Glutamate Dehydrogenase Activity in Roots: Distribution in a Seedling and Storage Root, and the Effects of Red and Far-red Illuminations

Pisum seedling and Pastinaca storage roots contained high glutanrate dehydrogenase (GDH) activity in areas of reported rapid growth and high phytoctrome content. A similar distribution was observed for malate dehydrogenase. Freeze-thawings of mitochondrial preparations from Pisum roots always resulted in increases of GDH specific activity; however, the observed increases were much larger with basal than apical sections. Both intact and freeze-thawed mitochondrial preparations from seedling roots exhibited increases in GDH activity with time after isolation. In intact mitochondrial preparations from roots of etiolated seedlings, an increase in malate dehydrogenase activity was observed similar to that of GDH activity; however, no increased malate dehydrogenase activity was noted in preparations from light-grown seedlings. Illuminating Pisum seedlings with far-red light slowly increased GDH activity in roots over a period of two weeks. Since these observed increases were not due to direct exposure of roots to light, other factors were likely involved.     

Water potential is maintained during water deficit in Nicotiana tabacum expressing the Escherichia coli glutamate dehydrogenase gene

Expression of bacterial gdhA (glutamate dehydrogenase; GDH; E.C. 1.4.1.1) genes in transgenic plants fundamentally alters plant growth, herbicide tolerance and metabolite profiles. The aim was to correlate gdhA expression with water potential during deficit using transgenic Nicotiana tabacum cv. ‘SR1’ (tobacco). Expression of GDH activity from the transgene was significantly correlated with high water potentials during deficit, both after 5 days of water deprivation (R = 0.91) and after 6 h after re-watering on day 6 (R = 0.72). GDH expression may provide a tool to alter the response of plants to periodic water deficit.     

Effects of temperature on germination and mitochondrial dehydrogenases in two soybean (Glycine max) cultivars

The effects of eight germination temperatures from 10°C to 35°C on germination and dehydrogenase activities of two soybean (Glycine max [L.] Merr.) cultivars were investigated after 48 h of seedling growth. Axis fresh weights of cv. Chippewa increased as germination temperature increased from 10°C to 35°C. In contrast, axis fresh weights for the cv. Wells increased more slowly with increasing temperature and reached a maximum at c. 25°C. In general, in vitro activities of glutamate dehydrogenase (GDH), NADP-isocitrate dehydrogenase (NADP-ICDH), and malate dehydrogenase (MDH) from the axes of cv. Chippewa correlated well with increases in axis fresh weights. GDH and MDH activities from axes of the cv. Wells also reflected increases in axis fresh weights although the correlation was not as evident as for the cv. Chippewa. NADP-ICDH activity from ‘Wells’ axes was highest at 35°C even though germination was poor at this temperature. GDH and MDH activities from cotyledons of both cultivars were not correlated with axis weight increases. No GDH activity was detected in ‘Wells’ cotyledons from seeds germinated at 35°C.     

Response of Ammonia Assimilation in Cucumber Seedlings to Nitrate Stress

The influence of increased nitrate concentration—14 (control) and 140 mmol L⁻¹ (T)—in hydroponic culture on ammonia assimilation in cucumber (Cucumis sativus L. cv. Xintaimici) seedlings was investigated. The results showed that NH₃ accumulation in the roots and leaves of T seedlings increased significantly, indicating that NH₃ toxicity might be involved in nitrate stress. Under control conditions, GS and GOGAT activity were much higher in the leaves than in the roots, whereas GDH activity was much higher in the roots than in the leaves. Correlation analysis showed that NH₃ concentration had a strong negative linear relationship with GDH activity in the roots but had a strong negative linear relationship with GS and GOGAT activity in the leaves. These results indicate that NH₃ might be assimilated primarily via GDH reaction in the roots and via GS/GOGAT cycle in the leaves. Short-term nitrate stress resulted in the increase of GS and GOGAT activity in the roots and GDH activity in the leaves of T seedlings, indicating possible shifts in ammonia assimilation from the normal GDH pathway to GS/GOGAT pathway in the roots and from the normal GS/GOGAT pathway to the GDH pathway in the leaves under nitrate stress, but with the increase of treatment time, GS, GOGAT, and GDH activity in the roots and leaves of T seedlings decreased possibly due to low water potential and NH₃ toxicity.     

Seedling age and cytokinin effects on glutamate dehydrogenase activity and nitrogen assimilation in maize leaves

Glutamate dehydrogenase (GDH) activity, protein and total nitrogen contents in the secondary leaves of maize(Zea mays L. cv. Ganga Safed-2) seedlings increased during early seedling growth and then declined after reaching a peak level at either 10 d (GDH) or 12 d (metabolites). While the effect of kinetin on enzyme activity was statistically insignificant, benzyladenine supplied with nutrient solution increased GDH activity in secondary leaves of both 10-d as well as 14-d seedlings. However, both growth regulators increased the contents of total soluble proteins, total nitrogen, chlorophyll(a+b) and carotenoids in both 10 and 14-d old leaves.     

Inducible Formation of Glutamate Dehydrogenase in Rice Plant Roots by the Addition of Ammonia to the Media

The activity of glutamate dehydrogenase (l-glutamate: NAD oxidoreductase, EC 1.4.1.2.; GDH) of rice plants changes in response to the nitrogen source supplied to the culture solution. The activity of NADH-GDH(aminating) in roots is rapidly increased by the addition of ammonia, whereas the activity in shoots is much less affected by nitrogen supply. The activity increased with increasing concentration of ammonia at least up to 14.3 mM. In roots GDH activity was found in both the mitochondrial and soluble fractions. The increase of NADH-GDH activity caused by the ammonia treatment occurs mainly in the latter fraction. The new band with GDH activity was detected on the zymogram of polyacrylamide gel electrophoresis and this inducible enzyme is active with both NAD and NADP. On the other hand, the constitutive enzyme activity active with NAD is also increased by the ammonia treatment. The increase of enzyme activity is prevented by the addition of cycloheximide or chloramphenicol to culture medium. The incorporation of ¹⁴C-leucine(U) into GDH proteins was also studied using polyacrylamide gel electrophoresis. Higher radioactivity was found in induced samples than in non-induced ones. These results show that the increase of GDH activity in roots by ammonia treatment seems to depend on de novo protein synthesis.     

Effect of NaCI on nitrate reductase,glutamate dehydrogenase and glutamate synthase inVigna radiata calli

The effect of NaCI stress on the activities of nitrate reductase (NR), glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT) in callus lines ofVigna radiata which differ in salt resistance, was studied at weekly intervals upto 28 d of growth. After 28 d, the NaCI resistant callus (selected at 300 mM NaCI) at NaCI concentrations higher than 200 mM maintained higher NR activity than non-selected line. NaCI stress also affects aminating and deaminating activities of GDH. The NADH-GDH activity in the presence of NaCI was higher in the resistant than non-selected line. On the other hand, NAD-GDH activity in both the lines was completely inhibited after 7 d of growth. The increased activity of NADH-GDH in resistant calli may play a vital role in protecting the cells from toxic effect of increased endogenous level of ammonia which probably accumulates due to efficient NO₃ ⁻ reduction. NADH-GOGAT activity was found to decrease under salt stress in both the callus lines. Nitrogen assimilation in salt-resistant calli under salt stress was found to be characterized by high NR and NADH-GDH activities, concomitantly with low GOGAT activity. The authors are grateful to DST and CSIR for financial assistance.     

Effect of dalargin on the activity of cytochrome oxidase and glutamate dehydrogenase in rat spinal cord dissociated cultured neurons

Dissociated cultured neurons from the rat embryo spinal cord were grown for six days in the presence of dalargin, the synthetic analog of leu-enkephalin. Then the activities of two enzymes of energy metabolism, cytochrome oxidase (CO) and glutamate dehydrogenase (GDH), were studied in these neurons using quantitative cytochemical technique. Dalargin, which possesses the properties of nerve growth factor, enhanced the nerve cell growth and increased the activity of the above enzymes, with GDH activity being increased more significantly. According to the classical standpoint, increased GDH activity under conditions of acute energy deflciency favors the invoivement of some amino acids in a citric acid cycle for subsequent reproduction. One can suggest, in this relation, that the increased energy production caused by the enhanced nerve cell growth in the presence of dalargin was partially compensated by the amino acid splitting. The results allow us to suggest that the effect of dalargin (growth factor) on the nerve cells is similar to the effects of the extremal factors, and requires additional energy to be supplied.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 95–99, March–June, 1996.     

Responses of amino acid metabolizing enzymes from plants differing in salt tolerance to NaCl

Summary This paper reports the effects of NaCl on the in vivo activity of glutamate dehydrogenase (GDH) and glutamic-oxaloacetic transaminase (GOT) and on the in vitro activity of GDH, both enzymes having been isolated from plants differing in salt tolerance. The plants investigated were Vicia faba (salt-sensitive), Atriplex nitens and Atriplex calotheca (more or less salt-tolerant), and Atriplex halimus (halophyte) grown at various NaCl concentrations. GDH and GOT isolated from various salt-tolerant plants grown at low NaCl concentrations were inhibited in a similar way. At high NaCl concentrations, the enzyme activities remain at constant values only in the Atriplex species. GOT was more impaired by NaCl than GDH. In the case of GOT, the double reciprocal plot indicated the type of a noncompetitive inhibition. The in vitro effect of NaCl on the activity of GDH from the differentially salt-tolerant plants was of a different kind, i.e. GDH isolated from V. faba was clearly inhibited by NaCl, whereas NaCl stimulated the activity of GDH from all Atriplex species investigated. Kinetic analysis showed that substrate inhibition of GDH from A. nitens and A. calotheca grown at non-saline conditions could be removed by NaCl. Inhibition by high NaCl concentrations at low substrate concentrations was removable by increasing substrate concentrations. Moreover, the inhibition at low substrate concentrations was shown to be competitive. GDH lost this regulatory property when the plants were pretreated with 500 mM NaCl. GDH from A. halimus also possessed this control, but in contrast to A. nitens and A. calotheca, activity and control of GDH isolated from A. halimus were stimulated by pretreating the plants with 500 mM NaCl. The results showed that DDH isolated from the salt-tolerant Atriplex species was adapted to high NaCl concentrations of the tissue. Possible mechanisms of the interactions between GDH from salt-tolerant Atriplex species and NaCl are discussed.     

Silicon improves salinity tolerance and affects ammonia assimilation in maize roots

The present study was conducted to evaluate the effect of different salt concentrations (50 and 200 mM NaCl) on growth, permeability properties (electrolyte leakage, cell viability) and activity of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in roots of maize seedlings. Both salt concentrations significantly affected growth and permeability properties of maize seedling roots and this negative effect increased with concentration of salt and duration of experiments. On the other hand salinity induced only small changes in the activities of GS and GDH, usually small increase in the activity was observed. To characterise the possible protective effect of silicon (Si) on maize roots exposed to saline stress, different concentrations of Si were simultaneously applied to both, low (50 mM) and high (200 mM) salt concentrations. Possible protective effects of Si on studied parameters were analysed in time range of 3 days treatment with the most positive effect on salt-induced root growth inhibition at high salt concentration and electrolyte leakage. The results show significant increase in GDH activity under all the tested conditions, although the mechanisms underlying this increase have not been elucidated. The results indicate that silicon may ameliorate the salt-induced root growth inhibition and increase the plant vigour at stressful conditions.     

Expression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development

We investigated the effects of genetic modification of nitrogen metabolism via the bacterial glutamate dehydrogenase (GDH) on plant growth and metabolism. The gdhA gene from Escherichia coli encoding a NADPH-GDH was expressed in tobacco plants under the control of the 35 S promoter. The specific activity of GDH in gdhA plants was 8-fold of that in E. coli. Damage caused by spray application of 1.35 mM of phosphinothricin (PPT) herbicide, a glutamine synthetase (GS) inhibitor, was less pronounced in gdhA plants as compared with the control plants which suggests that the introduced GDH can assimilate some of the excess ammonium, at least during GS inhibition. However, gdhA plants were susceptible to 2.7 mM PPT. Biomass production was consistently increased in gdhA transgenic plants grown under controlled conditions and in the field. Total free amino acids and total carbohydrates were increased in gdhA plants grown in the greenhouse suggesting that both nitrogen and carbon metabolism were altered. We conclude that the modifications in transgenic plants may result from both increased nitrogen efficiency and altered gene expression and metabolism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Impact of manipulation of glycerol/diol dehydratase activity on intestinal microbiota ecology and metabolism

Glycerol/diol dehydratases (GDH) are enzymes that catalyse the production of propionate from 1,2-propanediol, and acrolein from glycerol. Acrolein reacts with dietary carcinogenic heterocyclic amines (HCA), reducing HCA mutagenicity, but is itself also an antimicrobial agent and toxicant. Gut microbial GDH activity has been suggested as an endogenous acrolein source; however, there is limited information on the potential of the intestinal microbiota to have GDH activity, and what impact it can have on the intestinal ecosystem and host health. We hypothesized that GDH activity of gut microbiota is determined by the abundance and distribution of GDH-active taxa and can be enhanced by supplementation of the GDH active Anaerobutyricum hallii, and tested this hypothesis combining quantitative profiling of gdh, model batch fermentations, microbiota manipulation, and kinetic modelling of acrolein formation. Our results suggest that GDH activity is a common trait of intestinal microbiota shared by a few taxa, which was dependent on overall gdh abundance. Anaerobutyricum hallii was identified as a key taxon in GDH metabolism, and its supplementation increased the rate of GDH activity and acrolein release, which enhanced the transformation of HCA and reduced fermentation activity. The findings of this first systematic study on acrolein release by intestinal microbiota indicate that dietary and microbial modulation might impact GDH activity, which may influence host health.     

Molecular and biochemical characterisation of a Teladorsagia circumcincta glutamate dehydrogenase

A full length cDNA encoding glutamate dehydrogenase was cloned from Teladorsagia circumcincta (TcGDH). The TcGDH cDNA (1614 bp) encoded a 538 amino acid protein. The predicted amino acid sequence showed 96% and 93% similarity with Haemonchus contortus and Caenorhabditis elegans GDH, respectively. A soluble N-terminal 6xHis-tagged GDH protein was expressed in the recombinant Escherichia coli strain BL21 (DE3) pGroESL, purified and characterised. The recombinant TcGDH had similar kinetic properties to those of the enzyme in homogenates of T. circumcincta, including greater activity in the aminating than deaminating reaction. Addition of 1 mM ADP and ATP increased activity about 3-fold in the deaminating reaction, but had no effect in the reverse direction. TcGDH was a dual co-factor enzyme that operated both with NAD⁺ and NADP⁺, GDH activity was greater in the deaminating reaction with NADP⁺ as co-factor and more with NADH in the aminating reaction.     

Enzyme Activities in Urtica dioica: Effects of Daylength and Leaf Age on Glutamate Dehydrogenase, Aspartate Aminotransferase and Alanine Aminotransferase

Enzymes, important to protein synthesis, were investigated in young and old leaves of Urtica dioica. The plants, divided into two groups, were exposed to either 18-hour or 12-hour photo-periods. One group of plants from each photoperiodic regime was subjected to an irradiance of 28 W × m^-2, and the other group of plants to 42 W × m^-2. The enzymes investigated were glutamate dehydrogenase (GDH), aspartate aminotransferase (glutamate-oxaloacetate transaminase, GOT), and alanine aminotransferase (glutamate-pyruvate transaminase, GPT), GDH and GOT were determined by means of electrophoretic separation on polyacrylamide and spectrophotometric measurements. GPT was determined only by the latter method. Plants exposed to 18-hour photoperiods showed much higher GDH activity than did those exposed to 12-hour photoperiods. The activity of GDH also increased with leaf age. Besides one uniform NAD⁺-dependent GDH, two other NAD⁺-independent enzymes, showing GDH activity, were identified on polyacryl-amide gel electrophoresis. The distribution of NADH and NAD⁺-dependent GDH activity between young and old leaves was similar under different growth conditions. The activity of GOT was insensitive to environmental changes. The results regarding GPT indicate that this enzyme responded to different photoperiods in the same way as GDH. A correlation coefficient of 0.928 was obtained for the relationship between GDH and GPT activity.     

Effect of applied nitrate on enzymes of ammonia assimilation in nodules ofCicer arietinum L.

Summary The relationship between N₂-fixation, nitrate reductase and various enzymes of ammonia assimilation was studied in the nodules and leaves ofC. arietinum. In the nodules of the plants growing on atmospheric nitrogen, maximum activities of glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), asparagine synthetase (AS) and aspartate aminotransferase (AAT) were recorded just prior to maximum activity of nitrogenase. In nitrate fed plants, the first major peak of GDH and AS coincided with that of nitrate reductase in the nodules. With the exception of AS, application of nitrate decreased the activities of all these enzymes in nodules but not in leaves. Activities of GS, GOGAT and AAT were affected to much greater extent than that of GDH. On comparing the plants grown without nitrate and those with nitrate, the ratios of the activities of GDH/GS and GDH/GOGAT in nitrate given plants, increased by 4 and 12 fold, respectively. The results presented in this paper suggest that in nodules of nitrate fed plants, assimilation of ammonia via GDH assumes much greater importance.     

Arginase, glutamine synthetase and glutamate dehydrogenase activities in moist chilled and warm-incubated walnut kernels

The activities of arginase, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were studied in both moist chilled (5°C) and warm (27°C) incubated walnut (Juglans regia. L) kernels to asses whether the non-germinability of dormant kernels is associated with failure in amino acid metabolism. Warm-incubated kernels showed low germination (25%), whereas cold-stratified kernels displayed germination up to 61%. Arginase activity increased about twofold in imbibed kernels. It remained at a high level in cold-stratified kernels from mid-period of incubation onwards; however, in warm-incubated kernels the activity declined after an initial increase so that by 20 days, it was negligible. No significant differences in GS activity occurred between cold-stratified and warm-incubated kernels, but the activity of GDH was significantly more in kernels incubated at warm conditions. Thin-layer chromatographic separation of polyamines revealed greater ammonia, spermidine and an unknown polyamine accumulation in warm-incubated kernels. Thus, the declined rate of walnut kernel germination under warm conditions is mainly correlated with rapid inactivation of arginase, greater levels of ammonia and alterations in kernel polyamine composition. The enhanced activity of GDH in warm-incubated kernels implies that catabolic deamination of amino acids and their subsequent respiration is the favored pathway ongoing under warm conditions. This situation compromises germination-specific metabolism of amino acids which likely to operate better at lower temperatures during cold stratification of kernels.     

Induction of glutamate dehydrogenase in the ovine fetal liver by dexamethasone infusion during late gestation

Glucocorticoids near term are known to upregulate many important enzyme systems prior to birth. Glutamate dehydrogenase (GDH) is a mitochondrial enzyme that catalyzes both the reversible conversion of ammonium nitrogen into organic nitrogen (glutamate production) and the oxidative deamination of glutamate resulting in 2-oxoglutarate. The activity of this enzyme is considered to be of major importance in the development of catabolic conditions leading to gluconeogenesis prior to birth. Ovine hepatic GDH mRNA expression and activity were determined in near-term (130 days of gestation, term 147 +/- 4 days) control and acutely dexamethasone-treated (0.07 mg(-1) hr(-1) for 26 hr) fetuses. Dexamethasone infusion had no effect on placental or fetal liver weights. Dexamethasone infusion for 26 hr significantly increased hepatic GDH mRNA expression. This increased GDH mRNA expression was accompanied by an increase in hepatic mitochondrial GDH activity, from 30.0 +/- 7.4 to 58.2 +/- 8.1 U GDH/U CS (citrate synthase), and there was a significant correlation between GDH mRNA expression and GDH activity. The generated ovine GDH sequence displayed significant similarity with published human, rat, and murine GDH sequence. These data are consistent with the in vivo studies that have shown a redirection of glutamine carbon away from net hepatic glutamate release and into the citric acid cycle through the forward reaction catalyzed by GDH, i.e., glutamate to oxoglutarate.     

Regulation of glutamate dehydrogenases during morphogenesis of Schizophyllum commune

The specific activities of two glutamate dehydrogenases (GDH), one requiring nicotinamide adenine dinucleotide (NAD) and the other specific for nicotinamide adenine dinucleotide phosphate (NADP), varied during growth of Schizophyllum commune as a function of the stage of the life cycle and the exogenous nitrogen source. During basidiospore germination on either glucose-NH(3) or glucose-glutamate medium, NADP-GDH increased six- to eightfold in specific activity, whereas NAD-GDH was depressed. During dikaryotic mycelial growth on either nitrogen source, the two GDH increased in a 1:1 ratio, whereas, during homokaryotic mycelial growth on glucose-NH(3), NADP-GDH activity was depressed and NAD-GDH increased six- to eightfold. Homokaryotic mycelium cultured on glucose-glutamate medium yielded high NADP-GDH activities and normal NAD-GDH activities. Intracellular NH(3) concentration and NADP-GDH activities were inversely related during spore germination and homokaryotic mycelium growth, whereas guanosine-5'-triphosphate (GTP) and l-glutamine specifically inhibited NAD- and NADP-GDH respectively in vitro. GTP inhibition was shown in extracts from cells at all stages of the life cycle. Basidiospore germling extracts contained an NADP-GDH essentially resistant to l-glutamine inhibition.