Although the electron microscope has allowed detailed visualization of cell structure, microscopy alone is not sufficient to define the functions of the various components of eukaryotic cells. To address many questions concerning the function of subcellular organelles, it is necessary to isolate the organelles of eukaryotic cells in a form that can be used for biochemical studies. This is usually accomplished by differential centrifugation a method developed largely by Albert Claude, Christian de Duve, and their colleagues in the 1940s and 1950s to separate the components of cells on the basis of their size and density. The first step in subcellular fractionation is the disruption of the plasma membrane under conditions that do not destroy the internal components of the cell. Several methods are used, including sonication (exposure to high-frequency sound), grinding in a mechanical homogenizer, or treatment with a high-speed blender. All these procedures break the plasma membrane and the endoplasmic reticulum into small fragments, while leaving other components of the cell (such as nuclei, lysosomes, peroxisomes and mitochondria) intact. The suspension of broken cells (called a lysate or homogenate) is then fractionated into its components by a series of centrifugations, with an ultracentrifuge used to rotate samples at very high speeds (over 100,000 rpm), producing forces up to 500,000 times greater than gravity. This force causes cell components to move toward the bottom of the centrifuge tube and form a pellet (a process called sedimentation) at a rate that depends on their size and density, with the largest and heaviest structures sedimenting most rapidly (Figure 1.40). Usually the cell homogenate is first centrifuged at a low speed, which sediments only unbroken cells and the largest sub- cellular structures the nuclei. Thus, an enriched fraction of nuclei can be recovered from the pellet of such a low-speed centrifugation while the other cell components remain suspended in the supernatant (the remaining solution). The supernatant is then centrifuged at a higher speed to sediment mitochondria, chloroplasts, lysosomes, and peroxisomes. Recentrifugation of the supernatant at an even higher speed sediments fragments of the plasma membrane and the endoplasmic reticulum. A fourth centrifugation at a still higher speed sediments ribosomes, leaving only the soluble portion of the cytoplasm (the cytosol) in the supernatant.
Subcellular fractionation Cells are disrupted (lysed) and subcellular components are separated by a series of centrifugations at increasing speeds. Following each centrifugation, the organelles that have sedimented to the bottom of the tube are recovered in the pellet. The supernatant (remaining solution) is then recentrifuged at a higher speed to sediment the next-largest organelles.
The fractions obtained from differential centrifugation correspond to enriched, but still not pure, organelle preparations. A greater degree of purification can be achieved by density-gradient centrifugation, in which organelles are separated by sedimentation through a gradient of a dense substance, such as sucrose. In velocity centrifugation, the starting material is layered on top of the sucrose gradient (Figure 1.41). Particles of different sizes sediment through the gradient at different rates, moving as discrete bands. Following centrifugation, the collection of individual fractions of the gradient provides sufficient resolution to separate organelles of similar size, such as mitochondria, lysosomes, and peroxisomes.
Velocity centrifugation The sample is layered on top of a gradient of sucrose, and particles of different sizes sediment through the gradient as discrete bands. The separated particles can then be collected in individual fractions of the gradient, which can be obtained simply by puncturing the bottom of the centrifuge tube and collecting drops.
Equilibrium centrifugation in density gradients can be used to separate subcellular components on the basis of their buoyant density, independent of their size and shape. In this procedure, the sample is centrifuged in a gradient containing a high concentration of sucrose or cesium chloride. Rather than being separated on the basis of their sedimentation velocity, the sample particles are centrifuged until they reach an equilibrium position at which their buoyant density is equal to that of the surrounding sucrose or cesium chloride solution (Figure 1.42). Such equilibrium centrifugations are useful in separating different types of membranes from one another and are sufficiently sensitive to separate macromolecules that are labeled with different isotopes. For example, equilibrium centrifugation can be used to separate vesicles derived from rough and smooth endoplasmic reticulum, which are involved in the synthesis of proteins and lipids, respectively. Vesicles derived from the rough endoplasmic reticulum are covered with ribosomes (see Figure 1.7). Because ribosomes contain large amounts of RNA, vesicles from the rough endoplasmic reticulum are denser, allowing their separation in density gradients.
Equilibrium centrifugation Vesicles derived from the rough endoplasmic reticulum (ER), which is engaged in protein synthesis and covered with ribosomes, can be separated from those derived from the smooth ER by equilibrium centrifugation. Because ribosomes contain large amounts of RNA, vesicles from the rough ER are denser than those from the smooth ER and band at a heavier buoyant density.