Figure 8–6 Cell fractionation by centrifugation. Repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components. In general, the smaller the subcellular component, the greater the centrifugal force required to sediment it. Typical values for the various centrifugation steps referred to in the figure are: low speed: 1000 times gravity for 10 minutes medium speed: 20,000 times gravity for 20 minutes high speed: 80,000 times gravity for 1 hour very high speed: 150,000 times gravity for 3 hours steep(e.g., 5–20%) sucrose gradient (e.g., 20–70%) sample is sedimented through a steep density gradient that contains a very high concentration of sucrose or cesium chloride. Each cell component begins to move down the gradient as in Figure 8–7A, but it eventually reaches a position where the density of the solution is equal to its own density. At this point, the component floats and can move no farther. A series of distinct bands is thereby produced in the centrifuge tube, with the bands closest to the bottom of the tube containing the components of highest buoyant density (Figure 8–7B). This method, called equilibrium sedimentation, is so sensitive that it can separate macromolecules that have incorporated heavy isotopes, such as 13C or 15N, from the same macromolecules that contain the lighter, common isotopes (12C or 14N). In fact, the cesium-chloride method was developed in 1957 to separate the labeled from the unlabeled DNA produced after exposure of a growing population of bacteria to nucleotide precursors containing 15N; this classic experiment provided direct evidence for the semiconservative replication of DNA (see Figure 5–5).
Cell Extracts Provide Accessible Systems to Study Cell Functions
Studies of organelles and other large subcellular components isolated in the ultracentrifuge have contributed enormously to our understanding of the functions of different cell components. Experiments on mitochondria and chloroplasts purified by centrifugation, for example, demonstrated the central function of these organelles in converting energy into forms that the cell can use. Similarly, resealed vesicles formed from fragments of rough and smooth endoplasmic reticulum
Figure 8–7 Comparison of velocity sedimentation and equilibrium sedimentation. (A) In velocity sedimentation, subcellular components sediment at different speeds according to their size and shape when layered over a solution containing sucrose. To stabilize the sedimenting bands against convective mixing caused by small differences in temperature or solute concentration, the tube contains a continuous shallow gradient of sucrose, which increases in concentration toward the bottom of the tube (typically from 5% to 20% sucrose). After centrifugation, the different components can be collected individually, most simply by puncturing the plastic centrifuge tube with a needle and collecting drops from the bottom, as illustrated here.
(B) In equilibrium sedimentation, subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches their surroundings. Although a sucrose gradient is shown here, denser gradients, which are especially useful for protein and nucleic acid separation, can be formed from cesium chloride. The final bands, at equilibrium, can be collected as in (A).
(microsomes) have been separated from each other and analyzed as functional models of these compartments of the intact cell.
Similarly, highly concentrated cell extracts, especially extracts of Xenopus laevis (African clawed frog) oocytes, have played a critical role in the study of such complex and highly organized processes as the cell-division cycle, the separation of chromosomes on the mitotic spindle, and the vesicular-transport steps involved in the movement of proteins from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane.
Cell extracts also provide, in principle, the starting material for the complete separation of all of the individual macromolecular components of the cell. We now consider how this separation is achieved, focusing on proteins.