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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 5.2Purification of Cells and Their Parts

Most animal and plant tissues contain a mixture of cell types. However, an investigator often wishes to study a pure population of one type of cell. In some cases, cells differ in some physical property that allows different cell types to be separated. White blood cells (leukocytes) and red blood cells (erythrocytes), for instance, have very different densities because erythrocytes have no nucleus; thus these cells can be separated on the basis of density. Since most cell types cannot be differentiated so easily, other cell-separation techniques have had to be developed. Similarly, it is essential to isolate quantities of each of the major subcellular organelles to study their structures and metabolic functions in detail.

Flow Cytometry Separates Different Cell Types

A flow cytometer can identify different cells by measuring the light they scatter, or the fluorescence they emit, as they flow through a laser beam; thus it can sort out cells of a particular type from a mixture. Indeed, a fluorescence-activated cell sorter (FACS), an instrument based on flow cytometry, can select one cell from thousands of other cells (Figure 5-21). For example, if an antibody specific to a certain cell-surface molecule is linked to a fluorescent dye, any cell bearing this molecule will bind the antibody and will then be separated from other cells when it fluoresces in the FACS. Once sorted from the other cells, the selected cell can be grown in culture.

Figure 5-21. Fluorescence-activated cell sorter (FACS).

Figure 5-21

Fluorescence-activated cell sorter (FACS). A concentrated suspension of cells is allowed to react with a fluorescent antibody or a dye that binds to a particle or molecule such as DNA. The suspension is then mixed with a buffer (the sheath fluid), the (more...)

Image biotech.jpgThis procedure is commonly used to purify the different types of white blood cells, each of which bears on its surface one or more distinctive proteins and thus will bind monoclonal antibodies specific for that protein. Such FACS separations are more difficult to conduct on cultured cells or cells from animal tissues, which interact with adjacent cells and are surrounded by an extracellular matrix. Samples must be treated with proteases to degrade the extracellular-matrix proteins and cell-surface proteins that attach cells in tissues to one another; these proteases usually also degrade the distinctive cell-surface “marker” proteins that distinguish one cell type from another.

Other uses of flow cytometry include the measurement of a cell’s DNA and RNA content and the determination of its general shape and size. The FACS can make simultaneous measurements of the size of a cell (from the amount of scattered light) and the amount of DNA it contains (from the amount of fluorescence from a DNA-binding dye).

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 cell’s interior. Many cells can then be broken by stirring the cell suspension in a high-speed blender or by exposing it to highfrequency sound (sonication). 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. 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.

Because the plasma membrane is highly permeable to water but poorly permeable to the salts and other small molecules (solutes) within cells, osmotic flow can be enlisted to help rupture cells. Recall that water flows across a semipermeable membrane, such as the plasma membrane, from a solution of high water (low solute) concentration to one of low water (high solute) concentration until the water concentration on both sides is equal. Consequently, when cells are placed in a hypotonic solution (i.e., one with a lower salt concentration than that of the cell interior), water flows into the cells (Figure 5-22). This osmotic flow causes the cells to swell and then more easily rupture. Conversely, in a hypertonic solution (i.e., one with a higher salt concentration than that of the cell interior), water flows out of cells, causing them to shrink. When cells are placed in an isotonic solution (i.e., one with a salt concentration equal to that of the cell interior), there is no net movement of water in or out of cells. For this reason, an isotonic solution is best for preserving normal cell structure.

Figure 5-22. Response of animal cells to the osmotic strength of the surrounding medium.

Figure 5-22

Response of animal cells to the osmotic strength of the surrounding medium. Sodium, potassium, and chloride ions do not move freely across the cell membrane, but water does. (a) When the medium is isotonic, there is no net flux of water into or out of (more...)

Disrupting the cell produces a mix of suspended cellular components, the homogenate, from which the desired organelles can be retrieved. 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.

Different Organelles Can Be Separated by Centrifugation

In Chapter 3 we discussed the principles of centrifugation and the uses of centrifugation techniques for separating proteins and nucleic acids. Similar approaches are used for separating and purifying the various organelles, which differ in both size and density.

Most fractionation procedures begin with differential centrifugation at increasingly higher speeds (Figure 5-23), also called differential-velocity centrifugation. The different sedimentation rates of various cellular components make it possible to separate them partially by centrifugation. Nuclei and viral particles can sometimes be purified completely by such a procedure. After centrifugation at each speed for an appropriate time, the supernatant is poured off and centrifuged at higher speed. Each pelleted fraction can be resuspended and further separated by equilibrium densitygradient centrifugation (discussed next).

Figure 5-23. Cell fractionation by differential centrifugation.

Figure 5-23

Cell fractionation by differential centrifugation. Generally, the cellular homogenate is first filtered or centrifuged at relatively low speeds to remove unbroken cells. Then centrifugation of the homogenate at a slightly faster speed or for a longer (more...)

Differential centrifugation does not yield totally pure organelle fractions. One method for further purifying fractions is equilibrium density-gradient centrifugation, which separates cellular components according to their density. The impure organelle fraction is layered on top of a solution that contains a gradient of a dense nonionic substance, such as sucrose or glycerol. The tube is centrifuged at a high speed (about 40,000 rpm) for several hours, allowing each particle to migrate to an equilibrium position where the density of the surrounding liquid is equal to the density of the particle. In typical preparations from animal cells, the rough endoplasmic reticulum (density = 1.20 g/cm3) separates well from the Golgi vesicles (density = 1.14 g/cm3) and from the plasma membrane (density = 1.12 g/cm3). (The higher density of the rough endoplasmic reticulum is due largely to the ribosomes bound to it.) This method also works well for resolving lysosomes, mitochondria, and peroxisomes in the initial mixed fraction obtained by differential centrifugation (Figure 5-24).

Figure 5-24. Separation of organelles from rat liver by equilibrium density-gradient centrifugation.

Figure 5-24

Separation of organelles from rat liver by equilibrium density-gradient centrifugation. For example, the material deposited as a pellet by centrifugation at 15,000 g (see Figure 5-23) can be resuspended and layered on a density gradient composed of layers (more...)

Since each organelle has unique morphological features, the purity of organelle preparations can be assessed by examination in an electron microscope (Figure 5-25). Alternatively, organelle-specific marker molecules can be quantified. For example, the protein cytochrome c is present only in mitochondria, so the presence of this protein in a fraction of lysosomes would indicate its contamination by mitochondria. Similarly, catalase is present only in peroxisomes; acid phosphatase, only in lysosomes; and ribosomes, only in the rough endoplasmic reticulum or the cytosol.

Figure 5-25. Electron micrographs of purified rat liver organelles.

Figure 5-25

Electron micrographs of purified rat liver organelles. Seen here are (a) nuclei, (b) rough endoplasmic reticulum, sheared into smaller vesicles termed microsomes, and (c) peroxisomes. Note that the rough endoplasmic reticulumis studded with ribosomes (more...)

Organelle-Specific Antibodies Are Useful in Preparing Highly Purified Organelles

Cell fractions often contain more than one type of organelle even after differential and equilibrium density-gradient centrifugation. Such fractions can be further purified by immunological techniques, using monoclonal antibodies for various organelle-specific membrane proteins. One example is the purification of a particular class of cellular vesicles whose outer surface is coated with the protein clathrin (Figure 5-26). An antibody to clathrin, bound to a bacterial carrier, can selectively bind these vesicles in a crude preparation of membranes, and the whole antibody complex can then be isolated by low-speed centrifugation. A recently developed technique uses tiny metallic beads coated with specific antibodies. Organelles that bind to the antibodies, and thus are linked to the metallic beads, are recovered from the preparation by adhesion to a small magnet on the side of the test tube.

Figure 5-26. Immunological purification of clathrin-coated vesicles.

Figure 5-26

Immunological purification of clathrin-coated vesicles. (a) A suspension of membranes from rat liver is incubated with an antibody specific for clathrin, a protein that coats the outer surface of certain cytoplasmic vesicles. To this mixture is added (more...)

All cells contain a dozen or more different types of small 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 separate from one another by centrifugation techniques. Immunological techniques are particularly useful for purifying specific classes of such vesicles. Fat and muscle cells, for instance, 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 fuse with the cell-surface membrane, a process critical to maintaining the appropriate concentration of sugar in the blood (see Figure 5-7). These vesicles can be purified using an antibody that binds to a segment of the GLUT4 protein that faces the cytosol.

SUMMARY

  •  Flow cytometry can identify different cells based on the light they scatter or the fluorescence they emit. The fluorescence-activated cell sorter (FACS) is particularly useful in separating different types of white blood cells (see Figure 5-21). A cell’s DNA and RNA content also can be measured with a FACS.
  •  Disruption of cells by vigorous homogenization, sonication, or other techniques releases their organelles. When placed in a hypotonic solution, cells swell, thereby weakening the plasma membrane and making it easier to rupture.
  •  Sequential differential-velocity centrifugation of a cell homogenate yields partially purified organelles that differ in mass (see Figure 5-23).
  •  Equilibrium density-gradient centrifugation, which separates cellular components according to their density, can further purify cell fractions obtained by differential centrifugation.
  •  Because the membrane surrounding each type of organelle contains organelle-specific proteins, immunological techniques are very useful in purifying organelles and vesicles, particularly those that have a similar size and density.
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Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21492

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