LECTURE-2 - The Mitochondria Lecture 2 • Dr. Leeuwenburgh...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: The Mitochondria Lecture 2 • Dr. Leeuwenburgh • • • • Home page: http://grove.ufl.edu/~cleeuwen Email: [email protected] Office Center for Exercise Science Office Hours 9:00-12:00 Thursdays Atomic Mass 1 Atomic Mass 12 Part 1 Mitochondrial Structure/Function 1 Electron micrograph of the swimming muscle (longissimus dorsi) of a Harbor seal: white lines denote the cell membrane or the sarcolemma of three different muscle cells; the yellow arrows point at subsarcolemmal mitochondria; the white arrows point at the interfibrillar mitochondria and the blue arrows show a lipid droplet. Mitochondrial Sub-Populations Age differently • Lesnefsky EJ, Gudz TI, Moghaddas S, Migita CT, IkedaSaito M, Turkaly PJ, Hoppel CL. Aging decreases electron transport complex III activity in heart interfibrillar mitochondria by alteration of the cytochrome c binding site. Mol Cell Cardiol. 2001 Jan;33(1):37-47. Characteristics Characteristics • Occupy large space in cytosol • Mitochondria depicted as stiff, but….this is not true • Elongated cylinders – microtubules: may determine unique orientation and distribution – example Liver contains 1000 to 2000 mitochondria per cell – heart (large mitochondria) – mitochondrial plasticity (exercise training) • Aging alters cardiac physiology and structure and enhances damage during ischemia and reperfusion. • Aging selectively decreases the rate of oxidative phosphorylation in the interfibrillar population of cardiac mitochondria (IFM) located among the myofibers, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Structure of a Mitochondrion Defects in import of Enzymes with Age 2 Characteristics • Matrix Contain -1. Enzymes to metabolize Pyruvate 2. Metabolize Fatty Acids 3. Acetyl CoA oxidation (CO2 + NADH) • Inner membrane -ETC, embedded in the inner mitochondrial membrane • Outer membrane -Porins 5000 Dalton or less - small proteins Cardiolipin • Double phospholipid -cardiolipin • contains four fatty acid groups and makes the membrane impermeable to certain ions. Chemiosmotic Coupling • Process to harness energy by the mitochondria • membrane-bound proton pumps (H+; membranegenerating an electrochemical proton pump • Energy from the H+ flow used by the enzyme ATPase synthase Chemiosmotic-Coupling • 1. Inner membrane pumps H+ out of the matrix when electrons are transported along the chain • 2. if large enough electrochemical proton gradient is present, protons flow in reverse direction through the complex and drive ATP synthesis Continue • Inner membrane control metabolites and selected inorganic ions by carrier protiens • Inner membrane is impermeable to H+, OHanions and cations (due to cardiolipin) cardiolipin) • Electrochemical Proton gradient- back flow gradientof H+ down this gradient used to drive ATP synthase. synthase. 3 Mitochondria/Chloroplast • Both Converts energy to forms used to drive cellular reactions • Chloroplast vs. Mitochondria – Chloroplast derive energy from sunlight photosystem – Chloroplast makes Oxygen and carbohydrates – Mitochondria are energy-converting organelles energyenergy retrieved from carbon fuel – Mitochondria consumes Oxygen and oxidize CHO and FFA Mitochondria • “Electrons are carried from food to mitochondrial membranes • Glycolysis: Glucose to pyruvate only 2 Glycolysis: ATPs produced • Glucose Oxidative Phosphorylation -30 ATP Summary Mitochondria • Mitochondria serve as the major sites for energy production within the cell. They contain an outer membrane with a smooth contour and an inner membrane with infoldings called cristae • 1) Membrane sacs with folded inner partitions • 2) Mito’s Generate energy from substrates (food) which transforms into usable ATP • Mitochondria = “powerhouse of the cell” • Matrix = central area in the mitochondria • Does Mitochondria have DNA? Mitochondria-Genetic Composition Mitochondrial DNA (mtDNA) (mtDNA) Double -Stranded circular DNA molecules 16,569 Base Pairs 22 tRNAs 2 Ribosomal RNAs and 13 Polypeptides of the Resp. Chain are Resp. synthesized in the mitochondria • Mitochondrial Deletions and Aging • • • • • 4 Continue • Complex I -25 polypeptides 7 made by mtDNA • Complex II - 0 encoded by mtDNA • Complex III - 1 of 11 • Complex IV - 3 of 13 • Complex V - 2 of 12 Why do Mitochondria and Chloroplast have their own genetic system • Since it is costly” – more than 90 proteins – ribosomal proteins – aminoacyl -tRNA synthases • Why ? not clear • Evolution, reached some sort of “in between aerobic-anaerobic balance aerobicpending on % oxygen concentration (and DNA transfer was not completed? Bioenergetics Part II Triglycerides • Triacylglycerol or Triglycerides insoluble in water -nonpolar. nonpolar. • Stored in adipocytes • Example: heart mitochondria very dependent on FFA, therefore storage of fat droplets near mitochondria 5 Fatty Acids and Glycogen • Fatty acids -contain 6x more energy compared to glycogen and would last for a ~ a month • Glucose - Glycogen - last about one day • During day - Glucose + Fatty acids provide your acetyl CoA • Overnight fast - Acetyl CoA from FFA • After a meal - from glucose Glycogen • Large branched polymer of Glucose contained in granules in the cytoplasm • Glycogen -glucose-phosphate -glycolysis glucose• -6 C which becomes 2x3 carbon pyruvate in the cytosol • Transported to the matrix Pyruvate Dehydrogenase • Pyruvate Dehydrogenase complex (3 enzymes, five coenzymes, 2 regional proteins. • Pyruvate into acetyl CoA produces CO2 and NADH + H 6 Alcohol dehydrogenase Hydrogen Atoms and Electron Transfer • Hydryde ion (H-) → H atom + electron • NAD+ + Hydryde ion → NADH • Hydrogen atoms are used for transfer of electron not just electron (electron and proton) • Hydrogen loses electron → Oxidized • Proton gains electron → Reduced (Hydrogen) Example: Example: • CH3-CH2-OH (Ethanol) + NAD+ CH3- CH2(oxidized form binds to enzyme) → (enzyme alcohol dehydrogenase) dehydrogenase) CH3-HC=O + NADH (reduced) + H+ CH3• named for the substrate that loses it’s H’s it’ H’ • enzyme catalyses oxidation of substrate and reduction of cofactor Nicotinamide adenine dinucleotide Flavin adenine dinucleotide (riboflavin;B2) Nicotinamide NAD+ accepts two electrons only one proton NADH + H+ (H+ free proton) + Cofactors or Coenzymes? 7 Electron transfer • Protons and Electrons are separated -recombined with NADH • Electrons are transferred from NADH to oxygen -through 3 large respiratory enzyme complexes Oxidation versus Reduction • A molecule is said to be oxidized when it loses electrons and to be reduced when it gains electrons. • A reducing agent is thus an electron donor; an oxidizing agent is an electron acceptor. • Although oxygen is the final electron acceptor in the cell, other molecules can act as oxidizing agents. A single molecule can be an electron acceptor in one reaction and an electron donor in another. – 1. NAD and FAD can become reduced by accepting 2 electrons from hydrogen atoms removed from other molecules. – 2. NADH + H+, and FADH2, in turn, donate these electrons to other molecules in other locations within the cells. – 3. Oxygen is the final electron acceptor (oxidizing agent) in a chain of oxidationoxidationreduction reactions that provide energy for ATP production. Oxidation • Does not imply that oxygen participates in the reaction • Named because oxygen has a great tendency to accept electrons (strong oxidizing agent) • This is exploited by cells; oxygen acts as the final electron acceptor in a chain of oxidation-reduction reactions (makes oxidationATP) Chemiosmotic Theory Chemiosmotic Energy into ATP • Inner mitochondrial membrane and Matrix • Electrons are removed from NADH + H+ FADH2 FADH • ADP + Pi → ATP by Ox. Phos. Phos. • Electrons are passes to oxygen to form water • NADH + H+ mainly produced by matrix mainly • Some made in cytosol (shuttle) 8 NADH + H+ • Electrons carried from high energy state to lower energy state. Why? • Electrons from enzyme compound transferred to Oxygen • Passes 2 electrons to the first of more than 15 electron carriers • How is this possible?? • Electrons have high energy → to low energy → basically they lose energy on their trip • protein-metal-electron-transfer protein- metal- electron- Oxygen Radicals and Mitochondria Part III Reactive Oxygen species and the Respiratory Chain SOD1 H2O2 H + FeS C1 bb cyt c III cyt c a a3 CuB O2- O2 CoQ IMS Q0 Qi FeS N-2 SDH FeS N-1, N-3, N-4 FMN Succinate O2 NADH Complex I Complex II Complex III Complex IV O2SOD2 H2O2 MATRIX Melov S. PhD 9 “Radical” Reactions O2 + e <=> O2•Equation 1 Equation 2 Equation 3 Equation 4 Heart Mitochondria Manganese SOD units/mg protein 150 100 50 0 6-Months 24-Months O2 + 2e + 2H+ → H2O2 Complex II O2•- + O2•- + 2H+ →SOD→H2O2 + O2 H2O2+ 2GSH → GPX → H2O + GSSG + ROH 2H2O2 + → Catalase → 2H2O + O2 Fe2+ + H2O2 → HO• + HO- + Fe3+ O2•- + NO• → ONOOO2•- + Mn+ → O2 + M(n-1)+ Equation 5 Equation 6 * 50% Equation 7 Equation 8 GPX (nmol/min/mg protein) Age (Months) 50 * 25% 25 0 6-Months 24-Months Age (Months) Complex I, II, III,IV • Complex I (NADH-Ubiquinone reductase complex), • Complex II (succinate dehydrogenase complex). succinate • Ubiquinone, also known as coenzyme Q, accepts electrons from both complexes and is sequentially reduced, one electron at a time, to ubisemiquinone and ubiquinol • Complex III (ubiquinol-cytochrome c reductase), cytochrome c, • Complex IV (cytochrome c oxidase) – Function is to reduce O2 to H2O. Cytochrome oxidase is estimated to account for 90-95% of the total oxygen uptake in most cells • What happens to other 1-5% • O2-•, H2O2, HO• , HO2 • (hydroperoxyl radical) • Respiring cells avoid O2• formation 99-95% of the time Which complex is responsible for free radical Leak? • This electron is thought to come from the one-electron reduction of ubiquinone, which generates the reactive intermediate ubisemiquinone formed by Complex III. • Instead of accepting another electron and proton to form ubiquinol, ubisemiquinone may leak its unpaired electron to O2, forming O2•-. FREE RADICAL LEAK 10 Complex Damage with Age and Injury (IR) • Study of aging damage to complex III will help clarify the contribution of altered electron transport in IFM to increased oxidant production during aging, formation of the aging cardiac phenotype, and the relationship of aging defects to increased damage following ischemia. Each interacts specifically only with the carrier adjacent to it depending on affinity for electrons • Components present in different amounts • All expressed per NADH-dehydrogenase complex (heart) – – – – 3 molecules b-c1 complex (Complex III) 7 molecules cytochrome oxidase (complex IV) 9 molecules cytochrome c 50 molecules ubiquinone Isolation of Mitochondria Study of Mitochondria Function • Intact mitochondria (isolation procedures) – Respiratory Chain is inaccessible Functional Inside-Out mitochondria particles can be isolated Oxygen Consumption and Oxygen Radical Measurements • Oxygen consumption can be measured under State 4 conditions, where there is no addition of ADP and therefore no active oxidative phosphorylation and therefore a low amount of oxygen consumption. • Sub mitochondrial particles – Ultrasound – Outer Surface -have tiny speres attached to stalks – Sub-mitochondrial particles are inside-out vesicles of inner membranes- readily provided with membrane – Impermeable metabolites that would be normally in matrix can be made accessible 11 Oxygen Consumption and Oxygen Radical Measurements • Under State 3 conditions ADP is added which actively stimulates oxidative phosphorylation and therefore oxygen consumption • Besides oxygen consumption it is also relevant to measure oxygen radicals under State 3 and State 4 conditions Low Osmolarity High Osmolarity for shrinking Matrix and outer-M Density Gradient C Study of Mitochondria Function Experiments 1960 • Add NADH, ADP and Pi → Oxygen (electrons) → ATP • Experiment-1960- if you remove tiny speres no ATP will be formed • spheres equal = F1 ATPase • Put back -ATP produced again • ATP synthase makes-up 15% of protein of the inner membrane ATP synthase • Able to convert the electrochemical energy stored in a transmembrane ion gradient directly into phosphate bond energy in ATP 12 Experiment 1974 • Liposomes made from phopholipids • Purified Bacteriorhodopsin -light (proton pump) • Purified ATP synthase • Data Strongly suggest • I. Proton Translocation • II. ATP synthesis is a separate event Respiratory Chain Experiment • The respiratory Chain pumps protons across the inner mitochondrial membrane • isolated mitochondria + substrate + H+ flow initially blocked through ATP synthase • injection of O2 1 to 2 seconds respiration occurs • sudden acidification of medium ↑ H+ Experiment • Sub mitochondrial-particle • What happens with the pH in the medium? • Selected Articles by JOH and Co-workers – Rats, Mice, and Aging Mitochondrial biogenesis and increases in mitochondrial volume; Effects of Exercise Training • • • • • • • • • • • 1. Young, J.C., Chen, M., and Holloszy, J.O. 1983. Maintenance of the adaptation of skeletal muscle mitochondria to exercise in old rats. Med Sci Sports Exerc 15:243246. 2. Fitts, R.H., Troup, J.P., Witzmann, F.A., and Holloszy, J.O. 1984. The effect of ageing and exercise on skeletal muscle function. Mech Ageing Dev 27:161-172. 3. Higuchi, M., Cartier, L.J., Chen, M., and Holloszy, J.O. 1985. Superoxide dismutase and catalase in skeletal muscle: adaptive response to exercise. J Gerontol 40:281-286. 4. Holloszy, J.O., Smith, E.K., Vining, M., and Adams, S. 1985. Effect of voluntary exercise on longevity of rats. J Appl Physiol 59:826-831. 5. Vailas, A.C., Pedrini, V.A., Pedrini-Mille, A., and Holloszy, J.O. 1985. Patellar tendon matrix changes associated with aging and voluntary exercise. J Appl Physiol 58:1572-1576. 6. Garthwaite, S.M., Cheng, H., Bryan, J.E., Craig, B.W., and Holloszy, J.O. 1986. Ageing, exercise and food restriction: effects on body composition. Mech Ageing Dev 36:187-196. 7. Holloszy, J.O., and Smith, E.K. 1986. Longevity of cold-exposed rats: a reevaluation of the "rate-of-living theory". J Appl Physiol 61:1656-1660. 8. Craig, B.W., Garthwaite, S.M., and Holloszy, J.O. 1987. Adipocyte insulin resistance: effects of aging, obesity, exercise, and food restriction. J Appl Physiol 62:95-100. 9. Holloszy, J.O., and Smith, E.K. 1987. Effects of exercise on longevity of rats. Fed Proc 46:1850-1853. 10. Holloszy, J.O. 1988. Exercise and longevity: studies on rats. J Gerontol 43:B149-151. 13 Young, J.C., Chen, M., and Holloszy, J.O. 1983. Maintenance of the adaptation of skeletal muscle mitochondria to exercise in old rats. Med Sci Sports Exerc 15:243-246. • Rats were exercised by means of swimming for 3h/d, 5d/wk beginning at age 6 months and studied at ages 9 and 24 months. • The levels of activity of the four mitochondrial enzymes used as indicators of the capacity for aerobic metabolism were significantly increased in epitrochlearis muscle in both the 9-month and 24-monthold swimmers. • Neither the 24-month-old sedentary rats nor the 24-month-old swimmers had significantly lower muscle enzyme levels than the comparable 9-month-old rats. • Thus, it appears that aging does not result in a progressive decline in the capacity of muscle for aerobic metabolism or a progressive impairment in the ability to maintain an increase in muscle mitochondrial enzymes in response to chronic exercise in healthy rats. • With age (if you exercise) you can maintain your aerobic capacity Mitochondria Acute Exercise • Morphological changes due to Fluid retention • Bizarre invaginations • Enlarged mitochondria -swelling • Mitochondrial division? Figure 1 Figure 4 Not made de novo Figure 6 14 Mitochondria Growth-Division • • • • 1. Growth 2. Division of Existing Mitochondria 3. Fusion also possible Sequence Examples: – organelle doubles in mass then divides – Could happen in contracting skeletal muscle – Number of mitochondria could increase 5-fold Exercise Training Increases Mitochondrial “Volume” Dr. Holloszy • • • • Increase in mitochondria 1) Kreb’s cycle enzymes 2) Electron transfer (NADH shuttle’s) 3) Beta-Oxidation pathways Adaptations of Skeletal Muscle • Greater reliance on fat oxidation • Reduces utilization of muscle glycogen and blood glucose • Decrease lactate production (at given intensity) • Results into increased ability to perform prolonged strenuous exercise. Apoptosis Theory of Aging Mitochondria and Apoptosis • Recently, it has become clear that mitochondria play a key role in regulating apoptosis. Upon receiving a death-inducing signal, there is a disruption of the mitochondrial inner transmembrane potential which results in the opening of the mitochondrial permeability transition pore and the release of caspase-activating proteins, such as cytochrome c (a protein normally found in the intermembrane space) and apoptosis-inducing factor (AIF), into the cytosol. 15 Mitochondria and Turnover with Age • Terman A, Dalen H, Eaton JW, Neuzil J, Brunk UT. Exp Gerontol. 2003 Aug;38(8):863-76. Mitochondrial recycling and aging of cardiac myocytes: the role of autophagocytosis. • The mechanisms of mitochondrial alterations in aged post-mitotic cells, including formation of so-called 'giant' mitochondria, are poorly understood. • To test whether these large mitochondria might appear due to imperfect autophagic mitochondrial turnover, we inhibited autophagocytosis in cultured neonatal rat cardiac myocytes with 3methyladenine • PMID: 12915208 [PubMed - in process] Review Article • Brunk UT, Terman A The mitochondriallysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis. Eur J Biochem. 2002 Apr;269(8):1996-2002. • PMID: 11985575 Mitochondria’s Role as a Sink for Calcium Uptake Scientists have focused on physiological mechanisms underlying processes of aging, rather than on the large array of debilitating and costly disorders that so commonly emerge during the latter half of the life-spans of human beings. The reason for this decision is illustrated in the figure above. The “one disorder at a time” approach has limited power to delay death; rather, it is through deciphering the biological underpinnings of processes of aging that scientists will likely discover ways to extend the human life-span. This problem is being attacked from several angles, and we are seeing some emerging themes. -CR increases stress-resistance -Dauer-phases (time-outs from reproduction while awaiting more favorable conditions) -Defects in insulin/insulin like growth factor 1 signaling pathways -Endocrine regulation of critical life-span controlling functions -Lab vs. Natural environment -DNA damage and oxygen radicals and DNA polymerases (repair) -Scores on vocabulary tests appear to improve with age, and older people often outperform younger ones in interpersonal tasks. -The practical and ethical ramifications of research on aging. For example, there will be a challenge to society in the future and is increasing life-span compatible with the needs of humanity. 16 ...
View Full Document

This note was uploaded on 05/19/2011 for the course BCH 3218 taught by Professor Johnsteward during the Fall '08 term at University of Florida.

Ask a homework question - tutors are online