Osmoregulation in Dunaliella. Intracellular Distribution of Enzymes of Glycerol Metabolism

Dunaliella tertiolecta was disrupted mechanically and resolved by centrifugation into chloro­plast- and cytosol-enriched fractions. Intact chloroplasts could not be isolated because peripheral extensions of the single large chloroplast reached almost to the flagellar pole of the cell. The chloroplast envelope was closely appressed to the plasmalemma and, because of this and its dimensions, was vulnerable to mechanical damage to the cell.Distribution of enzymes of the glycerol cycle between the two fractions was compared with that of two marker enzymes, phosphoenolpyruvate carboxylase (cytosol) and ribulose bisphosphate carboxylase (chloroplast). The two reversible steps of the cycle were found to be spatially separated; glycerol-3-phosphate dehydrogenase (NAD-specific) was located in the chloroplast whereas glycerol dehydrogenase (NADP-specific) was located in the cytosol. The distribution of the two irreversible enzymes, glycerol phosphate phosphatase and dihydroxyacetone kinase is uncertain. These enzymes might occur about equally in both major compartments (cytoplasm and chloroplast) or be mitochondrial or they might be loosely associated with a membrane system. Implications of these results for regulation of the glycerol cycle are discussed.