Nitrogen-metabolizing Enzymes From Bean Leaves (Phaseolus vulgaris L.): Stability «in vitro» and Susceptibility to Proteolysis

Abstract

Summary The inactivation «in vitro» was investigated for four enzymes involved in nitrogen metabolism. Nitrate reductase (EC 1.6.6.1) was very unstable in extracts of bean leaves. Glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1) showed intermediate stabilities, and glutamate dehydrogenase (EC 1.4.1.2) proved to be very stable. Inactivation of both nitrate reductase and glutamine synthetase was accelerated in extracts from young leaves after addition of trypsin, chymotrypsin, extract from senescing leaves, or extract from cotyledons of germinating seeds. Glutamate synthase inactivation remained unchanged in presence of peptide hydrolases, while glutamate dehydrogenase was slowly inactivated by trypsin and chymotrypsin, but not by cotyledon or leaf extract. The inactivating factor from cotyledons and from leaves was precipitable with ammonium sulfate, heat labile and excluded by Sephadex G-25. The relative resistance to proteolysis was similar for nitrate reductase, glutamine synthetase and glutamate dehydrogenase, regardless of the peptide hydrolase source. Nitrate reductase was always the least stable and glutamate dehydrogenase the most stable of the three enzymes mentioned. It remains open by which factor(s) glutamate synthase was inactivated. Stability of glutamine synthetase decreased with age of the extracted leaves, and no increase in total endopeptidase activity was observed. Enzyme inactivation was delayed after addition of caScin or of heat inactivated extract. It appears likely that quality and quantity of other proteins present in the extract influence the inactivation of a particular enzyme.