needed for growth, but some animals are incapable of producing specific fatty acids and must obtain these in the diet. For example, humans have a dietary requirement for linoleic acid (18:2 6) and linolenic acid (18:3 3). s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 53 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 53 Fatty acids are oxidized in mitochondrial -oxidation Fatty acids are an important fuel for many tissues, such as the mammalian heart, which typically derives more than 70% of its energy from fatty acid oxidation. The fatty acid oxidation pathway occurs primarily in the mitochondria and results in the production of acetyl CoA. Depending on the conditions, this acetyl CoA can be oxidized by mitochondria or be diverted to other pathways. Fatty acids can have many structures, differing in chain length, branching patterns, and desaturation. These variations require side reactions to convert the fatty acids to forms that can enter -oxidation. We will focus on the pathway for oxidation of palmitate, but along the way we will identify some of the alternate pathways used to process other fatty acids. Because the actual substrate for -oxidation is fatty acyl CoA, cells must first convert fatty acids to their CoA esters using a fatty acyl CoA synthase. Short and medium chain fatty acids are able to enter the mitochondria directly, where they are activated by a mitochondrial fatty acyl CoA synthase. Palmitate, which cannot cross into mitochondria, is oxidized by the mitochondria by a multistep process involving activation and transport. The fatty acid is converted to fatty acyl CoA. Next, the enzyme carnitine palmitoyl transferase-1, or CPT-1, replaces the CoA with carnitine, forming fatty acyl carnitine, which is carried into the mitochondria, where another enzyme, CPT-2, converts it back to fatty acyl CoA. This elaborate transport scheme provides an extra level of control over long chain fatty acid oxidation. By regulating the activity of CPT-1, cells control how much fatty acid can enter the mitochondria for catabolism. Once inside the mitochondria, fatty acids enter the -oxidation pathway (Figure 2.32). This is a cyclical pathway that sequentially cuts pairs of carbons off the end of the fatty acid in the form of acetyl CoA. The shortened fatty acid returns to the pathway, and the cycle is repeated until the entire fatty acid is broken down to acetyl CoA. With each trip through the pathway, reducing equivalents are produced at two enzymatic steps: fatty acyl CoA dehydrogenase produces FADH2, and -hydroxyacyl CoA dehydrogenase produces NADH. About 30% of the energy liberated from fatty acids is derived from the reducing equivalents produced in -oxidation. The remaining 70% derives from oxidation of acetyl CoA in the TCA cycle. H C H H C H C C O O H H Fatty acid ATP Activation Fatty acyl CoA synthase Pe ar so CoASH AMP+PPi H C H H C H C C S-CoA O H H Fatty acyl CoA Le No ar tF n or in Di Re g s O tri r bu tio n Thiolysis n Oxidation Fatty acyl CoA dehydrogenase FAD FADH2 H C H H C H C C O S-CoA Enoyl CoA Enoyl CoA hydratase Hydration H2O H C OH H C C C O sa So le lu t Oxidation H C C O S-CoA H H H -Hydroxyacyl CoA NAD+ NADH +H+ O C H C H C O S-CoA -hydroxyacyl dehydrogenase H C H io n Thiolase -Ketoacyl CoA CoASH H + HC C S-CoA S-CoA H H Fatty acyl CoA Acetyl CoA s O Figure 2.32 Fatty acid oxidation Fatty acids are activated in the cytoplasm to form fatty acyl CoA. Once transported into mitochondria, fatty acyl CoA enters the -oxidation pathway. Oxidation, hydration, oxidation, and thiolysis produce acetyl CoA, reducing equivalents, and a fatty acyl CoA shortened by two carbons. The shortened fatty acyl CoA reenters the -oxidation pathway and the cycle repeats until the fatty acid is reduced to acetyl CoA units. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 54 54 PART ONE The Cellular Basis of Animal Physiology Ketogenesis 2 Acetyl CoA Ketolysis 2 Acetyl CoA CoASH GTP Thiolase CoASH Thiolase Acetoacetyl CoA Acetoacetyl CoA Succinate Pe ar so HMG CoA synthase Acetyl CoA CoASH HMG-CoA HMG CoA lyase Succinyl CoA synthase -Ketoacyl CoA transferase Acetyl CoA Acetoacetate Succinyl CoA Acetoacetate NADH + H+ -HBDH -HBDH NADH + H+ NAD+ -Hydroxybutyrate Figure 2.33 Ketone metabolism Acetyl CoA can be converted to the ketone bodies acetoacetate and -hydroxybutyrate. This reaction normally occurs in specific ketogenic tissues, such as liver. Ketone bodies are released through the blood for uptake by ketolytic tissues, such as brain. Acetyl CoA is resynthesized at the cost of one GTP to regenerate the substrate succinyl CoA. Fatty acids can be converted to ketone bodies Fatty acids are valuable sources of energy, but under some conditions they must first be processed into ketone bodies: acetone, acetoacetate, and -hydroxybutyrate (Figure 2.33). Ketone bodies provide a fuel for tissues that cannot use fatty acids directly. The mammalian brain usually relies on glucose oxidation for energy, but after an extended period of food deprivation, glucose levels may decline, forcing tissues to rely more on lipid stores. Since the brain cannot use fatty acids directly, the liver converts the fatty acids to ketone bodies, which can be transported into the brain and oxidized. The first step in ketone body synthesis, or ketogenesis, is the production of acetoacetyl CoA from two molecules of acetyl CoA, catalyzed by thiolase. This is the same enzyme used in the final step of -oxidation, but in ketogenesis it operates in the reverse direction. After condensation with another acetyl CoA and subsequent hydrolysis, acetoacetate is formed. Acetoacetate can then be converted to -hydroxybutyrate by the enzyme -hydroxybutyrate dehydrogenase ( -HBDH), or it can break down spontaneously to form acetone. In the target tissues, ketolysis reconverts -hydroxybutyrate and acetoacetate to acetyl CoA. Acetoacetate is ac- Le No ar tF n or in Di Re g s O tri r bu tio sa n H H C O C n NAD+ -Hydroxybutyrate tivated into the CoA ester, then hydrolyzed by thiolase to form two acetyl CoA molecules. Ketone bodies are a useful alternative to fatty acids for many animals. Although some energy is lost in the complete cycle of ketogenesis and ketolysis, for some tissues, particularly under starvation conditions, ketone bodies are the only metabolic energy source available. Chondrichthians (sharks and their relatives) in fact appear biochemically predisposed to using ketone bodies as their "lipid" fuel. Unlike in other vertebrates, their muscles are unable to use fatty acids directly, instead relying on ketone bodies as a fuel for energy. Triglyceride is the major form of lipid storage Most fatty acids in animal cells are esterified to a glycerol backbone. A monoacylglyceride has a single fatty acid esterified to glycerol, typically at the first position of the glycerol molecule. Diacylglyceride has fatty acids esterified to the first and second position of glycerol. Triglyceride has three fatty acids esterified to the glycerol backbone (Figure 2.34). Each of these terms--monoacylglycerides, diacylglycerides, and triglycerides--refers to a class of molecules. For example, hundreds of chemically distinct triglyceride molecules can be constructed by using different fatty acids in each of the three positions on the glycerol backbone. So le lu t H C O O C CH2 CH2 O H C O C CH2 O H CH2 CH2 CH2 io n Glycerol Fatty acids s CH3 CH3 CH3 Figure 2.34 Triglycerides Triglycerides are composed of three fatty acids esterified to a glycerol backbone. Fatty acids can vary in chain length and number of double bonds. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 55 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 55 Pe ar so Triglycerides are vital energy stores for animals, and can be found in high concentrations in lipid storage tissues in the form of lipid droplets. In insects, a tissue called the fat body is the main site of lipid storage. Many other invertebrates, such as molluscs and crustaceans, store lipid in a large hepatopancreas. Vertebrates store triglyceride in liver, muscle, and adipose tissue, such as blubber. Adipocytes, the cells within adipose tissue, store triglyceride when an animal is well fed, then release lipids when the animal needs extra fuel. Triglyceride synthesis, or lipogenesis, is a multistep process overlapping with phospholipid synthesis (Figure 2.35). Each fatty acid is activated into its CoA ester by fatty acyl CoA synthase. Starting with glycerol 3-phosphate, the fatty acids are added sequentially to carbon 1, then carbon 2, forming a phosphatide. After removal of the phosphate group, diacylglycerol is formed. Addition of a third fatty acid completes the triglyceride molecule. Triglyceride breakdown, or lipolysis, requires enzymes called lipases that attack the triglyceride molecule, breaking the bond between the fatty acid and the glycerol backbone. Hormone-sensitive lipase liberates fatty acids from triglycerides and diacylglycerides. Another lipase, monocyglyceride lipase, completes the breakdown of the triglyceride by releasing the last fatty acid from the glycerol backbone. The liberated fatty acids are either used directly within the cell or transferred to the circulation for uptake by other tissues that use them for energy metabolism. The balance between triglyceride synthesis and degradation is carefully controlled within animals. Lipolysis does not directly generate energy, but lipogenesis requires energy. As with other pathways we have discussed, if both synthesis and degradation occurred simultaneously, cells would waste energy. Dihydroxyacetone phosphate NADH+H+ NAD+ CoASH CoA OH Glycerol3P OH OH Pi Glycerol ATP ADP Fatty acid Fatty acid CoASH CoASH Le No ar tF n or in Di Re g s O tri r bu tio sa n n Pi Phosphatidic acid Phospholipid synthesis OH Diacylglycerol CoASH Figure 2.35 Phospholipids predominate in biological membranes In addition to their role in energy metabolism, fatty acids are vital components of the phospholipids used to produce biological membranes. Animal cells produce two classes of phospholipids: phosphoglycerides and sphingolipids (Figure 2.36). Phosphoglycerides are constructed from phosphatides, an intermediate in triglyceride synthesis. In phospholgylceride synthesis, the phosphate group links the phosphatide to a polar head Triglyceride synthesis Glycerol 3-phosphate, produced from glycolysis (dihydroxyacetone phosphate) or glycerol, is the acceptor for two sequential additions of activated fatty acids (fatty acyl CoA). The formation of triglyceride requires dephosphorylation and addition of another fatty acid group to the third and last position on the glycerol backbone. So le lu t io n s group, such as serine, choline, ethanolamine, and inositol. The physical properties of a phosphoglyceride are determined by the properties of both the fatty acids (chain length, saturation) and the polar head group. Although sphingolipids are chemically very different from phosphoglycerides, they have similar three-dimensional shapes. The backbone of a sphingolipid is sphingosine. With its long aliphatic 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 56 56 PART ONE The Cellular Basis of Animal Physiology Choline + Phosphate (a) Phosphoglycerides Pe ar so Glycerol Fatty acid Fatty acid Choline Phosphate + Sphingosine Le No ar tF n or in Di Re g s O n makes a specific combination of sphingolipids, providing a kind of cellular signature that other cells can recognize. During development, when cells move throughout the body to begin the process of tissue formation, sphingolipid signatures provide migrating cells with landmarks. When the migrating cells find the correct sphingolipid signature, they cease migration and differentiate to form tissues. Phospholipids are broken down by phospholipases, many of which are important in cell signaling cascades. Each type of phospholipase attacks a specific region of a phospholipid molecule. Phospholipase A (PLA) breaks the ester bonds that connect the fatty acids to the glycerol backbone. PLA1 releases the fatty acid from the first carbon of glycerol, whereas PLA2 releases the fatty acid from the second position. Phospholipases B and C break different phosphodiester bonds between the polar head group and the glycerol backbone. When PLB attacks phosphatidyl inositol, inositol and phosphatide are produced. When PLC attacks the same phospholipid, inositol phosphate and diacylglyceride are released. Regulation of PLC, an important enzyme in signal transduction pathways of many cells, will be discussed in more detail in Chapter 3: Hormones and Cell Signaling. Fatty acid tri (b) Sphingolipids Figure 2.36 Phospholipids Phospholipids, including (a) phosphoglycerides and (b) sphingolipids, share a similar three-dimensional structure. They are built on different backbones: glycerol for phosphoglycerides and sphingosine for sphingolipids. r bu tio sa Steroids share a multiple ring structure Steroids are a collection of lipid molecules that share a basic aromatic structure of four hydrocarbon rings. The steroid cholesterol is found in many cellular membranes and is part of the lipoprotein complexes that transport lipids through the blood. It is also a precursor for synthesis of the vertebrate steroid hormones. Although invertebrates don't possess steroid hormones, some use a steroid-like hormone, ecdysone, to control maturation and development. The pathways of steroid synthesis involve nonsteroid intermediates (Figure 2.37). Steroid synthesis begins when acetate is used to produce mevalonate, the precursor for activated isoprene. Activated isoprene is the precursor for many familiar molecules, such as carotenoids and vitamins A, E, and K. Ubiquinone, a type of quinone, is an important component in mitochondrial energy production. Activated isoprene is also used to produce isoprenoids that act as hormones, including insect juvenile hormone and pheromones. Further along the pathway of cholesterol synthesis n So le lu t io n chain, its structure is similar to monoacylglycerol. Ceramide is formed when a fatty acid is esterified to sphingosine, creating a structure that resembles diacylglycerol. Many different types of sphingolipid can be constructed by attaching different polar head groups to ceramide. When phosphocholine is attached to ceramide, sphingomyelin is formed. Carbohydrates can be attached to ceramide to form neutral glycolipids and gangliosides. Sphingolipids are most often found on the extracellular side of the cell membrane. Each cell s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 57 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 57 is squalene, a steroid used by sharks to aid in buoyancy. Cholesterol is the precursor for the many steroid hormones that we discuss in later chapters. Acetate Vitamin K Mevalonate Vitamin A Pe ar so Mitochondrial Metabolism Ubiquinone Activated isoprene Carotenoids Mitochondria process metabolites generated in the cytoplasm, breaking them down to capture their chemical energy in the form of ATP. The main point of entry for mitochondrial energy-producing pathways is acetyl CoA, which as you've learned earlier in this chapter is produced in many pathways (Figure 2.38). Acetyl CoA enters the cyclical tricarboxylic acid cycle (TCA cycle) and is oxidized to form reducing equivalents (NADH, FADH2), which provide the fuel for mitochondrial ATP production. Isoprenoids Squalene Vitamin E Vitamin D Cholesterol Bile acids The TCA cycle uses acetyl CoA to generate reducing equivalents Once acetyl CoA is produced within mitochondria, its fate depends on intracellular conditions. When cells need energy, acetyl CoA enters the TCA cycle, where its oxidation ultimately leads to ATP production. The TCA cycle (Figure 2.39) consists of eight enzymes that collectively catalyze the following reaction: Acetyl CoA 2CO2 3NAD 3NADH Le No ar tF n or in Di Re g s O tri r bu tio GDP Pi FAD FADH2 GTP n Pregnenolone Corticosterone Progesterone Testosterone Aldosterone Cortisol Estradiol Figure 2.37 Steroid biosynthesis This simplified pathway of steroid synthesis illustrates the many intermediates that are used by cells. The four dehydrogenases in the TCA cycle produce reducing equivalents: NADH is produced by isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, and malate dehydrogenase; FADH2 is produced by succinate dehydrogenase. Most of the ATP produced through acetyl CoA oxidation comes from the subsequent oxidation of NADH and FADH2. The TCA cycle also produces one molecule of GTP, which is energetically equivalent to ATP. This reaction, catalyzed by succinyl CoA synthase, is an example of substrate-level phosphorylation. The TCA cycle is not really an isolated pathway so much as a collection of enzymes acting on a pool of metabolites exchanged with other pathways. When intermediates are removed for other reactions, cells use anaplerotic pathways to regenerate the intermediates. Cells control the rate of the TCA cycle in three ways: by regulating the concentrations of reactants (substrates and products), the levels of the sa n enzymes, and the catalytic activity of enzymes. Tissues that use a lot of energy, such as heart and brain, have high levels of the TCA enzymes. In many tissues, the flux through the TCA cycle is affected by the levels of acetyl CoA and oxaloacetate, as well as other intermediates in the cycle. When tissues have abundant energy, they typically use acetyl CoA and intermediates as biosynthetic substrates. Acetyl CoA is an important substrate in fatty acid synthesis, and oxaloacetate is a substrate for glucose synthesis. When biosynthetic reactions deplete these substrates, the rate of the TCA cycle declines. Allosteric effectors also regulate the TCA cycle. Calcium, frequently elevated So le lu t Glucose Lactate Pyruvate io n Dietary sucrose Dietary starch Glycogen s Amino acids Amino acids Fatty acids Ketone bodies Acetyl CoA Figure 2.38 Acetyl CoA production from diverse metabolites 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 58 58 PART ONE The Cellular Basis of Animal Physiology pass electrons directly to ubiquinone. For example, the TCA cycle enzyme Citrate synthase succinate dehydrogenase is actually complex II of the ETS. An FAD group Oxaloacetate Citrate within its structure becomes reduced Malate during oxidation of succinate, formAconitase dehydrogenase NADH ing FADH2. The enzyme in turn passes the electrons from FADH2 to Malate Isocitrate ubiquinone. Once reduced, ubiquinone transfers its electrons to complex III, which in turn transfers NADH electrons to cytochrome c. Complex Fumarase Isocitrate dehydrogenase IV, or cytochrome c oxidase, acCO2 cepts electrons from cytochrome c and passes them on to molecular Fumarate 2-Oxoglutarate oxygen. Complete reduction of oxyFADH2 NADH gen (O2) requires four electrons Succinate 2 -Oxoglutarate from cytochrome c and consumes CO2 dehydrogenase dehydrogenase four protons to produce two water GTP molecules. Succinate Succinyl CoA Complexes I, III, and IV are also Succinyl CoA proton pumps. When these comsynthase plexes transfer energy to the next Figure 2.39 Tricarboxylic acid cycle The enzymes of the TCA cycle oxidize carrier, enough free energy remains acetyl CoA to release its energy in the form of reducing equivalents (3 NADH, 1 FADH2) to pump protons out of the mitoand 1 GTP. chondrial matrix. Fewer protons are pumped in the oxidation of during periods of high metabolic demand, stimuFADH2 because these enzymes pass their electrons lates isocitrate dehydrogenase and 2-oxoglutarate directly to ubiquinone, bypassing the protondehydrogenase, increasing the rate of NADH propumping complex I. The proton gradient formed duction to help the cell meet its energy demands. by the ETS has both electrical and chemical components: a pH gradient ( pH) and a membrane potential ( ). This proton motive force is potential The ETS generates a proton gradient, heat, energy that can be used to drive other processes, and reactive oxygen species such as ATP synthesis. Mitochondria use reducing equivalents as the subThe ETS converts much of the energy liberstrate for oxidative phosphorylation, a complex ated from NADH oxidation to the proton motive pathway that combines oxidation by the electron force. Some energy is "lost" in the formation of two transport system (ETS) with phosphorylation by-products: heat and reactive oxygen species (Figure 2.40). The ETS builds an electrochemical (ROS). Conditions that increase electron flow and gradient that can be used to drive ATP synthesis oxygen consumption also increase heat producand energy-dependent transport processes. tion. ROS production is an inevitable consequence Found within the inner mitochondrial membrane, of electron movement throught the ETS. Usually the ETS consists of four multisubunit proteins more than 99% of the electrons that enter the ETS (complexes I, II, III, and IV) and two electron carmake the journey all the way to the end of the riers (ubiquinone, cytochrome c). chain, forming water. However, a few are stolen Although electrons can enter the ETS in several from the ETS by molecular oxygen to form superways, each pathway converges on the first mobile oxide (O2 ), a potent ROS that can attack chemical carrier, ubiquinone. The NADH produced in the bonds, damaging macromolecules such as lipids, TCA cycle passes electrons to complex I, which in proteins, and DNA. Cells possess vigorous antioxturn reduces ubiquinone. Several FADH2-linked enidant defense mechanisms to inactivate superoxzymes found in the inner mitochondrial membrane ide before it can cause damage. The enzyme Acetyl CoA Pe ar so Le No ar tF n or in Di Re g s O tri r bu tio sa n n So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 59 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 59 Electron transport system Phosphorylation H+ H+ Electron Q Complex II FADH2 H+ O2 Complex III Cyt C H+ H+ Intermembrane space Pe ar so Complex I NADH + H+ H+ NAD+ Complex IV Complex V Mitochondrial inner membrane FAD H+ H2O ADP + Pi H+ ATP Mitochondrial matrix Succinate Figure 2.40 Oxidative phosphorylation Complex I collects electrons from NADH produced by various mitochondrial dehydrogenases. Complex II, or succinate dehydrogenase, transfers electrons from succinate to FAD. Both complex I and II, as well as other FAD-link dehydrogenases not shown, transfer electrons to ubiquinone superoxide dismutase, or SOD, consumes superoxide to produce hydrogen peroxide (H2O2), which is less toxic than superoxide. Other antioxidant enzymes, such as catalase and glutathione peroxidase, consume H2O2, preventing it from causing cellular damage. Le No ar tF n or in Di Re g s O tri r bu tio sa n n Fumarate (Q). Electron transfer continues through complex III, cytochrome c, and finally complex IV, cytochrome oxidase. During electron transport, complexes I, III, and IV also pump protons out of the mitochondrial matrix, creating the proton motive force ( p). The mitochondrial F1FO ATPase (complex V) uses p to fuel ATP synthesis. The F1FO ATPase uses the proton motive force to generate ATP To this point we have discussed how mitochondria build p but not how it is used to produce ATP. Mitochondria possess an ATP synthase, usually called the F1FO ATPase, that uses the energy contained in p to drive the phosphorylation of ADP. (Although it normally functions in the direction of ATP synthesis, the F1FO ATPase is reversible and able to break down ATP under some conditions.) The F1FO ATPase possesses a proton-pumping region and a catalytic region. When protons pass through the enzyme, which spans the mitochondrial inner membrane, the energy is used to catalyze the synthesis of ATP. Oxidative phosphorylation is the combination of oxidation by the ETS and phosphorylation by F1FO ATPase. Note that there is no physical linkage between oxidation and phosphor- ylation; the two processes are functionally coupled through a mutual dependence on p. The rate of ATP synthesis by the F1FO ATPase depends on the magnitude of p and the availability of the substrates ADP and inorganic phosphate (Pi). When cells are rapidly hydrolyzing ATP, [ADP] and [Pi] increase, accelerating the rate of the F1FO ATPase reaction. To understand how this process is regulated, consider what happens in a muscle that goes from rest to exercise. At rest, the rate of ATP breakdown is slow and [ATP] builds up while [ADP] and [Pi] decline. The ETS builds p to its maximum because the ATPase, the major drain on the gradient, is inhibited. With little flux through the ETS, the rate of respiration is low. This is the biochemical reason why you breathe less when resting. When muscle activity begins, ATP is hydrolyzed and the concentrations of ADP and Pi increase. With the stimulation of the ATP synthase, p is depleted and ETS accelerates to replenish the gradient. We increase our oxygen consumption during exercise because of this linkage between ATP synthesis and oxidation. The functional linkage between oxidation and phosphorylation depends on the integrity of the inner mitochondrial membrane. All membranes So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 60 60 PART ONE The Cellular Basis of Animal Physiology are somewhat permeable to protons, but the inner mitochondrial membrane is relatively resistant to proton leak. If the protons pumped out of the mitochondria by the ETS were to leak back into the mitochondria, p would be dissipated, causing two effects on oxidative phosphorylation. First, the ETS would continue at a high rate, pumping protons and consuming oxygen in a futile effort to rebuild p. Second, the reduction in p would prevent the mitochondria from producing ATP. Mitochondria that show high rates of respiration with no ATP production are considered uncoupled. While this state is disastrous for energy production, it is an important mechanism to produce heat. Some mammals have specific proteins that facilitate the movement of protons across the inner membrane. As you will learn in Chapter 13: Thermal Physiology, these uncoupling proteins are important in mammals that experience cold stress, such as newborns and hibernators. Myofibril ADP + Pi ATP CPK Creatine Phosphocreatine Pe ar so Creatine Phosphocreatine CPK Outer membrane ATP ADP + Pi Creatine phosphokinase enhances energy stores and transfer Some of the energy stored first as ATP is used to produce other high-energy phosphate compounds of equivalent energy, such as phosphocreatine. A vertebrate muscle, for example, may have 510 more phosphocreatine than ATP, serving as an energy store. When the muscle begins to work at high intensity, ATP is consumed to support muscle activity. The resulting decrease in [ATP] and increase in [ADP] drive the creatine phosphokinase (CPK) reaction in the direction of ATP production, at the expense of phosphocreatine. As muscle activity continues, the phosphocreatine pool is gradually depleted, allowing muscles to preserve ATP at normal levels for longer periods. Whereas the existing pool of ATP is sufficient for only a few seconds of activity, the large phosphocreatine pool allows muscle to maintain ATP levels and sustain contractions for a much longer duration. In addition to bolstering energy stores, phosphocreatine is a component of the phosphocreatine shuttle, a pathway that improves the efficiency of energy transfer within the cell (Figure 2.41). The cycle begins with ATP produced by the mitochondria. CPK on the outer mitochondrial membrane uses this ATP to phosphorylate creatine. The phosphocreatine diffuses from the mitochondria to the myofibrils where another CPK uses the phosphocreatine to regenerate ATP. This CPK Le No ar tF n or in Di Re g s O tri r bu tio sa n n Inner membrane ADP ATP Mitochondrion Figure 2.41 Phosphocreatine shuttle Phosphocreatine is produced by the mitochondrial enzyme creatine phosphokinase (CPK) and diffuses to the myofibrils, where another CPK enzyme regenerates the ATP. shuttle improves the efficiency of energy transfer two ways. First, creatine and phosphocreatine are smaller molecules than the adenylates and have higher diffusion coefficients. Second, the absolute concentrations of creatine and phosphocreatine are much greater than those of the adenylates, allowing much steeper intracellular gradients to form, which accelerates the rates of diffusion. Integration of Pathways of Energy Metabolism The metabolic traits exhibited by whole animals can be traced back to the cellular pathways of energy metabolism. Put simply, in order to remain in energetic balance, cells must produce ATP at rates that match the ATP demand. Consider the situation in a mammalian heart cell. We know from rates of oxygen consumption that the heart consumes ATP at a rate of about 30 mol/min/g. Since the concentration of ATP doesn't change during normal activity, the heart cell must also produce ATP at the same rate (30 mol/min/g). Since the concentration of ATP is only about 5 mol/g, at a metabolic rate of 30 mol/min/g, the heart turns over the entire ATP pool several times per minute. So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 61 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 61 Pe ar so At this turnover rate, a cell that produces ATP at a rate only 10% less than the rate of demand would be depleted of ATP within two minutes. At the cellular level, the balance between energyconsuming pathways and energy-producing pathways is orchestrated by the diverse regulators of metabolism, such as adenylates, redox balance, Ca2 , and carbon supply. These regulators "inform" the cell of energy needs, and the metabolic pathways respond accordingly. What is interesting about multicellular animals is that the needs of the cell are often superceded by the needs of the whole organism. A liver cell, for example, not only responds to its own metabolic needs but produces energy substrates for the entire animal. When glucose levels are low, the liver increases the rates of gluconeogenesis and glycogenolysis, releasing glucose to the blood for use in other tissues. This altruistic response is imposed on the liver cell by hormones that affect the catalytic properties of the enzymes of intermediary metabolism, largely through covalent modification, such as phosphorylation. We will discuss the nature of hormones in the next chapter, Hormones and Cell Signaling. The sum of the cellular metabolic properties yields the whole animal metabolic patterns. Variations in animal metabolic rate are central to many problems in animal physiology. You learned in Chapter 1 about allometric scaling of metabolic rate: large animals have lower mass-specific metabolic rates than small animals. In later chapters you will learn how animals control metabolic rate to survive challenging conditions, such as environmental hypoxia, hypothermia, and dehydration. In the accompanying feature, we describe the ways physiologists measure whole animal metabolic rate in the lab and in the field (see Box 2.2, Methods and Model Systems: Metabolic Rate). Metabolic strategies in animals address the constant fluctuations in nutrient availability, energy demand, and environmental conditions. Ensuring the correct flow of energy requires exquisite control of the pathways of intermediary metabolism. Opposing pathways must be reciprocally regulated to avoid a futile cycle: simultaneous synthesis and degradation of a metabolite. Similarly, the various alternatives must be utilized in a way that takes into consideration the advantages and disadvantages of each class of fuel. These choices are also influenced by long-term and short-term metabolic priorities of the cells and organisms. Oxygen and ATP control the balance between glycolysis and OXPHOS Glycolysis produces 2 mol of ATP for every mol of glucose, but glucose oxidation yields 36 mol of ATP per mol of glucose. Despite the differences in energy yield, glycolysis and oxidative metabolism both play important roles in energy metabolism. Glycolysis, in addition to being able to operate without oxygen, can produce ATP at much greater rates than can oxidative metabolism. The conditions that allow such high rates also require the pathway to be somewhat inefficient. In contrast, oxidative metabolism is very efficient in conserving chemical energy, but to do so, it is necessarily slow. Think of glycolysis as a high-performance race car--useful to get somewhere fast but not the best car for gas mileage. Oxidative metabolism, by contrast, is the fuel-efficient compact car. Like some suburban families, the cell maintains both types of engines in working order, to be called upon for different needs. Le No ar tF n or in Di Re g s O tri r bu tio sa n n Physical properties of fuels influence fuel selection Each of the major metabolic fuels displays physical properties that influence how the fuel is stored and used. Carbohydrate is stored as large granules of glycogen, coated with water molecules that make up its hydration shell. Glycogen particles can be so large that they interfere with cellular processes. Some tissues, such as the mantle of bivalve molluscs, can safely accumulate high levels of glycogen, but if glycogen reached this high level in a muscle it would prevent the muscle from contracting normally. Although glycogen is readily mobilized, its physical properties prevent most cells from storing high levels. In contrast, lipid is stored at much higher levels in the form of anhydrous, amorphous droplets of triglyceride. These physical differences, coupled with the energy content of a given mass of stored fuel, affect fuel selection. Cells can obtain about 10 times more ATP from lipid than from the same mass of hydrated glycogen particles. Given the advantage of lipid as an energy store, you might wonder why animals use glycogen at all. Recall that glycogen can be mobilized much faster than lipid, and plays a vital role under conditions in which energy is required very quickly, as in the "fight-orflight" response. Most cells use a combination of lipid and carbohydrate fuels to balance the advantages and disadvantages of each fuel. So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 62 62 PART ONE The Cellular Basis of Animal Physiology BOX 2.2 METHODS AND MODEL SYSTEMS Metabolic Rate resting metabolic rate (RMR) must ensure that the animal is unstressed and inactive at a neutral ambient temperature and has digested its most recent meal. While this approach yields important information about the physiological hardwiring of an animal, it may not be the best estimate of the animal's metabolic rate under normal conditions. Ecological physiologists are often more interested in long-term metabolic rate of freeranging animals in the natural setting. One of the most common approaches to measuring field metabolic rate is the doubly labeled water method. Most water in the body is composed of the most common isotopes of hydrogen (1H) and oxygen (16O). To initiate a doubly labeled water experiment, the animal of interest is captured and injected small volumes of water composed of less common isotopes of hydrogen (e.g., 2H) and oxygen (18O). Over time, the labeled hydrogen is lost from the body primarily as water, through evaporation, respiration, and excretion. Likewise, labeled oxygen is also lost in water, but some is exchanged with the O in CO2. Thus, the difference between the loss of labeled oxygen and labeled hydrogen reflects the rate of CO2 production. This method works very well in air-breathing animals, but in water breathers, the rates of water flux are much too great to detect the impact of CO2 production on isotope ratios. References q Hulbert, A. J., and P. L. Else. 2004. Basal metabolic rate: History, composition, regulation, and usefulness. Physiological and Biochemical Zoology 77: 869876. q Nagy, K. A. 2005. Field metabolic rate and body size. Journal of Experimental Biology 208: 16211625. Pe ar so If metabolism is literally the sum of all chemical reactions, then what exactly is meant by metabolic rate and how is it measured? Metabolic rate is the overall flux through the pathways of energy production, which matches the rate of energy consumption. In the presence of oxygen, the pathways that lead to production of ATP consume carbon fuels and oxygen (O2), and produce heat, carbon dioxide (CO2), and water. Many physiological questions revolve around changes in metabolic rate, and many approaches have been developed to measure the substrates and products of metabolism. Direct calorimetry assesses metabolic rate in terms of heat production, measured in energy units (joules). For purely pragmatic reasons (direct calorimetry requires expensive, specialized equipment), this is the least common way to measure metabolic rate. A more common approach to measuring metabolic rate is indirect calorimetry, in which the researcher measures the rate of O2 consumption or CO2 production. To infer a metabolic rate from these measurements, it is important to recognize where O2 is consumed (largely in the electron transport system) and where CO2 is produced (primarily in the TCA cycle). The quantitative relationship between these three estimates of metabolic rate--heat production, O2 consumption, and CO2 production--depends on many factors. You learned earlier in this chapter that the ratio of CO2 produced/O2 consumed reflects the metabolic fuel. Likewise, the oxycaloric relationships (O2 consumed/ joules released) depend on the nature of the fuel. Each of these approaches for measuring metabolic rate requires that the researcher hold an animal under controlled conditions and ensure that it remains in a constant, stable condition. A researcher interested in Le No ar tF n or in Di Re g s O tri r bu tio sa n n So le lu t The main way the cells regulate the balance between fatty acids and carbohydrates is through the mitochondrial enzyme pyruvate dehydrogenase (PDH). This enzyme is regulated allosterically by ATP, acetyl CoA, and NADH. When cells have fatty acids available, their oxidation increases concentrations of ATP, NADH, and acetyl CoA. These metabolites inhibit PDH, sparing pyruvate for gluconeogenesis. When energy stores are depleted, the concentrations of NADH, ATP, and acetyl CoA tend to decrease, which lessens the inhibition of PDH. These same metabolites also influence the phosphorylation state of PDH by regulating the activities of PDH kinase (PDHK) and PDH phos- phatase (PDHP). ATP, NADH, and acetyl CoA each activate PDHK, causing PDH to be converted to its inactive, phosphorylated form. The activity of PDHP, in contrast, is governed primarily by Ca2 . High [Ca2 ] stimulates PDHP, converting PDH to its active dephosphorylated form. io n s Fuel selection can be calculated from the respiratory quotient Each pathway for oxidation of fuels demonstrates characteristic relationships between the amount of (1) ATP produced, (2) oxygen consumed, and (3) CO2 generated. The reason these parameters differ 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 63 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 63 Pe ar so among fuels can be traced back to the pathways of degradation. Ratios of different combinations of these three parameters provide important information about fuel selection. The differences in the ratio of ATP produced to oxygen consumed (the ATP/O ratio) can be traced to the reliance on FAD-linked enzymes. Each time an NADH molecule is produced in the mitochondria, oxidative phosphorylation can produce 3 molecules of ATP and consume 1 atom of oxygen (ATP/O 3). When a molecule of FADH2 is produced, only 2 molecules of ATP can be generated while consuming the same 1 atom of oxygen (ATP/ O 2). Carbohydrate oxidation uses predominantly NADH-linked enzymes, whereas lipid oxidation relies more heavily on FAD-linked enzymes. Because of this difference, carbohydrate yields more ATP for a given volume of oxygen. This difference has an effect on the fuel preference of at least some animals that live at low oxygen levels. For example, the heart of most humans uses lipid as a major fuel. In contrast, humans that have adapted to high altitude, such as the natives of the high Andes, rely more heavily on glucose oxidation. Of course, in more extreme hypoxia and anoxia, animals have little choice but to rely on glycolysis. Differences in the ratio of CO2 produced to O2 consumed, known as the respiratory quotient (RQ), arise from the pathways of oxidation. Glucose has six carbons, and oxidizing it completely to 6 CO2 yields 2 NADH in the cytoplasm, 2 NADH via PDH, 6 NADH via the TCA cycle, and 2 FADH2 via succinate dehydrogenase. The 12 reducing equivalents consume 12 atoms of oxygen, or 6 molecules of O2. Thus, carbohydrate oxidation yields an RQ of 1 (6 mol of CO2 to 6 mol of O2). In contrast, oxidation of fatty acids generates an RQ of about 0.7, although the exact number depends on the specific fatty acid. Consider the pathway of palmitate oxidation. As a 16-carbon fatty acid, it generates 16 mol of CO2 per mol of palmitate. Seven cycles of -oxidation are required to break palmitate into 8 molecules of acetyl CoA, yielding 7 FADH2 and 7 NADH. Oxidation of the 8 acetyl CoA in the TCA cycle yields 24 NADH and 8 FADH2. Oxidation of the 46 reducing equivalents consumes 23 mol of O2, giving an RQ of 0.7 (16 mol of CO2 to 23 mol of O2). Because of these characteristic relationships between RQ and fuel oxidation, measurement of CO2 production and O2 consumption of whole animals can provide important insight into the pathways that are being used to support energy metabolism. Energetic intermediates regulate the balance between anabolism and catabolism A cell activates pathways of energy production when it needs energy, but when energy is abundant it stimulates anabolic pathways, storing nutrients or producing building blocks for cell growth or cell division. How do cells actually sense the need for energy and regulate the transition between catabolism and anabolism? Many of the pathways we have discussed are sensitive to the cellular indices of energetic status, primarily acetyl CoA, adenylates, and NADH. Changes in the concentration of these products reflect energy status and cause compensatory changes in metabolic pathways. When cells are "energy-rich," the concentrations of acetyl CoA, NADH, and ATP are relatively high and the concentrations of CoA, NAD , ADP, and AMP are low. Consider how these metabolites stimulate gluconeogenesis while inhibiting glycolysis (Figure 2.42). By matching the Glucose Le No ar tF n or in Di Re g s O tri r bu tio sa Glycolysis n Fructose 6-phosphate +ADP +AMP PFK ATP AMP n So le lu t ATP PK Acetyl CoA Pyruvate FBPase Fructose 1,6-biphosphate Gluconeogenesis Phosphoenol pyruvate PC +Acetyl CoA io n Oxaloacetate s PDHa PDHb ATP Acetyl CoA NADH +NADH +Acetyl CoA +ATP Acetyl CoA Figure 2.42 Reciprocal regulation of glucose metabolism High-energy compounds, such as ATP, acetyl CoA, and NADH, inhibit glycolysis and stimulate gluconeogenesis at key regulatory enzymes. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 64 64 PART ONE The Cellular Basis of Animal Physiology rates of ATP synthesis with rates of ATP utilization, cells are able to defend ATP concentrations within a narrow range. Animals also use other metabolites to reciprocally regulate opposing pathways. When metabolic conditions induce cells to commit to fatty acid synthesis, the increases in the levels of malonyl CoA block fatty acid oxidation by inhibiting CPT-1. Cells further separate anabolism and catabolism using tissue specializations. The liver has the enzymes for ketogenesis but cannot break down ketone bodies. Muscles have the enzymes for ketolysis but cannot synthesize ketone bodies. In fact, the control of energy metabolism in complex animals reflects a division of labor such that some tissues become servile to others. Liver and adipose tissue perform important functions for whole animal metabolic balance, giving lower priority to their own cellular and metabolic needs. Pe ar so 2 themselves from the environment, giving them control over intracellular conditions. Second, membranes help cells organize intracellular pathways into discrete subcellular compartments, including organelles. Separation of processes also requires specific mechanisms to transfer molecules across the membranes. Many complex physiological properties and responses depend on cellular transport and transfers between subcellular compartments. Ultimately, the cellular properties that determine physiological function are controlled through regulation of gene expression. C ONC EPT C HEC K 8. Compare the four types of macromolecules. Discuss how variation in structure arises in each class. 9. Distinguish between the following types of reactions: anabolic, catabolic, amphibolic, anaplerotic. 10. How is oxidation coupled to phosphorylation in mitochondrial oxidative phosphorylation? 11. Compare the pathways of glucose metabolism (synthesis and degradation) under (a) high versus low energy conditions and (b) normal versus low oxygen conditions. Le No ar tF n or in Di Re g s O tri r bu tio sa n n Membrane Structure Cellular membranes are composed of phospholipids, other lipids, and diverse proteins. The fluid mosaic model, shown in Figure 2.43, illustrates the structural features of biological membranes. Each of the two layers is a sheet of lipid molecules arranged side by side. The surfaces of the lipid bilayer are composed of the polar head groups of phospholipids, and the internal hydrophobic core is composed of long fatty acid chains of the phospholipids attached through van der Waals forces. The lipid profile affects membrane properties Animals produce specialized membranes with unique molecular signatures by varying the structures and proportions of the different types of lipid. Much of the variation can be attributed to the profile of phosphoglycerides, the most abundant of which are phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Recall from earlier in this chapter that each of these phospholipids is really a class of molecules with constituent fatty acids that differ in chain length and saturation. Although phosphoglycerides are the most abundant molecules in the bilayer, membranes also possess other lipids, including sphingolipids, glycolipids, and cholesterol, as well as many proteins. Glycolipids resemble phospholipids, but with complex carbohydrate modifications that impart a negative charge to the polar head group. Nerve cells possess high concentrations of sphingolipids and glycolipids because they alter the electrical properties of the membrane. Glycolipids are also important in communication between cells. 12. Under what conditions is it more advantageous to use carbohydrate rather than lipid as a metabolic fuel? So le lu t Cell Physiology io n In the opening essay, we discussed how the evolutionary origins of animals required a new relationship between cells to foster multicellular relationships. Cellular membranes1 perform two main roles that are central to multicellularity and cellular function. First, they allow cells to isolate Cellular membranes refer to all of the membranes within a cell, including the plasma membrane (or cell membrane) that surrounds the cell and the membranes that form the organelles. 1 s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 65 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 65 Sugar residues of glycoprotein Glycoprotein The unusual ability of cholesterol to increase fluidity while decreasing permeability provides animals with an important mechanism for controlling membrane properties. Phospholipid bilayer Pe ar so While the fluid mosaic model illustrates the general relationships between lipid and protein variation in membrane proteins, it underemPeripheral Cholesterol proteins Integral phasizes the spatial variation seen proteins in membrane lipids (Figure 2.45). Figure 2.43 Fluid mosaic model Membranes are composed of lipids The inner and outer layers of the such as phospholipids, cholesterol, and glycolipids. Proteins can be embedded in phospholipid bilayer typically posthe lipid bilayer. Each of the elements moves laterally within the membrane, sess different types of lipids. PE giving it a functional fluidity. and PS are found almost exclusively in the inner leaflet, whereas Cholesterol has an unusual role in memPC is concentrated in the outer leaflet. Glycolipids branes. Although it is absent from some memare found only in the outer leaflet of the membranes, such as mitochondrial membranes, brane. Membranes also possess discrete regions cholesterol can compose almost half the lipid comthat are enriched in cholesterol and glycolipids. ponent of other membranes. Cholesterol influThese lipid rafts serve two important functions. ences membrane properties in complex ways Their molecular composition causes a slight thickbecause of the way it integrates into the lipid biening of the lipid bilayer, which recruits phospholayer (Figure 2.44). One end of the molecule interlipids with longer chain fatty acids and proteins acts with phospholipids near the polar head with relatively long transmembrane domains. Begroups, filling gaps between phospholipids to recause of the distinct molecular composition of lipid duce the permeability to low-molecular-weight rafts, they can act as microcompartments within solutes. Cholesterol also disrupts the interactions the cell, providing an additional way to spatially between fatty acids, enhancing membrane fluidity. organize pathways. Channel protein Lipid membranes are heterogeneous Le No ar tF n or in Di Re g s O tri H3C CH3 CH3 n r bu tio CH2 CH CH2 CH2 sa CH n CH2 CH3 Environmental stress can alter membrane fluidity An essential component of the fluid mosaic model is the ability of the constituents to move throughout the membrane. Phospholipids and proteins can rotate in position as well as move laterally through the membrane. Membrane fluidity depends on the properties of the membrane lipids, which are influenced by the physical environment. Cells regulate the fluidity of the membrane by controlling the nature of lipids to achieve the appropriate degree of molecular movement. Low temperature, for example, can strengthen the van der Waals forces between membrane lipids, restricting molecular movement within the membrane (Figure 2.46). Since this can adversely affect membrane function, many animals actively So le lu t HO Cholesterol io n s Figure 2.44 Unusual membrane properties of cholesterol Cholesterol strengthens the interactions between phospholipid polar head groups while disrupting the interactions between fatty acid tails. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 66 66 PART ONE The Cellular Basis of Animal Physiology Extracellular fluid Cholesterol Glycolipids Figure 2.45 Membrane heterogeneity Cellular membranes are heterogeneous in composition. Most cells maintain distinct profiles in inner and outer monolayers, sometimes exchanging phospholipids between layers. Lipid rafts are regions of the plasma membrane that accumulate cholesterol and glycolipids, thickening the membrane. These thicker regions preferentially recruit proteins with longer transmembrane domains. Pe ar so Cytoplasm Lipid raft remodel their membranes to compensate for the effects of the physical environment. By altering the membrane lipid profile, they can keep membrane fluidity constant. We will discuss this pattern of membrane regulation, called homeoviscous adaptation, in Chapter 13: Thermal Physiology. Membranes possess integral and peripheral proteins Protein is an important constituent of most cellular membranes, in some cases making up more than half the mass of the membrane. Integral membrane proteins are tightly bound to the membrane, either embedded in the bilayer or spanning the entire membrane. Peripheral membrane proteins have a weaker association with the lipid bilayer, typically binding to integral membrane proteins or glycolipids. The different relationships between the bilayer and membrane proteins are shown in Figure 2.47. Le No ar tF n or in Di Re g s O tri r bu tio sa n n Membrane proteins have important structural and regulatory roles within cells. They contribute to structural support by linking the intracellular cytoskeleton to the extracellular matrix. Many of the intrinsic membrane proteins are receptors that are part of complex signaling pathways. Because membranes are physical barriers to the free movement of many vital organic and inorganic solutes, cells use integral proteins to transport molecules across membranes. Transport Across Cellular Membranes Many cellular processes depend on the ability to move molecules across membranes. A cell must be able to bring nutrients across its plasma membrane and expel end products. Hormones synthesized within the cell must be processed and packaged for secretion. All animal cells must be able to move specific ions across the plasma membrane to control their ionic and osmotic properties. Specific integral membrane proteins mediate these transport processes. The kinetic properties of a transporter are similar to those of enzymes, with an affinity constant (Km) and a maximal velocity (Jmax). The three main classes of membrane transport are passive diffusion, facilitated diffusion, and active transport. These classes of transport are distinguished by the direction of transport, the nature of the carriers, and the role of energy in the process (Figure 2.48). So le lu t io n Lipid-soluble molecules cross membranes by passive diffusion Cold Warm s Liquid crystalline Gel Figure 2.46 Temperature and membrane fluidity Temperature alters the fluidity of membranes by changing the interactions between phospholipids. Although membranes are barriers to the movement of many molecules, some molecules cross membranes without a transporter. The ability of a molecule to freely cross a biological membrane depends on its hydrophobicity. Many molecules, such as steroid hormones, are freely soluble in lipid. When these molecules encounter a cell membrane, they dissolve into the lipid bilayer and escape to the other side. Both influx and efflux oc- 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 67 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 67 Pe ar so cur simultaneously, but the net movement (influx minus efflux) depends on the concentration gradient. The net movement of molecules is from high concentration to low concentration. The steeper the concentration gradient, the greater the rate of movement across the membrane. This type of transport is called passive diffusion. No specific transporters are required, and no energy, beyond the concentration gradient itself, is required. Peripheral protein P P Glycolipid anchor -barrel Single pass -helix Multiple pass -helix Peripheral protein Membrane proteins can facilitate the diffusion of impermeant molecules Hydrophilic molecules cross membranes by other pathways that involve specific transport proteins. If the concentration gradient is favourable, the Outside of cell molecule may cross the membrane Lipid-soluble by facilitated diffusion. As with solute passive diffusion, no energy beyond Concentration that of the concentration gradient is gradient required to drive transport, but with facilitated diffusion a protein is required to carry the molecule across Energy the membrane. Three main types of proteins carry out facilitated diffusion: ion channels, porins, and perPassive Channel Permease Active meases (see Figure 2.49). diffusion transport Ion channels are membrane Facilitated diffusion proteins that form pores through Inside of cell which only specific ions may pass, and only when the channel is open. Figure 2.48 Modes of membrane transport The mechanisms of The channels are specific to one or transport across cellular membranes depend on the lipid solubility of the solute as sometimes two ions. Ca2 channels, well as the direction and magnitude of the concentration gradient. Passive diffusion for instance, possess a structure that needs no carrier, as lipid-soluble solutes move freely across the membrane. Facilitated diffusion carries impermeant solutes across the membrane on protein allows the free movement of Ca2 , carriers, including channels (either ion channels or porins) and permeases. Solutes but does not allow other cations can also be transported by active transport, which can move molecules against a such as Mg2 , K , or Na to cross at concentration gradient. appreciable rates. The specificity of transport is due to a structural component of the IP3, is present. Voltage-gated channels are opened or closed in response to membrane potenchannel known as the selectivity filter. The chantial. For example, K channels in muscle and neunel can be opened in response to cellular condirons open when the membrane depolarizes. tions. Ligand-gated channels are opened when Mechanogated channels are regulated through specific regulatory molecules are present. One im2 interactions with the subcellular proteins that portant ligand-gated channel is the Ca channel sensitive to inositol triphosphate (IP3); this channel make up the cytoskeleton. Changes in cell shape, such as cell swelling, alter the arrangement of the induces the release of Ca2 stores when its ligand, Le No ar tF n or in Di Re g s O tri r bu tio sa n n Integral membrane proteins Membrane proteins can demonstrate many different types of relationships with membranes. Each membrane protein has within its structure hydrophobic regions that interact favorably with the bilayer. Depending on the protein, these regions can be -helices or -barrels. Transmembrane proteins span the entire bilayer, exposing regions to both sides of the membrane. Often these exposed regions possess modifications, such as the carbohydrate chains of glycoproteins. Peripheral membrane proteins are not embedded within the membrane but associate with exposed regions of integral membrane proteins. Figure 2.47 So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 68 68 PART ONE The Cellular Basis of Animal Physiology (a) Voltage-gated Figure 2.49 Carriers involved in facilitated diffusion Channels are proteins that mediate facilitated diffusion of ions and other metabolites. Channels typically exist in either a closed or an open conformation. They are opened by specific triggers, including specific ligands, voltage conditions, or physical associations with structural elements. cytoskeleton. Upon sensing the changes in the cytoskeleton, mechanogated channels may open or close. Porins are large channels that function in similar ways to ion channels but permit the passage of much larger molecules. Mitochondria have a porin in the outer membrane that facilitates the transfer of low-molecular-weight molecules from the cytoplasm to the mitochondria. Aquaporins are water channels in the plasma membranes; each aquaporin molecule can transport 3 billion water molecules per second. Some aquaporins, called aquaglyceroporins, are also capable of transporting nonwater molecules, such as glycerol and possibly urea. Aquaporins may also be involved in transport of gases across membranes. The third type of protein that facilitates diffusion is a permease. Rather than creating a pore for a molecule, a permease functions more like an enzyme. It binds the substrate and then undergoes a conformation change that causes the carrier to release the substrate to the other side. Several tissues possess glucose permease, a transporter that allows glucose to enter cells, passing from high concentration to low concentration. Unlike porins and ion channels, permeases can become saturated with substrate at high concentration, such that the transport process depends on how quickly the permease can carry its substrate across the membrane. Pe ar so (b) Ligand-gated (c) Mechanogated Le No ar tF n or in Di Re g s O tri r bu tio sa n n Active transporters use energy to pump molecules against gradients In passive and facilitated diffusion, molecules can move only from high concentration to low concen- tration. In contrast, cells use active transport to move molecules across membranes against concentration gradients. Two main forms of active transport are distinguished by the source of the energy that drives the process. In primary active transport, the carrier protein uses an exergonic reaction to provide the energy to transport a molecule. The other form of active transport, called secondary active transport, couples the movement of one molecule to the movement of a second molecule. The most common primary active transporters use the hydrolysis of ATP to provide the necessary energy. Three general classes of ATP-dependent transporters, or ATPases, mediate primary active transport: P-type ATPases, F-type (or V-type) ATPases, and ABC transporters. P-type ATPases use ATP hydrolysis to pump specific ions across membranes. For example, animal cells have a Na /K ATPase in the cell membrane that extrudes Na from the cell in exchange for K . Many tissues have Ca2 ATPases to transport Ca2 across membranes. F-type and V-type ATPases are structurally related ATPases that pump H across membranes using the energy of ATP hydrolysis. The mitochondrial F-type ATPase operates in reverse, using H movements down electrochemical gradients to provide the energy for ATP synthesis. The V-type ATPases allow cells and organelles to extrude protons to acidify a compartment, such as the lumen of the lysosome or the inside of the stomach. The ABC transporters carry large organic molecules across the cell membrane. Cells often use ABC transporters to export toxins from the cell. The multidrug resistance protein, an important ABC transporter, is often linked to types of cancers So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 69 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 69 Pe ar so that become resistant to chemotherapy. Some cancerous cells survive chemotherapy by transporting the toxic drug out of the cell before the chemotherapeutic agent can kill it. Secondary active transport uses the energy held in the electrochemical gradient of one molecule to provide the energy to drive another molecule against its gradient. If the molecules move in opposite directions, the carrier is called an antiport, or exchanger. For example, red blood cells use a Cl /HCO3 exchanger (also called band 3) to drive the transport of these ions across the membrane. The direction of ion movement by this carrier depends on the relative gradients of the two ions. Alternatively, a symport, or cotransporter, is used to move molecules in the same direction. For example, intestinal cells use a Na -glucose cotransporter to import glucose against its concentration gradient, driven by a greater inward Na electrochemical gradient. All of these transport processes influence chemical gradients across membranes, but only a subset of transporters affect the electrical gradient. Carriers that transport uncharged molecules, such as the glucose permease, are termed electroneutral carriers. Similarly, carriers such as the Cl /HCO3 exchanger that exchange two ions of the same charge are also electroneutral. In contrast, the carriers that transfer a charge across the membrane are called electrogenic carriers. When we discuss Na /K ATPase throughout this text, keep in mind that it is an electrogenic carrier because it exchanges 3 Na for 2 K ions. ganisms. Electrical signaling is not, however, a unique property of nerve and muscle cells. Several other types of cells use electrical signals, including fertilized eggs and hormone-secreting cells. The interior of the membrane is electronegative at rest Membrane potential can be measured using a microelectrode. Microelectrodes consist of a thin recording electrode encased in a very fine-tipped glass pipette that can be inserted through the cell membrane into the cell. The microelectrode is connected via a voltmeter to a reference electrode that is immersed in the solution outside the cell. The voltmeter measures the voltage drop across the circuit caused by the membrane potential (Vm). In most animal cells, the membrane potential is between 5 and 100 mV. By convention, the membrane potential is expressed relative to the voltage outside the cell. Thus, the negative value for Vm means that the interior of the cell membrane is more electronegative than the exterior of the cell membrane. Le No ar tF n or in Di Re g s O tri r bu tio sa n n Ionic concentration gradients and permeability establish membrane potential Membrane Potential All animal cells maintain a voltage difference across their cell membranes, as well as some organelle membranes, such as the mitochondrial membrane. This voltage difference represents a source of potential energy that cells can harness to move molecules across membranes. The voltage difference is termed the resting membrane potential difference, or the membrane potential, for short. In addition to using the membrane potential as a source of energy, excitable cells use changes in membrane potential as communication signals. As we discuss in Chapters 4 and 5, this property is particularly important for nerve and muscle cells, and thus the membrane potential is critical for allowing the coordinated movements of cells and or- Only two factors are required to establish a potential difference across a membrane: a concentration gradient for an ion and a membrane that is permeable to that ion. Consider a situation where two solutions are separated by a membrane that is impermeable to ions (Figure 2.50). Assume that the interior of the cell contains 100 mM KCl and 10 mM NaCl, and the extracellular fluid contains 100 mM NaCl and 10 mM KCl. The concentration gradient for K (100 mM inside the cell and 10 mM outside the cell) favors outward movement, whereas the concentration gradient for Na (100 mM outside and 10 mM inside the cell) favors inward movement. There is no gradient for the movement of Cl (because the concentration of Cl is 110 mM both inside and outside the cell). The solutions on either side of the membrane are also electroneutral, with equal numbers of anions and cations. To create a membrane potential, we insert channels that allow the passage of K , but no other ion. The concentration gradient will cause K to move out of the cell along the concentration gradient, creating a local region of electronegativity on So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 70 70 PART ONE The Cellular Basis of Animal Physiology Cl ion K+ ion Na+ ion Inside cell: 100 mM KCl, 10 mM NaCl Outside cell: 10 mM KCl, 100 mM NaCl Pe ar so Inside cell + + + + + + + + + + + + + + 1 + + + + + + No permeability to any ion Concentration gradient Outside cell + + + + + + + + + + + + + + + Electrical gradient + + + + Figure 2.50 the inner face of the membrane (where K left) and a local region of electropositivity on the outer face of the membrane (where K appeared). This excess negative charge at the inside face of the membrane generates an electrical force that tends to draw positive charges back into the cell. As more K leaves the cell, the electrical force gradually increases to a level that exactly balances the driving force from the K concentration gradient. Potassium ions continue to move across the membrane, but their inward and outward fluxes exactly balance each other. The potential difference across the membrane under these equilibrium conditions is termed the equilibrium potential for that ion (Eion). Because only a single ion can move across the membrane in this hypothetical example, the equilibrium potential is equivalent to the resting membrane potential (Eion Vm). For a given concentration gradient, it is possible to calculate the equilibrium potential for an ion using the Nernst equation, Le No ar tF n or in Di Re g s O + + + 2 K+ channels open, K+ exits cell n + + + + + + + + + + + + + + + ++ + + ++ + + + + + + + + 3 Outward concentration gradient eventually equals inward electrical gradient Potassium movements and membrane potential tri r bu tio sa n Eion 3X4 outside RT ln zF 3X4 inside where R is the gas constant, T is the temperature (Kelvin), z is the valence of the ion, F is the Faraday constant (23,062 cal/V-mol), and [X] is the molar concentration of the ion. In our example, [K ]outside 10 mM and [K ]inside 100 mM, resulting in EK 60 mV. In other words, the force driving the outward movement of K resulting from its 10-fold concentration gradient can be exactly balanced by a 60 mV excess of negative charge inside the membrane (a membrane potential of 60 mV). The equilibrium potential for a particular ion is often termed its reversal potential, because the direction of ion movement across the membrane changes when the voltage difference across the membrane exceeds this level. In the case of our hypothetical example, net K flux was down its concentration gradient from the inside to the outside of the cell until the membrane potential difference reached 60 mV. If the membrane potential were to become even more negative, the net movement of K would be from the outside of the cell back to the inside--against its concentration gradient. Note that the actual number of ions that need to move across the membrane before So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 71 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 71 Pe ar so reaching the equilibrium potential is actually very small (less than 1/100,000 of the total K ions within a typical cell), and does not result in a measurable change in the overall K concentration either inside or outside the cell. It is important to emphasize that the membrane potential is the result of extremely small differences in the number of charged molecules immediately adjacent to the membrane--a difference that is too small to detectably affect the overall ion concentration of the cytoplasm or extracellular fluid. The localization of the charge difference immediately adjacent to the membrane arises because the cell membrane acts as a capacitor. A capacitor is a device containing two electrically conductive materials separated by an insulator, a very thin layer of a nonconducting material. Electrical charges can interact with each other across the insulator if the layer is sufficiently thin. In a cell, the cytoplasm and the extracellular fluid are conducting materials, whereas the lipid bilayer of the cell membrane is the insulator. The excess positive charge along the outside of the membrane attracts the excess negative charge along the intracellular face of the membrane. These electrical interactions can only occur across very small distances, and do not affect ions in the bulk phase of the cytoplasm or extracellular fluid. Thus, the membrane potential occurs only in the area immediately adjacent to the membrane. and permeabilities of all of the relevant ions (see Box 2.3, Mathematical Underpinnings: The Goldman Equation). The Goldman equation essentially represents the sum of the equilibrium potentials for all of the relevant ions, with a weighting factor that takes into account the relative permeabilities of the ions. The influence of each ion over the overall membrane potential is thus proportional to its permeability. For example, resting neurons are more permeable to K than to the other ions, and as a result, K plays the major role in setting the resting membrane potential of neurons. Le No ar tF n or in Di Re g s O tri r bu tio sa n n The Na /K ATPase establishes concentration gradients Active pumping of Na and K ions by the electrogenic Na /K ATPase is responsible for establishing the concentration gradients for these ions across the cell membrane. Ultimately it is these concentration gradients (along with the selective permeability of the membrane) that establish the membrane potential. The Na /K ATPase is also responsible for maintaining the resting membrane potential. Although most membranes are only sparingly permeable to Na at rest, a small amount of Na does leak into the cell down its electrochemical gradient, while K leaks out. Without appropriate compensation, these ion movements would result in the dissipation of the Na and K concentration gradients that are needed to establish the membrane potential. Cells use the Na /K ATPase to compensate for the leakage of Na and K ions. If you poison the Na /K ATPase with a drug called ouabain, the membrane potential difference of the cell slowly decays over the course of a few hours, eventually reaching a value of 0 mV. Potassium plays the major role in establishing membrane potential In our hypothetical example neither Na nor Cl affected the membrane potential because there was no concentration gradient for Cl and because the membrane was not permeable to either of these ions. Of course, the situation in real cells is not so simple, since there are several ions that differ in concentration between the inside and the outside of the cell, and real membranes have varying degrees of permeability to multiple ions. For most cells, the primary ions that affect the membrane potential are K , Na , and Cl because they can move across membranes and there are differences in their intracellular and extracellular concentrations. A modification of the Nernst equation, the Goldman-Hodgkin-Katz Constant Field equation (usually referred to as the Goldman equation) can be used to calculate the resting membrane potential based on the concentrations So le lu t Changes in membrane permeability alter membrane potential Because ion permeability is a major factor involved in establishing the resting membrane potential, changes in ion permeability cause changes in membrane potential. Excitable cells such as neurons and muscle cells alter the permeability of their membranes to generate changes in membrane potentials. We can use the Nernst equation to predict the nature of the ion movements following changes in membrane permeabilities. For example, in mammalian neurons the concentration io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 72 72 PART ONE The Cellular Basis of Animal Physiology BOX 2.3 MATHEMATICAL UNDERPINNINGS The Goldman Equation The two terms for K permeability also cancel out, leaving an equation that is equivalent to the Nernst equation for potassium (with the exception of the valence term, z, which is neglected because potassium has a valance of 1). Notice, however, that if we do the same exercise assuming that the membrane is permeable only to chloride, we end up with an equation that is similar to the Nernst equation, except that it neglects the valence and has the intracellular concentration in the numerator and the extracellular concentration in the denominator, rather than the other way around. Recall that one of the rules of logarithms is that ln x ln (1/x). By inverting the ratio of the concentrations of chloride, the Goldman equation takes into account the fact that chloride has a valence of 1. In addition to providing an estimate of the resting membrane potential, the Goldman equation allows the estimation of the membrane potential during electrical signaling. For example, when a large number of Na channels open within the membrane (as is the case during signaling in nerve cells), the permeability of the membrane to Na increases greatly. In the case of neural signaling, this increase in Na permeability is so large that PNa becomes much greater than PK and PCl. Under these conditions, the Goldman equation is dominated by the term for Na , and the membrane potential approaches the equilibrium potential for Na as calculated by the Nernst equation. Pe ar so Em RT PK 3 K 4 o ln F PK 3 K 4 i The Goldman-Hodgkin-Katz Constant Field equation (often simply referred to as the Goldman equation or GHK), allows the estimation of the membrane potential based on the concentrations, valences, and relative permeabilities of a series of ions. Since most plasma membranes under resting conditions have appreciable permeability only to postassium, sodium, and chloride, the Goldman equation can be written as follows: where Pion is the permeability of the membrane to that ion and [ion]o and [ion]i represent the extracellular and intracellular concentrations, repectively, of a given ion. From the Goldman equation, the impact of ion permeability on the membrane potential is clear. Any ion with a low permeability has little effect on the membrane potential, even if there is a large concentration gradient across the membrane for that ion. Notice that if the permeability of the membrane for an ion is zero, then the term for that ion drops out of the equation. For example, consider the case of a membrane that is impermeable to Na and Cl . In this case, the Goldman equation simplifies to Em Le No ar tF n or in Di Re g s O tri RT PK 3 K 4 o ln F PK 3 K 4 i n PNa 3 Na 4 i PNa 3Na 4 o PCl 3Cl 4 o PCl 3Cl 4 i of Na is typically about 10-fold greater outside the cell, so ENa 58 mV. In contrast, K concentration outside the cell is only about 1/40 of that inside the cell, so EK 90 mV. The resting membrane potential of neurons is typically about 70 mV. If the Na permeability of the membrane increases (as a result of the opening of Na channels), Na will enter the cell, because both the electrical and concentration gradients favor inward Na movement until the membrane potential reaches the equilibrium potential for Na of 58 mV. The resulting inward Na movement causes a reduction in the membrane potential termed a depolarization (Figure 2.51). In contrast, if the K permeability of the membrane increases (as a result of the opening of K channels) K will move out of the cell, because both its concentration and electrical gradients favor outward r bu tio sa n K movement until the membrane potential reaches the equilibrium potential for K of 90 mV. The loss of positive charges from the interior of the cell results in a hyperpolarization. As we discuss in later chapters, many cells use cycles of depolarization, hyperpolarization, and repolarization as communication signals. So le lu t io n s Subcellular Organization Eukaryotes rely on complex intracellular organization to orchestrate the many processes required for life. Central to the diversity in physiological function is the ability of individual cells to perform specific roles for the tissue and animal. We gain a clearer understanding of complex physiological systems by studying the role of the various cellular compartments that contribute to the process. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 73 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 73 Pe ar so Em 3K 4o RT ln F 3K 4i [K ]i [Na ]i [Cl ]i Experimentally, it is easier to measure the relative permeability of ions, rather than the absolute permeability. Hence, the Goldman equation is often rewritten after dividing each term by PK. PNa>PK 3Na 4 i PNa>PK 3Na 4 o PCl>PK 3Cl 4 o PCl>PK 3Cl 4 i For a cell such as a squid giant axon, the following values can be used to calculate the membrane potential: 400 mM and [K ]o 50 mM and [Na ]o 0.04 51 mM and [Cl ]o 0.45 Substituting these values into the Goldman equation predicts the membrane potential of this squid giant axon to be 60 mV at rest, which is a good approximation of the measured resting membrane potential. Returning to the Nernst equation, we can also calculate the equilibrium potentials for each of these ions. Using the concentrations relevant to the squid giant axon, the equilibrium potential is 75 mV for K , 55 mV for Na , and 60 mV for Cl . These equilibrium potentials establish the "boundary conditions" for the membrane potential. That is, the membrane potential Le No ar tF n or in Di Re g s O 560 mM PNa / PK PCl / PK n 20 mM 440 mM cannot be more negative than 75 mV or more positive than 55 mV because there are no chemical gradients large enough to produce larger membrane potential differences. At rest, the membrane does not quite reach the equilibrium potential for K because of the competing effects of Na , but because Na permeability is relatively low its influence is small, and the membrane potential is close to the K equilibrium potential. Note that the squid giant axon also has appreciable permeability to Cl (about half that of K ). In fact, some cell membranes (e.g., in muscle cells) are more permeable to Cl than they are to K . However, even in this case, K plays the major role in establishing the membrane potential. The Na /K ATPase actively pumps Na and K ions to establish their concentration gradients. The K concentration gradient sets the resting membrane potential difference, and Cl ions passively distribute themselves across the membrane in response. Thus, in the case of Cl ions, the intracellular and extracellular Cl levels are a consequence rather than a cause of the resting membrane potential. Reference Mitochondria are the powerhouse of the cell Mitochondria are complex organelles, possessing intricate networks of membranes (Figure 2.52). The innermost compartment is the mitochondrial matrix, delimited by the inner mitochondrial membrane. The outer mitochondrial membrane surrounds the organelle and creates another compartment called the intermembrane space. Each of these compartments has its own complement of enzymes and performs different functions for the mitochondria and the cell. The matrix houses the enzymes and metabolites of the TCA cycle. The inner mitochondrial membrane, which is often highly convoluted, holds the enzymes of oxidative phosphorylation and all the transporters necessary to move metabolites in and out of the mitochondria. About 80% of the mass of the inner membrane is protein, the highest protein content tri r bu tio sa q Hodgkin, A. L., and A. F. Huxley. 1952. A quantitative description of membrane current and its application to conduction and excitation in the nerve. Journal of Physiology 117: 500544. n of any biological membrane in animals. Mitochondria organize the inner membrane into layers, or lamellae, that are tightly folded. In some tissues, as much as 70 m2 of mitochondrial inner membrane can be folded into a 1-cm3 volume of mitochondria. Mitochondrial structure varies greatly among cell types. Many cells, such as liver, contain hundreds of individual oblong mitochondria scattered throughout the cell. These individual mitochondria are rapidly transported throughout the cell. Some cells organize their mitochondria into networks of interconnected organelles called the mitochondrial reticulum, which is constantly remodeled by enzymes that mediate its fission and fusion. Earlier in this chapter you learned that mitochondria possess the enzymes of oxidative phosphorylation, and make most of the ATP a cell So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 74 74 PART ONE The Cellular Basis of Animal Physiology Na+ channel K+ Na+ K+ channel Open Na+ Open K+ channels mV mV 50 100 Hyperpolarization and depolarization The gradients of Na and K across the cell membrane largely determine the resting membrane potential. When specific ion channels open, the movement of ions changes the membrane potential. If K moves out of the cell, the magnitude of the membrane potential increases (hyperpolarization). If Na moves into the cell, the magnitude of the membrane potential decreases (depolarization). requires. Cells frequently respond to changes in energy demand by altering their levels of mitochondria, using both biosynthetic and degradative pathways. Most of the genes required for synthesis of mitochondrial proteins are located in the nucleus. Mitochondrial biogenesis requires that each of these genes be expressed in unison to produce the hundreds of proteins needed for new mitochondria or an extension of the mitochondrial reticulum. Mitochondrial biogenesis also requires replication of mitochondrial DNA (mtDNA) and synthesis of additional mitochondrial membranes. Degradative pathways control the levels of mitochondria and mitochondrial proteins. Damaged mitochondrial fragments are engulfed by autophagosomes and degraded in lysosomes. Cells that fail to destroy defective mitochondria suffer energy shortfalls and eventually cell death. It has an important role in maintaining cell structure, acting as a frame upon which the cell membrane is mounted. It gives the cell its characteristic external shape and also supports and organizes intracellular membranes. Organelle networks such as the endoplasmic reticulum and Golgi apparatus are mounted on the cytoskeleton. The cytoskeleton is dynamic in structure, under constant reorganization. Apart from its structural roles, the cytoskeleton is an important participant in many cellular processes, including signal transduction. The cytoskeleton is constructed from three types of fibers: microfilaments, microtubules, and intermediate filaments. These proteins are long strings of monomers connected end-to-end to form a polymer. Microtubules are large, stiff tubes composed of the protein tubulin. Microfilaments are small, flexible chains of actin. Intermediate filaments, so named because they are intermediate in size, are composed of many types of monomers. Most cells possess each of these cytoskeletal elements, but many cells are richer in one particular type. For example, the tails of sperm are largely microtubules, muscles are largely actin Figure 2.51 Le No ar tF n or in Di Re g s O 50 Depolarization 100 Time (msec) Time (msec) mV Pe ar so +50 0 channels n +50 0 +50 0 50 100 Time (msec) Hyperpolarization tri r bu tio sa n So le lu t io n s The cytoskeleton controls cell shape and directs intracellular movement The cytoskeleton is a network of protein-based fibers that extends throughout the cell (Figure 2.53). 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 75 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 75 Mitochondrial reticulum Myofibril Microtubules (green) Pe ar so Outer membrane Nucleus Inner membrane Mitochondrial cross-section Matrix Microfilaments (red) Figure 2.52 Mitochondrial structure Mitochondria are found in almost every cell type, but with many different appearances. Muscle mitochondria exist as a network extending throughout the muscle myofibrils. In cross-section they appear as individual organelles, but three-dimensional reconstructions show the reticulum structure. Inside the mitochondria the highly folded inner membrane can be seen. polymers, and skin is rich in the intermediate filament keratin. Other proteins work in conjunction with the cytoskeleton to conduct many types of movement. These proteins, called motor proteins, are mechanoenzymes that use the energy of ATP hydrolysis to walk along the cytoskeleton. Myosin is the motor protein that walks along actin polymers; kinesin and dynein move on microtubules. In Chapter 5: Cellular Movement and Muscles, we discuss the structure and function of the cytoskeleton and motor proteins in the context of cellular and intracellular movement. Le No ar tF n or in Di Re g s O tri r bu tio sa (b) n Contact site (a) Intermediate filaments Nucleus n Figure 2.53 Three protein fibers of the cytoskeleton Panel (a) shows microtubules (green) and microfilaments (red). Panel (b) shows intermediate filaments. So le lu t The endoplasmic reticulum and Golgi apparatus mediate vesicular traffic Cells have layers of membranous organelles extending around the nucleus to the periphery of the cell (Figure 2.54). The first layer, the endoplasmic reticulum (ER), is the gateway to the other compartments. Proteins are made in the ER, folded, and then sent to their final destinations in the plasma membrane, the Golgi apparatus, lysosomes, and endosomes. The vehicle that carries proteins between compartments is a vesicle, a small membranebound organelle. Some vesicles are surrounded by a shell of coat proteins, such as clathrin, coat protein complex I (COP-I), and COP-II. These proteins help form the vesicle, but they also have an important influence on where the vesicle is sent. Cells are often illustrated in ways that suggest that vesicles drift freely throughout the cytoplasm. In reality, vesicles are carried throughout the cell by motor proteins moving on cytoskeletal tracks. For example, vesicles coated with COP-I may be carried toward the Golgi apparatus, whereas vesicles coated with COP-II may be sent to the ER. Coat proteins and other vesicle membrane proteins influence which motor protein is io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 76 76 PART ONE The Cellular Basis of Animal Physiology Damaged mitochondrion Autophagosome Lysosome Cytoplasm Phagocytosis Extracellular fluid Autophagy Late endosome Pe ar so Endoplasmic reticulum Pinocytosis Plasma membrane Early endosome Membrane protein cycling Figure 2.54 Intracellular traffic Vesicles move throughout the cell, transferring membranes and vesicle contents between compartments. bound. If a vesicle binds myosin it will be carried on microfilaments, but if it binds dynein it will be carried on microtubules. Protein kinases and protein phosphatases regulate vesicular traffic by altering the cytoskeleton, motor proteins, or vesicle proteins. These processes ensure that vesicles and their contents are sent to the correct location at the correct time. Many types of intracellular sorting pathways use the ER-Golgi network. Most cells produce proteins, and sometimes other molecules, for release from the cell. This process, called exocytosis, begins in the ER. Proteins are made here and packaged into vesicles that move through the Golgi apparatus, ultimately fusing to the plasma membrane to release the vesicle contents to the extracellular space. In the reverse pathway, endocytosis, vesicles form at the plasma membrane, engulfing liquid droplets (pinocytosis) or large particles (phagocytosis). The same pathways of endocytosis and exocytosis regulate the proteins found in the plasma membrane, such as membrane transporters and channels. When transporters are no longer needed, they can be removed from the membrane and stored in vesicles until needed again. Conversely, when a secretory vesicle fuses to the plasma membrane, its internal contents are expelled but the vesicle membrane, both lipid and integral proteins, disperses into the plasma membrane. Cells control the numbers and types of proteins in the plasma membrane through endocytosis and exocytosis. Vesicles rich in transporters fuse to the plasma membrane to increase transport capacity. Conversely, regions of the plasma membrane are extracted during vesicle formation to remove transporters for storage or degradation. Vesicles in transit can be directed to other compartments to assist in processing their contents. Endosomes act as clearinghouses for vesicles, collecting them and then redistributing their contents and membrane proteins into new vesicles that are sent to their correct locations. They send damaged proteins and foreign materials to lysosomes for proteolytic degradation. Once vesicles reach their destination, another series of proteins mediate the fusion of vesicles with target membranes. The pathways of intracellular sorting allow animal cells to control many of the processes we have considered throughout this chapter, including secretion, ingestion, and membrane transport. Le No ar tF n or in Di Re g s O Golgi network Storage vesicle n Secretory vesicle tri r bu tio sa n So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 77 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 77 Another function of these pathways, specifically the secretory pathway, is to build and maintain a fibrous network outside the cells: the extracellular matrix. Proteins and glycoproteins Simple structures Carbohydrate Collagen Complexes Collagen monomes Cross links Collagen fibril Elastin fiber Collagen Fibronectin Pe ar so The extracellular matrix mediates interactions between cells Elastin Cells are organized into a threedimensional tissue by a network of Heparin fibers called the extracellular maGAGs and proteoglycans trix. The proteins used to build the matrix are synthesized by the ER, Simple structures Complexes packaged into vesicles, and sent out Hyaluronan of the cell using the secretory pathHyaluronan way. During transit through the Golgi apparatus, suites of enzymes modify Aggrecan Core protein Keratan sulfate aggregate the proteins, adding branched chains of sugars. As you learned earlier in Aggrecan this chapter, glycosylation alters the properties of the proteins in many Chondroitin sulfate Aggrecan ways. In the extracellular matrix, water binds to the hydrophilic sugars to Figure 2.55 Extracellular matrix components The extracellular matrix create a gel-like coating that fills the is composed of combinations of proteins and glycoproteins, glycosaminoglycans (GAGs) and proteoglycans. Many of the individual molecules, shown in the left column, space between cells. Extracellular matrix macromol- can be combined into more complex macromolecules, shown on the right. The protein ecules can be proteins, simple gly- components are shown in green and the GAG components in blue. coproteins, glycosaminoglycans, or lently attached to proteins to form proteoglycans. combinations of both, known as proteoglycans Cartilage is composed primarily of aggrecan, a (Figure 2.55). Collagen is a long, stiff fiber formed proteoglycan that incorporates more than 100 glyas a triple helix of three separate collagen glycocosaminoglycans into its structure. Many proteoprotein monomers. Elastin is a small protein that glycans link the different extracellular matrix is linked together into an intricate web. When the proteins together to form a network. network is stretched it acts like a rubber band, The extracellular matrix can be simple in providing the tissue with elasticity. Many extracelstructure and composed of only a few proteins, or lular matrix components are linked together by it can be organized into an extensive network. The the glycoprotein fibronectin. Each fibronectin molextracellular matrix is more than just the cement ecule binds other fibronectins as well as different that connects cells together. Many specialized matrix components to form a fibrous network. structures such as the insect exoskeleton, verteHyaluronan is a glycosaminoglycan composed brate skeleton, and molluscan shells are modified of thousands of repeats of the disaccharide gluextracellular matrices secreted by specific cells. curonic acid-N-acetylglucosamine. With its hydraFor example, bone and cartilage are tissues tion shell, it forms a noncompressible gel that acts formed from the extracellular matrix of osas a cushion between cells. Hyaluronan fills the teoblasts and chondroblasts, respectively. The spaces between joints of land animals, easing basal lamina (Figure 2.56), or basement memmovement. Other glycosaminoglycans, such as 2 brane, is a type of extracellular matrix found in chondroitin sulfate and keratan sulfate, are covamany tissues, where it acts as a solid support that 2 helps anchor cells. It is designed and maintained Keratan is a GAG of the extracellular matrix, whereas keratin is an intermediate filament protein of the cytoskeleton. primarily by specialized cells called fibroblasts. Le No ar tF n or in Di Re g s O tri r bu tio sa n n So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 78 78 PART ONE The Cellular Basis of Animal Physiology Epithelial cell Connective tissue: Collagen Blood vessel Hyaluronan Macrophage Fibroblast Plasma membrane Collagen Proteoglycans Membrane protein Figure 2.56 Basal lamina In many tissues, fibroblasts produce a thick layer of extracellular matrix called the basal lamina. Some cells use the basal lamina as a foundation, but other cells and blood vessels use it as a porous frame. Cells use various strategies to modulate both the matrix properties and their relationship with the matrix. First, most types of extracellular matrix components can be made many ways. For instance, mammals have 20 different collagen genes, so in principle a collagen trimer can be constructed 8000 (203) ways. Even though most of these possible variants are never constructed, it illustrates the potential for variation in one of the many components of the extracellular matrix. Second, variations occur in the type and position of carbohydrate groups of simple glycoproteins and proteoglycans. Each variation influences the physical properties of the extracellular matrix protein. By controlling which proteins are made and how they are modified by glycosylation, cells determine which building blocks are available to build the extracellular matrix. Cells control which proteins are released to the extracellular space using the secretory pathway discussed in the previous section. Secreting the extracellular matrix components from the cell is really only one step in building a tissue. The cells also produce integral membrane proteins called matrix receptors to connect them to the extracellular matrix. Integrins are an important class of plasma membrane receptors that bind the cytoskeleton on the inside of cells and bind the extracellular matrix on the outside of cells. A cell changes its association with the extracellular matrix by changing the types of integrins in its membrane, mediated by endocytosis and exocytosis. Cells can also break down the extracellular matrix by secreting proteases called matrix metalloproteinases. By controlling both the production of the matrix and its degradation, cells can regulate their ability to move throughout a tissue. For example, when blood vessels grow, they use ma- Pe ar so Le No ar tF n or in Di Re g s O tri r bu tio sa n n trix metalloproteinases to break down the extracellular matrix of the local cells to allow the blood vessels to penetrate into new regions of the tissue. Physiological Genetics and Genomics The nature of physiological diversity, whether in the response of an individual or in the variations arising over evolutionary time, resides in the genes: how they differ between species and how they are regulated in individual cells. Homeostatic regulation depends on the ability of the cell to put the right protein in the proper place at the proper time with the appropriate activity. Cells have many mechanisms to control the rates of synthesis of specific proteins. RNA polymerases read the genes, producing mRNA in the process of transcription. Once RNA is made, it is used as a template to produce protein in the process of translation. Cells can control the levels of both RNA and protein using mechanisms that target rates of synthesis and degradation. So le lu t io n Nucleic acids are polymers of nucleotides s The two types of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are structurally similar but perform different functions within the cell. DNA is the genetic blueprint for building cells. RNA reads the information encoded by the DNA and interprets it to make proteins. Cells produce three main forms of RNA: transfer RNA (tRNA), ribosomal RNA (rRNA), and messenger RNA (mRNA). Certain molecules of RNA complex with proteins to form riboproteins. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 79 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 79 Pe ar so Both RNA and DNA are polymers of nucleotides. All nucleotides are composed of a nitrogenous base attached to a sugar linked to a phosphate. RNA and DNA differ in the type of sugar in the nucleotide: ribonucloetides contain ribose whereas deoxyribonucleotides possess deoxyribose. Both RNA and DNA are synthesized from combinations of four types of nucleotides that differ in the nature of their nitrogenous bases. Three of the four nitrogenous bases, the pyrimidine cytosine and the purines adenine and guanine, are found in nucleotides of both RNA and DNA. The fourth nitrogenous base is another pyrimidine: uracil in RNA and thymine in DNA. The ribonucleotides are ATP, UTP, CTP, and GTP. The deoxyribonucleotides are dATP, dTTP, dCTP, and dGTP. In many cases, the nucleotide sequence in DNA and RNA is represented using one-letter codes. Thus, A refers to the residue derived from the nucleotide ATP (in RNA) or dATP (in DNA), C is CTP/dCTP, G is GTP/dGTP, T is dTTP, and U is UTP. Nucleic acids form from long polymers of nucleotides linked by phosphodiester bonds that form between the phosphate of one nucleotide and the sugar of the adjacent nucleotide. The end of the polymer that terminates with a phosphate group is deemed the 5-prime end (5); the other end terminates with a sugar and is the 3 end. The nucleic acid has a polarity, conferred by its 5 and 3 ends, that is an important consideration when discussing the biochemical processes involved in nucleic acid function. Le No ar tF n or in Di Re g s O tri r bu tio 3 n DNA is a double-stranded -helix packaged into chromosomes DNA usually exists within cells as a double-stranded polymer (Figure 2.57) in which hydrogen bonds connect the two strands. Each specific nucleotide can form hydrogen bonds with only one other nucleotide. Three hydrogen bonds form between G and C, whereas two hydrogen bonds form between A and T. When one strand of DNA encounters another complementary strand, hydrogen bonds form between the strands, creating a double-stranded molecule. The two strands anneal in an antiparallel arrangement, with the 5 end of one strand associated with the 3 end of the other strand. sa 3 Double-stranded DNA twists into an -helix with two topological features: a minor groove and a major groove. The two strands of DNA appear as ridges, separated by a trough. These contours between two strands compose the minor groove. The major groove results from the twisting pattern of the -helix. Every 10 base pairs, a distance of about 3.6 nm, the helix completes a full turn, forming the major groove that resembles a saddle. Variations in nucleotide sequence cause subtle regional alterations in the shape of DNA and the topology of the major and minor grooves. This structural variation is information that is used by the DNA-binding proteins to attach to the correct location to regulate expression of specific genes. The DNA in animal cells is highly compressed into tight structures with the aid of DNA-binding proteins called histones. If you were to unwind the DNA in a single mammalian cell, the strands would stretch several meters. The long strands of DNA wrap twice around the barrel-shaped histones until a structure resembling a strand of pearls is formed. These strands are then twisted and folded into highly compressed arrangements, which has two main advantages to cells. First, it allows the cell to fit large amounts of DNA into the small volume. Second, coating DNA with histones helps reduce the damage caused by radiation and chemicals. However, in this compressed configuration DNA is biochemically inert; it cannot function as a template for RNA synthesis (transcription) 5 n C T G A G A T C Nucleotide Phosphate So le lu t 3 5 3 5 (b) Ribbon diagram Minor groove Sugar (deoxyribose) Major groove io n s Hydrogen bonds 5 Sugarphosphate backbone (a) Schematic model (c) Space-filling model DNA structure Each strand of DNA binds to another, complementary strand. Hydrogen bonds form between specific base pairs. Two bonds form between A and T. Three bonds form between C and G. The double-stranded DNA is twisted into an -helix, forming a minor groove between strands. The major groove reflects the period of the twisting of the helix. Figure 2.57 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 80 80 PART ONE The Cellular Basis of Animal Physiology or DNA synthesis (replication). Cells must use histone-modifying enzymes to release histones from DNA, thereby regulating gene expression. DNA is organized into genomes The entire collection of DNA within a cell is called the genome. Within the nucleus, the genome is divided into separate segments of DNA called chromosomes. Within chromosomes are the genes, which possess the DNA sequences that are used to produce all the different types of RNA, including the mRNA that encodes proteins. Each gene also possesses regions of DNA called promoters that determine when the gene is expressed. Many genes are divided into multiple sections on the same chromosome. The sections that encode RNA are known as exons, and the interspersed DNA sections are called introns (Figure 2.58). In most animals, genes account for less than half of the genome. The majority of the genome is a mixture of different types of random and repetitious DNA, much of which serves no known function and is often called junk DNA. Across the animal kingdom, genome size ranges more than 6000-fold (Figure 2.59). The smallest genome is found in one of the simplest animals; placozoans, a relative of sponges, have only about 0.02 pg of DNA per cell. The largest genome in animals, about 133 pg/cell, belongs to the African marbled lungfish. Surprisingly, there is lit- Pe ar so tle relationship between the size of the genome and the complexity of the animal. For example, both the largest and the smallest vertebrate genomes are found in fish. The pufferfish genome is only about 0.3% the size of the lungfish genome. There is also no relationship between the number of chromosomes and the complexity of the animal. Humans possess 46 chromosomes. Some deer have only 6, whereas carp may have more than 100. Transcriptional control acts at gene regulatory regions The rate of synthesis for many proteins is proportional to the levels of mRNA. Historically, mRNA Mammals Birds Le No ar tF n or in Di Re g s O Telomeres Centromere n Reptiles Frogs Salamanders Lungfish Teleosts Chondrichthians Agnathans Nonvertebrate chordates Crustaceans Insects tri r bu tio sa Arachnids Myriapods Molluscs Annelids (a) Chromosome Introns n Echinoderms Tardigrades Flatworms Rotifers Nematodes Cnidarians Sponges So le lu t 102 101 1 io n 10 102 s 103 Promoter Exons Genome size (pg) Gene (b) Gene Figure 2.59 Genome sizes in the animal kingdom The genome size in animals can vary widely, and there is no relationship between genome size and complexity. Bar lengths reflect the range in the sizes of genomes measured in picograms (pg). Figure 2.58 Chromosomes and genes Chromosomes possess structural regions, such as centromeres and telomeres, in addition to noncoding regions and genes. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 81 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 81 Pe ar so Nucleosome levels were measured using northern blots, but recent advances in genomics and engineering have led to the development of techniques for assessing complex changes in the levels of mRNA for thousands of genes simultaneously. At any point in time, most of the genome of a cell is wrapped around histones and rolled into nucleosomes (Figure 2.60). Under these conditions the genes are quiescent, unable to bind the transcriptional machinery. When the gene product is required, the chromatin must be remodeled to allow transcriptional activators access to the regulatory regions of the gene. Transcriptional regulators, both DNA-binding proteins and coactivators, associate with each other to form regulatory complexes on the promoter. The transcription initiation complex assembles near a specific region of the promoter designated as the transcription start site, typically a sequence of TATA (the TATA-box). Once the complex assembles, the process of mRNA synthesis can begin. Histone Le No ar tF n or in Di Re g s O Histone n tri Activator Coactivator General transcription factors Transcription start site r bu tio Transcriptional regulators bind TATA RNA polymerase Histone remodeling sa n Figure 2.60 Transcriptional regulation Quiescent DNA is tightly wrapped around histones. Remodeling of chromatin gives DNA-binding proteins access to gene control regions. The general transcription factors allow RNA polymerase II to bind to initiate transcription. Other DNA regulatory proteins, such as the activators and coactivators shown here, increase the likelihood that the transcriptional machinery will assemble. Cells can regulate the rate of mRNA synthesis by altering the conformation of the gene and changing the ability of the transcriptional machinery to assemble. Sometimes gene expression is induced by stimulation of the enzymes that remodel chromatin. These enzymes work by altering the structure of the histones that organize DNA into nucleosomes. Histones can be modified by acetylation, methylation, and phosphorylation. For example, when a histone acetyl transferase (HAT) adds an acetyl group to a critical lysine in a histone, this induces a change in structure that permits remodeling of chromatin to favor gene expression. The gene can be silenced by a histone deacetylase (HDAC) that removes the acetyl group. Once the regulatory regions within the gene are exposed, the transcriptional machinery is able to assemble. Transcription factors may bind to sites close to, or distant from, the transcriptional start site. Some transcription factors introduce bends into the DNA that bring critical regions of the gene in close proximity. Other transcription factors bind coactivators, which serve as docking sites for other proteins. Eventually, the general transcription factors are assembled, the RNA polymerase is recruited, and the process of transcription can begin. The entire process depends critically on the interactions between dozens of proteins. Consequently, cells can finetune the process by regulating the ability of different proteins to interact, typically by changes in protein phosphorylation. The phosphorylation state can affect the transfer of a transcription factor between the cytoplasm and the nucleus. It can also alter the ability of transcriptional regulators to interact with DNA or other proteins, both stimulatory and inhibitory proteins. Since each gene is regulated by dozens of transcription factors, the combinations of regulatory conditions are endless. The primary mRNA transcript possesses sequences that will eventually code for the protein (exons) as well as other sequences that are interspersed between exons (introns). It must first be processed in a way that removes introns and splices together exons. Next, the spliced RNA must be polyadenylated; long strings of 200 or more ATP residues are added to the 3 end of the transcript to produce the poly A tail that is characteristic of mRNA. Once these post-transcriptional modifications are completed, the mature mRNA is exported to the cytoplasm. So le lu t io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 82 82 PART ONE The Cellular Basis of Animal Physiology RNA degradation influences RNA levels Controlling transcription is one important mechanism for cells to alter RNA levels; another is to vary the rate of RNA degradation. RNA is degraded by nucleases called RNases. An RNase can attack the end of the RNA (exonucleases) or internal sites (endonucleases), preventing the mRNA from acting as a template for protein synthesis. Cells have ways to preferentially degrade or protect individual mRNAs. A long poly A tail protects an mRNA from degradation. Soon after release into the cytoplasm, exonucleases nibble off the ends of the poly A tail. The mRNA can still be translated into protein at this point. Once the exonucleases shorten the tail to about 30 bases, the RNA is attacked by an endonuclease, causing enough damage to prevent the protein from being translated. Other processes accelerate the rate of mRNA degradation. Some mRNAs are unstable, existing in the cytoplasm for only a few minutes before becoming degraded. These unstable mRNAs have long stretches of A and U bases within their 3 untranslated regions (3 UTR). These AU-rich regions recruit proteins that accelerate mRNA degradation. The ability to accelerate RNA degradation is essential in many cells, particularly those that produce regulatory proteins. Once a signaling protein is no longer needed, the RNase machinery can rapidly degrade the mRNA to prevent it from being translated. Cells can also reduce the rate of RNA degradation. Stabilizing proteins can bind to specific regions in the poly A tail or other regions of the mRNA to prevent RNase attack. This allows the cell to maintain a pool of preformed mRNA available for immediate use if cellular conditions demand the gene product. Pe ar so Le No ar tF n or in Di Re g s O tri r bu tio sa n n called a codon. The 5 end of the mRNA recruits proteins called initiation factors, in combination with a methionine tRNA (tRNAMET) and a ribosome. The complex moves down the mRNA chain until it reaches the sequence AUG, which is the start codon. Another amino acyl tRNA is recruited, and the ribosome catalyzes the formation of a peptide bond between the amino acids to begin the process of elongation. In most circumstances, proteins called elongation factors enter the ribosome and accelerate the catalytic cycle. In a typical animal cell, each individual ribosome can add an amino acid to the chain at a rate of one to two per second. The process continues until the ribosomal complex reaches a stop codon, a nucleotide sequence that is incapable of binding any amino acyl tRNA. At any point in time, a single mRNA may be translated by many ribosomes bound all along the mRNA. Cells can control the rate of translation using nonspecific mechanisms that affect all translation within the cell, as well as specific mechanisms that influence only a subset of mRNAs. Many of the initiation factors and elongation factors are regulated through protein phosphorylation. In addition, each of these factors can bind inhibitory proteins. Such mechanisms allow cells to mount global changes in translation rates. Many types of mRNA possess sequences that act to regulate their translation. For example, sequences in the 3 UTR and 5 UTR bind proteins that alter the ability of the mRNA to be translated. Cells rapidly reduce protein levels through protein degradation Once proteins are synthesized, they remain in the cell until they are degraded. Just as cells use degradation to control mRNA levels, they use protein degradation to control protein levels. Some proteins are removed only when they sustain enough damage to become dysfunctional. The structural changes in damaged proteins recruit enzymes that mark the protein for degradation. These enzymes transfer a small protein called ubiquitin to the damaged protein. Once the ubiquitination machinery has attached a ubiquitin chain to the damaged protein, the protein is bound by a multiprotein complex called the proteasome. Proteolytic enzymes within the proteasome degrade the ubiquitin-tagged proteins to amino acids. So le lu t io n Global changes in translation control many pathways Once an mRNA arrives in the cytoplasm, the process of translation can begin with the assistance of ribosomes and amino acyl tRNAs. Ribosomes, complexes of rRNA and proteins, catalyze the formation of peptide bonds between amino acids in the growing protein. The amino acids are provided in the form of amino acyl tRNA. Each amino acid uses a specific tRNA that can bind to a specific set of three nucleotides on the mRNA s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 83 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 83 Pe ar so Earlier we discussed how some types of mRNA are preferentially degraded. Many of these unstable mRNAs encode proteins that are also subject to accelerated degradation. Proteins such as cell cycle regulators and transcription factors can be ubiquitinated even in the absence of structural damage. Characteristic amino acid sequences within the proteins recruit the ubiquitination machinery. Often the recognition sequences can be phosphorylated, altering their ability to be subjected to rapid degradation. Collectively, cells use these regulatory processes to control the levels of mRNA and protein. They enable cells to modify cellular properties in response to changing environmental and physiological conditions. Cells are also able to modulate their physiological response by altering the types of proteins they express. Animals, particularly vertebrates, can draw upon isoforms of proteins with subtly different properties that provide cells with alternative strategies to meet environmental and physiological challenges. Primary transcript E1 E2 E3 E4 E5 E6 Intron E1 E2 E3 E4 E5 E6 E1 E3 E4 E5 E6 E1 E3 E5 (a) Alternate splicing LDH-A LDH-a Protein variants arise through gene duplications and rearrangements Protein isoforms provide a cell with flexibility in structure and function. A suite of proteins can be created with distinct properties. Isoforms can be produced through multiple mechanisms involving single genes, different alleles, or different genes (Figure 2.61). Variations in protein structure can arise when the primary mRNA from a gene is connected together using different combinations of exons, a process known as alternative splicing. For example, more than 40 different isoforms of fibronectin can result from a single gene. Each isoform of fibronectin binds different combinations of extracellular matrix molecules. Within any population of animals, there is some variation in the exact sequence of specific genes. As a consequence, a diploid individual may possess two different versions of the same gene, one arising from the mother and one from the father. These different forms of the same gene are alleles. If the gene encodes an enzyme, the isoforms are also called allozymes. Often the differences in allozyme structure have little effect on function. Because they are functionally neutral, natural selection does not remove them from the population. However, in some cases the regulatory Le No ar tF n or in Di Re g s O 1 1 n (b) Allelic variation LDH-A LDH-B tri r bu tio sa (c) Gene families Figure 2.61 n Origins of protein variants Cells are able to produce protein isoforms in many different ways. Cells can splice exons in different combinations to create distinct proteins. Often the same gene can occur in different sequences within a population. Some individuals can have two different versions of the same gene (A or a) on chromosomes inherited from each parent. Gene duplications can lead to extra gene copies in different loci. These genes can diverge to encode different enzymes (A and B). So le lu t io n or catalytic properties of allozymes may be subtly different. Often different allozymes predominate in two populations of animals. For example, if a specific allozyme functions better in the cold, that gene might occur at a higher frequency in populations of animals exposed to the cold. Other types of isoforms are encoded by separate genes that arose from ancestral gene duplications. Figure 2.62 shows some of the ways that genes can become duplicated. During the process of meiosis, long stretches of DNA may be s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 84 84 PART ONE The Cellular Basis of Animal Physiology Pe ar so (a) Homologous recombination (equal crossover) Le No ar tF n or in Di Re g s O (b) Unequal crossover (c) Mobile elements n Gene duplications Gene recombination can provide cells with extra copies of genes. In contrast to equal crossover, (a) where homologous regions of chromosomes are exchanged, unequal crossover (b) provides one chromosome with extra genetic material. (c) Cells also possess many different kinds of mobile elements that can move or duplicate genes between chromosomes. Figure 2.62 tri will be endowed with extra copies of the duplicated genes. These extra copies could kill the cell or, if neutral or beneficial, get transmitted to the next generation. Another way that genes can become duplicated is through mobile elements. Many organisms possess genes that are capable of jumping from one chromosome to another. In most cases, the mobile element encodes a transposase, the enzyme required to cut the DNA from one strand and insert it into another. Occasionally, other genes become trapped in the mobile elements. When the mobile elements move, the other genes are carried along, endowing the recipient chromosome with the extra copy. Genetic recombination does not always lead to production of extra copies of entire genes. In some cases, fragments of genes are moved from one gene and inserted into a completely different gene. A protein may possess domains within its structure that resemble regions of otherwise unrelated proteins. For instance, hundreds of different proteins can bind ATP using a protein structure called an ATP-binding cassette. This structure, which appears in all living organisms, probably arose only once, or a few times, billions of years ago. Its appearance in so many different genes and in all taxa is likely due to genetic recombination events that moved this region from one gene to another. r bu tio sa Ancient genome duplications contribute to physiological diversity Gene duplications provide organisms with extra copies of redundant DNA that can accumulate mutations and diverge to endow the organisms with novel capacities. The key to achieving the opportunity for specialization is obtaining the raw material: a nonlethal extra copy of a gene. At several points in the evolution of animals, whole genomes were duplicated. Many of the duplicated genes were eventually lost, but many were retained and diverged to form gene families. Many of the anatomical and functional specializations of vertebrates are a result of these genomic duplications. Often, if a particular gene is found in a single copy in an invertebrate, there are four isoforms in vertebrates. This "rule-of-four" reflects ancestral genome duplications; each single gene locus was duplicated, giving two copies of all genes, then reduplicated, giving four copies of all genes. The individual genes within the duplicated genomes n So le lu t transferred from one chromosome to another. In most cases, two chromosomes exchange homologous regions and no gain or loss of genes occurs. This process of shuffling gene combinations is one of the advantages of sexual reproduction. Occasionally, the machinery of homologous recombination misidentifies homologous regions. Unequal crossover results, and one chromosome donates an end to another chromosome. The progeny derived from the gamete that lost the chromosomal region would not likely survive. However, the progeny from the recipient gamete io n s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 85 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 85 Pe ar so underwent mutation, selection, and drift to diverge into distantly related genes. After a period of divergence, some individual genes duplicated again. The newly duplicated genes were more closely related to each other than to their distant ancestors, creating gene clusters. When did these genome duplications occur? A possible answer comes from phylogenetic analyses of a family of genes involved in development, the Hox family. The first genome duplication probably occurred just before the jawless vertebrates, or agnathans, diverged from the vertebrate lineage. The second duplication coincided with the development of jaws. The primitive chordates such as amphioxus have a single cluster of Hox genes, the agnathan lamprey has two or sometimes three clusters, and the more recent jawed vertebrates, from sharks to humans, possess at least four clusters of Hox genes. In each case, genome duplications coincided with important revolutions in morphological and physiological complexity. These original genome duplications in the vertebrate lineage probably occurred more than 300 million years ago. Many modern animals have experienced relatively recent genome duplications, including many examples of frogs and fish that gained an extra set of chromosomes to become tetraploids. In some cases, tetraploid populations exist within diploid species; not nearly enough time has passed within the tetraploid lineage for the duplicated genes to diverge. The common carp, however, became tetraploid about 15 million years ago. Its closest relative, the grass carp, has half the number of chromosomes. Many genes that are in single copy in other vertebrates are found in pairs in common carp. While the pairs have di- Le No ar tF n or in Di Re g s O 2 n verged in structure, they have not yet become different in function. Over many generations, the duplicated genes can follow many fates. The duplicated gene might incur mutations in the promoter or coding region that prevent it from being transcribed, rendering it a pseudogene. In some cases, one copy of the gene mutates and diverges, resulting in a protein with distinct properties. In other cases, both copies mutate and diverge, resulting in a pair of proteins with overlapping functions. These genetic processes, originating early in animal evolution and operating at the level of individual cells, provide animals with physiological flexibility. The integration of different cell types into complex physiological systems is an important reason why animals have radiated into so many diverse species over the course of evolution. CO NCE P T CH E CK 13. Compare the categories of membrane transport in terms of energy requirements and direction of transport in relation to chemical gradients. 14. Discuss the composition of biological membranes. What are the unique properties of each type of lipid? 15. How can cells alter the fluidity of membranes, and why is this capacity important to cellular function? 16. Summarize the roles of the different subcellular compartments within a cell, and discuss how they influence physiological function. 17. Discuss the origins of genetic variation. How does genetic variation provide physiological flexibility? tri r bu tio sa Summary Chemistry n So le lu t io n k All biological systems depend on kinetic and potential energy. k Food webs are essentially transfers of chemical energy between organisms. k Molecules possess thermal energy, which is reflected in molecular movement, and many metabolic processes in cells are mechanisms for capturing and transferring this energy. k Cells can also store energy in the form of electrochemical gradients. Gravitational energy and elastic storage energy are used in locomotion. k Covalent bonds, which arise when two atoms share electrons, are strong in comparison to weak bonds, including hydrogen bonds, van der Waals forces, and hydrophobic interactions. k Weak bonds control the three-dimensional structure of macromolecules. They form and break in response to modest changes in temperature. s 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 86 86 PART ONE The Cellular Basis of Animal Physiology k Solute concentration imposes osmotic challenges. Organisms must modulate their biological solutions to regulate the ionization of water into H and OH . k Changes in proton concentration, or pH, alter many molecular properties. As a result, animals have many physiological mechanisms to regulate pH, including pH buffers. branes. Steroids and their precursors fulfill many roles within cells, and steroid hormones are particularly important in cell signaling. k Cells oxidize fatty acids for energy using the mitochondrial -oxidation pathway, which generates reducing equivalents and acetyl CoA. The rate of -oxidation is governed by the availability of fatty acids and the rate of transport into the mitochondria using the carnitine shuttle. k Fatty acids can be synthesized by the enzyme fatty acid synthase, for use in biosynthesis or energy storage. When energy is needed, lipases can break down triglycerides to release the fatty acids. k Under some conditions, such as starvation, fatty acids can be converted to ketone bodies for use in tissues that cannot use fatty acids directly. k Most oxidative fuels can be converted to acetyl CoA within mitochondria. When acetyl CoA enters the tricarboxylic acid cycle, acetyl CoA is oxidized to produce reducing equivalents, NADH and FADH2. k Oxidation of reducing equivalents by the electron transport system generates a proton gradient, heat, and reactive oxygen species. k The mitochondria F1FO ATPase, or ATP synthase, uses the proton motive force to generate ATP. Phosphorylation is coupled to oxidation through a shared dependence on the proton motive force. Biochemistry k Enzymes are organic catalysts, usually proteins, that speed reactions by reducing the activation energy barrier. k Enzyme reaction velocity (V) and substrate affinity (Km) depend on the physicochemical environment, such as the temperature, ion composition, and pH of the solution. Pe ar so k Cells control reaction rates by changing the concentration of reactants, the levels or activities of enzymes, or the concentration of substrates and products. k Competitive inhibitors compete for the enzyme active site. Allosteric regulators bind at locations distant from the active site, altering enzyme kinetics. Many enzyme and nonenzyme proteins are regulated by covalent modification. For example, protein kinases use ATP to attach phosphate groups to specific amino acid residues, and protein phosphatases remove phosphate groups. k Cells use combinations of enzymes and enzymatic regulation to construct and maintain complex metabolic pathways. k Proteins, carbohydrates, and lipids have important roles in structure and metabolism. Animals store excess carbohydrate as glycogen. Glucose can be produced from noncarbohydrate precursors using gluconeogenesis. Glucose can be broken down to pyruvate (glycolysis) or further oxidized to CO2. k Most animals use lactate dehydrogenase to balance redox and dispose of pyruvate. Anoxiatolerant animals can use other pathways for oxidizing NADH in the absence of oxygen, some of which provide additional ATP. k Phospholipids, including phosphoglycerides and sphingolipids, are used to make cell mem- Le No ar tF n or in Di Re g s O tri r bu tio sa n n k Under some circumstances, mitochondria can become uncoupled, leading to the production of heat instead of ATP. k The balance between biosynthesis and catabolism is regulated by energetic intermediates such as ATP, NADH, and acetyl CoA. Without this regulation, the two processes could occur simultaneously, leading to loss of energy in futile cycles. So le lu t io n s k Metabolic regulation also determines which fuels are oxidized under which conditions. Cell Physiology k Membranes allow cells to create permeability barriers that help them to define environments. Membranes are heterogeneous combinations of 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 87 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 87 phospholipids, cholesterol, and numerous integral and peripheral proteins. k The nature of the lipid membrane influences fluidity, an important determinant of protein function. are the most important component of the resting membrane potential. Changes in membrane permeability alter the membrane potential in ways that cells use to communicate. k Many aspects of animal physiology can be traced back to cellular processes. k The basic structure of cells--including the mitochondria, cytoskeleton, extracellular matrix, and secretory networks--can be regulated and remodeled to serve many purposes. k The ability to follow developmental programs, or respond to physiological and environmental challenges, resides in the genes. Physiological change begins in many cases with the ways cells control genes. k Cells and tissues are remodeled using processes from transcriptional control to post-translational regulation. k Evolutionary processes, including gene and genome duplications, provide the raw material for achieving physiological diversity. Pe ar so k While some hydrophobic molecules can cross membranes by passive diffusion, membrane proteins are required for transport of most molecules. k Some transporters, such as ion channels, facilitate the diffusion of impermeant molecules down concentration gradients by creating pores. k Active transporters use energy to pump molecules against gradients. k The electrochemical gradients that exist across cellular membranes are produced by active transporters and used to drive diverse physiological processes. k The interior of the plasma membrane is electronegative, with a membrane potential between 5 and 100 mV. Potassium gradients Review Questions 1. How does the density of water change in relation to temperature? How do these properties affect animals that live in marine and freshwater environments? 2. If the enzymatic reaction A B C D is near equilibrium, then the mass action ratio is close to the equilibrium constant. What happens to the mass action ratio if you add more enzyme? What happens when you add more of A? What do you need to know to predict what would happen if temperature changed? Le No ar tF n or in Di Re g s O tri r bu tio sa n n 3. What metabolic conditions can affect the values of the respiratory quotient? 4. What metabolic conditions affect the relationship between ATP produced and oxygen consumed? 5. Trace the path of a protein hormone, such as insulin, from its gene in the nucleus to secretion out of the cell. 6. Discuss the mechanism by which cells can use transporters to change their osmotic and ionic properties. So le lu t io n s Synthesis Questions 1. A type of protein comes in six different forms. Each form can dimerize with the other. How many unique homodimers and heterodimers can be formed from these six proteins? 2. Many animals maintain metabolites at concentrations near the Km value for metabolic enzymes. For example, the concentration of pyruvate is often close to the Km value for LDH. Why might this be advantageous, in terms of kinetic regulation? 3. Describe, in chemical terms, how antacids work. 4. Why do your hands get wrinkled if you spend too much time in the bathtub? Would the same 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 88 88 PART ONE The Cellular Basis of Animal Physiology Quantitative Questions 1. What is the proton concentration of a solution at pH 7.4? At what temperature would this solution be neutral? 2. Calculate the basis for an RQ 1 for carbohydrate oxidation. Why does palmitate oxidation give an RQ 0.7? 3. What rate of oxygen consumption would you expect in a tissue with a metabolic rate of 30 mol ATP/ min? Pe ar so thing happen when you swim in the ocean? Describe these environments using the terminology of osmolarity and tonicity. 5. Many physiological processes require a change in the levels of proteins, such as membrane transporters. Discuss the processes that cells can use to change the protein levels. Discuss how the subcellular compartment influences this pathway. 6. Other physiological processes require changes in the activities of proteins. While this can arise through changes in the levels of proteins, it can also change through regulation of protein function. Discuss the various ways that cells can alter the activity of enzymes or transporters. 7. Discuss the ways in which a cell is able to alter its interactions with other cells. For Further Reading See the Additional References section at the back of the book for more references related to the topics in this chapter. Chemistry These texts provide good overviews of the chemical and physical underpinnings of cell biology and biochemistry. Becker, W. M., L. J. Kleinsmith, and J. Hardin. 2003. The world of the cell, 5th ed. San Francisco: Benjamin Cummings. Le No ar tF n or in Di Re g s O tri r bu tio sa n n This book looks at how animals and other organisms alter macromolecules in relation to environmental stress. Hochachka, P. W., and G. N. Somero. 2002. Biochemical adaptation. Oxford: Oxford University Press. These two books present differing views of the history of the discovery of the structure of DNA. Sayre, A. 1975. Rosalind Franklin & DNA. New York: Norton, 1975. Watson, J. 2001. The double helix: A personal account of the discovery of the structure of DNA. New York: Touchstone Books. Lehninger, A. L., D. L. Nelson, and M. M. Cox. 1999. Principles of biochemistry, 3rd ed. New York: Worth. This text is a good primer for understanding the factors that affect protein structure. Branden, C., and J. Tooze. 1991. Introduction to protein structure. New York: Garland Science. Biochemistry These publications provide good background on the interactions between energy, chemical bonds, and water. Bryant, R. G. 1996. The dynamics of water-protein interactions. Annual Review of Biophysics and Biomolecular Structure 25: 2953. Thornton, R. M. 1998. The chemistry of life. Menlo Park, CA: Benjamin Cummings. Westof, E. 1993. Water and biological macromolecules. Boca Raton, FL: CRC Press. This book, written by two pioneers in comparative biochemistry, explores the metabolic basis of biological diversity. Although the focus is on animals, they also consider other organisms that exemplify biochemical strategies for survival in adverse environments. So le lu t io n s Hochachka, P. W., and G. N. Somero. 2002. Biochemical adaptation. Oxford: Oxford University Press. Arthur Kornberg's autobiography gives his perspective on the history of the study of metabolic biochemistry. Kornberg, A. 1991. For the love of enzymes: The odyssey of a biochemist. Cambridge, MA: Harvard University Press. 8140606_CH02_p020-089.qxd 10/11/08 7:47 PM Page 89 CHAPTER 2 Chemistry, Biochemistry, and Cell Physiology 89 Lehninger is one of the standard undergraduate textbooks in biochemistry, with particularly good sections on metabolism and metabolic regulation. Nelson, D. L., and M. M. Cox. 2000. Lehninger principles of biochemistry. New York: Worth. Pe ar so Cell Physiology Ohno's early book outlines his perspective on the importance of gene duplication in the evolution of biological diversity. More recently, in a series of papers, a number of authors bring the field up-todate, incorporating recent evidence of the role of genome duplications in origins of gene families and cellular diversity. Ohno, S. 1970. Evolution by gene duplication. Heidelberg: Springer Verlag. Various authors. 1999. Gene duplication in development and evolution. Seminars in Cell and Developmental Biology 10: 515563. An excellent overview of transport and transporters. Stein, W. D. 1990. Channels, carriers and pumps: An introduction to membrane transport. San Diego: Academic Press. The original book by Sir D'Arcy Wentworth Thompson, written in 1917, was one of the first to examine how physiology was influenced by mathematics and physics. Thompson, D. W. 1961. On growth and form. Abridged edition edited by J. T. Bonner. Cambridge: Cambridge University Press. These two textbooks cover the breadth of cell and molecular biology, with excellent illustrations. The strength of Alberts is its comprehensive nature, while Becker is very readable. Alberts, B., A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2002. Molecular biology of the cell. New York: Garland Science. Becker, W. M., L. J. Kleinsmith, and J. Hardin. 2002. The world of the cell. San Francisco: Benjamin Cummings. This comprehensive review of the ATP synthase does an excellent job of explaining how the enzyme works in the context of structural models of its function. Boyer, P. D. 1997. The ATP synthase--A splendid molecular machine. Annual Review of Biochemistry 66: 717749. This book discusses the nature of evolutionary and physiological variation from the perspective of cell and developmental biology. Gerhart, J., and M. Kirschner. 1997. Cells, embryos and evolution. New York: Blackwell Science. Le No ar tF n or in Di Re g s O tri r bu tio sa n n So le lu t io n s ...
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This note was uploaded on 10/05/2011 for the course BIO 203, CH taught by Professor Lacey,simmerling,deng,hanson during the Fall '10 term at SUNY Stony Brook.

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