Laboratoire de Physiologie Végétale Métabolique, ERA Centre National de la Recherche Scientifique 799, Université de Paris-Sud, Bâtiment 430, 91405 Orsay cedex, France.
Plant Physiol. 1982 Apr;69(4):848-52. doi: 10.1104/pp.69.4.848.
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 mumoles 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 mum at a glutamine concentration higher than 800 mum. K(m) values for alpha-ketoglutarate and ferredoxin were 330 and 5.5 mum, respectively. Asparagine and oxaloacetate could not be substituted for glutamine and alpha-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.
水稻叶片（Oryza sativa L. cv Delta）依赖铁氧还蛋白的谷氨酸合酶（EC 1.4.7.1）经硫酸铵分级、DEAE-纤维素层析、Sephacryl S-300 凝胶过滤和铁氧还蛋白-Sepharose 亲和层析等步骤纯化 206 倍，终比活为 35.9 微摩尔谷氨酸形成/分钟/毫克蛋白。纯化的酶在聚丙烯酰胺凝胶电泳中产生单一蛋白质带。通过 Sepharose 6B 凝胶过滤估计天然酶的分子量为 224,000 道尔顿。在十二烷基硫酸钠-聚丙烯酰胺凝胶中对解离酶进行电泳，得到一条单一的蛋白质带，对应于 115,000 道尔顿的亚基分子量。因此，结论是谷氨酸合酶由两条具有相同分子量的多肽链组成。分光光度分析表明，该酶不含铁-硫和黄素。最适 pH 为 7.3。该酶对谷氨酰胺具有负协同性（Hill 数为 0.70），当谷氨酰胺浓度高于 800 µm 时，其 K(m) 值从 270 µm 增加到 570 µm。α-酮戊二酸和铁氧还蛋白的 K(m) 值分别为 330 µm 和 5.5 µm。天冬酰胺和草酰乙酸分别不能替代谷氨酰胺和α-酮戊二酸。吡啶核苷酸不能作为电子供体检测到酶活性。偶氮丝氨酸和几种二价阳离子是有效的抑制剂。纯化的酶通过二硫苏糖醇稳定。
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