Enzymes of nitrogen assimilation in maize roots

111

Glutamate dehydrogenase (GDH) specific activity and function have been studied in cell suspension cultures of carrot (Daucus carota L. cv Chantenay) in response to carbon and nitrogen supply in the culture medium. The specific activity of GDH was derepressed in sucrose-starved cells concomitant with protein catabolism, ammonium excretion, and the accumulation of metabolically active amino acids. The addition of sucrose led to a rapid decrease in GDH specific activity, an uptake of ammonium from the medium, and a decrease in amino acid levels. The extent of GDH derepression was correlated positively with cellular glutamate concentration. These findings strengthen the view that the function of GDH is the catabolism of glutamate, which under conditions of carbon stress provides carbon skeletons for tricarboxylic acid cycle activity.If this view is correct, an inverse correlation between GDH activity and carbohydrate supply would be predicted. Here we report metabolic studies of the effect of carbohydrate supply on GDH activity and nitrogen metabolism in cultured carrot cells. MATERIALS AND METHODS Growth

Section: Biochemical Determinationsmentioning

confidence: 76%

Section: Biochemical Determinationsmentioning

confidence: 99%

Regulation of Glutamate Dehydrogenase Activity in Relation to Carbon Limitation and Protein Catabolism in Carrot Cell Suspension Cultures

Robinson¹,

Stewart²,

Phillips³

1992

111

“…The effect of carbon supply on GDH activity has been much studied and there is agreement that sugars exert a regulatory effect on this enzyme. In general GDH levels rise in response to carbon limitation and this effect is reversed by the addition of various sugars (16,21). The link between GDH activity and carbon starvation is therefore well established.…”

Section: Results and Discussion Ammonium Assimilationmentioning

confidence: 99%

The Role of Glutamate Dehydrogenase in Plant Nitrogen Metabolism

Robinson

Slade²,

Fox³

et al. 1991

283

153

In vivo nuclear magnetic resonance spectroscopy, in vitro gas chromatography-mass spectrometry, and automated 15N/13C mass spectrometry have been used to demonstrate that glutamate dehydrogenase is active in the oxidation of glutamate, but not in the reductive amination of 2-oxoglutarate. In cell suspension cultures of carrot (Daucus carota L. cv Chantenay), primary assimilation of ammonium occurs via the glutamate synthase pathway. Glutamate dehydrogenase is derepressed in carbonlimited cells and in such cells the function of glutamate dehydrogenase appears to be the oxidation of glutamate, thus ensuring sufficient carbon skeletons for effective functioning of the tricarboxylic acid cycle. This catabolic role for glutamate dehydrogenase implies an important regulatory function in carbon and nitrogen metabolism.

“…The The amination reaction of GDH was measured using a crude preparation desalted on Sephadex G-75 column (27) Mm NADH, respectively, in the absence of Ca2". Effect of 2 mM EGTA was also tested in the presence of 160 Mm NADH and I mM Ca2" (A).…”

Section: Resultsmentioning

confidence: 99%

“…The purified mitochondrial pellet was treated hypotonically using 1.5 ml of 20 mM Hepes-KOH buffer (pH 7.5). The (27). However, in critical experiments (NH4)2SO4 was used in a concentration range of 2 to 100 mm.…”

Section: Methodsmentioning

confidence: 99%

Characteristics of Glutamate Dehydrogenase in Mitochondria Prepared from Corn Shoots

Yamaya¹,

Oaks²,

Matsumoto³

1984

Self Cite

The amination of a-ketoglutarate (a-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca2+. The NADH-GDH purified 161-fold with ammonium sulfate, was also activated by Ca2" in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca2 had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca2".About 25% of the NADH-GDH activity was solubilized from purified mitochondria after a simple osmotic shock treatment, whereas the remaining 75% of the activity was associated with the mitochondrial membrane fraction. When the lysed mitochondria, mitochondrial matrix, or mitochondrial membrane fraction was used as the source of NADH-GDH, Ca2" had little effect on its activity. The mitochondrial fraction contained about 155 nanomoles Ca per milligram of mitochondrial protein, suggesting that the NADH-GDH in the mitochondria is already in an activated form with regard Ca2". In a simulated in vitro system using concentrations of 6A millimolar NAD, 0.21 millimolar NADH, 5 millimolar a-KG, and 5 millimolar glutamate thought to occur in the mitochondria, together with 1 millimolar Ca2", 10 and 50 millimolar NH4', and purified enzyme, the equilibrium of GDH was in the direction of glutamate formation.NADH-GDH2 is easily solubilized from mitochondria from roots (30), shoots (4, 25), and seeds (28) of various plants. Although it is the only mitochondrial enzyme with the potential for the assimilation of NH44, it has been suggested by Miflin and Lea (21) that the NH44 released by the conversion of glycine to serine in the mitochondria (3) is reassimilated via GS in the cytosol. They have also suggested that the principal function of mitochondrial GDH is the oxidation ofglutamate (21). However, a part of intramitochondrially generated NH44 from glycine is incorporated into glutamate by mitochondria isolated from pea shoots (I 1). Chou and Splittstoesser (7), Joy (14), and recently Furuhashi and Takahashi (10) Isolation of Mitochondria by Density Gradient Centrifugation. All reagents and buffer solutions were prepared with double glass-distilled H20 to minimize Ca24 contamination, and all operations were carried out at 4°C. Whole shoots of 5-d seedlings were harvested, washed in water, blotted dry with filter paper, and weighed. The shoots (40 to 60 g in fresh weight) were chopped with razor blades in extraction buffer consisting of 0.4 M mannitol, 0.1 M Hepes-KOH buffer (pH 7.5), 1 mM EDTA, 0.1% (w/v) BSA, and 0.6% (w/v) insoluble PVP, at a ratio of 1 g fresh weight per 3 ml of buffer. The chopped materials were ground with motar and pestle.The mitochondrial fraction was isolated by modification of the method described by Nishimura et al. (26) as described previously by us (32). The purified mitochondria after Percoll discontinuous density gradient were able to oxidize both malate and succinate with a high P/O and RCR ratios and appear to...