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Articles

Glutamate Synthase from Rice Leaves ¹

Laboratoire de Physiologie Végétale Métabolique, ERA Centre National de la Recherche Scientifique 799, Université de Paris-Sud, Bâtiment 430, 91405 Orsay cedex, France

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) from rice leaves (Oryza sativa L. cv Delta) was purified 206-fold with a final specific activity of 35.9 µmoles glutamate formed per min per milligram protein by a procedure including ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephacryl S-300 gel filtration, and ferredoxin-Sepharose affinity chromatography. The purified enzyme yielded a single protein band on polyacrylamide gel electrophoresis. Molecular weight of the native enzyme was estimated to be 224,000 daltons by Sepharose 6B gel filtration. Electrophoresis of the dissociated enzyme in sodium dodecyl sulfate-polyacrylamide gel gave a single protein band which corresponds to the subunit molecular weight of 115,000 daltons. Thus, it is concluded that the glutamate synthase is composed of two polypeptidic chains exhibiting the same molecular weight. Spectrophotometric analysis indicated that the enzyme is free of iron-sulfide and flavin. The pH optimum was 7.3. The enzyme had a negative cooperativity (Hill number of 0.70) for glutamine, and its K_m value increased from 270 to 570 µM at a glutamine concentration higher than 800 µM. K_m values for -ketoglutarate and ferredoxin were 330 and 5.5 µM, respectively. Asparagine and oxaloacetate could not be substituted for glutamine and -ketoglutarate, respectively. Enzyme activity was not detected with pyridine nucleotides as electron donors. Azaserine and several divalent cations were potent inhibitors. The purified enzyme was stabilized by dithiothreitol.

¹ Supported by Grant RCP 389 from the Centre National de la Recherche Scientifique and by Grant 78 70 445 from the Direction Générale à la Recherche Scientifique et Technique.

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