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)

When Escherichia coli was grown in a minimum medium with glucose as sole carbon source and a proper level of ammonia, NADP+ specific glutamate dehydrogenase (L-glutamate: NADP+ oxidoreductase (deaminating), ED 1.4.1.4) was induced. The enzyme was solubilized by French press treatment and purified to homogeneity by (NH4)2SO4 fractionation, heat treatment followed by DEAE-cellulose, hydroxylapatite and Bio-Gel chromatography with an overall yield of 30%. The enzyme proved to be heat stable and relatively resistant to protein denaturants. The optimum of enzymic activity for the reductive amination is at pH 8 and at pH 9 for the oxidative deamination. The activity is affected by adenine nucleotides. The molecular weight (about 250 000 for the native form and 46 000 for the inactive subunit) and amino acid composition, suggest strict similarities with the NADP+ enzyme from fungal origin.
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PMID:Glutamate dehydrogenase from Escherichia coli: induction, purification and properties of the enzyme. 23 98

We have constructed a series of deletions in the 5' non-coding sequences of the cloned Neurospora crassa am gene which specifies NADP specific glutamate dehydrogenase. All of the deletions begin at -4.4 kb with respect to the am transcription start site and extend for various distances toward the am gene. Using vectors with a truncated fragment of the am gene, we introduced these deletions into the chromosome upstream of am by transformation. Analysis of glutamate dehydrogenase expression in strains with the deletion mutations confirmed that there are two upstream regulatory sequences (URS) that control the expression of the am gene. The more distal of these elements (URSam beta) has been limited to the 157 bp between -1924 and -2081 with respect to the start of am transcription. The proximal element (URSam alpha) was limited to the 97 bp between -1296 and -1393. The DNA sequence of the entire region was determined. Within the sequences that contain the URS elements several regions of homology with yeast UAS sequences were found. Gel mobility assays with DNA fragments containing the URS elements indicated that sequences in both elements are bound by nuclear proteins from Neurospora. The interaction of these proteins and the DNA fragments was found to be specific.
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PMID:Nucleotide sequence and nuclear protein binding of the two regulatory sequences upstream of the am (GDH) gene in Neurospora. 216 25

The concentration-dependent association-dissociation tendency of purified bovine liver and rat liver glutamic dehydrogenase (GDH) has been demonstrated by high-performance liquid chromatographic gel filtration. In the concentration range of 100 to 1.0 micrograms bovine GDH/ml molecular species ranged from dimer and unimer to subunimeric forms. The dissociation process of the unimeric hexapeptide, consisting of six polypeptide chains, to the subunimeric tripeptide, consisting of three polypeptide chains, was irreversible without added ionic support, but reversible with added ionic support. In dilute Tris-HCl bovine liver GDH was dispersed to subunimeric sizes. Increasing the ionic strength in 20 mM phosphate as the mobile phase increased dissociation to a subunimeric tripeptide while sustaining as much as 80% of its activity. Activity of a eluting subunimer was verified by the inclusion of reaction substrates (NAD and glutamute) in the mobile phase and quantification of reaction products (NADH) in chromatograms. Gel filtration of GDH in the presence of GTP with NADH rendered a subunimeric tripeptide, largely independent of ionic strength or GDH concentration. Rat liver GDH, differing from bovine liver GDH, was dissociated by gel filtration to an active tripeptide independent of ionic or buffer conditions.
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PMID:Association-dissociation studies of bovine and rat liver glutamic dehydrogenase by high-performance liquid chromatography gel filtration. 317 32

The membrane phospholipid organization in monkey erythrocytes harbouring different developmental stages of the simian malarial parasite Plasmodium knowlesi was studied using phospholipase A2 from two different sources and Merocyanine 540 as the external-membrane probes. Experiments were done to confirm that the phospholipases did not penetrate into the infected cells or hydrolyse phospholipids during membrane isolation. The parasite-free erythrocyte membrane was isolated by differential centrifugation or by using the cationic beads Affi-Gel 731. The purity of the membranes was established by optical and electron microscopy, and by assaying the parasite-specific enzyme glutamate dehydrogenase. About 10% of the phosphatidylethanolamine and none of phosphatidylserine were hydrolysed by the phospholipases in intact normal monkey erythrocytes. However, accessibility of these aminophospholipids to the enzymes was significantly enhanced in the infected cells under identical conditions. The degree of this enhancement depended on the developmental stage of the intracellular parasite, but not on the parasitaemia levels in the infected monkeys, and increased with the parasite growth inside the cells. Analogously, Merocyanine 540 was found to label the trophozoite- or schizont-infected erythrocytes, but not the ring-infected or normal cells. These results demonstrate that the intracellular malarial parasite produces stage-dependent alterations in the membrane phospholipid organization of its host erythrocyte.
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PMID:An intracellular simian malarial parasite (Plasmodium knowlesi) induces stage-dependent alterations in membrane phospholipid organization of its host erythrocyte. 367 50

6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TADP) has been shown to react at the reduced diphosphopyridine nucleotide (DPNH) inhibitory site of bovine liver glutamate dehydrogenase with incorporation of 1 mol of reagent/mol of enzyme subunit [Batra, S. P., & Colman, R. F. (1984) Biochemistry 23, 4940-4946]. The modified enzyme had lost one of the six free sulfhydryl groups per enzyme subunit as detected by 5,5'-dithiobis(2-nitrobenzoate). In the unmodified enzyme digested with trypsin, six cysteinyl peptides labeled with [14C]iodoacetic acid were detected by high-performance liquid chromatography (HPLC), whereas only five were observed in the 6-BDB-TADP-modified enzyme. A cysteinyl peptide has been isolated from modified enzyme digested with trypsin and chymotrypsin. Purification of the nucleotidyl peptide was accomplished by chromatography on phenyl boronate-agarose, followed by gel filtration on Sephadex G-25 and Bio-Gel P-4 in 50 mM ammonium bicarbonate, pH 8.0. The modified peptides were finally purified by HPLC on a C18 column using 0.1% trifluoroacetic acid with an acetonitrile gradient. By comparison of the amino acid composition and N-terminal residue of the isolated peptide with the known amino acid sequence of the enzyme, the peptide in the DPNH inhibitory site labeled by 6-BDB-TADP has been identified as the 19-membered fragment from Glu-311 to Lys-329. A unique residue, Cys-319, was identified as the reactive amino acid within the DPNH inhibitory site.
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PMID:Isolation and identification of cysteinyl peptide labeled by 6- [( 4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate in the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase. 371 40

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)
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PMID:Dirofilaria immitis: comparison of cytosolic and mitochondrial glutamate dehydrogenases. 395 79

Glutamic dehydrogenase (GDH) activity in rat heart was found to be 2.1 U/g of heart (wet wt). The mitochondrial glutamic dehydrogenase activity accounted for only 18% of the total. This percentage of the total activity in heart mitochondria was not altered by nagarse treatment, acetone extraction, sonication in Triton X-100, and extraction with buffer containing a protease inhibitor. The remainder of the activity was present in the cytosol. Cytosolic GDH activity differed from mitochondrial GDH activity by its pH curve, stability to heat, Arrhenius plot, and the effect of different nucleotides. Acetone extraction of the mitochondria resulted in GDH that was stable to heat and had a shallow temperature activation curve resembling cytosolic GDH. Acetone extraction of cytosolic GDH inactivated it. The cytosolic activity was purified 288-fold and the mitochondrial activity 100-fold. Purified cytosolic and mitochondrial GDH enzymes had different monomeric molecular weights on sucrose density gradient centrifugation. Gel filtration of cytosolic and mitochondrial GDH also showed different monomeric molecular weights. We conclude that rat heart GDH exists in two forms with different physical and kinetic characteristics. The majority of GDH activity in rat heart is cytosolic. The mitochondrial enzyme has a lipid-soluble component that can be removed with acetone without destroying its activity.
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PMID:Glutamic dehydrogenase activity in rat heart: demonstration of two forms of enzyme activity. 672 Sep 7

An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.
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PMID:Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron. 736 28

Expression of the Neurospora crassa am (NADP-specific glutamate dehydrogenase) gene is controlled by two upstream enhancer-like elements designated URSam alpha and URSam beta. URSam alpha is localized between - 1.3 and - 1.4 kb with respect to the major transcriptional start site. Deletion of a 90 bp sequence containing this element resulted in the loss of approximately 50% of normal glutamate dehydrogenase expression. Gel mobility shift analysis indicated that a nuclear protein from Neurospora binds in a specific manner to sequences within the 90 bp fragment. We have now used a combination of ion-exchange and affinity chromatography to purify this nuclear protein, which we call Am Alpha Binding protein (AAB). The activity was monitored by gel shift analysis. The protein was purified more than 14,000-fold with a yield of approximately 7%. The purified protein appears as a heteromer on denaturing polyacrylamide gel electrophoresis, with only two strong bands visible in silver-stained preparations. One band has an apparent molecular mass of 40 kDa, the other appears as a doublet with an apparent molecular mass of 30 kDa. DNAse I protection analysis indicated a protected region consisting of 30 bp, which contains a CCAAT pentanucleotide motif. Mutagenesis of the CCAAT motif abolished the binding of AAB to the DNA fragment.
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PMID:Purification of a heteromeric CCAAT binding protein from Neurospora crassa. 750 Sep 55

We have used gel mobility shift assays to scan 1.7 kb of 5' non-coding sequence of the am (glutamate dehydrogenase) gene of Neurospora crassa for binding by partially fractionated Neurospora proteins. Using genetic analysis this region had been shown to play an important role in the control of glutamate dehydrogenase (GDH) expression. Gel mobility shift analysis identified three regions to which Neurospora proteins bind specifically. Two of these corresponded to the two elements previously defined by genetic analysis (URSam alpha and URSam beta). The third protein binding site appears to be unrelated to am gene expression. Competition experiments showed that the proteins that bind to the URSam alpha and URSam beta elements are different. The URSam alpha element was shown to contain two independent binding sites for the URSam alpha binding protein(s). Both fragments contain a CCAAT motif, suggesting that URSam alpha binding protein(s) may be members of one of the CCAAT-binding protein families. The effect of deletion of either the URSam alpha or URSam beta elements on catabolite induction of am expression was also determined. Both elements appear to act as constitutive enhancers of gene expression.
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PMID:Sequential gel mobility shift scanning of 5' upstream sequences of the Neurospora crassa am (GDH) gene. 812 95


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