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At least 559 records · Page 31Linked to original sources
PubMed1980

Blockade of methamphetamine-induced depression of tyrosine hydroxylase by GABA transaminase inhibitors.

Tyrosine hydroxylase (TH) levels in the rat neostriatum are decreased by chronic treatment with methamphetamine. GABAergic neurons could potentially interact with the nigrostriatal dopaminergic neurons in either the neostriatum or the substantia nigra; therefore, the GABA transaminase inhibitors, amino-oxyacetic acid, gamma-acetylenic GABA and ethanolamine-O-sulfate, were evaluated for possible influences on the methamphetamine-induced decrease in TH. TH was measured by the procedure of Nagatsu et al. (1964). Methamphetamine (10 mg/kg, s.c.) was given every 6 h for 24 h. Thirty-six h after initiation of the methamphetamine treatment, neostriatal TH activity was approximately 70% of control. Concurrent administration of amino-oxyacetic acid (20 mg/kg, i.p.) or gamma-acetylenic GABA (15 mg/kg, i.p.) with methamphetamine completely blocked the TH depression. Dose-response curves were constructed for amino-oxyacetic acid and gamma-acetylenic GABA. A single intraventricular injection of ethanolamine-O-sulfate (400 micrograms/rat), 2-6 h before initiating the methamphetamine regimen, also completely blocked the TH depression. These data suggest that the striatonigral or other GABAergic systems are involved in the regulation of the functional state of the nigrostriatal dopaminergic neurons, and that enhanced GABAergic function will antagonize the effects of high doses of methamphetamine.

4-Aminobutyrate Transaminase↗
PubMed1982

Inhibition of acute feeding responses to systemic 2-deoxyglucose or insulin in rats pretreated with the GABA-transaminase blocker ethanolamine-O-sulfate (EOS).

Acute feeding responses to 2-deoxyglucose (750 mg/kg) or insulin (12 U/kg) were examined 24 hr after intracisternal injection of the GABA-transaminase inhibitor ethanolamine-O-sulfate (EOS, 400 micrograms) in female rats. EOS pretreatment completely abolished acute feeding responses to both challenges. These findings complement recent research showing that central EOS can reverse chronic overeating in several experimental preparations. The present results are consistent with previous indications that EOS treatment may induce a metabolic shift away from brain glucose utilization, thus making glucoprivation irrelevant as a metabolic challenge. An alternative possibility is that EOS-induced increases of brain GABA may offset specific neural mechanisms through which these glucoprivic agents normally induce feeding.

4-Aminobutyrate Transaminase↗
PubMed1982

Platelet GABA-transaminase in affective illness.

Platelet gamma-aminobutyric acid (GABA)-transaminase was measured in 18 bipolar patients, 59 normal volunteers, and 9 pairs of monozygotic twins. A significantly lower mean activity was observed in the bipolar group, although lower female bipolar values may account for most of the difference. Enzyme activity was correlated in monozygotic twins, suggesting genetic influences on enzyme activity. Values obtained in the postprandial state were significantly lower than values in the fasting state. These data are discussed in light of other evidence of altered GABA metabolism in affective illness.

4-Aminobutyrate Transaminase↗
PubMed1985

Changes in serum transaminases, SDH and liver morphology after treatment with trypanocidal diamidines in mice.

The effects of 2 recently developed trypanocidal agents, Diamidinophenylindol (DAPI) and Diimidazolinophenylindol (DIPI) at doses of 10-30 micrograms/g intraperitoneally (i.p.) on serum GOT, GPT and sorbitol dehydrogenase (SDH) levels and on liver morphology have been investigated in mice. Pentamidine served as reference drug. Both agents caused dose-dependent increases in serum transaminases and SDH, and discrete morphological changes of the liver, e.g., fatty degenerations, azinoperipheral vacuolisation and alterations of the nuclei, at least at the higher dosage.

Alanine Transaminase↗
PubMed1984

Enzyme-activated inhibition of bacterial D-amino acid transaminase by beta-cyano-D-alanine.

beta-Cyano-D-alanine is an efficient suicide substrate (Ki = 10 microM) of D-amino acid transaminase. This apparent inactivation is temperature dependent: it is irreversible at 10 degrees C or below and becomes progressively reversible at higher temperatures. Since at higher temperatures the apparent reactivation process predominates over the inactivation reaction, the reactivation process is considered to be endothermic. The nature of this reversibility suggests the formation of a heat labile bond between the inhibitor molecule and a nucleophilic group on the enzyme.

Alanine↗
PubMed1998

Fenofibrate modifies transaminase gene expression via a peroxisome proliferator activated receptor alpha-dependent pathway.

Fibrates modify the expression of genes implicated in lipoprotein and fatty acid metabolism via the peroxisome proliferator-activated receptor alpha(PPARalpha), leading to reductions in serum triglycerides and cholesterol. The expression of certain genes regulated by PPARalpha have been shown to be modified in a species dependent manner. Aspartate aminotransferase (AspAT or GOT) and alanine aminotransferase (AlaAT or GPT) are enzymes involved in intermediate metabolism in all cells and in hepatic gluconeogenesis. These enzymes are also widely used as serum markers of possible tissue damage. This study investigated whether fenofibrate could modify the expression of liver AspAT and/or AlaAT and thus possibly alter transaminase levels independently of a cytotoxic effect. In human Hep G2 cells, fenofibrate increased cytosolic AspAT (cAspAT) activity by 40% and AlaAT activity by 100%, as well as both mRNAs. Nuclear run on assays showed that this effect was, at least in part, transcriptional. Increases in mRNA were also observed in human hepatocyte cultures at concentrations of the drug attained in patients. In C57BL/6 mice, fenofibrate decreased cAspAT and cAlaAT mRNA, while these effects were abolished in PPARalpha knock-out mice. In conclusion, fenofibrate has been shown to modify cAspAT and AlaAT gene expression in a species and PPARalpha dependent manner. This is the first demonstration that cAspAT and AlaAT activities may be pharmacologically altered, independently of a toxic phenomenon.

Alanine Transaminase↗
PubMed2003

Modeling and molecular dynamics of glutamine transaminase K/cysteine conjugate beta-lyase.

The homodimeric, pyridoxal 5'-phosphate (PLP)-dependent enzyme glutamine transaminase K/cysteine conjugate beta-lyase (GTK/beta-lyase) has been implicated in the bioactivation of chemopreventive compounds. This paper describes the first homology model of rat renal GTK/beta-lyase and its active site residues, deduced from molecular dynamics (MD) simulations of the binding mode of 13 structurally diverse cysteine S-conjugates and amino acids after Amber-parametrization of PLP. Comparison with Thermus thermophilus aspartate aminotransferase (tAAT) and Trypanosoma cruzi tyrosine aminotransferase (tTAT), used as templates for modeling GTK/beta-lyase, showed that the PLP-binding site of GTK/beta-lyase is highly conserved. Binding of the ligand alpha-carboxylate-group occurred via the conserved residues Arg(432) and Asn(219), and Asn(50) and Gly(70). Two pockets accommodated the various ligand side chains. A small pocket, located directly above PLP, was of a highly hydrophobic and aromatic character. A larger pocket, formed partly by the substrate access channel, was more hydrophilic and notably involved the salt bridge partners Glu(54) and Arg(99*) (* denotes the other subunit). Ligand-binding residues included Leu(51), Phe(71), Tyr(135), Phe(373) and Phe(312*), and pi-stacking interactions were often observed. Tyr(135) and Asn(50) were prominent in hydrogen bonding with the sulfur-atom of cysteine S-conjugates. The observed binding mode of the ligands corresponded well with their experimentally determined inhibitory potency toward GTK/beta-lyase. The current homology model thus provides a starting point for further validation of the role of active site residues in ligand-binding by means of mutagenesis studies. Ultimately, insight in the binding of ligands to GTK/beta-lyase may result in the rational design of new ligands and selective inhibitors.

Amino Acid Sequence↗
PubMed1992

Partial reactions of bacterial D-amino acid transaminase with asparagine substituted for the lysine that binds coenzyme pyridoxal 5'-phosphate.

In bacterial D-amino acid transaminase (EC 2.6.1.21) replacement of Lys-145, which is covalently linked to the coenzyme pyridoxal 5'-phosphate in the wild-type enzyme, by an Asn residue gave a mutant enzyme (K145N) that slowly performed each half-reaction, as determined by spectral measurements. With the wild-type enzyme, the kinetics of these events were so rapid that pre-steady-state conditions were needed for their determination. The internal aldimine between coenzyme and Lys-145 was rapidly reduced with NaCNBH3 in the wild-type enzyme, whereas in the mutant enzyme the coenzyme, which is not covalently linked to the protein, was more resistant to reduction; the reduced forms of both wild-type and mutant enzymes were inactive. With large amounts of the K145N mutant enzyme and either amino acid or keto acid substrate alone, the formation of some reaction intermediates, i.e., the external aldimine with D-alanine and the ketimine with alpha-ketoglutarate, can be measured by conventional spectroscopy. Suicide substrates also induced slow spectral shifts of the E-PLP form of the enzyme. For the K145N enzyme, exogenous amines affected only the rate of the transaldimination but not the removal of the alpha-proton of the substrate. These results suggest that in the mutant enzyme some amino acid side chain other than Lys-145 performs this function. In order to identify this site, the K145N mutant enzyme was completely inactivated by the radiolabeled suicide substrate D-serine. Peptide mapping of tryptic digests showed that Lys-267 was the modified site.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1989

Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase.

Each of the three cysteinyl residues per subunit in D-amino acid transaminase from a thermophilic species of Bacillus has been changed to a glycine residue (C142G, C164G, and C212G) by site-directed mutagenesis. The mutant enzymes were detected by Western blots and a stain for activity. After purification to homogeneity, each mutant protein had the same activity as the wild-type enzyme. Thus, none of the Cys residues are essential for catalysis. Each protein when denatured showed the expected titer of two SH groups per subunit. In the native state, each of the three mutant proteins exhibited nearly the same slow rate of titration of SH groups as the wild-type protein with about one SH group titratable over a period of 4 h. Conversion of Ser-146, adjacent to Lys-145 to which the coenzyme pyridoxal phosphate is bound, to an alanine residue (S146A) does not alter the catalytic activity but has a significant effect on the SH titration behavior. Thus, three to four of the six SH groups of S146A are titratable by DTNB. The rapid SH titration of S146A is prevented by the presence of D-alanine. This finding suggests that the change of Ser-146 to Ala at the active site promotes the exposure and rapid titration of a Cys residue in that region. The rapid SH titration of S146A by DTNB is accompanied by a loss of enzyme activity. Two of the mutant enzymes, C142G and S146A, lose activity at 4 degrees C and also upon freezing and thawing. The mutant enzymes C164G and C212G show the same degree of thermostability as the wild-type enzyme.

Bacillus↗
PubMed1996

Interaction of pyridoxal 5'-phosphate with tryptophan-139 at the subunit interface of dimeric D-amino acid transaminase.

The crystal structure of dimeric bacterial D-amino acid transaminase shows that the indole rings of the two Trp-139 side chains face each other in the subunit interface about 10 angstroms from the coenzyme, pyridoxal 5'-phosphate. To determine whether it has a role in the catalytic efficiency of the enzyme or interacts with the coenzyme, Trp-139 has been substituted by several different types of amino acids, and the properties of these recombinant mutant enzymes have been compared to the wild-type enzyme. In the native wild-type holoenzyme, the fluorescence of one of the three Trp residues per monomer is almost completely quenched, probably due to its interaction with PLP since in the native wild-type apoenzyme devoid of PLP, tryptophan fluorescence is not quenched. Upon reconstitution of this apoenzyme with PLP, the tryptophan fluorescence is quenched to about the same extent as it is in the native wild-type enzyme. The site of fluorescence quenching is Trp-139 since the W139F mutant in which Trp-139 is replaced by Phe has about the same amount of fluorescence as the wild-type enzyme. The circular dichroism spectra of the holo and the apo forms of both the wild-type and the W139F enzymes in the far-ultraviolet show about the same degree of ellipticity, consistent with the absence of extensive global changes in protein structure. Furthermore, comparison of the circular dichroism spectrum of the W139F enzyme at 280 nm with the corresponding spectral region of the wild-type enzyme suggests a restricted microenvironment for Trp-139 in the latter enzyme. The functional importance of Trp-139 is also demonstrated by the finding that its replacement by Phe, His, Pro, or Ala gives mutant enzymes that are optimally active at temperatures below that of the wild-type enzyme and undergo the E-PLP --> E-PMP transition as a function of D-Ala concentration with reduced efficiency. The results suggest that a fully functional dimeric interface with the two juxtaposed indole rings of Trp-139 is important for optimal catalytic function and maximum thermostability of the enzyme and, furthermore, that there might be energy transfer between Trp-139 and coenzyme PLP.

Catalysis↗
PubMed1998

Substrate inhibition of D-amino acid transaminase and protection by salts and by reduced nicotinamide adenine dinucleotide: isolation and initial characterization of a pyridoxo intermediate related to inactivation.

D-Amino acid transaminase, a pyridoxal phosphate (PLP) enzyme, is inactivated by its natural substrate, D-alanine, concomitant with its alpha-decarboxylation [Martinez del Pozo, A., Yoshimura, T., Bhatia, M. B., Futaki, S., Manning, J. M., Ringe, D., and Soda, K. (1992) Biochemistry 31, 6018-6023; Bhatia, M. B., Martinez del Pozo, A., Ringe, D., Yoshimura, T., Soda, K., and Manning, J. M. (1993) J. Biol. Chem. 268, 17687-17694]. beta-Decarboxylation of d-aspartate to d-alanine leads also to this inactivation [Jones, W. M., van Ophem, P. W., Pospischil, M. A., Ringe, D., Petsko, G., Soda, K., and Manning, J. M. (1996) Protein Sci. 5, 2545-2551]. Using a high-performance liquid chromatography-based method for the determination of pyridoxo cofactors, we detected a new intermediate closely related to the inactivation by d-alanine; its formation occurred at the same rate as the inactivation and upon reactivation it reverted to PLP. Conditions were found under which it was characterized by ultraviolet-visible spectral analysis and mass spectroscopy; it is a pyridoxamine phosphate-like compound with a C2 fragment derived from the substrate attached to the C'-4 of the pyridinium ring and it has a molecular mass of 306 consistent with this structure. In the presence of d-serine, slow accumulation of a quinonoid intermediate is also related to inactivation. The inactivation can be prevented by salts, which possibly stabilize the protonated aldimine coenzyme complex. The reduced cofactor, nicotinamide adenine dinucleotide, prevents D-aspartate-induced inactivation. Both of these events also are related to formation of the novel intermediate.

Alanine↗
PubMed2000

Reversible dissociation/association of D-amino acid transaminase subunits: properties of isolated active dimers and inactive monomers.

The crystal structure of dimeric D-amino acid transaminase shows that the two Trp-139 sites are located in a hydrophobic pocket at the interface between the subunits and that the two indole side chains face one another and are within 10 A of coenzyme. This enzyme prefers an aromatic character at position 139, as previously demonstrated by the finding that Phe-139 but no other substitution tested provides the maximum degree of thermostability and catalytic efficiency. Here we show that an equilibrium between active dimers and inactive monomers can be demonstrated with the W139F mutant enzyme, whereas with the wild-type enzyme the subunit interface is so tight that a study of this equilibrium is precluded. We show how the processes of dimerization of monomers and dissociation of dimers to monomers are controlled. Lower pH (5.0) favors monomer formation from dimers. Gel filtration and activity analysis show that at higher pH (7.0) the monomers combine to form active dimers with a K(d) of 0.17 microM. This assembly process is relatively slow and takes several hours for completion, thereby permitting accurate measurement of kinetics and equilibrium parameters. Absorption and circular dichroism spectra of dimers and monomers are significantly different, indicating that the environment around the cofactor is very likely altered between them. The circular dichroism peak of the W139F dimer at 418 nm is less negative than that of the wild-type enzyme in accordance with its lower visible absorbance; the circular dichroism peak of the W139F monomer at 418 nm is more negative than that of the wild-type enzyme. The dissociation of dimers to monomers has also been studied by taking advantage of these spectral differences, thus permitting the rates of the dissociation and the reassociation to be calculated and compared. 2-Mercaptoethanol assists in the conversion of monomers to dimers. The results here describe dissociation/reassociation in the dimeric enzyme under native conditions without denaturants.

Chromatography, Gel↗
PubMed1979

Immunocytochemical localization of gamma-aminobutyric acid transaminase at cellular and ultrastructural levels.

gamma-Aminobutyric acid transaminase (GABA-Tase; 4-aminobutyrate:2-oxaglutarate aminotransferase, EC 2.6.1.19) immunoreactivity in the rat's cerebellum was studied by light and electron microscopy with indirect immunofluorescence and peroxidase-antiperoxidase methods. Evidence is presented for neuronal and neuroglial compartments of GABA-Tase. Labeled neurons included stellate, basket, Purkinje, and Golgi cells of the cortex and a few large neurons in the deep nuclei. Labeled neuroglia included those surrounding Purkinje cells, their radial fibers in the molecular layer, and astrocytes in the granular layer and deep nuclei. No evidence for sagittal microzonation was found. At the ultrastructural level, GABA-Tase immunoreactive sites were localized to cell surface membranes, intracellular organelles, and the cytoplasmic matrix. GABA-Tase immunoreactivity at synapses could be localized precisely to pre- and postsynaptic membranes in gamma-aminobutyric acid (GABA)-containing as well as non-GABA-containing neurons. Specific label was absent from tissues treated with normal rabbit preimmune sera. GABA-Tase labeling was more intense in tissues from animals anesthetized with ether than with barbiturates and after formaldehyde fixation without glutaraldehyde. Increased GABA-Tase immunoreactivity was observed on treatment with colchicine, GABA with oxamic acid, GABA, harmaline, norepinephrine and glutamate, or diazepam (in order of decreasing effectiveness). Serotonin produced no detectable change, and apomorphine and muscimol decreased the immunoreactivity.

4-Aminobutyrate Transaminase↗
PubMed1955

Glutamic-oxaloacetic transaminase activity of serum in mice with viral hepatitis.

Experimental infectious mouse hepatitis is associated with an increase in glutamic-oxaloacetic transaminase activity of the serum (SGO-T). A relationship appears to exist between the rise in SGO-T activity and (a) the size of the virus inoculum, (b) the blood virus titer, and (c) the degree of liver necrosis. Trauma to the liver following partial hepatectomy results in a rise in SGO-T activity in mice. Although mouse hepatitis differs from human hepatitis in the incubation period, histological changes, and natural course, both infections bring about comparable changes in SGO-T activity.

Animals↗
PubMed1979

Mating-type effect on cis mutations leading to constitutivity of ornithine transaminase in diploid cells of Saccharomyces cerevisiae.

Cis-acting regulatory mutations have been isolated that affect L-ornithine transaminase (OTAse), an enzyme catalyzing the second step of arginine breakdown in yeast. These mutations lead to constitutive synthesis of OTAse at various levels. Two different types of mutations have been recovered, both of which are tightly linked to the structural gene (cargB) for this enzyme. One type behaves as a classical operator-constitutive mutation similar to the cargB+O---1 mutation previously described (DUBOIS et al. 1978). The second type is peculiar in two respects: the higher level of constitutive OTAse synthesis and the expression of constitutivity in diploid cells. These mutations are designated cargB+Oh. They behave as usual operator-constitutive mutations in diploid strains homozygous for mating type (a/a or alpha/alpha), but the constitutivity is strongly reduced in a/alpha diploid cells.

Diploidy↗
PubMed1975

Effect of different levels of gossypol on transaminase activity, on nonessential to essential amino acid ratio, and on iron and nitrogen retention in rats.

Metabolic experiments with rats fed rations containing varying levels of free gossypol (from 3 to 109 mg/100 g) showed that nitrogen retention was not affected by gossypol while iron absorption decreased as the levels of gossypol in the ration increased. This in turn resulted in lower hematocrit and hemoglobin values and lower levels of iron in the liver. The levels of glutamic-oxaloacetic and glutamic-pyruvic transaminases, an indication of liver necrosis, increased in blood serum and decreased in liver when gossypol was fed. The ratio of nonessential to essential amino acids in both serum and liver increased with increasing levels of gossypol in the diet showing that, in spite of an equalized available lysine intake, the cottonseed pigment was capable of binding this and/or other essential amino acids. In all cases, weight gain was adversely affected by the level of gossypol used.

Alanine Transaminase↗
PubMed1994

Purification and amino- and carboxyl-terminal amino acid sequences of alanine-glyoxylate transaminase 1 from human liver.

In order to confirm the amino acid sequence predicted from the nucleotide sequence of cDNA and also to elucidate the intracellular localization and molecular evolution, human liver alanine-glyoxylate transaminase 1 (AGT1) was purified and subjected to partial amino acid sequence determination, with special attention to posttranslational modification. The enzyme was purified to homogeneity from the 10,000 x g supernatant of human liver homogenate. The purified enzyme showed only a single protein band at about 43 kDa on SDS-PAGE, indicating that it is a homodimer of two identical subunits, because the native enzyme has a molecular mass of about 80 kDa. Both the amino- and carboxyl-terminal peptides of the enzyme were isolated from a cyanogen bromide digest of the S-carboxyl-methylated protein and subjected to amino acid sequence determination. The alpha-amino group of the amino-terminal peptide was shown to be blocked by an acetyl group. The carboxyl-terminal sequence contained a putative N-glycosylation sequence (-Asn-Ala-Thr-), the only one present in the whole molecule, but this sequence was normally determined, indicating that the enzyme is not N-glycosylated. Purdue et al. [J. Cell Biol. 111, 2341-2351 (1990)] have reported that Pro-11, Gly-170, and Ile-340 in normal human AGT1 were replaced by Leu, Arg, and Met, respectively, in a patient with primary hyperoxaluria type 1. We confirmed that residue-11 was Pro. Both the amino- and carboxyl-terminal sequences of the enzyme showed extensive similarity with those of rat liver mitochondrial serine-pyruvate aminotransferase and the small chain of hydrogenase from a thermophilic unicellular cyanobacterium, Synechococcus PCC 6716.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine Transaminase↗