Glutamate Dehydrogenase Isoenzymes Research Articles

The role of methylation of glutamate dehydrogenase gene promoters (<i>GDH1</i> and <i>GDH2</i>) in the regulation of their expression in corn leaves under hypoxia

The regulation of glutamate dehydrogenase, an enzyme that is involved in both nitrogen and carbon metabolism, and also links between the tricarboxylic acid cycle and the -aminobutyric acid shunt, has been studied. It was found that oxygen deficiency-induced changes in glutamate dehydrogenase activity in maize leaves (Zea mays L.) are to increase its catalytic activity by more than twice. Differential expression of genes was studied by real-time PCR in GDH1 and GDH2, which encode the - and -subunits of glutamate dehydrogenase, respectively, in the maize genome. Decreased relative level of gene transcripts GDH2 was accompanied by an increase in the expression activity of the gene GDH1. This, in turn, presumably promoted the amination reaction of 2-oxoglutarate. In the promoter of the gene GDH2, the presence of two CpG islands 404 and 383 bp in size was found. Gene promoter GDH1 does not contain a single CpG island; however, 38% of the CpNpG and CpNpN sites of the total number of studied dinucleotides in its composition were found. To assess the influence of the degree of methylation of individual CpG dinucleotides that are part of the promoter regions of genes GDH1 and GDH2 on their expression under hypoxic conditions, a comparative analysis of the dynamics of the transcriptional activity of the genes of - and -subunits of glutamate dehydrogenase from the methyl status of their promoters was carried out. Inversely proportional superposition of changes in the methylation profile of gene promoters GDH1 and GDH2 and transformation of the level of expression of these genes shows their correlation. The data obtained as a result of methyl-specific PCR indicate that an increase in the proportion of methylated CpG dinucleotides leads to a decrease in the amount of mRNA of the gene GDH2, while a decrease in this value for the gene GDH1 causes the induction of its functioning. Methylation of promoter regions of glutamate dehydrogenase genes regulates their transcriptional activity in maize leaves in vivo under conditions of oxygen deficiency. Thus, the little data on the molecular mechanisms of regulation of the synthesis of glutamate dehydrogenase isoenzymes were supplemented by new results on the role of the degree of methylation of gene promoters GDH1 and GDH2 glutamate dehydrogenases in their differential expression during maizes adaptation to hypoxia.

How to easily detect plant NADH-glutamate dehydrogenase (GDH) activity? A simple and reliable in planta procedure suitable for tissues, extracts and heterologous microbial systems

Plant NADH glutamate dehydrogenase (GDH) is an intriguing enzyme, since it is involved in different metabolic processes owing to its reversible (anabolic/catabolic) activity and due to the oligomeric nature of the enzyme, that gives rise to several isoforms. The complexity of GDH isoenzymes pattern and the variability of the spatial and temporal localization of the different isoforms have limited our comprehension of the physiological role of GDH in plants. Genetics, immunological, and biochemical approaches have been used until now in order to shed light on the regulatory mechanism that control GDH expression in different plant systems and environmental conditions. We describe here the validation of a simple in planta GDH activity staining procedure, providing evidence that it might be used, with different purposes, to determine GDH expression in plant organs, tissues, extracts and also heterologous systems.

Structural Properties of the RNA Synthesized by Glutamate Dehydrogenase for the Degradation of Total RNA

Glutamate dehydrogenase (GDH)-synthesized RNA, a nongenetic code-based RNA is suitable for unraveling the structural constraints imposed on the regulation (transcription, translation, siRNA etc.) of metabolism by genetic code. GDH-synthesized RNAs have been induced in whole plants to knock out target mRNA populations thereby producing plant phenotypes that are allergen-free; enriched in fatty acids, essential amino acids, shikimic acid, resveratrol etc. Methods applied hereunder for investigating the structural properties of GDH-synthesized RNA included purification of GDH isoenzymes, synthesis of RNA by the isoenzymes, reverse transcription of the RNA to cDNA, sequencing of the cDNA, computation of the G+C-contents, profiling the stability through PCR amplification compared with genetic code-based DNA; and biochemical characterization of the RNAs synthesized by individual hexameric isoenzymes of GDH. Single product bands resulted from the PCR amplification of the cDNAs of GDH-synthesized RNA, whereas several bands resulted from the amplification of genetic code-based DNA. The cDNAs have wide G+C-contents (35% to 59%), whereas genetic code-based DNA has narrower G+C-contents (50% to 60%). The GDH β6 homo-hexameric isoenzyme synthesized the A+U-rich RNAs, whereas the a6, and α6 homo-hexameric isoenzymes synthesized the G+C-rich RNAs. Therefore, the RNA synthesized by GDH is different from genetic code-based RNAs. In vitro chemical reactions revealed that GDH-synthesized RNA degraded total RNA to lower molecular weight products. Therefore, GDH-synthesized RNA is RNA enzyme. Dismantling of the structural constraints imposed on RNA by genetic code liberated RNA to become an enzyme with specificity to degrade unwanted transcripts. The RNA enzyme activity of GDH-synthesized RNA is ubiquitous in cells; it is readily induced by treatment of plants with mineral nutrients etc. and may simplify experimental approaches in plant enzymology and molecular biology research projects.

Getting into mitochondria.

The human mitochondrial glutamate dehydrogenase isoenzymes (hGDH1 and hGDH2) are abundant matrix-localized proteins encoded by nuclear genes. The proteins are synthesized in the cytoplasm, with an atypically long N-terminal mitochondrial targeting sequence (MTS). The results of secondary structure predictions suggest the presence of two α-helices within the N-terminal region of the MTS. Results from deletion analyses indicate that individual helices have limited ability to direct protein import and matrix localization, but that there is a synergistic interaction when both helices are present [Biochem. J. (2016) 473: , 2813-2829]. Mutagenesis of the MTS cleavage sites blocked post-import removal of the presequences, but did not impede import. The authors propose that the high matrix levels of hGDH can be attributed to the unusual length and secondary structure of the MTS.

Electrophoretic Studies of Isozymes of Glutamate Dehydrogenase (EC 1.4.1.3) (GDH) and Aspartate aminotransferase (EC: 2.6.1.1) AspAT (GOT) inSesuvium portulacastrum(L.), An Associate Halophyte.

Sesuvium portulacastrum L. (Aizoaceae) is a pioneer, psammophytic associate halophyte of subtropical, Mediterranean regions. It dominates coastal and warmer zones of the world. Apart from being utilized as a vegetable and forage by local people, it is also utilized for bioreclamation of saline soil in the arid and semiarid regions. Coastal soils has poor nitrogen content whereas halophytes which inhabit these areas have high protein content due to the ability to conserve nitrogen and recycle it through their body metabolism. In present investigation, Sesuvium portulacastrum (L.) is used as a model system representing an associate halophyte with efficacy in Nitrogen utilization in saline conditions. The plant has adapted to nitrogen stress by evolving efficient nitrogen assimilating isoenzymes. Isoenzymes of glutamate dehydrogenase (GDH) and Aspartate aminotransferase (AspAT) are separated electrophoretically from the leaves and roots of Sesuvium portulacastrum L. Presence of Isozymes for nitrogen assimilating and distributing enzymes like GDH and AspAT indicates the physiological adaptation of plants growing in saline nitrogen deficient conditions. The same study would be extended to other important enzymes of Nitrogen metabolism to get an insight in efficacy of such halophytes to conserve available Nitrogen from saline soils and help in phytoremediation of saline soils.

Glutamate dehydrogenase isoenzyme 3 (GDH3) of Arabidopsis thaliana is regulated by a combined effect of nitrogen and cytokinin

In higher plants, NAD(H)-glutamate dehydrogenase (GDH; EC 1.4.1.2) is an abundant enzyme that exists in different isoenzymic forms. In Arabidopsis thaliana, three genes (Gdh1, Gdh2 and Gdh3) encode three different GDH subunits (β, α and γ) that randomly associate to form a complex array of homo- and heterohexamers. The modification of the GDH isoenzyme pattern and its regulation was studied during the development of A. thaliana in the gdh1, gdh2 single mutants and the gdh1-2 double mutant, with particular emphasis on GDH3. Investigations showed that the GDH3 isoenzyme could not be detected in closely related Arabidopsis species. The induction and regulation of GDH3 activity in the leaves and roots was investigated following nitrogen deprivation in the presence or absence of sucrose or kinetin. These experiments indicate that GDH3 is likely to play an important role during senescence and nutrient remobilization.

Deamination role of inducible glutamate dehydrogenase isoenzyme 7 in Brassica napus leaf protoplasts

Glutamate dehydrogenase (GDH) is a ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate to glutamate. In Brassica napus, GDH isoenzymes 1 and 7 are hexamers of β and α subunits, respectively and the isoenzyme profile in leaves is known to change on wounding. Here, parallels were sought between the effects of wounding and protoplast isolation because of the possible relevance of changes in GDH activity to the perturbed metabolism in recalcitrant B. napus protoplasts. When leaf protoplasts of B. napus were isolated, GDH7 isoforms predominated. Transcription of GDH2, which encodes the GDH α subunit, was activated and translation of the GDH2 mRNA was also activated to synthesize α subunit polypeptides. When detached leaves absorbed either acidic 5 mM jasmonic acid or salicylic acid solutions via petioles, GDH7 isoenzymes were activated and the GDH isoenzyme patterns were similar to those of protoplasts. Salicylic acid β-glycosides were generated soon after treatment with the pectinase–cellulase enzyme solution and peaked at 1 h. NMR spectroscopic analysis of protoplasts and unstressed leaves incubated with 5 mM 15NH 4Cl showed that the change in GDH isoenzyme profile had no effect on ammonium assimilation. Protoplast isolation changed the redox state with NAD(P)H and oxidized glutathione levels increasing, and ascorbate, dehydroascorbate, NAD(P) and glutathione decreasing. ATP content in protoplasts declined to 2.6% of that in leaves, while that in wounded leaves increased by twofold. It is concluded that GDH7 does not support net amination in vivo and it is suggested that the increase in GDH7 activity is a response to oxidative stress during protoplast isolation.

Organ-specific expression of glutamate dehydrogenase (GDH) subunits in yellow lupine

Glutamate dehydrogenase (GDH, EC 1.4.2-4) is present in yellow lupine ( Lupinus luteus cv. Juno) in many isoforms. The number and banding pattern of isoenzymes varies with respect to plant organ and developmental stage. To better understand the complex nature of GDH regulation in plants, the levels of GDH transcripts, enzyme activity and isoenzyme patterns in germinating seeds and roots of yellow lupine were examined. The analysis of GDH cDNA sequences in lupine revealed three mRNA types, of which two encoded the β-GDH subunit and one encoded the α-GDH subunit (corresponding to the GDH1( GDH3) and GDH2 genes, respectively). The relative expression of GDH1 and GDH2 genes was analyzed in various lupine organs by using quantitative real-time PCR. Our results indicate that different mRNA types were differently regulated depending on organ type. Although both genes appeared to be ubiquitously expressed in all lupine tissues, the GDH1 transcripts evidently predominated over those of GDH2. Immunochemical analyses confirmed that, during embryo development, varied expression of two GDH subunits takes place. The α-GDH subunit (43 kDa) predominated in the early stages of germinating seeds, while the β-GDH subunit (44 kDa) was the only GDH polypeptide present in lupine roots. These results firmly support the hypothesis that isoenzyme variability of GDH in yellow lupine is associated with the varied expression of α and β subunits into the complexes of hexameric GDH forms. The presence of several isogenes of GDH in yellow lupine may explain the high number (over 20) of its molecular forms in germinating lupine.

Subcellular localization of Saccharomyces cerevisiae glutamate dehydrogenase isoenzymes

Reductive amination of 2‐oxoglutarate by glutamate dehydrogenase (GDH) is a primary pathway for glutamate synthesis in Saccharomyces cerevisiae. There are three GDH isoenzymes: NADP‐dependent Gdh1p and Gdh3p and NAD‐dependent Gdh2p. Knowing the subcellular localization of the GDH enzymes is vital to understanding the role of compartmentation in glutamate homeostasis. Previous studies suggested cytosolic, nuclear, and/or mitochondrial localization, but the precise location of the enzymes remains unclear. Wild‐type and mutant (gdh3Δ) cells were grown on lactate to induce mitochondrial biogenesis, spheroplasted, and homogenized. Then, crude mitochondrial and extramitochondrial fractions were isolated by sedimentation. Analysis of cytochrome c oxidase as a mitochondrial marker and hexokinase as a cytosolic marker revealed limited cross‐fractional contamination. Measurement of NADP‐dependent and NAD‐dependent GDH activity conclusively demonstrated extramitochondrial localization of Gdh1p and Gdh2p enzyme activity, respectively. Comparison of data from wild‐type as compared to gdh3Δ cells implies extramitochondrial localization for Gdh3p. These data indicate that in S. cerevisiae 2‐oxoglutarate generated within the mitochondria must be transported out for conversion to glutamate by GDHs. (Supported by NIH grant GM069372 [P. J. T.] and a gift from the Grilly family [M.C.P.]).

Stress-induced changes in glutamate dehydrogenase activity imply its role in adaptation to C and N metabolism in lupine embryos

The modifying effect of sucrose on glutamate dehydrogenase (GDH) activity and isoenzyme pattern was investigated in isolated embryos of lupine (Lupinus luteus L.), cultured in vitro in a medium with sucrose (+S) or without sucrose (-S) and exposed to cadmium (Cd) and lead (Pb) stress. Sucrose starvation of lupine embryos led to a rapid increase in the specific activity of GDH, immunoreactive beta-polypeptide and it was accompanied by appearance of new cathodal isoforms of enzyme. This suggests that isoenzymes induced in lupine embryos by sucrose starvation combine into GDH hexamers with the predominance of beta-GDH subunits synthetized under GDH1 gene control. The addition of sucrose to the medium caused an opposite effect. Along with upregulation of catabolic activity of GDH by sucrose starvation, activity of proteolytic enzymes was also induced. These data can point to regulatory mechanism implying a sucrose dependent repression of the GDH1 gene according to the mechanism of catabolic repression. Treatment of embryos with Cd(2+) or Pb(2+) resulted in ammonium accumulation in the tissues, accompanied by an increase in anabolic activity of GDH and activity of anodal isoenzymes, in both (+S) and (-S) embryos without new de novo synthesis of alpha subunit proteins. Thus, GDH isoenzyme profiles may reflect the physiological function of GDH, which appears to be an important link of metabolic adaptation in cells, aimed at using carbon sources other than sugar during carbohydrate starvation (catabolic activity of GDH) and protecting plant tissues against ammonium accumulated because of heavy metal stress (anabolic activity of GDH).

The human GLUD2 glutamate dehydrogenase: Localization and functional aspects

In all mammals, glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is encoded by a single gene ( GLUD1 in humans) which is expressed widely (housekeeping). Humans and other primates also possess a second gene, GLUD2, which encodes a highly homologous GDH isoenzyme (hGDH2) expressed predominantly in retina, brain and testis. There is evidence that GLUD1 was retro-posed <23 million years ago to the X chromosome, where it gave rise to GLUD2 through random mutations and natural selection. These mutations provided the novel enzyme with unique properties thought to facilitate its function in the particular milieu of the nervous system. hGDH2, having been dissociated from GTP control (through the Gly456Ala change), is mainly regulated by rising levels of ADP/ l-leucine. To achieve full-range regulation by these activators, hGDH2 needs to set its basal activity at low levels (<10% of full capacity), a property largely conferred by the evolutionary Arg443Ser change. Studies of structure/function relationships have identified residues in the regulatory domain of hGDH2 that modify basal catalytic activity and regulation. In addition, enzyme concentration and buffer ionic strength can influence basal enzyme activity. While mature hGDH1 and hGDH2 isoproteins are highly homologous, their predicted leader peptide sequences show a greater degree of divergence. Study of the subcellular sites targeted by hGDH2 in three different cultured cell lines using a GLUD2/EGFP construct revealed that hGDH2 localizes mainly to mitochondria and to a lesser extent to the endoplasmic reticulum of these cells. The implications of these findings for the potential role of this enzyme in the biology of the nervous system in health and disease are discussed.

Systemic Neurochemical Alterations in Schizophrenic Brain: Glutamate Metabolism in Focus

We have used a systemic approach to establish a relationship between enzyme measures of glial glutamate and energy metabolism (glutamine synthetase and glutamine synthetase-like protein, glutamate dehydrogenase isoenzymes, brain isoform creatine phosphokinase) and two major glial proteins (glial fibrillary acidic protein and myelin basic protein) in autopsied brain samples taken from patients with schizophrenia (SCH) and mentally healthy subjects (23 and 22 cases, respectively). These biochemical parameters were measured in tissue extracts in three brain areas (prefrontal cortex, caudate nucleus, and cerebellum). Significant differences in the level of at least one of the glutamate metabolizing enzymes were observed between two studied groups in all studied brain areas. Different patterns of correlative links between the biochemical parameters were found in healthy and schizophrenic brains. These findings give a new perspective to our understanding of the impaired regulation of enzyme levels in the brain in SCH.

Redox and translational regulation of glutamate dehydrogenase α subunits in Brassica napus under wounding stress

Glutamate dehydrogenase (GDH) is a ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate to glutamine. In Brassica napus, GDH isoenzymes 1 and 7 are hexamers of β and α subunits, respectively. When leaves of B. napus were sliced into narrow strips, seven GDH isoforms appeared with increases in both amination and deamination activity. Isoform profiles ceased changing from 1.5 to 2.5 h after wounding. Treatment with jasmonic acid (JA), salicylic acid (SA), ethylene, and oligosaccharides (OGs) changed the profiles. Ammonium ions at the same concentration as the signal substances (5 mM) caused only slight induction of the GDH7 isoenzyme. Ethylene was not released until 2 h after wounding, whereas JA and OGs were generated soon after wounding and peaked at 30 and 60 min after wounding, respectively. Accordingly, the wounding of leaves first releases OGs. Transcription of GDH2 , which encodes the GDH α subunit, was activated by wounding, whereas translation of the GDH2 mRNA was repressed. The GDH7 isoform was activated by the reduction of disulfide bonds in the GDHα subunits, but wounding did not cause synthesis of new α subunits. In leaves, wounding changed the redox state; it decreased NAD(P)H, ascorbic acid, and glutathione levels and increased dehydroascorbic acid. These results indicate that the GDH2 gene is regulated both at the transcriptional and translational levels and that the α subunit proteins are regulated posttranslationally via redox mechanisms. Thus, GDH isoenzyme profiles may reflect the redox state of leaves under oxidative stress.

Effect of olanzapine treatment on platelet glutamine synthetase-like protein and glutamate dehydrogenase immunoreactivity in schizophrenia

According to contemporary views, the glutamatergic system is implicated in the pathogenesis of schizophrenia, and atypical neuroleptics exert their effects (at least partially) through the glutamatergic system. Immunoreactive glutamate-metabolising enzymes, such as glutamine synthetase-like protein (GSLP) and two glutamate dehydrogenase isoenzymes (GDH), have been discovered in human platelets. The amount of GSLP in the platelets of 40 chronic patients with schizophrenia was found to be significantly higher than in 33 controls (consistent with our previous finding of increased amounts of GSLP in the prefrontal cortex of chronic schizophrenia patients). Moreover, survival analysis of the group of patients treated with olanzapine for 28 weeks showed that the larger amount of GSLP measured in platelets before treatment, the shorter the treatment time needed to achieve a positive clinical response (defined a priori as ≥20% reduction in PANSS total score from the initial level before the treatment). Hence, GSLP level may serve as a predictor of the treatment duration to achieve a positive outcome with olanzapine. Both GSLP and GDH were found significantly changed in the course of treatment; hence, treatment with olanzapine influences the amounts of glutamate-metabolising enzymes in the platelets of chronic schizophrenia patients.

Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae

The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn 54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.

The modifying effect of sucrose on glutamate dehydrogenase (GDH) activity in lupine embryos treated with inhibitors of RNA and protein synthesis

The modifying effect of sucrose on GDH activity and isoenzyme pattern in isolated embryos of lupine subjected to treatments with inhibitors of RNA synthesis (transcription inhibitors: actinomycin D and cordycepin) and protein synthesis (cycloheximide and chloramphenicol) was investigated. Sucrose starvation of embryos caused the increase of total activity of GDH(NADH-GDH and NAD-GDH) more than twice. Supply of sucrose to sucrose starved embryos caused a reduction of enzyme activity by 40 %. Electrophoretic analysis showed the presence of ca 17 isoenzymes of glutamate dehydrogenase in embryos grown for 72 h in medium with sucrose, while sucrose starvation increased the number of isoenzymes up to 22 forms. Addition of sucrose to sucrose starved embryos after 24 h of cultivation caused the inhibition of synthesis of new isoenzymes. This down-regulation by sucrose was blocked when sucrose was added together with cycloheximide, CHX, (0.025 mM). Treatment of sucrose fed and sucrose starved embryos with cycloheximide (0.020 mM) inhibited protein synthesis by 58 % and 24 %, respectively. The addition of cycloheximide (0.025 mM) to sucrose starved embryos decreased by 60 % NADH-GDH and by 30 % NAD-GDH activity and reduced the spectrum of isoenzymes. CHX treatment did not lead to a significant reduction of enzyme activity and isoenzyme pattern in sucrose fed embryos. The chloramphenicol (CMP) treatment (1 mM) stimulated the total GDH activity, 2.5 fold and 1.5 fold, in sucrose fed and sucrose starved embryos, respectively. Addition of CMP (10 mM) to the media did not affect GDH activity. Both concentrations of CMP caused no significant changes in the isoenzymatic pattern of enzyme in sucrose starved embryos, but induction of new isoenzymes was observed in sucrose fed embryos treated with CMP. In the case of using RNA synthesis inhibitors only cordycepin inhibited the total GDH activity (by ca 25 %) but neither actinomycin D or cordycepin caused any changes in isoenzyme pattern of GDH. The possible mechanism of sugar-mediated regulation of GDH activity is discussed.

Nucleotide-Dependent Isomerization of Glutamate Dehydrogenase in Relation to Total RNA Contents of Peanut

The physiological function of glutamate dehydrogenase (GDH) was investigated by treating germinating peanut (Arachis hypogaea L.) seeds with nucleoside triphosphate (NTP) solutions in order to alter the isoenzyme distribution patterns. The free nucleosides and nucleotides of the GTP-treated peanut were the highest [8.7 μmol g−1(f.m.)], and they decreased through the ATP-treated peanut [5.8 μmol g−1(f.m.)], and CTP-treated peanut [5.5 μmol g−1(f.m.)], to the UTP-treated peanut [4.1 μmol g−1(f.m.)]. The combination of 4 NTPs induced 20 % higher content of Pi [173 nmol g−1(f.m.)] than in the control, but the combined ATP+UTP treatment induced the lowest (93.0 nmol g−1(f.m.)] Pi. The 4 NTP treatment also induced the highest number of GDH isoenzymes (28) followed by the purine NTP treatments (15 to 20), but the pyrimidine NTP treatments and the combined purine + pyrimidine NTP treatments induced the lowest numbers ( ATP > CTP > GTP.

Biomass Enhancement in Maize and Soybean in Response to Glutamate Dehydrogenase Isomerization

The relationship between nutrient composition, crop biomass, and glutamate dehydrogenase (GDH) isoenzyme pattern was investigated in soybean (Glycine max) and maize (Zea mays) by monitoring the nutrient induced isomerization of the enzyme from the seedling stage to the mature crop. GDH was extracted from the leaves of the plants, and the isoenzymes were fractionated by isoelectric focusing followed by native polyacrylamide gel electrophoresis. The isomerization Vmax values for soybean GDH, similar to maize GDH increased curvilinearly from 200 - 400 &mu;mol mg-1 min-1 as the inorganic phosphate nutrient applied to the soil decreased from 50 - 0 mM. In soybean, combinations of N and K, P, or S nutrients induced the acidic and neutral isoenzymes, and gave biomass increases 25 - 50 % higher than the control plant. GDH isoenzymes were suppressed in soybean that received nutrients without N, K, or P and accordingly the biomass was about 30 % lower than the control. Treatment of maize with NPK nutrients increased the GDH Vmax values from 138.9 at the vegetative to 256.4 &mu;mol mg-1 min-1 at the reproductive phase, and suppressed the basic isoenzymes, but induced both the acidic and neutral isoenzymes thereby inducing seed production (27.0 &plusmn; 1.4 g per plant); whereas both the acidic and basic isoenzymes were suppressed in the control maize, and seeds did not develop. Simultaneous induction of the acidic, neutral, and basic isoenzymes of GDH indicated the occurrence of senescence. Therefore in maize and soybean, the induction of the acidic and basic isoenzymes of GDH led to the enhancement of biomass.

Glutamine synthetase and glutamate dehydrogenase in the prefrontal cortex of patients with schizophrenia

Basing primarily on the facts of altered levels of glutamate neurotransmitter, its receptors and transporters in schizophrenic brain, the “glutamatergic hypothesis” of schizophrenia has been broadened into the field of brain glutamate metabolism. Significantly changed levels of glutamine synthetase (GS) and glutamate dehydrogenase (GDH), the key enzymes involved in glutamine–glutamate cycling between neurons and glia, have been found in the prefrontal cortex (area 10) of patients with schizophrenia compared to controls ( P<.01). The data were obtained by enzymatic activity determinations as well as immunoreactivity level evaluations for GS, glutamine synthetase-like protein (GSLP), and three GDH isoenzymes in brain extracts by immunoblotting using specific polyclonal and monoclonal antibodies. Inverse changes in amounts of proteins of GS and GSLP, as well as elevation in amounts of GDH isoenzymes have been observed in schizophrenia. The presented results provide evidence for the impairment of glutamate metabolism and, in turn, abnormalities in functioning of the glutamate–glutamine cycle in the frontal cortex of patients with schizophrenia.

Purification of Glutamate Dehydrogenase Isoenzymes and Characterization of Their Substrate Specificities

Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, α, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06–0.8 mM of each of UTP, ATP, GTP, and CTP, 0–100.0 mM NH4Cl, 0–50.0 mM α-ketoglutaratr (α-KG), 0–0.2 mM NADH, 0–10.0 mM CaCl2 5 units of DNase 1, antibiotics, and ∼5 µg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16°C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and α-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for the isoenzymes in RNA synthesis reaction. The product RNA was up to 2 µg µg−1 GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [α-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.