Starch Degradation in Spinach Leaves: ISOLATION AND CHARACTERIZATION OF THE AMYLASES AND R-ENZYME OF SPINACH LEAVES.

The properties of two amylase activities which differ in their substrate specificity and subcellular location as well as a chloroplast-associated R-enzyme (debranching activity) are reported. An extrachloroplastic amylase is resolved by gel filtration chromatography into two activities of 80,000 and 40,000 daltons. Both extrachloroplastic activities hydrolyze amylopectin and shellfish glycogen and only slowly hydrolyze rabbit liver glycogen, beta-limit amylopectin, and amylose. In contrast, the major chloroplastic amylase attacks all of these glucans at comparable rates. Glucan hydrolysis by both the extrachloroplastic and chloroplastic amylase generates not only maltose but appreciable amounts of other oligosaccharides, whereas maltotetraose hydrolysis produces glucose, maltose, and maltotriose. The action patterns displayed by the amylase activities indicate that both are endoamylases, although they lack the typical Ca(2+) requirement or heat stability of seed endosperm alpha-amylases. Dithiothreitol, glutathione (oxidized or reduced), ascorbate, dehydroascorbate, and dithiothreitol plus thioredoxin have no effect on either the chloroplastic or extrachloroplastic amylase activities.The chloroplastic R-enzyme debranches amylopectin, beta-limit amylopectin, pullulan, and alpha-limit dextrins, but not rabbit liver glycogen. An increase in extinction coefficient and lambda(max) is detected when the debranched amylopectin and beta-limit amylopectin form a complex with I(2)-KI. Based on these properties, the chloroplastic R-enzyme is similar in enzymic activity to the R-enzyme observed in endosperm tissue.