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PubMed1998

Identification of an NAD+ binding site of brain glutamate dehydrogenase isoproteins by photoaffinity labeling.

Photoaffinity labeling with [32P]nicotinamide 2-azidoadenosine dinucleotide (2N3NAD+) was used to identify the NAD+ binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. In the absence of photolysis, 2N3NAD+ is a substrate for the GDH isoproteins. When the enzymes were covalently modified by photolysis in the presence of saturating amounts of photoprobe, about 50% inhibition of the GDH activities was observed. Photoinsertion of probe was increased by GTP or glutarate and decreased by NAD+ or ADP. With the combination of immobilized boronate affinity chromatography and reversed-phase HPLC, photolabel-containing peptides generated with trypsin were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as the region containing the sequence, CIAVGXSDGSIWNPDGIDPK for both GDH isoproteins, corresponding to Cys270 through Lys289 of the amino acid sequence of well known bovine liver GDH. The X indicates a position for which no phenylthiohydantoin-derivative could be assigned. The missing residue, however, can be designated as a photolabeled glutamate since the sequences including the glutamate residue in question have a complete identity with those of the other GDH species known. Photolabeling of these peptides was prevented by the presence of NAD+ during photolysis. These results demonstrate selectivity of the photoprobe for the NAD+ binding site and suggest that the peptide identified using the photoprobe is located in the NAD+ binding domain of the brain GDH isoproteins. Both amino acid sequencing and compositional analysis identified Glu275 as the site of photoinsertion.

Amino Acid Sequence↗
PubMed2002

Glutamine-, glutamine synthetase-, glutamate dehydrogenase- and pyruvate carboxylase-immunoreactivities in the rat dorsal root ganglion and peripheral nerve.

Supporting glial cells of the peripheral nervous system include satellite cells of dorsal root ganglia and Schwann cells of peripheral nerves. In the central nervous system, glial cells contain enzymes related to the tricarboxylic acid and glutamine cycles: pyruvate carboxylase, glutamate dehydrogenase, and glutamine synthetase. The present study used immunohistochemistry in the rat peripheral nervous system to determine the cellular distribution of these enzymes along with glutamine. In dorsal root ganglia and peripheral nerves, glutamine and glutamine related enzymes were enriched in satellite and Schwann cells. In the dorsal root ganglia, immunoreactive satellite cells surrounded neurons of all sizes. In peripheral nerve, immunoreactive Schwann cells were most easily observed surrounding large diameter, myelinated axons. These Schwann cells contained immunoreactivity in their cell bodies, nodes of Ranvier, and the rim of cytoplasm outside the myelin sheath. Myelin sheaths were non-immunoreactive. The peripheral glial tricarboxylic and glutamine cycles may be used to produce glutamine for neuronal cell uptake and conversion to glutamate for synaptic transmission. Alternatively, these cycles may function in peripheral glia similar to central nervous system astrocytes for supporting the energy demands of neurons.

Animals↗
PubMed1990

Kinetics of NH(4) Assimilation in Zea mays: Preliminary Studies with a Glutamate Dehydrogenase (GDH1) Null Mutant.

In higher plants it is now generally considered that glutamate dehydrogenase (GDH) plays only a small or negligible role in ammonia assimilation. To test this specific point, comparative studies of (15)NH(4) (+) assimilation were undertaken with a GDH1-null mutant of Zea mays and a related (but not strictly isogenic) GDH1-positive wild type from which this mutant was derived. The kinetics of (15)NH(4) (+) assimilation into free amino acids and total reduced nitrogen were monitored in both roots and shoots of 2-week-old seedlings supplied with 5 millimolar 99% ((15)NH(4))(2)SO(4) via the aerated root medium in hydroponic culture over a 24-h period. The GDH1-null mutant, with a 10- to 15-fold lower total root GDH activity in comparison to the wild type, was found to exhibit a 40 to 50% lower rate of (15)NH(4) (+) assimilation into total reduced nitrogen. Observed rates of root ammonium assimilation were 5.9 and 3.1 micromoles per hour per gram fresh weight for the wild type and mutant, respectively. The lower rate of (15)NH(4) (+) assimilation in the mutant was associated with lower rates of labeling of several free amino acids (including glutamate, glutamine-amino N, aspartate, asparagine-amino N, and alanine) in both roots and shoots of the mutant in comparison to the wild type. Qualitatively, these labeling kinetics appear consistent with a reduced flux of (15)N via glutamate in the GDH1-null mutant. However, the responses of the two genotypes to the potent inhibitor of glutamine synthetase, methionine sulfoximine, and differences in morphology of the two genotypes (particularly a lower shoot:root ratio in the GDH1-null mutant) urge caution in concluding that GDH1 is solely responsible for these differences in ammonia assimilation rate.

Journal Article↗
PubMed1997

Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press

Journal Article↗
PubMed1984

Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am132 host strain which contains a deletion encompassing the cloned fragment. Every one of 33 transformants tested showed extreme meiotic instability: less than 1 or 2% am+ progeny were obtained in initial or successive backcrosses between am+ transformants and am132 or in intercrosses between am+ progeny. Furthermore, am+ progeny from backcrosses gave a high proportion of auxotrophic (am) mitotic segregants during vegetative growth. These results indicate that the am+ character is not stably integrated into chromosomal DNA in any of the transformants tested. Nuclear DNAs from six transformants were analyzed by Southern hybridization. All six transformants contained sequences homologous to pJR2. Four showed restriction fragments expected for unmodified pJR2, but most showed additional bands. Southern blots of undigested DNAs showed that the plasmid sequences are present predominantly in high-molecular-weight form (larger than 20 kilobases). Southern blots showed that auxotrophic (am) progeny from a backcross to am132 had lost restriction bands corresponding to free plasmid but retained additional bands, apparently integrated into chromosomal DNA in a nonfunctional manner. Considered together, these results are most reasonably interpreted as follows: recombinant plasmids containing the am+ gene can replicate autonomously in N. crassa, the free plasmids are present in oligomeric or modified form or both, and plasmid sequences also integrate at multiple sites in the deletion host but in a nonfunctional manner. An alternate interpretation--that tandem repeats of the plasmid are integrated into chromosomal DNA but eliminated during meiosis--cannot be completely excluded. However, stable integration of the am gene can be obtained under a variety of other conditions, viz., using the am gene cloned in a phage lambda vector (J. A. Kinsey and J. A. Rambosek, Mol. Cell. Biol. 4:117-122, 1984), using derivatives of pJR2, or using pJR2 to transform a frameshift mutant.

DNA Replication↗
PubMed1977

Lack of a regulatory function for glutamine synthetase protein in the synthesis of glutamate dehydrogenase and nitrite reductase in Escherichia coli K12.

Synthesis of glutamine synthetase (GS) in anaerobic batch cultures of Escherichia coli was repressed when excess NH4+ was available, but derepressed during growth with a poor nitrogen source. In wild-type bacteria there was only a weak inverse correlation between the activities of GS and glutamate dehydrogenase (GDH) during growth in various media. No positive correlations were found between the activities of GS and nitrite reductase, or between GS and cytochrome c552: both of these proteins were synthesized normally by mutants that contained no active GS. Although activities of GS and GDH were low in two mutants that are unable to synthesize cytochrome c552 or reduce nitrite because of defects in the nirA gene, the nirA defect was separated from the GS and GDH defects by transduction with bacteriophage P1. Attempts to show that catabolite repression of proline oxidase synthesis could be relieved during NH4+ starvation also failed. It is, therefore, unlikely that nitrite reduction or proline oxidation by E. coli are under positive control by GS protein. The regulation of the synthesis of enzymes for the utilization of secondary nitrogen sources in E. coli, therefore, different from that in Klebsiella aerogenes, but is similar to that in Salmonella typhimurium.

Culture Media↗
PubMed2000

Electrochemical studies of the effect of lanthanide ions on the activity of glutamate dehydrogenase.

Using the electrochemical method based on the "diffusion" layer concept of the convective system, the effect of lanthanide ions La3+ and Eu3+ on the activity of glutamate dehydrogenase (GLDH) has been determined. In suitable concentrations, the lanthanide ions La3+ and Eu3+ can activate the GLDH in the reductive amination of alpha-Ketoglutarate (alpha-KG). The activation mechanism would be that the lanthanide ions could interrupt the binding of NAD+ to GLDH by combining preferentially to NAD+. This mechanism was proposed by the voltammetric studies.

Electrochemistry↗
PubMed1979

The effects of methanol on the glutamate dehydrogenase reaction at 0 degrees C.

The effects of 0-30% methanol (vol/vol) on the Km an Vm values for both the forward and reverse directions of the L-glutamate dehydrogenase reaction were determined at 0 degrees C. The decrease in temperature alone had very little effect on these parameters. However, in the forward reaction, 30% methanol resulted in a 14-fold decrease in the Km value for glutamate, a slight decrease in the Km value for NADP, and a thirty-fold decrease in Vm. Substrate inhibition by glutamate was observed at concentrations greater than 4 mM. In the reverse reaction, 30% methanol caused a decrease in the Km values for alpha-ketoglutarate and ammonia and a 10-fold decrease in Vm. Substrate inhibition by both alpha-ketoglutarate and NADPH was observed at concentrations of either substrate above 0.03 mM. The dependence of Km for glutamate and Vm values for the forward reaction on methanol concentration suggests that they are similarly affected by methanol, in direct contrast to results obtained for NADP. Methanol appeared to cause a general tightening of complexes, which may arise from an effect on the "activities" of species in solution. The use of methanol not only allows for the study of reaction intermediates by slowing the reaction with the cryogenic method, but may also serve as a mechanistic probe by affecting several polarity as well as Km, Vm, and K1 values.

Animals↗
PubMed1994

Glutamate dehydrogenase isoenzymes in the bovine retina and human leukocyte.

In the present study, we investigated the enzymatical properties of the heat-labile and heat-stable glutamate dehydrogenases (GDHs) from the bovine retina and human leukocytes. Retinal GDHs were mitochondrial enzymes with an optimal pH of 7.5. Heat-labile (membrane-associated form) and heat-stable (soluble form) GDHs were found in the bovine retina as well as in the human leukocytes. At least 5 GDH isoenzymes were found in the retina and leukocytes by nondenaturing polyacrylamide gel electrophoresis. After heat treatment, the high-speed pellet obtained from centrifugation of the human leukocytes at 100,000 x g retained the 3 smaller isoenzymes but had lost the 2 larger isoenzymes. Both heat-labile and heat-stable GDH were found in the neuroretina, retinal pigment epithelium and optic nerve with high activities. Our results suggest that these tissues may be the main active sites of the GDH deficiency.

Animals↗
PubMed1997

Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: molecular characterization and phylogenetic implications.

The hyperthermophilic bacterium Thermotoga maritima, which grows at up to 90 degrees C, contains an L-glutamate dehydrogenase (GDH). Activity of this enzyme could be detected in T. maritima crude extracts, and appeared to be associated with a 47-kDa protein which cross-reacted with antibodies against purified GDH from the hyperthermophilic archaeon Pyrococcus woesei. The single-copy T. maritima gdh gene was cloned by complementation in a glutamate auxotrophic Escherichia coli strain. The nucleotide sequence of the gdh gene predicts a 416-residue protein with a calculated molecular weight of 45,852. The gdh gene was inserted in an expression vector and expressed in E. coli as an active enzyme. The T. maritima GDH was purified to homogeneity. The NH2-terminal sequence of the purified enzyme was PEKSLYEMAVEQ, which is identical to positions 2-13 of the peptide sequence derived from the gdh gene. The purified native enzyme has a size of 265 kDa and a subunit size of 47kDa, indicating that GDH is a homohexamer. Maximum activity of the enzyme was measured at 75 degrees C and the pH optima are 8.3 and 8.8 for the anabolic and catabolic reaction, respectively. The enzyme was found to be very stable at 80 degrees C, but appeared to lose activity quickly at higher temperatures. The T. maritima GDH shows the highest rate of activity with NADH (Vmax of 172 U/mg protein), but also utilizes NADPH (Vmax of 12 U/mg protein). Sequence comparisons showed that the T. maritima GDH is a member of the family II of hexameric GDHs which includes all the GDHs isolated so far from hyperthermophiles. Remarkably, phylogenetic analysis positions all these hyperthermophilic GDHs in the middle of the GDH family II tree, with the bacterial T. maritima GDH located between that of halophilic and thermophilic euryarchaeota.

Amino Acid Sequence↗
PubMed2005

Central nervous system hyperexcitability associated with glutamate dehydrogenase gain of function mutations.

OBJECTIVE: To describe seizure phenotypes associated with the hyperinsulinism/hyperammonemia syndrome (HI/HA), which is caused by gain of function mutations in the enzyme glutamate dehydrogenase (GDH). STUDY DESIGN: A retrospective review of records of 14 patients with HI/HA. RESULTS: Nine patients had seizures as the first symptom of HI/HA, and six had seizures in the absence of hypoglycemia. No electroencephalogram (EEG) background abnormalities were identified. In four patients, EEG recordings during seizures in the setting of normal blood glucose contained generalized epileptiform discharges. EEGs of three of these patients showed 0.5- to 2-second generalized irregular spike-and-wave discharge at 3 to 6 Hz corresponding to eye blinks, eye rolling, or staring. The EEG of the fourth patient consisted of 20 seconds of generalized regular spike-and-wave discharge at 3 Hz in the clinical context of staring and unresponsiveness. In two patients, seizure control worsened with carbamezapine or oxcarbezapine treatment. CONCLUSIONS: In patients with HI/HA, generalized seizures are common and can occur in the absence of hypoglycemia. The drugs carbamazepine and oxcarbazepine should be used with caution for treatment. Pathogenesis of epilepsy in these patients may be related to effects of GDH mutations in the brain, perhaps in combination with effects of recurrent hypoglycemia and chronic hyperammonemia.

Adolescent↗
PubMed2003

Effect of vigabatrin on glutamate dehydrogenase in the hippocampus of seizure prone gerbils.

Vigabatrin (VGB, gamma-vinyl-gamma-aminobutyric acid (GABA)), an irreversible inhibitor of GABA transaminase, increases regional inhibitory effects by elevating GABA concentration and reducing glutamate synthesis. In the present study, changes in glutamate dehydrogenase (GDH) activity and its immunoreactivity in the seizure prone gerbil hippocampus after treating VGB were investigated to identify the effect of VGB on energy and/or glutamate metabolism via GDH. In the VGB treated group, GDH immunoreactivity and its activity in the hippocampus were significantly decreased, as compared with those of controls. These findings suggest that VGB administration may suppress the development and spread of seizures not only by elevating the level of GABA, but also by affecting the glutamate signaling and energy metabolism in neurons.

Animals↗
PubMed1982

Effect of pH on the kinetics of bovine liver glutamate dehydrogenase self-association.

The temperature-jump technique with light-scattering detection was applied to the kinetic study on the self-association of bovine liver glutamate dehydrogenase (EC 1.4.1.3). The measurements were carried out for systems in phosphate buffer of differing pH, 6.3-7.8, and of differing phosphate concentration at 11.5 degrees C. The observed association rate constant decreased with increasing phosphate concentration at the respective pH values. This observation was interpreted in terms of the inhibition from self-association by specific binding of phosphate ion to the protein. According to this interpretation, the intrinsic association rate constant, k(0)a, and the binding constant of phosphate ion, Kp, were estimated. k(0)a varied with pH in a manner as described by a titration curve with pK of about 7, while Kp remained almost constant in the pH range studied.

Animals↗
PubMed1991

Identification of cysteine-319 as the target amino acid of 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate in bovine liver glutamate dehydrogenase.

The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) has been shown to react with bovine liver glutamate dehydrogenase in the region of the GTP-dependent NADH inhibitory site with incorporation of about 1 mol of reagent/mol of subunit [Ozturk, D. H., Safer, D., & Colman, R. F. (1990) Biochemistry 29, 7112-7118]. The modified enzyme was shown to contain only 5 free sulfhydryl groups upon 5,5'-dithiobis (2-nitrobenzoate) titration as compared with 6 in the unmodified enzyme. In the unmodified enzyme digested with trypsin, 6 cysteinyl peptides were detected by high-performance liquid chromatography upon treatment with iodo [3H]acetic acid. In contrast, only 5 (carboxymethyl)cysteinyl peptides were detected in 8-BDB-TA-5'-TP-modified enzyme. When carboxymethylated modified and unmodified enzymes were digested with thermolysin, 6 peptide sequences containing (carboxymethyl)cysteine were obtained in the unmodified enzyme, but only 5 were observed in the modified enzyme. The (carboxymethyl)cysteine which was absent in the modified enzyme was determined to be Cys-319, leading to the conclusion that 8-BDB-TA-5'-TP reacts with Cys-319, thereby preventing it from subsequent reaction with radioactive iodoacetate. It was previously reported that 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TA-5'-DP) modifies Cys-319 in this enzyme [Batra, S. P., & Colman, R. F. (1986) Biochemistry 25, 3508-3515].(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed2005

The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicotiana tabacum L. leaves.

To investigate the role of stress in nitrogen management in plants, the effect of pathogen attack, elicitors, and phytohormone application on the expression of the two senescence-related markers GS1 (cytosolic glutamine synthetase EC 6.3.1.2) and GDH (glutamate dehydrogenase, EC 1.4.1.2) involved in nitrogen mobilization in senescing leaves of tobacco (Nicotiana tabacum L.) plants, was studied. The expression of genes involved in primary nitrogen assimilation such as GS2 (chloroplastic glutamine synthetase) and Nia (nitrate reductase, EC 1.6.1.1) was also analysed. The Glubas gene, coding a beta-1,3-glucanase, was used as a plant-defence gene control. As during natural senescence, the expression of GS2 and Nia was repressed under almost all stress conditions. By contrast, GS1 and GDH mRNA accumulation was increased. However, GS1 and GDH showed differential patterns of expression depending on the stress applied. The expression of GS1 appeared more selective than GDH. Results indicate that the GDH and GS1 genes involved in leaf senescence are also a component of the plant defence response during plant-pathogen interaction. The links between natural plant senescence and stress-induced senescence are discussed, as well as the potential role of GS1 and GDH in a metabolic safeguard process.

Biomarkers↗
PubMed1978

Transient-state kinetics of L-glutamate dehydrogenase: mechanism of alpha-ketoglutarate inhibition in the burst phase.

Stopped-flow studies of the initial burst of NADPH production accompanying the oxidative deamination of L-glutamate by L-glutamate dehydrogenase and NADP+ were performed in the presence of alpha-ketoglutarate, a product of the reaction. Both binary enzyme-alpha-ketoglutarate and ternary enzyme--NADP+-alpha-ketoglutarate complexes are inhibitory in the burst presence of the enzyme-catalyzed reaction. Order-of-addition experiments show the binary complex to form rapidly, in the 3 ms dead time of the stopped-flow instrument. There is a distinct lag, however, in the achievement of the full ternary complex inhibitory effect unless the enzyme is preincubated with both NADP+ and alpha-ketoglutarate prior to initiation of the catalytic reaction with L-glutamate. The formation of an inhibitory enzyme--NADP+-alpha-ketoglutarate complex appears to be sufficiently slow to give a delayed kinetic response when alpha-ketoglutarate is added to the reaction system.

Animals↗