PARTIAL CHARACTERIZATION OF ENZYMES OF NITROGEN-METABOLISM IN CHLORELLA-AUTOTROPHICA SHIHIRA AND KRAUSS

Chlorella autotrophica shows a major decline in the intracellular conc entrations of glutamine synthetase (GS) isoenzymes, GS1 and GS2, and a marked increase in the concentration of NADPH-dependent glutamate deh ydrogenase (GDH) when nitrate is replaced by ammonium as the sole nitr ogen source. NADH-dependent GDH shows very little change under these c onditions. The apparent K(m) for ammonium of both GS isoenzymes is aro und 50 muM and that of NADPH-GDH is around 15 mm. There is little chan ge in the latter when the NADPH concentration in the assay medium is d ecreased, or when cells are harvested at different growth phases. In v itro kinetic analysis of GS1 and GS2 indicate low affinities of these isoenzymes for substrates other than ammonium. Both GS1 and GS2 requir e about 10 mm ATP, 20 mm Mg2+ and 70 mm glutamate for maximum activiti es. The rate response curves obtained for GS2 show nonlinear kinetics for glutamate and Mg2+. The double reciprocal plots for activity vs. s ubstrate concentration for GS2 show dual kinetics with respect to glut amate concentrations, with a low K(m) value of about 10 mm and a high K(m) value of 40 mm. The rate response curve for GS2 for Mg2+ is sigmo idal. The K(m) values for glutamate measured for aspartate aminotransf erase (AsAT) and alanine aminotransferase (AlAT) are in the same range as those measured for the two GS isoenzymes. The algal aminotransfera ses have very low K(m) values (18-55 muM) for 2-oxoglutarate.