Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.6.1.19 (GABA transaminase)
 808 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A homogeneous 4-aminobutyrate aminotransferase isolated from pig brain exhibits a kcat = 9.6 s-1 and contains only 1 mol of pyridoxal-5-P/mol of dimer. The equilibrium dissociation constant for pyridoxal-5-P tightly bound to the enzyme is 1 nM. Spectrophotometric titrations reveal that the enzyme binds a second molecule of pyridoxal-5-P with a KD = 3 microM. The reaction of enzyme containing 1 pyridoxal-5-P/dimer with the inhibitors NaBH4 and DL-gabaculine was studied by observing changes in the absorption spectrum of the bound coenzyme and by monitoring loss of catalytic activity. The native enzyme is inactivated by both inhibitors. Incubation of the resultant P-pyridoxyl aminotransferase and m-anthraniyl-PMP-aminotransferase with excess pyridoxal-5-P at 25 degrees C restores full catalytic activity. It is postulated that the dimeric enzyme contains two classes of catalytic binding sites, and that the binding site characterized by a weak affinity for pyridoxal-5-P (KD = 3 microM) becomes functional after specific chemical modification of the molecule of cofactor tightly bound to the protein (KD - 1 nM).
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PMID:4-Aminobutyrate aminotransferase. The presence of nonequivalent binding sites. 745 92

Mitochondrial 4-aminobutyrate aminotransferase in rat kidney can utilize pyruvate as the acceptor for the amino group of 4-aminobutyrate. Renal 4-aminobutyrate aminotransferase activity at saturating equimolar concentration of 4-aminobutyrate and 5 mM pyruvate is 42.8 +/- 2.5 mumol/g protein per h (mean +/- S.E.M.) or 70% of 4-aminobutyrate aminotransferase activity with equimolar alpha-ketoglutarate. 4-Aminobutyrate aminotransferase in brain does not transaminate with pyruvate. Since pyruvate is an important mitochondrial metabolite in kidney, net disposal of glutamate via the 4-aminobutyrate pathway is possible. The renal 4-aminobutyrate pathway in the rat has other distinctive features when compared with the pathway in rat brain. Most inhibitors of rat neuronal glutamate decarboxylase were ineffective against the renal form of the enzyme, but 20 mM semicarbazide inhibited the latter form by 80% (P < 0.001) in vitro and reduced renal 4-aminobutyrate content by 75% (P < 0.001) in vivo. In the presence of 20 mM semicarbazide, ammoniagenesis by rat renal cortex slices incubated in 1 mM glutamine was inhibited 26% (P < 0.01). Semicarbazide was proportionately less effective (15% inhibition) when ammonia-genesis was stimulated (+243%) in slices prepared from chronically acidotic animals, and was no deterrant to ammoniagenesis when non-acidotic slices were incubated in supraphysiologic concentrations of 10 mM glutamine. We conclude that whereas integrity of the renal 4-aminobutyrate pathway may contribute to glutamate disposal and thus ammoniagenesis under physiologic conditions, the pathway is a passive participant in the overall process of ammoniagenesis.
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PMID:The relationship of 4-aminobutyric acid metabolism to ammoniagenesis in renal cortex. 745 89

Alanine-glyoxylate aminotransferase (AGT) 2 is a pyridoxal 5'-phosphate dependent, mitochondrial enzyme which, in the rat, is expressed at a high level in the kidney. The amino acid sequences of nine tryptic and seven CNBr peptides of the rat kidney AGT2 were determined. Three overlapping cDNAs encoding the AGT2 were cloned on the basis of its partial amino acid sequences by means of a polymerase chain reaction-based approach involving rat kidney poly(A)+ RNA. The complete cDNA sequence comprised 1,919 bases, and contained a 1,536-base open reading frame which encodes a polypeptide of 512 amino acid residues with a putative presequence consisting of 39 amino acid residues at the amino terminus, giving a precursor protein with a molecular mass of 57,150 Da. The sequence of AGT2 exhibits significant homology with neither peroxisomal AGT1 from human liver nor mitochondrial AGT1 from rat liver. However, the sequence of AGT2 exhibited 30.8, 29.2, and 27.1% identity with those of Escherichia coli 4-aminobutyrate aminotransferase, rat ornithine aminotransferase, and Pseudomonas cepacia 2,2-dialkylglycine decarboxylase, respectively. The active site sequences were also well conserved among these aminotransferases. AGT2, thus, is more similar to the other aminotransferases than to AGT1. The results suggest that the rat kidney AGT2 may play a biological role in amino acid metabolism distinct from that of AGT1.
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PMID:Molecular cloning and sequencing of a cDNA encoding alanine-glyoxylate aminotransferase 2 from rat kidney. 759 50

A human brain cDNA library constructed in the lambda ZAP II vector was screened using a fragment of pig brain cDNA encoding 4-aminobutyrate aminotransferase (pGaba-t). A cDNA that encodes the human brain Gaba-t (hGaba-t) has been isolated from the library and sequenced. Using the GenBank and EMBL databases, comparison of the predicted amino-acid sequence of hGaba-t with the pig enzyme revealed 95.4% homology.
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PMID:Screening and sequence determination of a cDNA encoding the human brain 4-aminobutyrate aminotransferase. 772 Oct 88

The effects of chronic treatment with the specific, mechanism-based, irreversible inhibitors of 4-aminobutyrate aminotransferase (EC 2.6.1.19; GABA transaminase), ethanolamine O-sulphate (EOS), and 4-aminohexenoate [vigabatrin; gamma-vinyl-GABA (GVG)] on the extracellular concentrations of GABA in the hippocampus have been studied using in vivo microdialysis in conscious animals. Oral dosing [3 mg/ml of drinking water, giving doses of GVG of 194 +/- 38 mg/kg/day and of EOS of 303 +/- 42 mg/kg/day (mean +/- SD)] was followed by microdialysis at 2, 8, and 21 days. The basal outflow of GABA (in the range of approximately 1-2 pmol/30 microliters/30-min sample) after 2 and 8 days of treatment was not significantly different from that in control animals, but the 21-day treatment gave significant rises in the extracellular GABA concentration (up to approximately 6-8 pmol/30 microliters/30-min sample). Both inhibitors gave similar results. Depolarisation with 100 mM K+ gave large increases in GABA release in control (approximately 20-60 pmol/30 microliters/30-min sample) and treated animals. The 8- and 21-day-treated animals showed significant increases in the stimulated release compared with control animals (approximately 80-100 pmol/30 microliters/30-min sample). Excluding Ca2+ had no significant effect on either basal or stimulated release. The significant increases in K(+)-evoked release of GABA show that the increased intracellular pool of GABA is available for release, and this may be related to the anticonvulsant action of these compounds.
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PMID:The effect of chronic treatment with the GABA transaminase inhibitors gamma-vinyl-GABA and ethanolamine-O-sulphate on the in vivo release of GABA from rat hippocampus. 772 10

4-Aminobutyrate aminotransferase undergoes a reversible process of association/dissociation at low pH. At pH 5.0, monomeric species exist predominantly in solution as revealed by FPLC and time-dependent emission anisotropy measurements. The observed rotational correlation time at pH 5.0, phi obs = 25 ns, corresponds to a compact spherical unit of 52 kDa. An increase in the net charge of the macromolecule at pH 5.0 is responsible for destabilization of the dimeric structure, (WEL approximately 41.84 kJ/mol), but the dissociation of the protein does not perturb the secondary structure as revealed by CD measurements. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS), bound to hydrophobic sites of the enzyme, was used to monitor the kinetics of protein dissociation by stopped-flow spectroscopy. The dissociation of the dimeric structure at pH 5.0 was characterized by a relaxation time of 18 ms. The rate of association of monomeric subunits at pH 7.0 was too fast to be detected in the stopped-flow instrument. These observations have some bearing on the mechanism of reconstitution of dimeric structures of 4-aminobutyrate aminotransferase in the cell.
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PMID:Characterization of monomeric 4-aminobutyrate aminotransferase at low pH. 773 64

cDNA encoding human 4-aminobutyrate aminotransferase (aminobutyrate:2-oxoglutarate aminotransferase) was prepared by polymerase chain reaction using mRNA from human neuroblastoma cells as the template and oligonucleotides synthesized on the basis of the information obtained from direct protein sequencing. The cDNA-deduced sequence enabled peptides, sequenced by automated Edman degradation, to be aligned for confirmation of the complete primary structure. The results are compared with the recently published sequence of the rat enzyme deduced entirely from DNA sequencing [Medina-Kauwe, L. K., Tillakaratne, N. J. K., Wu, J.-Y. & Tobin, A. J. (1994) J. Neurochem. 62, 1267-1275]. Although the sequences are almost identical for most of their length, they differ in a segment of 36 residues. Almost complete identity of the two sequences is established if it is assumed that a frame-shift error was introduced into the reported rat cDNA sequence. The human cDNA was used to probe for the presence of 4-aminobutyrate aminotransferase mRNA in human tissues and a significant transcript was found in heart, placenta and in tissues usually associated with the expression of this enzyme.
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PMID:Primary structure and tissue distribution of human 4-aminobutyrate aminotransferase. 785 25

The unfolding of pig liver 4-aminobutyrate aminotransferase by urea has been investigated at equilibrium. The overall process was reversible as judged from the recovery of catalytic activity after dilution of urea-treated samples. Unfolding of the enzyme was monitored by circular dichroism and fluorescence spectroscopy. The steepness of the fluorescence and CD changes between 2 and 8 M urea, and the lack of any discernible plateau suggests that unfolding of the protein is a cooperative process. The unfolding of 4-aminobutyrate aminotransferase as a function of urea concentration was monitored by fluorescence measurements of the tryptophanyl residues. The kinetic results indicate that the aminotransferase unfolds in a single kinetic phase. Unfolded 4-aminobutyrate aminotransferase recovers immediately its catalytic activity upon dilution with buffers of neutral pH. Based on equilibrium and kinetic results, a two-state model for the unfolding of the aminotransferase is proposed.
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PMID:Unfolding of 4-aminobutyrate aminotransferase equilibrium and kinetic studies. 807 51

The amino acid sequences of two pyridoxal phosphate-dependent enzymes, namely glutamate decarboxylase and 4-aminobutyrate aminotransferase, were solved by a combination of direct protein sequencing and cDNA sequencing. In the case of glutamate decarboxylase from Escherichia coli, correct ordering of three internal peptides was achieved by sequencing a fragment of DNA generated by PCR. In the case of 4-aminobutyrate aminotransferase from pig liver, chemical studies on the reaction of the enzyme with a suicide inhibitor allowed a detailed description of the mechanism of inactivation and collection of partial sequence information on the protein. The sequence of the protein was completely reconstructed after analysis of further peptide fragments derived by proteolytic and chemical cleavages, with the help of the information from the cDNA-deduced sequence of pig brain 4-aminobutyrate aminotransferase performed by another group (Kwon, Park and Churchich [1992] J. Biol. Chem. 267, 7215-7216).
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PMID:Sequence studies on pyridoxal phosphate-dependent enzymes. 825 Nov 12

The enzyme 4-aminobutyrate aminotransferase (EC 2.6.1.19) isolated from Pseudomonas fluorescens was inhibited by the nucleotide ATP in an apparent competitive manner (Ki = 10.4 mM). This reversible effect was antagonized by the substrate GABA, whose apparent Km was increased from 0.6 mM to 2 mM in the presence of 20 mM ATP, suggesting that ATP interferes with GABA binding to the active site of the enzyme. The apparent Km with respect to the second substrate alpha-ketoglutarate was also increased, although to a lesser extent, whereas the cofactor pyridoxal 5'-phosphate was unable to influence the inhibition by ATP. The ATP structural analogues ADP, CTP and XTP were also able to inhibit the enzyme to a similar extent. These data indicate that GABA concentrations within the bacterial cell can be regulated by the action of ATP on 4-aminobutyrate aminotransferase. In addition, because the inhibition by ATP is similar to the inhibition of the enzyme from mammalian brain, the bacterial enzyme could provide a convenient source of the enzyme for studies of drug effects on brain GABA metabolism in vitro.
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PMID:Inhibition of 4-aminobutyrate aminotransferase from Pseudomonas fluorescens by ATP. 826 Sep 45


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