Osmoregulation in Dunaliella. Intracellular Distribution of Enzymes of Glycerol Metabolism

Dunaliella tertiolecta was disrupted mechanically and resolved by centrifugation into chloro­ plast* and cytosol-enriched fractions. Intact chloroplasts could not be isolated because peripheral extensions of the single large chloroplast reached almost to the flagellar pole o f the cell. The chloroplast envelope was closely appressed to the plasmalemma and, because o f this and its dimensions, was vulnerable to mechanical damage to the cell. Distribution o f enzymes o f the glycerol cycle between the two fractions was compared with that o f two marker enzymes, phosphoenolpyruvate carboxylase (cytosol) and ribulose bisphosphate carboxylase (chloroplast). The two reversible steps o f the cycle were found to be spatially separated; glycerol-3-phosphate dehydrogenase (N A D -specific) was located in the chloroplast whereas glycerol dehydrogenase (NA DP-specific) was located in the cytosol. The distribution o f 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 o f these results for regulation o f the glycerol cycle are discussed.