Purification and partial characterization of glutamate synthase from Rhodospirillum rubrum grown under nitrogen-fixing conditions.
Glutamate synthase, a key enzyme in ammonia assimilation, has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The purification procedure involves ion-exchange chromatography, affinity chromatography and gel filtration. The recovery in the procedure is high (62%) and the specific activity is 21 mumol of NADPH oxidized/min per mg. The enzyme is specific for its substrates, and no activity was demonstrated with NADH or NH4+ ions substituting for NADPH and glutamine respectively. The enzyme is composed of two dissimilar subunits with molecular masses of 53 and 152 kDa, and it is shown that Cl- ions have an effect on the aggregation of the enzyme. Km values for the substrates are: NADPH, 16 microM; 2-oxoglutarate, 10 microM; and glutamine, 65 microM. The enzyme is inhibited by amidotransferase inhibitors at micromolar concentrations. The role of the enzyme in the metabolic regulation of nitrogenase is discussed.
Carlberg
I
I
Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden.
Nordlund
S
S
D000596:Amino Acids / Q000494:pharmacology; D001377:Azaserine / Q000494:pharmacology; D002853:Chromatography, Liquid; D003980:Diazooxonorleucine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005970:Glutamate Synthase / Q000302:isolation & purification* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009586:Nitrogen Fixation; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D003980:Diazooxonorleucine; D009243:NAD; D009249:NADP; D001377:Azaserine; D005970:Glutamate Synthase