Glutamate Synthase from Rice Leaves

Abstract

Ferredoxin-dependent glutamate synthase (EC 1A.7.1) from rice leaves (Oryza sativa L. cv Delta) was purified 206-fold with a fmal specific activity of 35.9 pmoles glutamate formed per min per milligram protein by a procedure including ammonium sulfate fractionafton, DEAE-ceilulose chromatography, Sephacryl S-300 gel filtration, and ferredoxin-Sepharose affinity chromatography. The purfied 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 fidtration. 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 Glutamate synthase:glutamine:a-ketoglutarate aminotransferase (NADPH oxidizing; EC 2.6.1.53) was first identified in Aerobacter (31). The enzyme was found later in cultured cells and roots of higher plants, in which glutamine-amide group was reductively transferred to a-ketoglutarate using pyridine nucleotides to form two molecules of glutamate (5,7,21). A different form of glutamate synthase was detected in leaves (15); in these tissues, the enzyme was specific for reduced Fd and was inactive with pyridine nucleotides as electron donors. Fd-dependent glutamate synthase (EC 1.4.7.1) has been found in algae (3,16,20) and green tissues of higher plants (21,25,27). The enzyme from both roots and leaves is located in the plastid fraction (6,28, 33 18). A reaction mixture of 1 ml contained 22.5 ,umol phosphate buffer (KH2PO4-Na2HPO4), pH 7.3, 5 ,umol glutamine, 5 ,umol a-ketoglutarate, 0.02 ,Amol Fd or 0.33 ,lmol methyl viologen, 9 ,umol sodium dithionite dissolved in 0.05 ml of 190 mm NaHCO3. The reaction was started by the addition of Na2S204. After incubation at 30°C for 5 to 15 min, the reaction was stopped by heating the assay tubes at 100°C for 1 min in a water bath. The assay mixture was adjusted at pH 6.95, and centrifuged for 10 min at 2,000g to remove the precipitate. The supernatant was passed through a resin AG 1 x 8 column (3 x 40 mm). After washing with 3 ml of distilled water, glutamate was eluted from the column with 2 ml of 1 N acetic acid. An aliquot of the eluent was incubated with a modified ninhydrin solution (36) Mayhew (19). The ratio A420/A275 was 0.45. The concentration of Fd was determined using M extinction coefficient of 9,680 M-' cm-' (29).Preparation of Fd-CNBr Activated Sepharose 4B Column. CNBr Sepharose 4B gel (6.7 g) was suspended in 1 ml HCI and washed on a glass filter. The gel was then suspended in 100 mM NaHCO3, pH 8.3, containing 500 mm NaCl in a flask. To

Glutamate Synthase Isoforms in Rice

“…Fd-dependent glutamate synthase in rice green leaf tissue was purified to homogeneity by the method described previously (29 Affinity Column Chromatography. Glutamate synthase preparation obtained by standard extraction procedure was applied to Fd-Sepharose 4B column, equilibrated with the standard buffer containing 14 mm fi-mercaptoethanol.…”

Development of Nitrogen Assimilation Enzymes during Photoautotrophic Growth of Chenopodium rubrum Suspension Cultures

“…Vol. 74,1984 accepted to account for nitrate and ammonium assimilation in green leaf cells (6,13,14,18,(23)(24)(25)27). However, the metal> olism of the amino acids must be studied before the similarity of N metabolism of leaf cells and photoautotrophic cell cultures can be fully judged.…”

“…NR has the lowest activity ofall the enzymes we assayed and probably would be the rate-limiting catalyst in nitrate assimilation (6). GOGAT catalyzes the transfer of amide N to amino N, which requires two electrons, and may represent the rate-limiting step in accumulation of total reduced N (24,27). However, the product of the reaction catalyzed by GS, glutamine, can act as a donor of reduced N in some biosynthetic reactions and, as a result, not all ammonium may be processed via GOGAT (19,24).…”

“…GOGAT catalyzes the transfer of amide N to amino N, which requires two electrons, and may represent the rate-limiting step in accumulation of total reduced N (24,27). However, the product of the reaction catalyzed by GS, glutamine, can act as a donor of reduced N in some biosynthetic reactions and, as a result, not all ammonium may be processed via GOGAT (19,24). Since we found only a methyl viologen-dependentGOGAT, it seems likely that this activity would reside in the chloroplast and be driven by photosynthetically reduced ferredoxin (24,27).…”

“…However, the product of the reaction catalyzed by GS, glutamine, can act as a donor of reduced N in some biosynthetic reactions and, as a result, not all ammonium may be processed via GOGAT (19,24). Since we found only a methyl viologen-dependentGOGAT, it seems likely that this activity would reside in the chloroplast and be driven by photosynthetically reduced ferredoxin (24,27). This is the case for GOGAT in green leaf cells where the GS-GOGAT pathway for ammonium assimilation resides in the chloroplast (14).…”