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:2.6.1.19 (GABA transaminase)
808 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kinetics of resolution of the pyridoxamine phosphate form of the enzyme 4-aminobutyrate aminotransferase were monitored by fluorescence spectroscopy. 2 mol pyridoxamine phosphate are released/mol enzyme, indicating that two molecules of cofactor are involved in catalysis. The apoprotein is reconstituted by addition of pyridoxal phosphate; the apparent rate constant corresponding to the formation of active species is not a linear function of the concentration of cofactor. A multistep mechanism is proposed for the reconstitution of 4-aminobutyrate aminotransferase. A slow phase of reactivation of the aminotransferase is observed when the apoprotein is allowed to reconstitute in the presence of pyridoxal kinase, ATP and pyridoxal. The enzyme 4-aminobutyrate aminotransferase is a dimeric protein made up of subunits of identical molecular weight. It is characterized by a rotational relaxation time of 110 ns. The dimeric structure does not dissociate into subunits over a wide range of protein concentration (4--0.2 micrometer) at neutral pH.
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PMID:4-Aminobutyrate aminotransferase fluorescence studies. 64 27

Bilateral ischemia has been shown to alter the net brain levels of energy metabolites such as ATP, phosphocreatine, glucose, and glycogen. The amino acid neurotransmitter gamma-aminobutyric acid (GABA) exerts a tonic inhibitory influence on neural activity. The present studies were designed to evaluate the influence of elevated GABA levels on the metabolic sequelae of ischemia. The GABA transaminase inhibitor gamma-vinyl-GABA (GVG; vigabatrin) was administered to Mongolian gerbils before the production of a bilateral ischemic incident. GABA levels were elevated in all regions assayed. Levels of energy metabolites were also increased, an indication of reduced energy utilization. In control animals, in the absence of GVG, 1 min of bilateral ischemia produced decreases in the levels of all metabolites. In animals pretreated with GVG, the effects of 1 min of bilateral ischemia were attenuated. These data suggest that the level of ongoing activity may affect the response to an ischemic insult. Furthermore, GVG may have a clinical indication in reducing the effect of minor ischemic incidents.
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PMID:Elevated gamma-aminobutyric acid levels attenuate the metabolic response to bilateral ischemia. 149 8

The nucleotide ATP was shown to be a reversible inhibitor of partially purified gamma-aminobutyrate aminotransferase isolated from mouse brain. This inhibition was of the competitive type with respect to the substrate, gamma-aminobutyric acid (Ki = 3.7 +/- 0.6 mM), but was noncompetitive with respect to both the second substrate alpha-ketoglutarate and the cofactor pyridoxal 5'-phosphate. Two analogues of ATP, ADP and GTP, also gave rise to an inhibition gamma-aminobutyrate aminotransferase that was similar to that produced by ATP. These results are consistent with the view that mouse brain gamma-aminobutyric acid aminotransferase could be under regulatory control by ATP and certain other nucleotides within the mitochondria.
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PMID:Competitive inhibition of mouse brain gamma-aminobutyrate aminotransferase by ATP. 279 33

The dialdehyde of oxidized 1,N6-etheno-ATP and adenosine triphosphopyridoxal were used as probes of the catalytic site of 4-aminobutyrate aminotransferase. Both compounds react with lysine residues critically connected with aminotransferase activity. The binding of 1 mol of oxidized 1,N6-etheno-ATP per mol of enzyme or the binding of 1 mol of adenosine triphosphopyridoxal abrogates catalytic activity. The presence of substrate alpha-ketoglutarate (4 mM) prevents inactivation of the aminotransferase by either one of the ATP analogs. Reduction of the enzyme modified with oxidized 1,N6-etheno-ATP yields a chromophore which displays a maximum of emission at 415 nm and a fluorescent lifetime of 21.6 ns. The degree of exposure of the ethenoadenine ring to collisional encounters with the strong quencher KI was determined at pH 7.0. The ethenoadenine ring of the bound ligand is partially shielded from collisional encounters with the quencher. Steady-state emission anisotropy measurements of the bound ligand reveal that oxidized 1,N6-etheno-ATP is not rigidly attached to the protein matrix. It is postulated that the catalytic domain of 4-aminobutyrate aminotransferase is accessible to bulky reagents of greater length than the substrates 4-aminobutyrate and alpha-ketoglutarate.
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PMID:Active site modification of 4-aminobutyrate aminotransferase with ATP analogs. 312 Jul 74

Highly purified 4-aminobutyrate aminotransferase from pig brain is susceptible to phosphorylation by the purified cAMP-dependent protein kinase catalytic subunit. Up to 0.7 moles of phosphate from ATP-(gamma)-32P can be incorporated per mole of dimeric holoenzyme. Maximum phosphorylation was observed within about 90 minutes at 30 degrees C. Despite the extensive degree of phosphorylation observed, no kinetic property of the enzyme was perceptibly altered. Removal of cofactor had no detectable impact on the extent of phosphorylation but thermal inactivation of the enzyme increased and mild reduction with sodium borohydride decreased the phosphorylatability of the aminotransferase. It was possible to separate the enzyme into phospho and dephospho forms by the use of DEAE chromatography. Validation that the two fractions represented genuine aminotransferase was obtained by proteolytic peptide mapping. The phospho form of the enzyme was found to possess little or no aminotransferase activity while that of the dephospho form exhibited higher specific activity than the purified enzyme prior to phosphorylation. Furthermore, the dephospho form of the enzyme could not be detectably phosphorylated by reincubation with the kinase following DEAE chromatography unless it was subjected to thermal inactivation. The stoichiometry of phosphorylation of the fraction containing 32P from DEAE chromatography was approximately 1 mole/mole of dimer. These results suggest that the substrate for phosphorylation by the kinase is a form of the aminotransferase which is somehow inactivated during routine purification even when extensive precautions are taken to maximally preserve catalytic activity.
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PMID:In vitro phosphorylation of 4-aminobutyrate aminotransferase by cAMP dependent protein kinase. 370 Jul 75

Chronic acidosis evoked by a 7-day application of ammonium chloride in concentration of 2% increased the activity of glutamate decarboxylase (GAD) in renal homogenates of rats to approximately 160%. The enzyme activators, chlorides and adenosine triphosphate influenced in varying measures the GAD activity in renal homogenates of both controlled and acidotic animals. Whilst ATP was gradually loosing the activating effect, chlorides preserved it. The renal GAD is firmly bound on insoluble structures. The increase in GAD activity due to acidosis was accompanied by increasing permanence of this bind. After the substitution of ammonium chloride by drinking water, the return of the increased GAD activity to previous normal values lasted 7 days, whilst apparent normalization of the weight of experimental animals reoccurred on the first day. Subfractionation of the crude renal mitochondrial fraction by use of enzyme markers localized GAD in mitochondria. In renal homogenates the activities of GABA-transaminases were assessed. GABA-alpha-ketoglutarate transaminase was 5x more active than GABA-pyruvate transaminase. Acidosis resulted in augmentation of both transaminases--the first to 130%, the second to 160%. (Tab. 5, Fig. 3, Ref. 25.)
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PMID:[The effect of chronic acidosis on the activity of renal glutamate decarboxylase and GABA-transaminase]. 788 63

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

1. The modulatory effects of L-glutamate and its structural analogues, and of gamma-aminobutyric acid (GABA), on sympathetic co-transmission were studied in the rat isolated vas deferens exposed to electrical field stimulation (EFS). 2. Application of exogenous L-glutamate caused a concentration-dependent (1 microM-3 mM) inhibition of the rapid twitch component of the biphasic EFS contraction. However, L-glutamate (1 microM-3 mM) had a minimal effect on the phasic contraction induced by exogenous adenosine 5'-triphosphate (ATP, 150 microM) and noradrenaline (50 microM). Unlike L-glutamate, D-glutamate had no effect on the EFS contraction. 3. The L-glutamate-induced inhibition of the EFS contractions was significantly attenuated by the glutamate decarboxylase (GAD) inhibitor 3-mercapto-propionic acid (150 microM) and was abolished in the presence of the GABA transaminase (GABA-T) inhibitor, 2-aminoethyl hydrogen sulphate (500 microM). 4. The L-glutamate-induced inhibition of the electrically evoked contraction was not affected by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)(30 nM), reactive blue 2 (30 microM) or the GABAA receptor antagonist bicuculline (50 microM). However, the GABAB receptor antagonist 2-hydroxysaclofen (50 microM) significantly inhibited the L-glutamate effect. 5. Similar to L-glutamate, GABA also caused a concentration-dependent (0.1-100 microM) inhibition of the EFS contractions. This GABA-induced inhibition was not affected by either the GABAA receptor antagonist bicuculline (50 microM) or reactive blue 2 (30 microM). However, a significant attenuation of the GABA-mediated effect was recorded with the GABAB receptor antagonist 2-hydroxysaclofen (50 microM). Contractions of the vas deferens induced by exogenous ATP and noradrenaline were not affected by GABA (0.1-100 microM). 6. The L-glutamate analogues, N-methyl-D-aspartate (NMDA) (1 microM-1 mM) and quisqualate (Quis 0.1 microM-0.3 mM) had no effect, whilst kainate (Kain, 1 microM-1 mM) caused an inhibition of the EFS-induced contractions. Effects of Kain could be abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dioxine (CNQX, 10 microM). NMDA, Quis and Kain had no effect on the exogenous ATP- or noradrenaline-induced contractions. 7. It is concluded that the excitatory amino acid L-glutamate modulates the electrically evoked vas deferens contraction through conversion to the inhibitory amino acid GABA by a specific GABA transaminase. The GABA formed may then act on GABAB receptors and cause inhibition of the contraction through a presynaptic mechanism.
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PMID:Presynaptic modulation by L-glutamate and GABA of sympathetic co-transmission in rat isolated vas deferens. 876 4

Hypoxia may increase GABA levels in neurons by ATP depletion-induced activation of glutamate decarboxylase and by inhibiting GABA transaminase. Hypoglycemia, which also depletes ATP, reduces neuronal levels of GABA and its precursor glutamate. We examined whether differences in glutamate levels may contribute to these altered GABA levels in hippocampal slices. GABA levels were highly correlated with endogenous glutamate levels during both hypoxia and hypoglycemia (R=0.93 for combined data). Hypoxia maximally increased GABA levels (146+/-6.3% of control, S.E.M.) when glutamate remained above 90% of control levels and ATP was at 30% of control levels. Hypoglycemia with similar ATP levels and glutamate levels at 40% of control decreased GABA levels to 55% of control. Effects of inhibitors of glutamate decarboxylase and GABA transaminase suggested that increased synthesis and decreased catabolism may both contribute to increased hypoxic GABA levels. Immunocytochemical studies suggested that hypoxia increased GABA concentrations primarily in neurons and their processes, but not in glial cells. Severe hypoxic ATP depletion increased the release of both GABA and glutamate. Hypoxia increased GABA levels in neurons, while hypoglycemia with a similar severity of ATP depletion decreased GABA levels. Much of the difference may be related to lower levels of precursor glutamate during hypoglycemia. The twofold higher levels of neuroprotective GABA available for release during hypoxia may contribute to differences in the pathophysiology of these metabolic insults.
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PMID:Glutamate dependence of GABA levels in neurons of hypoxic and hypoglycemic rat hippocampal slices. 1072 84

We have developed defective herpes simplex virus 1 (HSV-1) vectors, based on amplicon plasmids with a replication-deficient mutant, as helper for the transfer of the glutamic acid decarboxylase (GAD67) or beta-galactosidase (beta-gal) gene as control directed by HCMV promoter into neuronal-like cells (PC12) and primary neurons. GAD67 protein was detected immunochemically, while GAD67 activity in virus-producing and nonproducing cell lines was detected enzymatically or by GABA release. Infection with GAD67-expressing amplicon vectors enhanced the resistance of PC12 cells to H(2)O(2). This protection was related to increased energy metabolism, as shown by MTT reduction and ATP level, and involved the GABA shunt, as shown by the reduction in ATP level seen in the presence of gamma-vinyl GABA (GVG), a specific GABA transaminase inhibitor. Level of glutathione (GSH), which requires ATP for its synthesis, was increased by the GAD67 transgene. The activity of glucose-6-phosphate dehydrogenase involved in the maintenance of the NADPH that can be used for the regeneration of the GSH pool, was increased by infection with amplicon vectors. Thus, replication-deficient HSV-1 and the GAD67 transgene have complementary neuroprotective effects and infection with GAD67-expressing amplicon vectors was able to protect nondifferentiated cortical neurons from glutamate toxicity mediated by oxidative stress. Such defective GAD67-expressing HSV-1, as neurotropic vector, should be helpful in neurodegenerative diseases implicating alterations of energy metabolism and oxidative stress in neuronal cells expressing GABA transaminase.
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PMID:Enhancement of neuronal protection from oxidative stress by glutamic acid decarboxylase delivery with a defective herpes simplex virus vector. 1463 8


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