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PubMed1999

Crystallization and preliminary x-ray diffraction studies of hyperthermostable glutamate dehydrogenase from Thermococcus profundus.

Recombinant glutamate dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus, was crystallized in the presence of both polyethylene glycol 8000 and lithium sulfate. Four types of crystals having different morphologies appeared in the crystallization trials; however, only one type was suitable for X-ray crystal structure analysis. The crystal belonged to the monoclinic space group P2(1) and the unit-cell parameters were a = 112.99, b = 163.70, c = 133.07 A, beta = 113.46 degrees at 110 K. The calculated V(M) value of 3.42 A(3) Da(-1) was acceptable when one hexamer of the enzyme, which was the physiological functional unit, occupied a crystallographic asymmetric unit. X-ray diffraction intensity data were collected to a resolution of 2.25 A with good statistics at the BL44B2 beamline of SPring-8.

Crystallization↗

PubMed1986

Dirofilaria immitis: comparison of cytosolic and mitochondrial glutamate dehydrogenases.

Two membrane-bound glutamate dehydrogenases were found in adult Dirofilaria immitis, an NAD-linked enzyme (EC 1.4.1.2) in the cytosol (C-GDH) and an enzyme equally reactive with NAD or NADP (EC 1.4.1.3) in the mitochondria (M-GDH). The cytosolic enzyme had a pH optimum of 7.8-8.0 and exhibited 30% more activity at 25 C than at 37 C (pH 8.0). The mitochondrial enzyme had a pH optimum at 8.4 and exhibited 27% more activity at 37 C than at 25 C (pH 8.4); it was also more sensitive to heat denaturation. Gel filtration of worm subfractions separated four peaks of C-GDH activity with molecular weights of approximately 610, 285, 180, and less than 100 thousand, and a single major peak of M-GDH activity with a molecular weight of about 335,000. When assayed at pH 8, 37 C, and 200 microM NADH, the Km for the substrate, alpha-ketoglutarate, was equivalent for the two enzymes, but the Km for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 microM NADH, 1 mM ADP approximately doubled and 1 mM ATP halved the velocity observed for each enzyme with no effector present. Under these assay conditions AMP, IDP, GDP, and GTP had opposite effects on the reaction velocities for the two enzymes. When the assay conditions were changed, the effects of added purine nucleotides varied, even directionally. Addition of up to 5 mM glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate Km of M-GDH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

TAT-mediated delivery of human glutamate dehydrogenase into PC12 cells.

Human glutamate dehydrogenase (GDH) gene was fused with a gene fragment encoding the nine amino acid (RKKRRQRRR) protein transduction domain of human immunodeficiency virus TAT protein in bacterial expression vector to produce genetic in-frame TAT-GDH fusion protein. The TAT-GDH protein can enter PC12 cells efficiently when added exogenously in culture media as determined by Western blot analysis and enzyme activities. Once inside the cells, the transduced denatured TAT-GDH protein showed a full activity of GDH indicating that the TAT-GDH fusion protein was correctly refolded after delivery into cells and the activities of GDH in the TAT-GDH fusion protein was not affected by the addition of the TAT sequence. TAT-GDH fusion protein and TAT itself showed no cytotoxicity in PC12 cells. Although the exact mechanism of transduction across a membrane remains unclear, the transduction activity of TAT-GDH into PC12 cells may suggest new possibilities for direct delivery of GDH into the patients with the GDH-deficient disorders.

Amino Acid Sequence↗

PubMed2000

Molecular basis and characterization of the hyperinsulinism/hyperammonemia syndrome: predominance of mutations in exons 11 and 12 of the glutamate dehydrogenase gene. HI/HA Contributing Investigators.

Glutamate dehydrogenase (GDH) is allosterically activated by the amino acid leucine to mediate protein stimulation of insulin secretion. Children with the hyperinsulinism/hyperammonemia (HI/HA) syndrome have symptomatic hypoglycemia plus persistent elevations of plasma ammonium. We have reported that HI/HA may be caused by dominant mutations of GDH that lie in a unique allosteric domain that is encoded within GDH exons 11 and 12. To examine the frequency of mutations in this domain, we screened genomic DNA from 48 unrelated cases with the HI/HA syndrome for exon 11 and 12 mutations in GDH. Twenty-five (52%) had mutations in these exons; 74% of the mutations were sporadic. Clinical manifestations included normal birth weight, late onset of hypoglycemia, diazoxide responsiveness, and protein-sensitive hypoglycemia. Enzymatic studies of lymphoblast GDH in seven of the mutations showed that all had reduced sensitivity to inhibition with GTP, consistent with an increase in enzyme activity. Mutations had little or no effect on enzyme responses to positive allosteric effectors, such as ADP or leucine. Based on the three-dimensional structure of GDH, the mutations may function by impairing the binding of an inhibitory GTP to a domain responsible for the allosteric and cooperativity properties of GDH.

Adenosine Diphosphate↗

PubMed1997

Regulation of peanut glutamate dehydrogenase by methionine sulphoximine.

Peanut glutamate dehydrogenase (GDH) was electrophoretically purified to homogeneity. Rotofor IEF fractionated the peanut GDH to 7 isoelectric (charge) isomers, which focused in the pH 5-8 range. Western blot analysis of the charge isomers using anti-GDH serum showed that methionine sulphoximine (MSX) treatment suppressed the b-subunit (69 KDa), but enhanced the a-subunit (45 KDa), and alpha-subunit (46 KDa) of the enzyme. The MSX-mediated suppression of the b-subunit increased the NH4+ Km value of the acidic charge isomers from 7.7 mM in the control to 50 mM in the MSX-treated peanut, and also increased the 2-ketoglutarate Km value of the basic charge isomers from 0.4 mM in the control to 7.0 mM in the MSX treatment. Therefore, the control peanut could salvage NH4+ with its GDH activity. But by increasing the NH4+ Km value, the MSX rendered the enzyme ineffective in NH4+ salvage. In the deamination direction, despite the enhancement of the a-, and alpha-subunits by MSX, the basic charge isomers of GDH had very high Km value for L-glu (50 mM), similar to that (25 mM) of the control. Thus, the GDHs of the control, and MSX treatment could not function in the deamination reaction in vivo. These results show that the treatment of peanut with MSX impaired the amination function of GDH.

Arachis↗

PubMed1975

Binding studies of NADPH to NADP-specific L-glutamate dehydrogenase from Saccharomyces cerevisiae.

Optical characteristics of enzyme-reduced coenzyme complexes of yeast NADP-specific glutamate dehydrogenase have been investigated in the presence and absence of product (L-glutamate) and in the presence or absence of phosphate. The phosphate effect, pointed out in a previous work, is found again: inorganic phosphate (Pi) destabilizes the binary complex (E - NADPH), the dissociation constant of which is equal to 14 muM, a value much higher than that determined in Tris-HCl buffer: Kd = 0.9 muM. Concerning the role of phosphate some assumptions are drawn up with respect to a similar behaviour of Pi toward yeast glutamate dehydrogenase and ADP toward the beef liver enzyme. In the same way, L-glutamate induces a stabilization of the binary complex; this latter effect is unchanged in the presence of phosphate, yet it is less marked than in the case of beef liver glutamate dehydrogenase. Protein fluorescence, nucleotide fluorescence and circular dichroism measurements allowed the determination of three identical and independent NADPH binding sites per hexameric active unit. In analogy with beef liver enzyme, it seems that yeast glutamate dehydrogenase is a good model to study anticooperativity in ligand binding.

Binding Sites↗

PubMed1995

Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability.

The glutamate dehydrogenase gene from the hyperthermophilic archaeon Pyrococcus furiosus has been functionally expressed in Escherichia coli under the control of the lambda PL promoter. The P. furiosus glutamate dehydrogenase amounted to 20% of the total E. coli cell protein, and the vast majority consisted of hexamers. Following activation by heat treatment, an enzyme could be purified from E. coli that was indistinguishable from the glutamate dehydrogenase purified from P. furiosus. Hybrid genes, that consisted of the coding regions for the homologous glutamate dehydrogenases from P. furiosus and the mesophilic bacterium Clostridium difficile, were constructed and successfully expressed in E. coli. One of the resulting hybrid proteins, containing the glutamate binding domain of the C. difficile enzyme and the cofactor binding domain of the P. furiosus enzyme, did not show a detectable activity. In contrast, the complementary hybrid containing the P. furiosus glutamate and the C. difficile cofactor binding domain was a catalytically active hexamer that showed a reduced substrate affinity but maintained efficient cofactor binding with the specificity found in the Clostridium symbiosum enzyme. Compared with the C. difficile glutamate dehydrogenase, the archaeal-bacterial hybrid is slightly more thermoactive, less thermostable but much more stable towards guanidinium chloride-induced inactivation and denaturation.

Archaea↗

PubMed2004

Morphology-associated expression of NADP-dependent glutamate dehydrogenases during yeast-mycelium transition of a dimorphic fungus Benjaminiella poitrasii.

Benjaminiella poitrasii, a dimorphic zygomycetous fungus possesses three glutamate dehydrogenases, one requiring NAD while the other two use NADP as a coenzyme. In the activity staining after electrophoresis on native polyacrylamide gel NAD- dependent glutamate dehydrogenase revealed the presence of one enzyme that was expressed in both, yeast- and mycelium-form cells. While in case of NADP- dependent glutamate dehydrogenase two distinct activity bands that were differentially expressed in yeast- and mycelium-form cells were seen. Interestingly, during yeast-mycelium transition and reverse, quantitative changes in form-specific native NADP-dependent glutamate dehydrogenase activities were seen. The biochemical data on temperature and pH optima, thermostability, and kinetic properties confirmed the presence of two NADP-dependent proteins in B. poitrasii, parent strain. The monomorphic mutant (Y-5, yeast form) showed NADP- glutamate dehydrogenase similar to parent yeast-form enzyme. For the first time the significance of differential expression of these enzymes during morphological transition in B. poitrasii has been suggested.

Enzyme Stability↗

PubMed1984

A kinetic study of the oxidative deamination of L-glutamate by Peptostreptococcus asaccharolyticus glutamate dehydrogenase using a variety of coenzymes.

The NAD+-specific glutamate dehydrogenase from Peptostreptococcus asaccharolyticus follows Michaelis-Menten kinetics in contrast to the enzyme from several other sources, and thus gives linear double-reciprocal plots of initial-rate data. The initial-rate parameters have been determined for this bacterial dehyrogenase in the direction of oxidative deamination. The use of alternative coenzymes leads to some conclusions about the order of substrate addition. An investigation of the pH dependence of this reaction reveals that the binding of oxidised coenzyme is independent of pH over the range 6-9. The kinetic data are consistent with an ordered addition of coenzyme prior to glutamate, the reverse of the mechanism derived with ox glutamate dehydrogenase in the presence of ADP.

Coenzymes↗

PubMed2004

Developmental expression of glutamate transporters and glutamate dehydrogenase in astrocytes of the postnatal rat hippocampus.

Glutamate is the major excitatory transmitter in the CNS and plays distinct roles in a number of developmental events. Its extracellular concentration, which mediates these activities, is regulated by glutamate transporters in glial cells and neurons. In the present study, we have used nonradioactive in situ hybridization, immunocytochemistry, and immunoblotting to show the cellular and regional expression of the high-affinity glutamate transporters GLAST (EAAT1) and generic GLT1 (EAAT2; glial form of GLT1) in the rat hippocampus during postnatal development (P1-60). The results of transporter expression were compared with the localization and activity pattern of glutamate dehydrogenase (GDH), an important glutamate-metabolizing enzyme. The study showed that both transporters and GDH were demonstrable at P1 (day of birth). The expression of GLAST (detected by in situ hybridization and immunocytochemistry) in the early postnatal development was higher than GLT1. Thereafter, the expression of both transporters increased, showing adult levels at between P20 and P30 (detected by in situ hybridization and immunoblotting). At these time points, the expression of GLT1 appeared to be significantly higher than the GLAST expression. GLT1 and GLAST proteins were demonstrable only in astrocytes. The increase of GDH activities (steepest increase from P5-P8), which were localized preferentially in astrocytes, was in agreement with the increase of transporter expression, preferentially with that of GLT1. These observations suggest that the extent of glutamate transporter expression and of glutamate-metabolizing GDH activity in astrocytes is intimately correlated with the formation of glutamatergic synapses in the developing hippocampus.

Aging↗

PubMed1984

Glutamic dehydrogenase from rat heart mitochondria. I. Purification and physical properties including molecular weight determination.

Glutamic dehydrogenase extracted with tris buffer from fresh freeze-thawed rat heart mitochondria was purified by ammonium sulphate fractionation, affinity chromatography on GTP agarose, hydroxyapatite chromatography and concentration using a molecular sieve. The final specific activity is 80 units/mg protein. Thin gel SDS electrophoresis of the purified enzyme preparation after reduction with dithiothreitol shows a major band with a molecular weight of 38 000 Daltons. Two minor bands are also present. Sucrose density gradient centrifugation reveals a molecular weight of 230 000 Daltons for unreduced mitochondrial GDH activity. By gel filtration rat heart mitochondrial glutamic dehydrogenase has a major peak at 230 000 Daltons, a minor peak at 300 000 Daltons and some larger molecular weight species. Rat liver mitochondrial glutamic dehydrogenase has a minor peak at 230 000, a major peak at 300 000 and some larger molecular weight species. The rat liver mitochondrial glutamic dehydrogenase predominance at 300 000 is unchanged by incubation, extraction and purification with rat heart mitochondria. The purified GDH is stable frozen at -10 degrees C in tris-HCl buffer with EDTA. It loses activity at 4 degrees C especially when stored in 0.2 M phosphate buffer. It also loses activity when dialyzed for 24 h. This loss of activity is not completely prevented by adding nucleotides to the buffer (AMP or ADP) but is decreased by their presence.

Animals↗

PubMed1983

[Mechanism of the glutamate dehydrogenase reaction].

The data concerning the chemical and kinetic mechanisms of the glutamate dehydrogenase reaction have been reviewed. Based on the differences between two catalytically active glutamate dehydrogenase conformations induced by the substrates as well as on some other evidence, it has been proposed that the amino groups of lysine residues 27 and 126 in the beef liver enzyme are interchangeable depending on the direction of the glutamate dehydrogenase reaction.

Animals↗

PubMed1968

Evidence for two species of glutamate dehydrogenases in Thiobacillus novellus.

When grown autotrophically in a thiosulfate-mineral salts medium, cells of the facultative chemoautotrophic bacterium, Thiobacillus novellus, produced two distinct glutamate dehydrogenases, one specific for nicotinamide adenine dinucleotide phosphate (NADP) and the other specific for nicotinamide adenine dinucleotide (NAD). When glutamate was supplied exogenously as the sole carbon source, the NAD-specific glutamate dehydrogenase was fully induced. Lower levels of the enzyme were found in bacteria grown in l-arginine, l-alanine, glucose, glycerol, lactate, citrate, or succinate. Arginine, histidine, and aspartate, on the other hand, caused a marked repression of the NADP-specific glutamate dehydrogenase activity. The NAD-dependent glutamate dehydrogenase was allosteric. Adenosine-5'-monophosphate and adenosine-5'-diphosphate acted as positive effectors. Both glutamate dehydrogenases were purified about 250-fold and were shown to be distinct protein with different physical properties.

Adenine Nucleotides↗

PubMed1997

Chloroquine is a potent inhibitor of glutamate dehydrogenase in liver and kidney-cortex of rabbit.

The effect of chloroquine and other antimalarial drugs on glutamate dehydrogenase activity was studied in liver and renal mitochondria as well as in kidney-cortex tubules of rabbit. In permeabilized mitochondria, with free access of substrates and drugs to glutamate dehydrogenase, 100 microns chloroquine decreased both glutamate synthesis and glutamate deamination by about 70 and 50%, respectively. Ki value was equal to 49 microns in both liver and renal mitochondria. Other antimalarials (amodiaquine, quinacrine, chinidine and chinine) showed much smaller effect on the enzyme activity. Both ADP and L-leucine, allosteric activators of glutamate dehydrogenase, did not abolish the inhibitory action of chloroquine. Moreover, when added at 200 microns concentrations all drugs besides chinine suppressed glutamate formation in kidney-cortex tubules while chloroquine and quinacrine inhibited also glutamate deamination. Furthermore, chloroquine at 500 microns concentration decreased significantly [14C]glutamate transport into kidney-cortex mitochondria. In view of these observations it seems likely that chloroquine and some other antimalarials may inhibit the rate of glutamate metabolism in both liver and kidney-cortex causing hepatoxicity and nephrotoxicity. A possible action of chloroquine as an inhibitor of glutamate dehydrogenase in Plasmodium falciparum during the clinical treatment of malaria is discussed.

Animals↗

PubMed1988

Immunohistochemical localization of glutamate dehydrogenase in rat liver: plasticity of distribution during development and with hormone treatment.

In adult rat liver, glutamate dehydrogenase is present in high concentrations around the terminal portal (zone 1) and hepatic (zone 3) veins, whereas its concentration is low in the intermediate zone. Although the size and staining intensity of the periportal glutamate dehydrogenase-positive compartment are less than those of the pericentral compartment, it can expand under appropriate endocrine conditions, leading to a homogeneous distribution. At birth, glutamate dehydrogenase is also homogeneously distributed. Glutamate dehydrogenase disappears from the periportal compartment during the first postnatal week and reappears in that compartment after weaning. These observations indicate an independent regulation of glutamate dehydrogenase levels in the periportal and pericentral zone. The size of the periportal glutamate dehydrogenase-containing zone is appreciably smaller than that of carbamoylphosphate synthetase, whereas the pericentral glutamate dehydrogenase-containing zone is appreciably larger than that of glutamine synthetase. The heterogeneous distribution of glutamate dehydrogenase suggests the possibility that, under normal conditions, deamination of glutamate prevails in the periportal compartment and amination of glutamate in the pericentral compartment.

Aging↗

PubMed1973

Regulation of Nicotinamide Adenine Dinucleotide Phosphate-specific Glutamate Dehydrogenase in Germinated Spores of Geotrichum candidum.

Germinating spores of Geotrichum candidum produce only a nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase. Synthesis of glutamate dehydrogenase was repressed by the presence of ammonia, whereas urea, glutamate, or glutamine were ineffective. The enzyme was not subject to catabolite repression and was localized in the cell sap fraction. The glutamate dehydrogenase has been purified 93-fold and showed maximal activity at pH 8.2 in the forward and reverse directions. When measuring the initial reaction rate at pH 7.2, a variety of tricarboxylic acid cycle intermediates displayed additive and unidirectional activation of the reductive amination reaction and inhibition of the oxidative deamination reaction. The modulating effects were pH-dependent and diminished at alkaline pH values. Substrate inhibition exerted by alpha-ketoglutarate was strongest at neutral pH.Rate-concentration plot with respect to alpha-ketoglutarate at pH 7.2 was sigmoidal, and the enzyme was entirely inactive at substrate concentrations lower than 1 mm. In the presence of fumarate, the enzyme did not exhibit the cooperative interaction described above and was markedly activated at alpha-ketoglutarate concentrations lower than 1 mm. The tricarboxylic acid cycle intermediates released the enzyme from substrate inhibition by alpha-ketoglutarate. Double reciprocal rate-concentration plots displayed noncompetitive inhibition between succinate and NADP and a mixed noncompetitive and competitive inhibition between succinate and glutamate. The binding order of substrates in the reductive amination reaction was NADPH, alpha-ketoglutarate, and subsequently ammonia.

Journal Article↗

PubMedDate not supplied

Molecular basis of human glutamate dehydrogenase regulation under changing energy demands.

Glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism, is located in the mitochondria although there is evidence for extramitochondrial localization of GDH. In the human, housekeeping and nerve tissue-specific isoforms, encoded by the GLUD1 and GLUD2 genes, have been identified. The two isoenzymes differ markedly in their baseline activities, allosteric regulation, and thermal stability. GTP potently inhibits GLUD1-derived GDH (IC(50) = 0.2 muM), whereas the GLUD2-derived isoenzyme is resistant to this compound. The GLUD2-derived GDH shows low basal activity and has the capacity to be activated fully by ADP or L-leucine. We used molecular biological tools to study the subcellular localization of GLUD1-derived GDH in cultured cells and the molecular basis of its regulation. COS7 cells, transfected with a GLUD1-pEGFP-N3 vector, revealed a GFP fluorescence pattern nearly identical to that of the mitochondrial marker pDsRed2-Mito. Site-directed mutagenesis of GLUD1 gene showed that replacement of Gly456 by Ala made the enzyme resistant to GTP (IC(50) = 2.8 +/- 0.15 microM) without altering its regulation by ADP. Substitution of Ser for Arg443 rendered the enzyme virtually inactive at its basal state, but fully responsive to ADP activation. The Arg443Ser mutant was more active at pH 7.0 than at pH 8.0. The Gly456Ala change therefore dissociated GLUD2-derived GDH function from GTP, whereas the Arg443Ser change made enzyme regulation possible without this inhibitor. These properties may allow the brain isoenzyme to function well under conditions of intracellular acidification and increased turnover of ATP to ADP, as occurs in synaptic astrocytes during excitatory transmission.

Adenosine Diphosphate↗

PubMed1991

Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus.

An NAD(P)-dependent glutamate dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme is a hexamer (subunit mass 45 kDa) which dissociates into lower states of association when submitted to gel filtration. Isoelectric focusing analysis of the purified enzyme showed a pI of 5.7 and occasionally revealed microheterogeneity. The enzyme is strictly specific for the natural substrates 2-oxoglutarate and L-glutamate, but is active with both NADH and NADPH. S. solfataricus glutamate dehydrogenase revealed a high degree of thermal stability (at 80 C the half-life was 15 h) which was strictly dependent on the protein concentration. Very high levels of glutamate dehydrogenase were found in this archaebacterium which suggests that the conversion of 2-oxoglutarate and ammonia to glutamate is of central importance to the nitrogen metabolism in this bacterium.

Archaea↗