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

Chlorella autotrophica shows a major decline in the intracellular concentrations of glutamine synthetase (GS) isoenzymes, GS1 and GS2, and a marked increase in the concentration of NADPH-dependent glutamate dehydrogenase (GDH) when nitrate is replaced by ammonium as the sole nitrogen source. NADH-dependent GDH shows very little change under these conditions. The apparent K(m) for ammonium of both GS isoenzymes is around 50 muM and that of NADPH-GDH is around 15 mm. There is little change in the latter when the NADPH concentration in the assay medium is decreased, or when cells are harvested at different growth phases. In vitro kinetic analysis of GS1 and GS2 indicate low affinities of these isoenzymes for substrates other than ammonium. Both GS1 and GS2 require about 10 mm ATP, 20 mm Mg2+ and 70 mm glutamate for maximum activities. The rate response curves obtained for GS2 show nonlinear kinetics for glutamate and Mg2+. The double reciprocal plots for activity vs. substrate concentration for GS2 show dual kinetics with respect to glutamate 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 sigmoidal. The K(m) values for glutamate measured for aspartate aminotransferase (AsAT) and alanine aminotransferase (AlAT) are in the same range as those measured for the two GS isoenzymes. The algal aminotransferases have very low K(m) values (18-55 muM) for 2-oxoglutarate.