Purification and properties of the cytoplasmic glucose-6-phosphate dehydrogenase from pea leaves.
A method involving affinity chromatography on the yellow dye Remazol Brilliant Gelb GL to highly purify the cytoplasmic isoenzyme of glucose-6-phosphate dehydrogenase from pea shoots is described. Purification is at least 6000-fold. The specific activity of the purified enzyme is 185 mumol NADP reduced/min per mg protein. The preparation was free from any contamination of chloroplastic isoenzyme. The purified enzyme retains its activity in the presence of reducing agents which, in contrast, inactivate the chloroplast enzyme. The state of activity of the cytoplasmic and the chloroplastic isoenzyme in illuminated or darkened pea leaves was investigated using specific antibodies. While upon illumination the chloroplastic isoenzyme was inactivated by 80 to 90%, we could not find any change in activity of the cytoplasmic glucose-6-phosphate dehydrogenase. ATP, ADP, NAD, NADH, and various sugar phosphates do not inhibit the enzyme activity. Only NADPH is a strong competitive inhibitor with respect to NADP, suggesting that the enzyme is regulated by feedback inhibition by one of its products. Mg2+ ions have no influence on the activity of the enzyme. The molecular weight has found to be 240,000 for the native enzyme and 60,000 for the subunit. Throughout the purification procedure the enzyme was very unstable unless NADP was present in the buffer.
D002736:Chloroplasts / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D003624:Darkness; D004229:Dithiothreitol / Q000494:pharmacology; D007887:Fabaceae / Q000201:enzymology* / Q000648:ultrastructure; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007120:Immunochemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008027:Light; D008970:Molecular Weight; D010946:Plants, Medicinal; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis
D007527:Isoenzymes; D013438:Sulfhydryl Compounds; D005954:Glucosephosphate Dehydrogenase; D004229:Dithiothreitol