SEARCH · PubMed Health

Results for “ASPARTATE AMINOTRANSFERASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources

PubMed1988

Induction of cytosolic aspartate aminotransferase by glucagon in primary cultured rat hepatocytes.

The activity and the mRNA content of cytosolic aspartate aminotransferase (EC 2.6.1.1) were examined in cultured rat hepatocytes. Addition of glucagon (1 x 10(-7) M) in the presence of dexamethasone (1 x 10(-7) M) caused about 2-fold increase in the activity and mRNA content. Dibutyryl cAMP (1 x 10(-4) M) could replace glucagon for this effect. Maximal induction of cytosolic aspartate aminotransferase mRNA was observed 8 h after their additions. Insulin (1 x 10(-7) M) did not inhibit the enzyme induction by glucagon or dibutyryl cAMP. These results suggest that the cytosolic aspartate aminotransferase gene is regulated by cAMP, and not by insulin.

Animals↗

PubMed1991

The in vitro-synthesized precursor and mature mitochondrial aspartate aminotransferase share the same import pathway in isolated mitochondria.

Both the precursor and the mature form of mitochondrial aspartate aminotransferase were synthesized in a cell-free coupled transcription/translation system directed by the recombinant expression plasmid pOTS-pmAspAT and pOTS-mAspAT, respectively. Both newly synthesized forms of the protein were imported into isolated mitochondria, with the precursor correctly processed to the mature form. In both cases the import process showed resistance to externally added pronase and was abolished in mitochondria treated with the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Moreover the imported products showed the same intramitochondrial localization as judged by a subfractionation procedure. In both cases import was time dependent and was completed in about 15 min. Finally a competitive inhibition of the import of the precursor of aspartate aminotransferase was found due to externally added purified aspartate aminotransferase.

Animals↗

PubMed1989

Kinetic isotope effect studies on aspartate aminotransferase: evidence for a concerted 1,3 prototropic shift mechanism for the cytoplasmic isozyme and L-aspartate and dichotomy in mechanism.

The C alpha primary hydrogen kinetic isotope effects (C alpha-KIEs) for the reaction of the cytoplasmic isozyme of aspartate aminotransferase (cAATase) with [alpha-2H]-L-aspartate are small and only slightly affected by deuterium oxide solvent (DV = 1.43 +/- 0.03 and DV/KAsp = 1.36 +/- 0.04 in H2O; DV = 1.44 +/- 0.01 and DV/KAsp = 1.61 +/- 0.06 in D2O). The D2O solvent KIEs (SKIEs) are somewhat larger and are essentially independent of deuterium at C alpha (D2OV = 2.21 +/- 0.07 and D2OV/KAsp = 1.70 +/- 0.03 with [alpha-1H]-L-aspartate; D2OV = 2.34 +/- 0.12 and D2OV/KAsp = 1.82 +/- 0.06 with [alpha-2H]-L- aspartate). The C alpha-KIEs on V and on V/KAsp are independent of pH from pH 5.0 to pH 10.0. These results support a rate-determining concerted 1,3 prototropic shift mechanism by the multiple KIE criteria [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106]. The large C alpha-KIEs for the reaction of mitochondrial AATase (mAATase) with L-glutamate (DV = 1.88 +/- 0.13 and DV/KGlu = 3.80 +/- 0.43 in H2O; DV = 1.57 +/- 0.05 and DV/KGlu = 4.21 +/- 0.19 in D2O) coupled with the relatively small SKIEs (D2OV = 1.58 +/- 0.04 and D2OV/KGlu = 1.25 +/- 0.05 with [alpha-1H]-L-glutamate; D2OV = 1.46 +/- 0.06 and D2OV/KGlu = 1.16 +/- 0.05 with [alpha-2H]-L-glutamate) are most consistent with a two-step mechanism for the 1,3 prototropic shift for this isozyme-substrate pair.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Transfer of C alpha-hydrogen of glutamate to coenzyme of aspartate aminotransferase during transamination reaction.

During the transamination reaction of mitochondrial aspartate aminotransferase, transfer of tritium from the alpha-position of glutamate to the pro-S position of C4' of pyridoxamine 5'-phosphate was detected. A fast mixing and quenching device had to be used in order to reduce the number of transamination cycles undergone by the enzyme and thus to minimize the accompanying exchange of label with water. The extent of transfer of label (mean value 1.5%; range 0.8-4%) indicates that the 1,3-prototropic shift follows a stepwise rather than a concerted mechanism and that a single acid/base group is responsible for the proton transfer. The actual extent of proton transfer has to be much higher because the rate of alpha-tritium exchange with solvent was only approximately 10% of that of the turnover of unlabeled substrate, reflecting either an isotope effect or a retention of the tritium label in the reaction center during tautomerization. Under the assumption of an isotope effect, the actual transfer may be estimated to be 13%. This value is consistent with the notion of Lys-258 acting as the proton transferring group in which case the maximal value of transfer in an active site not accessible to solvent during the 1,3-prototropic shift would be 33%. However, alternative mechanisms involving Tyr-70 or a water molecule enclosed in the active site serving as acid/base group cannot be excluded on the basis of the present results. Furthermore, in these investigations aspartate aminotransferase was found to catalyze also the exchange of tritium from the beta-position of glutamate, though at a rate 350 times slower than that of the alpha-exchange.

Animals↗

PubMed1995

Accumulation of the quinonoid intermediate in the reaction catalyzed by aspartate aminotransferase with cysteine sulfinic acid.

The pyridoxal phosphate form of aspartate aminotransferase from Escherichia coli catalyzes the irreversible conversion of L-cysteine sulfinate to the pyridoxamine phosphate form of the enzyme, bisulfite, and pyruvate. The addition of L-cysteine sulfinate to a solution containing a high concentration of enzyme (approximately 10 microM) yields a rapidly appearing red color (lambda max = 520 nm) which decays with a rate constant which is only about 1% of kcat (2-3 s-1 versus 250 s-1 at 15 degrees C, pH 7). The red color can be assigned to the quinonoid form of the enzyme substrate complex, which accumulates under these single turnover conditions. The rate of decay of this species is dependent on that for the decomposition of beta-sulfinylpyruvate (beta-SP), the initial product of the reaction between aspartate aminotransferase and L-cysteine sulfinate. Trapping beta-SP with morpholine or malate dehydrogenase plus NADH abolishes the transient red color; therefore, the intermediate accumulates by virtue of the reverse reaction of beta-SP with the pyridoxamine phosphate form of the enzyme. The association and dissociation rate constants of beta-SP with the pyridoxamine-5'-phosphate form of the enzyme are 2 x 10(7) M-1 s-1 and 400 s-1, respectively, at 15 degrees C. No red transient species is observed under these conditions when aspartate is substituted for L-cysteine sulfinate.

Aspartate Aminotransferases↗

PubMed1990

Trigonal crystals of porcine mitochondrial aspartate aminotransferase.

Crystals suitable for X-ray analysis of porcine mitochondrial aspartate aminotransferase in the closed conformation were obtained after the apoenzyme was reconstituted with N-5'-phosphopyridoxyl-L-aspartate, an inhibitor in which the cofactor is covalently bound to the substrate. This results in a crystal form that has not been encountered previously in studies of aspartate aminotransferases. The crystals belong to the trigonal space group P3121 (or the enantiomeric P3221) with unit cell dimensions alpha = b = 202.0 A, c = 58.0 A, alpha = beta = 90 degrees, gamma = 120 degrees and contain one dimer in the asymmetric unit.

Animals↗

PubMed1995

Use of protease sensitivity to probe the conformations of newly synthesised mutant forms of mitochondrial aspartate aminotransferase.

Sensitivity to digestion with pronase has been used to show that the precursor form of mitochondrial aspartate aminotransferase, the form lacking the N-terminal presequence, that with a deletion of the first 9 residues and mutants of the mature enzyme in which residue Cys-166 is mutated to alanine or serine, all retain unfolded conformations after synthesis in a reticulocyte lysate. In the presence of lysed mitochondria the various forms of mitochondrial aspartate aminotransferase retained their susceptibilities to pronase in a way that mirrored the efficiencies with which they are imported into intact mitochondria. The results are interpreted as showing that the presequence of mitochondrial aspartate aminotransferase is not uniquely required for interaction with cytosolic factors required to maintain the newly synthesised protein in a form competent for interacting with, and being imported into, mitochondria.

Alanine↗

PubMed1984

Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure.

Aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme that catalyses the transamination reaction: L-aspartate + 2-oxoglutarate----oxaloacetate + L-glutamate. The enzyme shuttles between its pyridoxal and pyridoxamine forms in a double-displacement process. This paper proposes a mechanism of action that delineates the dynamic role of the protein moiety of this enzyme. It is based on crystallographically determined spatial structures (at 2.8 A resolution) of the mitochondrial isoenzyme in its unliganded forms and in complexes with substrate analogues, as well as on model building studies. The enzyme is composed of two identical subunits, which consist of two domains. The coenzyme is bound to the larger domain and is situated in a pocket near the subunit interface. The proximal and distal carboxylate group of dicarboxylic substrates are bound to Arg386 and Arg292 , respectively, the latter residue belonging to the adjacent subunit. These interactions largely determine the substrate specificity of the enzyme. They not only position the substrate efficient catalysis but also bring about a bulk movement of the small domain that closes the active site crevice and moves Arg386 about 3 A closer to the coenzyme. The replacement of the epsilon-amino group of Lys258 by the alpha-amino group of the substrate in the aldimine bond to pyridoxal phosphate is accompanied by a tilting of the coenzyme by approximately 30 degrees. The released epsilon-amino group of Lys258 serves as a proton acceptor/donor in the 1,3- prototropic shift producing the ketimine intermediate. At this stage, or after hydrolysis of the ketimine bond, the coenzyme rotates back to an orientation between that in the "external" aldimine intermediate and that in the pyridoxal form. Throughout this process, the protonated pyridine nitrogen atom maintains a hydrogen bond to the beta-carboxylate group of Asp222 . Upon formation of the pyridoxamine form, the small domain moves back to its original position. The proposed mechanism is compatible with the known kinetic and stereochemical features of enzymic transamination.

Aspartate Aminotransferases↗

PubMed1979

Monoamine oxidase and aspartate aminotransferase in cellular fractions of thyroid gland in rats after hypophysectomy and TSH stimulation.

UNLABELLED: Monoamine oxidase (MAO) and aspartate aminotransferase activities in cellular fractions of thyroid gland in rats after hypophysectomy and TSH treatment were investigated. MAO and aspartate aminotransferase activities in thyroid mitochondria were decreased after hypophysectomy and significantly increased after daily injection of TSH during five days to hypophysectomized rats. In microsomes after hypophysectomy a similar decrease of MAO activity was found but TSH was without effect on this activity. IN CONCLUSION: it is evident that MAO and aspartate aminotransferase in thyroid mitochondria the enzymes which could be a source for hydrogen peroxide to catalyze thyroid hormone synthesis are under the regulatory influence of TSH.

Animals↗

PubMed1981

Independent quantitation of the mitochondrial and the cytosolic isoenzyme of aspartate aminotransferase in chicken tissues by radioimmunoassays.

Two specific and sensitive radioimmunoassays for the independent determination of the cytosolic and mitochondrial isoenzyme of aspartate aminotransferase from chicken are described. The assays use 125I-labeled aspartate aminotransferases as tracer antigens, rabbit antisera, and goat anti-rabbit IgG antisera for indirect immunoprecipitation. The detection limit for the cytosolic isoenzyme was 177 pg and for the mitochondrial isoenzyme 83 pg. The assays were used for measuring the concentrations of the aspartate aminotransferase isoenzymes in serum, cultured fibroblasts, and various tissues of chicken. Both isoenzymes in the different tissue extracts proved immunologically identical with the corresponding isoenzymes isolated from heart.

Animals↗

PubMed2006

Capillary electrophoresis with electrochemiluminescence detection for measurement of aspartate aminotransferase and alanine aminotransferase activities in biofluids.

A new sensitive assay for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in biofluids was developed, based on the separation and detection of alanine, glutamate, and aspartate using capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. The three amino acids were separated in 5 mM phosphate of pH 2.1 as background electrolyte, and detected on a 500 microm platinum disk electrode at 1.2V (versus Ag/AgCl) in the presence of 10 mM tris(2,2'-bipyridyl)ruthenium(II) dissolved in 80 mM phosphate of pH 10.5. A mass detection limit of 37.3 fmol (or 81.5 fmol) for glutamate, corresponding to the product in the enzyme reaction catalyzed by 1.24 x 10(-9)U AST (or 2.72 x 10(-9)U ALT) in a 30 min reaction period, was achieved. This assay was applied to investigate the cytotoxicity effect of ethanol on HepG2 cells and differentiating nonalcoholic steatohepatitis (NASH) from alcoholic liver disease, indicating that the technique is promising for the application in the cell biological and clinical fields.

Alanine↗

PubMed1990

Quantitative distributions of aspartate aminotransferase and glutaminase activities in the guinea pig cochlea.

Distributions of aspartate aminotransferase and glutaminase activities in the guinea pig cochlea have been examined with use of quantitative microchemical techniques to evaluate their roles in cochlear energy metabolism and neurotransmission. Other enzyme activities analyzed were those of choline acetyltransferase and malate dehydrogenase. It is concluded that aspartate aminotransferase activity appears to be especially concerned with cochlear energy metabolism, while glutaminase activity may function in transmitter metabolism in the guinea pig cochlea. Neither enzyme shows a clear association with the olivocochlear bundle.

Animals↗

PubMed1987

Separate enzymatic microassays for aspartate aminotransferase isoenzymes.

The properties of the cytosolic and mitochondrial isoenzymes of aspartate aminotransferase were studied using a commercial preparation of the cytosolic isoenzyme, a mitochondrial preparation, and whole brain homogenate. Based on these properties, microassays were developed and shown to be highly specific and quantitatively accurate for measuring the activity of either the cytosolic or mitochondrial isoenzyme in microgram quantities of tissue. The assays have been successfully applied to homogenates of a wide variety of tissues. They can be used to measure the activities of aspartate aminotransferase isoenzymes in sub-microgram samples of freeze-dried tissue.

Animals↗

PubMedDate not supplied

[Aspartate aminotransferase from chicken heart cytosol. Characterization of SH-groups].

Homogeneous aspartate aminotransferase has been prepared from chicken heart cytosol. The purification procedure includes fractionation with NH4-sulfate and with ethanol, chromatography on ion-exchange cellulose DE-32 and on hydroxylapatite. Crystallization of the enyme is described. The enzyme was shown to contain 4 SH-groups per protein subunit of molecular weight 50 000. Two of the SH-groups are fully buried, they can be blocked with thiol reagents only upon denaturation of the protein. One exposed SH-group is readily modified at alkaline pH by iodoacetamide, N-ethymaleimide or tetranitromethane, without any inhibition of enzymic activity; this group readily reacts also with 5,5,-ditthiobis (2-nitrobenzoate) and p-mercuribenzoate. One SH-group is semi-buried: it is inaccessible to the above-mentioned reagents at pH 8, but can be blocked by p-mercuribenzoate at pH about 5. Blocking with p-mercuribenzoate of two SH-groups-the exposed and the semi-buried one-lowers enzymic activity to 70% of the initial value. Syncatalytic modication of a SH-group observed in aspartate aminotransferase from pig heart cytosol does not occur in chicken enzyme.

Animals↗

PubMed1983

Catalytic activity of non-cross-linked microcrystals of aspartate aminotransferase in poly(ethylene glycol).

The molar activity of crystalline mitochondrial aspartate aminotransferase is decreased to 10% of that of the enzyme in solution. The activity was measured in suspensions of non-cross-linked microcrystals (average dimensions 22 microns X 5 microns X 0.8 microns) in 30% (w/v) poly(ethylene glycol). Kinetic tests ruled out the possibility that diffusion of the substrate in the crystals is rate-limiting. The observed decrease in catalytic efficiency can be attributed exclusively to crystal-packing effects. A direct inhibition by poly(ethylene glycol) is excluded because poly(ethylene glycol), with average Mr 6000, cannot penetrate the liquid channels of the crystals, owing to its large Stokes radius. The crystals examined were triclinic and of the same habit as those used for high-resolution X-ray-crystallographic analysis [Ford, Eichele & Jansonius (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2559-2563]. The catalytic competence of crystalline aspartate aminotransferase confirms the relevance of the spatial model of this protein for the elucidation of its mechanism of action.

Alanine↗

PubMed1989

[Arg292----Val] or [Arg292----Leu] mutation enhances the reactivity of Escherichia coli aspartate aminotransferase with aromatic amino acids.

Arg292 of E. coli aspartate aminotransferase was substituted with valine or leucine by site-directed mutagenesis. In comparison with the wild-type enzyme, either of the mutant enzymes showed a decrease by over 5 orders of magnitude of kcat/km values for aspartate and glutamate. This supports the contention that Arg292 is important for determining the specificity of this enzyme for dicarboxylic substrates. In contrast, mutant enzymes displayed a 5- to 10-fold increase in kcat/Km values for aromatic amino acids as substrates. Thus, introduction of an uncharged, hydrophobic side chain into position 292 leads to a striking alteration in substrate specificity of this enzyme, thereby improving catalytic efficiency toward aromatic amino acids.

Amino Acid Sequence↗

PubMed1995

Crystal structure of the closed form of chicken cytosolic aspartate aminotransferase at 1.9 A resolution.

The crystal structure of chicken cytosolic aspartate aminotransferase (cAATase; EC 2.6.1.1) has been solved and refined at 1.9 A resolution. Orthorhombic crystals, space group P2(1)2(1)2(1), a = 56.4 A, b = 126.0 A and c = 142.3 A, were grown from polyethylene glycol solutions in the presence of maleate, a dicarboxylic inhibitor that forms a Michaelis-like complex. The pyridoxal form of the enzyme was used for crystallization. Diffraction data were collected using synchrotron radiation. The structure of the new orthorhombic crystal form was solved by molecular replacement using the partially refined 2.8 A resolution structure of the high-salt crystal form as a search model. The final value of the crystallographic R-factor after rigid body and restrained least-squares refinement is 0.175 with very good model geometry. The two 2-fold-related subunits of cAATase have distinct environments in the crystal lattice. Domain movement is strictly hindered by the lattice contacts in one subunit, while the second one possesses conformational freedom. Despite their different environments, both subunits were found in the closed conformation with one maleate molecule tightly bound in each active site. The present study allows a detailed comparison of the highly refined structures of the aspartate aminotransferase isozymes, and thus provide better insight into the role of conserved and variable residues in substrate recognition and catalysis.

Animals↗

PubMed1991

The amino acid sequence of the aspartate aminotransferase from baker's yeast (Saccharomyces cerevisiae).

1. The single (cytosolic) aspartate aminotransferase was purified in high yield from baker's yeast (Saccharomyces cerevisiae). 2. Amino-acid-sequence analysis was carried out by digestion of the protein with trypsin and with CNBr; some of the peptides produced were further subdigested with Staphylococcus aureus V8 proteinase or with pepsin. Peptides were sequenced by the dansyl-Edman method and/or by automated gas-phase methods. The amino acid sequence obtained was complete except for a probable gap of two residues as indicated by comparison with the structures of counterpart proteins in other species. 3. The N-terminus of the enzyme is blocked. Fast-atom-bombardment m.s. was used to identify the blocking group as an acetyl one. 4. Alignment of the sequence of the enzyme with those of vertebrate cytosolic and mitochondrial aspartate aminotransferases and with the enzyme from Escherichia coli showed that about 25% of residues are conserved between these distantly related forms. 5. Experimental details and confirmatory data for the results presented here are given in a Supplementary Publication (SUP 50164, 25 pages) that has been deposited at the British Library Document Supply Centre, Boston Spa. Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

Amino Acid Sequence↗