bio B sat 18.10 - Disruption of Cells Releases Their...

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Unformatted text preview: Disruption of Cells Releases Their Organelles and Other Contents The initial step in purifying subcellular structures is to rupture the plasma membrane and the cell wall, if present. - First, the cells are suspended in a solution of appropriate pH and salt content, usually isotonic sucrose (0.25 M) or a combination of salts similar in composition to those in the cells interior. - Many cells can then be broken by o stirring the cell suspension in a high-speed blender or by o exposing it to ultrahigh-frequency sound ( sonication ). o Plasma membranes can also be sheared by special pressurized tissue homogenizers in which the cells are forced through a very narrow space between the plunger and the vessel wall. o As noted earlier, water flows into cells when they are placed in a hypotonic solution (see Figure 5-18). This osmotic flow causes cells to swell, weakening the plasma membrane and facilitating its rupture. - Generally, the cell solution is kept at 0 C to best preserve enzymes and other constituents after their release from the stabilizing forces of the cell.- Disrupting the cell produces a mix of suspended cellular components, the homogenate, from which the desired organelles can be retrieved. - Homogenization of the cell and dilution of the cytosol cause the depolymerization of actin microfilaments and microtubules, releasing their monomeric subunits, and shear intermediate filaments into short fragments.- Thus other procedures, described in Chapters 19 and 20, are used to study these important constituents. Because rat liver contains an abundance of a single cell type, this tissue has been used in many classic studies of cell organelles. However, the same isolation principles apply to virtually all cells and tissues, and modifications of these cell-fractionation techniques can be used to separate and purify any desired components. ------------------------------------------- 1- T h e homogenate resulting from disrupting cells is usually filtered to remove unbroken cells and - then centrifuged at a fairly low speed to selectively pellet the nucleus the largest organelle. - The undeposited material (the supernatant) is next centrifuged at a higher speed to sediment the mitochondria, chloroplasts, lysosomes, and peroxisomes. - Subsequent centrifugation in the ultracentrifuge at 100,000 g for 60 minutes results in deposition of the plasma membrane, fragments of the endoplasmic reticulum, and large polyribosomes. - The recovery of ribosomal subunits, small polyribosomes, and particles such as complexes of enzymes requires additional centrifugation at still higher speeds. 2 EXPERIMENTAL FIGURE 5-36 Differential centrifugation is a common first step in fractionating a cell homogenate- Only the cytosolthe soluble aqueous part of the cytoplasmremains in the supernatant after centrifugation at 300,000 g for 2 hours.0 Centrifugation Can Separate Many Types of Organelles In Chapter 3, we considered the principles...
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This note was uploaded on 05/29/2010 for the course MEDICEN 2100 taught by Professor Saba during the Spring '10 term at Columbia State Community College.

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bio B sat 18.10 - Disruption of Cells Releases Their...

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