Isolation and partial characterization of homogeneous glutamate synthase from Spinacia oleracea

Tamura, Goro; Oto, Michiei; Hirasawa, Masakazu; Aketagawa, Jun

doi:10.1016/0304-4211(80)90074-7

Cited by 23 publications

(11 citation statements)

References 10 publications

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“…In the final step, a significant amount of free proteins (99.3%) was eliminated by washing, and the eluted glutamate synthase was purified 206-fold with 24% recovery of initial total activity. The specific activity was 35.88 units/mg protein, which is similar to the results obtained for spinach leaf enzyme (30). The specific activities previously reported for the purified glutamate synthase range between 10 and 26, including plant (18, 33), nodule (1), fungus (12), and bacterial enzymes (17,22), except that a higher value (1,259 units/mg protein) was reported for Aerobacter enzyme (8).…”

Section: Resultssupporting

confidence: 75%

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Glutamate Synthase from Rice Leaves

Suzuki¹,

Gadal²

1982

Plant Physiol.

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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

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Section: Resultssupporting

confidence: 75%

“…The mol wt of the native enzyme was estimated to be 224,000 daltons by chromatography on a calibrated Sepharose 6B column. The mol wt is thus higher than that of other Fd-dependent glutamate synthases (145,000-180,000 daltons) from alga (3) and higher plants (18,30,34 (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

Glutamate Synthase from Rice Leaves

Suzuki¹,

Gadal²

1982

Plant Physiol.

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“…An elongated protein would give a higher mol wt estimate than a spherical globular protein. The mol wt of alfalfa nodule GOGAT is comparable to that of lupine (225 kD) (1), but higher than that reported for the Fddependent GOGATs from maize and spinach leaves and soybean nodules (145-180 kD) (11,23,24).…”

Section: Mol Wt and Isoelectric Pointmentioning

confidence: 58%

“…The Fd-GOGAT form has also been demonstrated in roots (20)(21)(22) and most recently in soybean (Glycine max L. Merr) nodules (23). The NAD(P)H-dependent GOGATs have been reported in roots (20), shoots (12,22,24), and nodules (2,9) and ranged in mol wt from 200 to 240 kD. Antibodies have been produced to rice (Oryza sativa L.) (22) and maize (Zea mays L.) (21) leaf Fd-GOGAT.…”

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confidence: 96%

Purification and Characterization of NADH-Glutamate Synthase from Alfalfa Root Nodules

Anderson¹,

Vance²,

Heichel³

et al. 1989

Plant Physiol.

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Glutamate synthase (GOGAT), a key enzyme in the pathway for the assimilation of symbiotically fixed dinitrogen (N2) 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 -800C 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 pi was estimated to be 6.6. The Km values for glutamine, a-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-GO-GAT antibodies cross-reacted with polypeptides of a similar molecular weight in a number of legume species. Westem 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.

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“…For a detemination of Fd-glutamate synthase activity, methyl viologen is used as a reducing dye (22,31,33). In pea etiolated shoots (13) and Chlamydomonas (4), methyl viologen-linked enzyme activity is associated with the NADH-glutamate synthase rather than with Fd-glutamate synthase.…”

Section: Discussionmentioning

confidence: 99%