Publications

• Purification and characterization of NADH-glutamate synthase from alfalfa root nodules.

  Anderson M.P., Vance C.P., Heichel G.H., Miller S.S.

  Glutamate synthase (GOGAT), a key enzyme in the pathway for the assimilation of symbiotically fixed dinitrogen (N(2)) into amino acids in alfalfa (Medicago sativa L.) root nodules, was purified and used to produce high titer polyclonal antibodies. Purification resulted in a 208-fold increase in sp ... >> More

  Glutamate synthase (GOGAT), a key enzyme in the pathway for the assimilation of symbiotically fixed dinitrogen (N(2)) into amino acids in alfalfa (Medicago sativa L.) root nodules, was purified and used to produce high titer polyclonal antibodies. Purification resulted in a 208-fold increase in specific activity to 13 micromole per minute per milligram of protein and an activity yield of 37%. Further purification to near homogeneity was achieved by fast protein liquid chromatography, but with substantial loss of activity. Enzymic activity was highly labile, losing 3% per hour even when substrates, stabilizers, and reducing agents were included in buffers. However, activity could be partially stabilized for up to 1 month by storing GOGAT at -80 degrees C in 50% glycerol. The subunit molecular weight of GOGAT was estimated at 200 +/-7 kilodaltons with a native molecular weight of 235 +/-16 kilodaltons, which suggested that GOGAT is a monomer of unusually high molecular weight. The pl was estimated to be 6.6. The K(m) values for glutamine, alpha-ketoglutarate, and NADH were 466, 33, and 4.2 micromolar, respectively. Antibodies were produced to NADH-GOGAT. Specificity of the antibodies was shown by immunotitration of GOGAT activity. Alfalfa nodule NADH-GOGAT antibodies cross-reacted with polypeptides of a similar molecular weight in a number of legume species. Western blots probed with anti-GOGAT showed that the high GOGAT activity of nodules as compared to roots was associated with increased levels of GOGAT polypeptides. Nodule NADH-GOGAT appeared to be highly expressed in effective nodules and little if any in other organs. << Less

  Plant Physiol. 90:351-358(1989) [PubMed] [EuropePMC]

• Kinetic mechanism of NADH-dependent glutamate synthase from lupin nodules.

  Boland M.J.

  From initial-rate studies, a partially random kinetic mechanism has been deduced for NADH-dependent glutamate synthase from lupin nodules. The mechanism involves compulsory binding of NADH as first substrate, followed by random-order binding of glutamine and 2-oxoglutarate. Patterns of inhibition ... >> More

  From initial-rate studies, a partially random kinetic mechanism has been deduced for NADH-dependent glutamate synthase from lupin nodules. The mechanism involves compulsory binding of NADH as first substrate, followed by random-order binding of glutamine and 2-oxoglutarate. Patterns of inhibition by glutamate substantiate the mechanism. Dithionite was incapable of acting as an alternative reducing substrate although it is known to reduce the flavine groups of the enzyme. The implications of these results are discussed. Published rate equations for this type of mechanism were found to be unsatisfactory for this enzyme and suitable new equations are produced. These equations should have general application where the obligatory first substrate binds very tightly. << Less

  Eur J Biochem 99:531-539(1979) [PubMed] [EuropePMC]

• Glutamate synthase: properties of the reduced nicotinamide adenine dinucleotide-dependent enzyme from Saccharomyces cerevisiae.

  Roon R.J., Even H.L., Larimore F.

  A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae. The enzyme is specific for NADH, glutamine, and alpha-ketoglutarate (K(m) values of 2.6 muM, 1.0 mM, and 140 muM, respectively) ... >> More

  A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae. The enzyme is specific for NADH, glutamine, and alpha-ketoglutarate (K(m) values of 2.6 muM, 1.0 mM, and 140 muM, respectively) and has a pH optimum between 7.1 and 7.7. The stoichiometry of the reaction has been determined as 2 mol of glutamate synthesized per mol of glutamine consumed. Glutamate synthase can be distinguished from either of the glutamate dehydrogenases of yeast on the basis of its substrate requirements and behavior during agarose gel and ion exchange chromatography. Variations in the specific activity of glutamate synthase, which occur in response to changes in the growth medium, are similar in character to those observed with the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase. << Less

  J. Bacteriol. 118:89-95(1974) [PubMed] [EuropePMC]

• Enzymes of nitrogen metabolism in legume nodules. Purification and properties of NADH-dependent glutamate synthase from lupin nodules.

  Boland M.J., Benny A.G.

  An NADH-dependent glutamate synthase has been purified 500-fold from the plant cytoplasm fraction of Lupinus angustifolius nodules. It consists of a single polypeptide chain, Mr 235000. The optimum pH is 8.5, at which Km values for 2-oxoglutarate, glutamine and NADH are 39 micrometer, 400 microme ... >> More

  An NADH-dependent glutamate synthase has been purified 500-fold from the plant cytoplasm fraction of Lupinus angustifolius nodules. It consists of a single polypeptide chain, Mr 235000. The optimum pH is 8.5, at which Km values for 2-oxoglutarate, glutamine and NADH are 39 micrometer, 400 micrometer and 1.3 micrometer respectively. The catalytic centre activity is of the order of 70 s-1 and is independent of pH between 6.5 and 9.5. Glutamate synthase is inhibited by glutamic acid, oxaloacetic acid, aspartic acid and asparagine, all competitive with 2-oxoglutarate; and by NAD+, which is competitive with NADH. There is evidence of two flavine prosthetic groups per enzyme molecule. << Less

  Eur J Biochem 79:355-362(1977) [PubMed] [EuropePMC]