Identification and subcellular localization of starch-metabolizing enzymes in the green alga Dunaliella marina

Enzymes of starch synthesis and degradation were identified in crude extracts of the unicellular green alga Dunaliella marina (Volvocales). By polyacrylamide gel electrophoresis and specific staining for enzyme activities, 4 multiple forms of starch synthase, 2 amylases, and at least 2 forms of a-glucan phosphorylase were visible. Using specific a-glucans incorporated into the gel before electrophoresis we have tentatively correlated a-amylase and ß-amylase with both hydrolytic activities. The activities of a-glucan phosphorylase and amylase(s) were measured quantitatively in crude extracts, and the concomitant action of a-glucan phosphorylase and amylase(s) was found to account for the fastest rate of starch mobilization observed in vivo. Isolated chloroplasts retained both typical plastid marker enzymes and ADPglucose pyrophosphorylase, starch synthase, amylase(s), and a-glucan phosphorylase to a similar percentage. Gel electrophoretic analysis followed by staining for enzyme activity of a stromal fraction resulted in a pattern of multiple forms of starch-metabolizing enzymes analogous to that found in a crude extract. We interpret the combined data as indicating the exclusive location in vivo of starch-metabolizing enzymes in chloroplasts of D. marina.