Organelles cell fractionation

Bowles DJ, Quail PH, Moire DJ, Hartnann GC. Use of markers in plant cell fractionation, in Plant Organelles (Reid E, ed.), Ellis Horwood, Chichester, UK, 1979, pp. 207-227. 

During cell fractionation, it is very important to analyze the purity of the fractions obtained. Whether or not the intended organelle is present in a particular fraction, and whether or not the fraction contains other components, can be determined by analyzing characteristic marker molecules. These are molecules that occur exclusively or predominantly in one type of organelle. For example, the activity of organelle-specific enzymes (marker enzymes) is often assessed. The distribution of marker enzymes in the cell reflects the compartmentation of the processes they catalyze. These reactions are discussed in greater detail here under the specific organelles. 

Intracellular Distribution and Development. When pea segments are homogenized and centrifuged to separate fractions containing wall material from particulate material and soluble supernatant, most of BS cellulase is found in the supernatant fraction while most BI cellulase is adsorbed to the wall (Table III). A minor part of both enzymes is particulate, i.e., retained in intact cell organelles and microsomes derived by vesiculation from cell membranes. In time-course studies of the distribution between cell fractions of total cellulase following auxin treatment (23), the activity appears first in the microsomes, and only... 

Cell fractionation techniques (Figure 2D) allow the study of cell organelles in a relatively intact form outside of cells. 

Cell Fractionation Simulation. The wall protein, cytosol and organelles of yeast each contain enzymes which are found nowhere else in the cell. Some examples of these enzymes include invertase in the walls, glycolytic pathway enzymes in the cytosol and fumarase in the mitochondria (13). A model of recovery of these enzymes is offered here. 

Although all eukaryotic cells have much in common, the ultrastructure of a plant cell differs firom that of the typical mammalian cell in three major ways. First, all living plant cells contain plastids. Second, the plasma membrane of plant cells is shielded by the cellulosic cell wall, preventing lysis in the naturally hypotonic environment but making preparation of cell fractions more difficult. Finally, the nucleus, cytosol, and organelles are pressed against the cell wall by the tonoplast, the membrane of the large, central vacuole that can occupy 80% or more of the cell s volume. 

Structure, function and turnover of autophagic vacuoles have been studied biochemically after enrichment of the organelles by cell fractionation... 

Sometimes biochemists must identify cell fractions by using reference biochemicals or markers —compounds found in high proportion in a specific organelle. Obviously, all fractions are best characterized when submitted to correlated biochemical and morphological analysis. 

Furthermore, the soluble fraction is probably the cell fraction that differs most from actual intracellular distribution. It contains numerous constituents, which in the intact cells are probably associated with other cellular organelles. The soluble fraction is arbitrarily defined as the supernatant of a pellet centrifuged either at 1,500,000 gx minutes or at 3,000,000 g x minutes. If this preparation is centrifuged longer (6,000,000 g X minutes), smaller particles are sedimented, and some of the glycolytic enzymes possibly could be associated with these small cell particles. 

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