Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.4.1.2 (glutamate dehydrogenase)
4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The concentration-dependent aggregation behaviour of purified ox liver and brain glutamate dehydrogenase preparations was compared with that of commercially-obtained preparations of the liver enzyme, which have recently been shown to have suffered proteolytic cleavage. Although there were no significant differences in these effects, the presence of 3 mM-GTP and 3 mM-NADH had markedly different effects on the two types of preparation. In this situation, at higher protein concentrations the commercially obtained preparations existed in a higher degree of aggregation than those which had not suffered proteolysis. Studies of the effects of GTP and NADH concentrations on the sedimentation coefficients at a fixed enzyme concentration suggested these effects to be largely due to differences in the affinities of the two preparations for nucleotides.
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PMID:Sedimentation properties of native and proteolysed preparations of ox glutamate dehydrogenase. 733 5

Bovine liver glutamate dehydrogenase is an allosteric enzyme which is activated by ADP. The affinity label adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] (AMPSBDB), a new ADP analog featuring a reactive group at a position equivalent to that of the pyrophosphate, reacts with this glutamate dehydrogenase to yield enzyme containing about 0.9 mol/mol of enzyme subunit. The reaction results in a time-dependent irreversible activation of the enzyme. Glutamate dehydrogenase (8.9 microM subunit) modified with 10-60 microM AMPSBDB is about 3.2-fold more active than native enzyme. The modified enzyme is still inhibited by GTP and by high concentrations of NADH, but is no longer activated by ADP. The addition to the reaction mixture of (a) NADH or alpha-ketoglutarate; (b) GTP + NADH; or (c) alpha-ketoglutarate + NADH has little effect on the functional changes produced by AMPSBDB; whereas, the reaction is prevented by ADP. Purification of labeled peptide from proteolytic and chemical digests of [2-3H]AMPSBDB-modified enzyme leads to identification of Arg459 as the target amino acid. We conclude that AMPSBDB functions as an ADP mimic covalently bound to Arg459 within the ADP activator site of the allosteric bovine liver glutamate dehydrogenase.
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PMID:Activation of bovine liver glutamate dehydrogenase by covalent reaction of adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] with arginine-459 at an ADP regulatory site. 791 68

The biochemical and cytotoxic activities of the IMP dehydrogenase (IMPDH) inhibitors benzamide riboside, tiazofurin, and selenazofurin were compared. These three C-nucleosides exert their cytotoxicity by forming an analogue of NAD, wherein nicotinamide is replaced by the C-nucleoside base. The antiproliferative activities of these three agents were compared in a panel of 60 human cancer cell lines. To examine the relationship of benzamide riboside and selenazofurin to tiazofurin, COMPARE computer analysis was performed, and correlation coefficients of 0.761 and 0.815 were obtained for benzamide riboside and selenazofurin, respectively. The biochemical activities of these agents were examined in human myelogenous leukemia K562 cells. Incubation of K562 cells for 4 hr with 10 microM each of benzamide riboside, selenazofurin and tiazofurin resulted in a 49, 71, and 26% decrease in IMPDH activity with a concurrent increase in intracellular IMP pools. As a consequence of IMPDH inhibition, GTP and dGTP concentrations were curtailed. These studies demonstrated that selenazofurin was the most potent of the three agents. To compare the cellular synthesis of NAD analogues of these agents, K562 cells were incubated with 10 microM each of benzamide riboside, tiazofurin and selenazofurin after prelabeling the cells with [2,8-3H]adenosine. The results demonstrated that benzamide riboside produced 2- and 3-fold more of NAD analogue (BAD) than tiazofurin and selenazofurin did. To elucidate the effects of the three compounds on other NAD-utilizing enzymes, the inhibitory activities of purified benzamide adenine dinucleotide (BAD), thiazole-4-carboxamide adenine dinucleotide (TAD) and selenazole-4-carboxamide adenine dinucleotide (SAD) were studied in commercially available purified preparations of lactate dehydrogenase, glutamate dehydrogenase and malate dehydrogenase. TAD and SAD did not inhibit these three dehydrogenases. Although BAD did not influence lactate and glutamate dehydrogenases, it selectively inhibited 50% of malate dehydrogenase activity at a 3.2 microM concentration. These studies demonstrate similarities and differences in the biochemical actions of the three C-nucleosides, even though they share similar mechanisms of action.
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PMID:Comparison of biochemical parameters of benzamide riboside, a new inhibitor of IMP dehydrogenase, with tiazofurin and selenazofurin. 794 41

Two "targeted bidentate" photoaffinity cross-linking reagents, the monoanhydride of 8-N3ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]8-N3ATP gamma BP) and the monoanhydride of 8-N3GDP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]8-N3GTP gamma BP), were developed for studying the inter- and intramolecular interactions of nucleotide-binding proteins. Experiments using these bidentate reagents with two photoactive groups led to specific cross-linking: [gamma-32P]8-N3GTP gamma BP and [gamma-32P]8-N3ATP gamma BP showed intersubunit cross-linking of glutamate dehydrogenase and [gamma-32P]8-N3GTP gamma BP appeared to cross-link the alpha- and beta-subunits of tubulin. The non-azido "monodentate" versions of these reagents, the monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]ATP gamma BP) and the monoanhydride of GDP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]GTP gamma BP), were also synthesized and characterized. The ability of these monodentate reagents with one photoactive group to serve as photoaffinity probes was established by photolabeling specifically the exchangeable GTP-binding domain of tubulin with [gamma-32P]GTP gamma BP and the ATP-binding domain of purified adenylate kinase and several nucleotide-binding proteins in human brain homogenate with [gamma-32P]ATP gamma BP.
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PMID:Synthesis and application of bidentate photoaffinity cross-linking reagents. Nucleotide photoaffinity probes with two photoactive groups. 831 86

The effects of pyridoxal 5'-phosphate (PalP) on ox liver glutamate dehydrogenase (94% inactivation by 1.8 mM reagent at pH 7 and 25 degrees C) have been compared with those of three analogues, 5'-deoxypyridoxal (96% inactivation), pyridoxal 5'-sulphate (97%) and pyridoxal 5-methylsulphonate (94%), in order to establish whether PalP acts as an affinity label for this enzyme. Like PalP and unlike pyridoxal, which is a much less potent inactivator, none of the analogues has a free 5'-OH group to cyclize with the aldehyde function. The result with 5'-deoxypyridoxal shows that a negative charge, such as that of the phosphate group, is not required for efficient inactivation. With all four reagents, addition of an excess of cysteine or lysine led to 90-100% re-activation over 3-20 h. Dialysis also caused reactivation to a similar extent. A combination of 2.15 mM NADH, 1 mM GTP and 10 mM 2-oxoglutarate gave complete protection against PalP, but only partial protection against the analogues. 5'-Deoxypyridoxal still caused 20-25% inactivation in the presence of the protection mixture. Absorbance measurements after reduction with NaBH4 show the characteristic features of a reduced Schiff's base and allowed estimation of the extent of reaction. With all the reagents the protection mixture decreased incorporation by about 1 mol/mol, but levels of incorporation without protection varied from about 2 mol/mol for PalP up to about 5 mol/mol for 5'-deoxypyridoxal. The labelling at additional sites may explain the residual inactivation in the presence of potent protecting agents.
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PMID:Is pyridoxal 5'-phosphate an affinity label for phosphate-binding sites in proteins?: The case of bovine glutamate dehydrogenase. 837 38

Photoaffinity labeling with [alpha-32P]8N3GTP and [gamma-32P]8N3GTP was used to identify the guanine binding domain of the GTP regulatory site within glutamate dehydrogenase (GDH). Without photolysis, 8N3GTP mimicked the regulatory properties of GTP on GDH activity with 8N3GTP exhibiting a Ki of 5 microM while the Ki for GTP was about 0.6 microM. Under optimal photolabeling conditions saturation of photoinsertion with 1 microgram of GDH revealed an apparent Kd of 9 +/- 4 microM for [gamma-32P]8N3GTP. Photolabeling with this analog could be competitively inhibited with GTP with an apparent Kd of 12 +/- 2 microM. Other nucleotides such as ATP and NAD(P)H could not reduce the amount of photoinsertion as effectively as GTP. ADP could decrease photoinsertion, but only at much higher concentrations. NAD(P)+, GDP, AMP, and GMP had little effect on photoinsertion. Divalent cations Mg2+ and Ca2+ also reduced photoinsertion significantly while the monovalent K+ and Na+ ions had no effect. Aluminum(III)-chelate or iron(III)-chelate affinity chromatography and reversed-phase HPLC were used to purify photolabel-containing peptides generated with either trypsin or chymotrypsin. This identified a portion of the guanine binding domain within the GTP regulatory site as the region containing the sequence Ile439 to Tyr454. Photolabeling of this peptide was prevented 91% by the presence of 300 microM GTP during photolysis. Lys445 was not identified in sequence analyses of the photolabeled peptides. Also, trypsin was unable to cleave the photolabeled peptide at this site. These results suggest that Lys445 may be the residue modified by [alpha-32P]8N3GTP.
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PMID:Identification of a guanine binding domain peptide of the GTP binding site of glutamate dehydrogenase: isolation with metal-chelate affinity chromatography. 843 45

A variety of metabolites have been found to elicit a form of inhibition or activation on an NAD-specific glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from Halobacterium halobium. The purified halophilic enzyme was tested with several compounds known to be allosteric modifiers of mammalian glutamate dehydrogenases to determine their effects on enzyme activity. GTP, ATP, ADP and AMP did not affect the enzyme, so these effectors of bovine glutamate dehydrogenase do not play a role in the regulation of the halophilic enzyme. However, the halophilic enzyme was subject to strong inhibition by TCA intermediates. When measuring the initial rate of the reaction, the oxidative deamination of L-glutamate was inhibited by TCA metabolites such as: fumarate, oxalacetate, succinate and malate; by substrate analogues such as: NADP+, D-glutamate and glutarate; and by dicarboxylic compounds such as adipate. On the other hand, all the amino acids tested were activators of this enzyme, except the D-isomer of the substrate L-glutamate that acted as an inhibitor. The relative effectiveness of each inhibitor or activator (Ki or Ka values) was correlated with the dipole moment (mu), HOMO and LUMO molecular orbital energies, optimal distance between two carboxyl groups, and hydrophobicity. Compounds with high dipole moment acted as good activators while compounds with low dipole moment were inhibitors. We have also found that the best activators were amino acids with no polar lateral chain.
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PMID:NAD-glutamate dehydrogenase from Halobacterium halobium: inhibition and activation by TCA intermediates and amino acids. 860 24

Photoaffinity labeling with [gamma-32P]8N3GTP (8-azidoguanosine triphosphate) was used to identify the guanine binding peptides of the GTT binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. 8N3GTP, without photolysis, mimicked the inhibitory properties of GTP on GDH I and GDH II activities. Saturation of photoinsertion of GDH isoproteins revealed an apparent Kd of 8 microM (GDH I) and 24 microM (GDH II) for [gamma-32P]8N3GTP. Ion exchange and reversed-phase high-performance liquid chromatography (HPLC) were used to isolate photolabel-containing peptides generated with trypsin. This identified a portion of the guanine binding domain within the GTP binding site is the region containing the sequence I-S-G-A-S-E-X-D-I-V-H-S-A-L-A-Y-T-M E-R (GDH I) and I-S-G-A-S-E-X-D-I-V-H-S-G-L-A-Y-T-M-E-R (GDH II). The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as a photolabeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other GDH species known. Also, trypsin was unable to cleave the photolabeled peptide at this site. Photolabeling of these peptides was prevented by the presence of GTP during photolysis, while other nucleotides could not reduce the amount of photoinsertion as effectively as GTP. These results demonstrate selectivity of the photoprobe for the GTP binding site and suggest that the peptide identified using the photoprobe is located in the GTP binding domain of the brain GDH isoproteins.
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PMID:Identification of a peptide of the guanosine triphosphate binding site within brain glutamate dehydrogenase isoproteins using 8-azidoguanosine triphosphate. 890 87

Bovine liver glutamate dehydrogenase has been crystallized as an abortive complex with glutamic acid, NADH, and an inhibitor, GTP. Crystals of this complex were grown using the sitting drop vapor diffusion method with PEG 8000 as the precipitant and diffract to better than 2.5 A resolution. The crystals belong to the space group P2(1) with an entire enzyme hexamer in the crystallographic asymmetric unit. Self-rotation and self-Patterson functions clearly define the orientation and position of this hexameric enzyme.
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PMID:Crystallization and characterization of bovine liver glutamate dehydrogenase. 935 94

The stimulatory effects of gabapentin on the activities of two types of glutamate dehydrogenase (GDH) isoproteins homogeneously purified from bovine brain have been studied at various conditions. When the effects of different gabapentin concentrations on GDH activities were studied in the direction of reductive amination of 2-oxoglutarate with NADPH as a coenzyme, a marked activation was observed for both isoproteins, whereas both isoproteins showed activation to a lesser extent with NADH as a coenzyme. Stimulatory effects of gabapentin on GDH activities in the direction of the oxidative deamination of glutamate were also observed, but to a much lesser extent than reductive amination. There were big differences between the two GDH isoproteins in their sensitivity to the action of gabapentin. The largest activation was observed with GDH II when NADPH was used as a coenzyme. Half-maximal stimulation was reached at around 1.5 mM. Gabapentin relieved the inhibition of GDH isoproteins by GTP and this resulted in an increase in the apparent activation by gabapentin in the presence of GTP. 2-Oxoglutarate was found to give rise to high substrate inhibition and gabapentin reduced the substrate inhibition in the presence of 0.2 mM NADH. Since there are neurodegenerative disorders in which GDH activity is decreased, the therapeutic modulation of the activity of this enzyme may be clinically useful.
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PMID:Activation of two types of brain glutamate dehydrogenase isoproteins by gabapentin. 959 7


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