H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 75
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In this example, material in the pelletfrom centrifugation at 15,000g (see Figure 5-36) is resuspendedand layered on a gradient of increasingly more dense sucrosesolutions in a centrifuge tube. During centrifugation for severalhours, each organelle migrates to its appropriate equilibriumdensity and remains there. To obtain a good separation oflysosomes from mitochondria, the liver is perfused with asolution containing a small amount of detergent before the tissueis disrupted.
During this perfusion period, detergent is taken intothe cells by endocytosis and transferred to the lysosomes,making them less dense than they would normally be andpermitting a “clean” separation of lysosomes from mitochondria.181tains a gradient of a dense nonionic substance (e.g., sucroseor glycerol). The tube is centrifuged at a high speed (about40,000 rpm) for several hours, allowing each particle to migrate to an equilibrium position where the density of thesurrounding liquid is equal to the density of the particle(Figure 5-37).Because each organelle has unique morphological features, the purity of organelle preparations can be assessedby examination in an electron microscope.
Alternatively,organelle-specific marker molecules can be quantified. Forexample, the protein cytochrome c is present only in mitochondria; so the presence of this protein in a fraction oflysosomes would indicate its contamination by mitochondria. Similarly, catalase is present only in peroxisomes; acidphosphatase, only in lysosomes; and ribosomes, only in therough endoplasmic reticulum or the cytosol.Organelle-Specific Antibodies Are Usefulin Preparing Highly Purified OrganellesCell fractions remaining after differential and equilibriumdensity-gradient centrifugation may still contain more thanone type of organelle. Monoclonal antibodies for variousorganelle-specific membrane proteins are a powerful toolfor further purifying such fractions. One example is the purification of coated vesicles whose outer surface is coveredwith clathrin (Figure 5-38).
An antibody to clathrin, boundto a bacterial carrier, can selectively bind these vesicles in acrude preparation of membranes, and the whole antibodycomplex can then be isolated by low-speed centrifugation.A related technique uses tiny metallic beads coated with specific antibodies. Organelles that bind to the antibodies, andare thus linked to the metallic beads, are recovered from thepreparation by adhesion to a small magnet on the side of thetest tube.All cells contain a dozen or more different types ofsmall membrane-limited vesicles of about the same size(50–100 nm in diameter) and density.
Because of their similar size and density, these vesicles are difficult to separatefrom one another by centrifugation techniques. Immunological techniques are particularly useful for purifying specific classes of such vesicles. Fat and muscle cells, forinstance, contain a particular glucose transporter (GLUT4)that is localized to the membrane of a specific kind of vesicle. When insulin is added to the cells, these vesicles fusewith the cell-surface membrane and increase the numberof glucose transporters able to take up glucose from theblood. As will be seen in Chapter 15, this process is critical to maintaining the appropriate concentration of sugarin the blood. The GLUT4-containing vesicles can be purified by using an antibody that binds to a segment of theGLUT4 protein that faces the cytosol.
Likewise, the varioustransport vesicles discussed in Chapter 17 are characterizedby unique surface proteins that permit their separation withthe aid of specific antibodies.182CHAPTER 5 • Biomembranes and Cell Architecture(a)(b)ClathrinBacterial cellCoatedvesiclesAntibody to clathrinProtein ACoated vesicle0.1 m▲ EXPERIMENTAL FIGURE 5-38 Small vesicles can bepurified by binding of antibody specific for a vesicle surfaceprotein and linkage to bacterial cells. In this example, asuspension of membranes from rat liver is incubated with anantibody specific for clathrin, a protein that coats the outersurface of certain cytosolic vesicles.
To this mixture is added asuspension of Staphylococcus aureus bacteria whose surfacemembrane contains protein A, which binds to the Fc constantregion of antibodies. (a) Interaction of protein A with antibodiesbound to clathrin-coated vesicles links the vesicles to thebacterial cells. The vesicle–bacteria complexes can then berecovered by low-speed centrifugation. (b) A thin-section electronmicrograph reveals clathrin-coated vesicles bound to an S. aureuscell. [See E.
Merisko et al., 1982, J. Cell Biol. 93:846. MicrographProteins Can Be Removed from Membranesby Detergents or High-Salt SolutionsIonic detergents bind to the exposed hydrophobic regionsof membrane proteins as well as to the hydrophobic coresof water-soluble proteins. Because of their charge, these detergents also disrupt ionic and hydrogen bonds.
At high concentrations, for example, sodium dodecylsulfate completelydenatures proteins by binding to every side chain, a property that is exploited in SDS gel electrophoresis (see Figure3-32). Nonionic detergents do not denature proteins and arethus useful in extracting proteins from membranes beforepurifying them. These detergents act in different ways at different concentrations.
At high concentrations (above theCMC), they solubilize biological membranes by formingmixed micelles of detergent, phospholipid, and integralmembrane proteins (Figure 5-40). At low concentrations(below the CMC), these detergents bind to the hydrophobicregions of most integral membrane proteins, making themsoluble in aqueous solution.Treatment of cultured cells with a buffered salt solutioncontaining a nonionic detergent such as Triton X-100 extractswater-soluble proteins as well as integral membrane proteins.As noted earlier, the exoplasmic and cytosolic domains of integral membrane proteins are generally hydrophilic and sol-Detergents are amphipathic molecules that disrupt membranes by intercalating into phospholipid bilayers and solubilizing lipids and proteins.
The hydrophobic part of adetergent molecule is attracted to hydrocarbons and mingleswith them readily; the hydrophilic part is strongly attracted towater. Some detergents are natural products, but most aresynthetic molecules developed for cleaning and for dispersing mixtures of oil and water (Figure 5-39). Ionic detergents,such as sodium deoxycholate and sodium dodecylsulfate(SDS), contain a charged group; nonionic detergents, such asTriton X-100 and octylglucoside, lack a charged group. Atvery low concentrations, detergents dissolve in pure water asisolated molecules.
As the concentration increases, the molecules begin to form micelles—small, spherical aggregates inwhich hydrophilic parts of the molecules face outward andthe hydrophobic parts cluster in the center (see Figure 2-20).The critical micelle concentration (CMC) at which micellesform is characteristic of each detergent and is a function ofthe structures of its hydrophobic and hydrophilic parts.courtesy of G. Palade.]5.5 • Purification of Cells and Their Parts183IONIC DETERGENTSOH3CHCCH2CH2COONaH3C(CH2)11OOHCH3ONaOSodium dodecylsulfate (SDS)Sodium deoxycholateCH3SHONONIONIC DETERGENTSHOCH2OH3CH3CCH3CCH3CH2CCH3O(CH2CH2O)9.5(Average)Triton X-100(polyoxyethylene(9.5)p-t-octylphenol)HO(CH2)7CH3OHHOOHOctylglucoside(octyl--D-glucopyranoside)▲ FIGURE 5-39 Structures of four common detergents.
Thehydrophobic part of each molecule is shown in yellow; thehydrophilic part, in blue. The bile salt sodium deoxycholate is anatural product; the others are synthetic. Although ionicdetergents commonly cause denaturation of proteins, nonionicdetergents do not and are thus useful in solubilizing integralmembrane proteins.uble in water. The membrane-spanning domains, however,are rich in hydrophobic and uncharged residues (see Figure5-12). When separated from membranes, these exposed hydrophobic segments tend to interact with one another, causing the protein molecules to aggregate and precipitate fromaqueous solutions.
The hydrophobic parts of nonionic detergent molecules preferentially bind to the hydrophobic seg-ments of transmembrane proteins, preventing protein aggregation and allowing the proteins to remain in the aqueous solution. Detergent-solubilized transmembrane proteins canthen be purified by affinity chromatography and other techniques used in purifying water-soluble proteins (Chapter 3).As discussed previously, most peripheral proteins arebound to specific transmembrane proteins or membraneMicellesConcentrationabove CMCDetergentConcentrationbelow CMC▲ FIGURE 5-40 Solubilization of integral membraneproteins by nonionic detergents. At a concentration higher thanits critical micelle concentration (CMC), a detergent solubilizeslipids and integral membrane proteins, forming mixed micellescontaining detergent, protein, and lipid molecules.
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