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.
Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden.
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Carlberg
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Nordlund
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