6.3—
Isolation

Microbodies appear to be very fragile, since homogenization of plant tissue damages them to such an extent that their constituent enzymes appear in soluble fractions after removal of chloroplasts and mitochondria by centrifugation. More gentle techniques of tissue breakage however allow microbodies to be isolated at a yield of usually about 10% of the organelles present in the whole tissue as judged by the solubilization of enzymes presumed to be present in the microbody. The method of breakage depends upon the tissue. Leaves are ground for a brief time with a mortar and pestle at 0–4°C in a buffer containing an osmoticum such as sucrose at a concentration of 0.4 to 0.8 M . Other tissues may be chopped or finely sliced with razor blades in a similar medium. The resulting homogenate is squeezed through cheesecloth and the brei centrifuged at a low speed to remove cell debris. The supernatant is then centrifuged at 6,000 to 10,000 g to obtain a pellet containing broken chloroplasts, microbodies and some mitochondria.

Microbodies are separated from the other organelles by layering this resuspended pellet fraction on a discontinuous or continuous density gradient of sucrose ranging in concentration from 1.3 to 2.5 M , and centrifuging in an ultracentrifuge for 3–4 hr at 40,000 g. Microbodies increase in density during this process by a loss of water and ultimately form a band in the gradient at a density of 1.24 to 1.26 g cm^–3 . This density is higher than that of other organelles and they are clearly separated from mitochondria which sediment at a desntiy of 1.16 to 1.19 g cm^–3 (Fig. 6.2). On the separation of the gradient into fractions

Figure 6.2
The distribution of protein in continuous (A) and
discontinuous (B) sucrose density gradients after
centrifugation of crude particles from castor bean endosperm.
(Reproduced with permission from Cooper & Beevers, 1969a.)

the microbodies are detected by assaying for specific 'marker' enzymes. Typical marker enzymes for peroxisomes are catalase, glycollate oxidase and hydroxypyruvate reductase, while those for glyoxysomes are catalse, ma late synthetase and isocitrate lyase. Mtitochondria are characterized by the presence of cytochrome c or succinic dehydrogenase, and chloroplasts by their chlorophyll pigments.

Much of the activity of the microbody marker enzymes is found in the soluble non-particulate fraction of gradients, indicating that the yield of micro-bodies is low. Despite these low yields, sufficient amounts of intact microbodies have been isolated from several plant tissues to be able to determine their metabolic functions. This has resulted in the distinguishing of at least two types of microbody, peroxisomes and glyoxysomes, with distinctly different enzyme complements and physiological functions.