Characteristics of Glutamate Dehydrogenase in Mitochondria Prepared from Corn Shoots

Abstract

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...

Estimation of Ammonium Ion Distribution between Cytoplasm and Vacuole Using Nuclear Magnetic Resonance Spectroscopy

“…Results of in vivo experiments have tended to support the view that GS is primarily responsible for NH4' assimilation (9,11,22), whereas in vitro mitochondrial GDH catalyzes NH4' assimilation in the presence of millimolar amounts of NH4' (31,32). The estimated cytoplasmic NH4' concentrations of approximately 10 uM in Table I contrast to the much lower concentrations estimated here.…”

“…The estimated cytoplasmic NH4' concentrations of approximately 10 uM in Table I contrast to the much lower concentrations estimated here. This discrepancy may be due to mixing of the mitochondria with large pools of vacuolar NH4' during extraction in the earlier study (31).…”

“…Published estimates of the concentration of NH4+ in maize leaf mitochondria (31), root cytoplasm (10), and soybean nodule cytosol (7,29) have been in the millimolar range, similar to the Km of GDH for NH4+. These results suggest that GDH could catalyze significant assimilation of NH4' in plants.…”

Characterization and Expression of NAD(H)-Dependent Glutamate Dehydrogenase Genes in Arabidopsis

“…In most cases, e.g. pea (Joy, 1973;Garland and Dennis, 1977), corn (Yamaya et al, 1984), isoenzyme 1 of grape (Loulakakis and Roubelakis-Angelakis, 1990a), and GDH3 of soybean (F.J. Turano, unpublished results), Ca2+ stimulation was observed only in the direction of the amination reaction. Furthermore, there appears to be an interaction between the leve1 of Ca2+ stimulation and NADH concentration, i.e.…”

“…The activity of severa1 NAD(H)-GDH isoenzymes has been shown to be stimulated by divalent cations, and is particularly sensitive to Ca2+ (Chou and Splittstoesser, 1972;Joy, 1973;Garland and Dennis, 1977;Yamaya et al, 1984;Itagaki et al, 1988;Loulakakis and Roubelakis-Angelakis, 1990a; F. J. Turano, unpublished results). In most cases, e.g.…”