SUMMARY: A method is described for the preparation of spheroplasts in high yield from , by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome oxidase activity and spectroscopically-detectable cytochromes ) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with , cytochrome oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mM-MgCl or 0.4 mM-EDTA. Mitochondria were apparently smaller and of lower buoyant density in gradients containing EDTA. The bulk of the total units of malate dehydrogenase and NADH: cytochrome oxidoreductase sedimented with mitochondrial whereas NADPH: cytochrome oxidoreductase was located in fractions containing no mitochondria. The distributions of mitochondrial enzymes were heterogeneous in populations of mitochondria separated on the basis of size or density. The possible origins of mitochondrial heterogeneity in extracts of are discussed with special reference to changes in the enzyme activities of cells during the cell cycle.


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