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PubMed1989

Isolation and properties of a liver mitochondrial precursor protein to aspartate aminotransferase expressed in Escherichia coli.

The precursor to rat liver mitochondrial aspartate aminotransferase has been expressed in Escherichia coli JM105 using the pKK233-2 expression vector. This mammalian natural precursor has been isolated as a soluble dimeric protein. The amino-terminal sequence and the amino acid composition of the isolated protein correspond to those predicted from the inserted cDNA (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). The isolated precursor contains bound pyridoxal phosphate and shows catalytic activity with a specific activity equal to that of the mature form of the enzyme. This precursor can also be processed by mitochondria into a form with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of mature enzyme. The isolation of this precursor as a stable and catalytically active entity indicates that the presequence peptide does not necessarily interfere with much of the folding and basic structural properties of the mature protein component.

Animals↗

PubMed1976

Kinetics of the interaction between aspartic aminotransferase and anions.

The kinetics of the interaction between deionized supernatant aspartic aminotransferase and various anions (cacodylate, phosphate and chloride) were studied by the temperature-jump technique. The anion concentration in the range covered by our experiments does not affect the transamination rate. On the other hand the conformational transition, recently observed at the active site of the enzyme, is hindered by an excess of anions. A single relaxation effect was observed at the enzyme chromophore wavelength in systems containing the aldimine form of the enzyme and the above anions. It is shown that this effect corresponds to the protonation of the chromophore. The relaxation times were of about 10 mus with phosphate, 20-100 mus with cacodylate and 1-2 ms with chloride. The pH and concentration dependence of this effect were studied. The fits of experimental data to a rate equations for various models were tested by a chi2 analysis. The best fit was obtained with models where anions bind rapidly to a site close to the chromophore, so that the pK of the chromophore is affected by anions binding. The rate of the observed relaxation considerably increased when the anion has buffering capacities; this indicates, in the case of cacodylate and phosphate, that the acidic component of the buffer directly exchanges a proton with the enzyme chromophore.

Animals↗

PubMed1990

Hormonal discrimination among transcription start sites of aspartate aminotransferase.

The promoter of the gene coding for the rat cytosolic aspartate aminotransferase was cloned from a Charon 4A genomic library. We have sequenced a 1.1-kilobase PstI-PstI fragment which contains the first exon of the gene, the beginning of the first intron and 682 base pairs of the 5' regulatory region (+1 being the A of the first ATG codon), which exhibits promoter activity. The promoter region is G + C rich, does not include any TATA-like element, but has 4 putative Sp1-binding sites and 6 regularly spaced CCAAT boxes. The promoter activity of the 5' regulatory region, as well as its sensitivity to glucocorticoids, were assessed by transient gene expression assays after fusion to the chloramphenicol acetyltransferase gene in the hepatoma cell lines HepG2 and Fao. Multiple transcription start sites were found on the gene over a short distance (55 base pairs), but they were differentially regulated by glucocorticoids as determined by both primer extension analysis and S1 mapping. In particular, transcription from 2 start sites was increased 15- to 18-fold, whereas transcription from the 3 other ones was increased 3-fold. In addition, three new start sites, below the detection limit in control cells, were highly induced. Therefore, a hormonal regulatory element can discriminate among closely related transcription start sites.

Animals↗

PubMed2005

Electron paramagnetic resonance and fluorescence studies of the conformation of aspartate aminotransferase bound to GroEL.

The interaction of the precursor to mitochondrial aspartate aminotransferase (pmAAT) with GroEL has been studied by electron paramagnetic resonance (EPR) and fluorescence spectroscopy. In the native protein, the spin probe was immobilized when attached to Cys166 at the domain interface, but was fully mobile when introduced at Cys(-19) in the N-terminal presequence peptide. Unfolding of the protein resulted in a highly mobile EPR spectrum for probes introduced at either site. However, the nitroxide group in GroEL-bound pmAAT showed either intermediate or high mobility depending on the spin probe used. Power saturation experiments indicated that the accessibility of the nitroxide side chain to Ni(EDDA) in the GroEL-pmAAT complex was higher than in the native state when in position 166 but lower when at position -19. Similar results were obtained in fluorescence quenching experiments. These data suggest that GroEL binds partly folded states of pmAAT with the presequence peptide probably in direct contact with GroEL. GroES and ATP, but not AMP-PNP or ADP, support refolding of pmAAT. During refolding, the rate of recovery of the native spectroscopic properties of labeled Cys166 is nearly identical to the rate-limiting reactivation step. Thus, correct docking of the large and small domains of pmAAT may be a key structural event in the regain of catalytic activity.

Aspartate Aminotransferases↗

PubMed1993

Regulation of the cytosolic aspartate aminotransferase housekeeping gene promoter by glucocorticoids, cAMP, and insulin.

The cytosolic aspartate aminotransferase (cAspAT) is a ubiquitous enzyme that displays liver-specific hormonal regulation. In the hepatoma cell line Fao, both the activity and the mRNA level of cAspAT are increased by glucocorticoids. This effect is potentiated by cAMP and inhibited by insulin. Using in vivo run-on experiments, we showed that these effectors act at the transcriptional level. A cAspAT gene fragment containing 2405 bp of the promoter was sequenced. Deletion fragments of this promoter were inserted upstream of the CAT gene, and the regulation of their activity was assayed following transfection in Fao cells. Stable transfection experiments established that the construct including the entire 2.405-kb fragment undergoes positive regulation by glucocorticoids and cAMP and negative regulation by insulin similar to the regulation of the endogenous gene. A physical separation of the positive and negative control elements is suggested by the fact that cAMP acted on the -682/-26-bp fragment (a 2-fold increase of the stimulation by dexamethasone), whereas the negative regulation by insulin (50% of the stimulation by dexamethasone) required the -1983/-1718-bp fragment. Both regions were required for maximal glucocorticoid activity (6-9-fold increase of CAT activity). We conclude that at least two regulatory regions, a proximal and a distal one, are required for full hormonal regulation of the cAspAT gene.

Animals↗

PubMed1986

Identification of coenzyme aldimine proton in 1H NMR spectra of pyridoxal 5'-phosphate dependent enzymes: aspartate aminotransferase isoenzymes.

The pyridoxal form of the alpha subform of cytosolic aspartate aminotransferase (EC 2.6.1.1) is fully active and binds pyridoxal 5'-phosphate via an aldimine formation with Lys-258 whereas the gamma subform is virtually inactive and lacks the aldimine linkage. Comparison of 1H NMR spectra between the alpha and gamma subforms suggested that peak 1 of the alpha subform at 8.89 ppm contains a resonance assignable to the internal aldimine 4'-H. Reaction with a reagent that cleaves or modifies the internal aldimine bond [(amino-oxy)acetate, L-cysteinesulfinate, NH2OH, NaBH4, or NaCNBH3] caused the disappearance of a resonance line at 8.89 ppm that possessed a broad line width and corresponded in intensity to a single proton. These reagents were also used successfully for the identification of the aldimine 4'-H resonance in the mitochondrial isoenzyme. In contrast to the cytosolic isoenzyme whose resonance for the 4'-H did not show any detectable change in chemical shift with pH, the corresponding resonance in the mitochondrial isoenzyme exhibited pH-dependent chemical shift change (8.84 ppm at pH 5 and 8.67 ppm at pH 8) with a pK value of 6.3, reflecting the interisozymic difference in the microenvironment provided for the internal aldimine. Validity of the signal assignment was further shown by the two findings: the resonance assigned to the 4'-H emerged upon conversion of the pyridoxamine into the pyridoxal form, and the resonance appeared upon reconstitution of the apoenzyme with [4'-1H]pyridoxal phosphate but not with [4'-2H]pyridoxal phosphate.

Aminooxyacetic Acid↗

PubMed1997

Macroaspartasemia as a cause of isolated elevation of aspartate aminotransferase--its biochemical and physiological characteristics.

OBJECTIVES: The increase of serum aspartate aminotransferase (AST) is generally found in hepatic, cardiac, muscular disease and hemolytic disorders of the red blood cell (RBC). The elevation of its activity is suspected in pathological conditions of these organs. However, instances without any of those conditions rarely exist. METHODS: The experimental samples were obtained from a normal person's hemolysed RBC, a hepatitis patient and a macroaspartatemic female's serum. They were studied with exclusion chromatography, electrophoresis of AST and changes of AST activity due to Polyethylene Glycol (PEG) and various conditions on storage. RESULTS: 1) The patterns of AST activity by exclusion chromatography are similar to the hemolysed RBC and the hepatitis's serum but differs by the isolated AST elevation. 2) The AST activity with addition of PEG and different anti-immunoglobulin subtypes to different serums are slightly decreased in hepatitis but markedly decreased with PEG and anti-IgG in macroaspartatemia. 3) The patterns of AST activity in electrophoresis are single band-cytosomal AST (cAST)-from hemolysed RBC and two bands-mitochondrial AST (mAST) and cAST-from hepatitis, the major being cAST and the minor mAST. Even though there are two bands, the major one is atypical and the minor corresponds to mAST in macroaspartatemia. 4) The changes of AST activity on storage according to time and temperature show to be stable over 4 weeks at room temperature and cooled condition, and 9 weeks under frozen state in macroaspartatase. CONCLUSION: Concluding from the above findings, macroaspartatemia is an enzyme-immunoglobulin complex composed of cAST with IgG. MacroAST might be stabler than usual AST at physical conditions.

Adult↗

PubMed1991

A longitudinal study of aspartate aminotransferase in human gingival crevicular fluid.

Previous studies have shown that aspartate aminotransferase (AST), an established serum marker for cardiac and liver damage in humans, appears in elevated concentrations in samples of gingival crevicular fluid (GCF) from ligated vs. non-ligated teeth in beagle dogs and in elevated quantities in cross-sectional GCF sampling, adjusted for collection time, from human sites with clinical signs of past or present periodontal disease as compared to healthy sites. This paper describes a longitudinal study in which AST was monitored quarterly over a 2-year period at 2 sites/tooth in 31 patients with mild to moderate adult periodontitis. In this study sample, 40 (2.6%) of 1536 sites exhibited confirmed loss of at least 2 mm of attachment during the 2-yr observation period. In comparison with healthy sites within the same patients, AST standardized to a 30-second collection interval (AST30) was elevated at these sites with new confirmed attachment loss, and at sites with past attachment loss or gingivitis in the absence of periodontitis. When both within- and between-patient variation were taken into account, observed odds-ratios associating enzyme with disease were higher for sites with new attachment loss (9-16 depending on test cut-point) than for sites with pre-study attachment loss (3-12), or gingivitis in the absence of periodontitis (5-8). AST in GCF is strongly related to human periodontal disease. The data are consistent with the hypothesis that the relationship is strongest during episodes of cumulative tissue breakdown, but the small numbers of sites with confirmed attachment loss during the study period, or with gingivitis in the absence of periodontitis, means that further clinical studies are necessary to clarify this issue.

Adult↗

PubMed2003

Reaction of aspartate aminotransferase with C5-dicarboxylic acids: comparison with the reaction with C4-dicarboxylic acids.

The reaction of Escherichia coli aspartate aminotransferase (AspAT) with glutamate and other C5-dicarboxylates was analyzed in order to compare its mechanism of action toward C5 substrates with that toward C4 substrates, which had been extensively characterized. The association of the amino-group protonated and unprotonated forms of glutamate (SH(+) and S, respectively) with the Schiff-base protonated and unprotonated forms of the enzyme (E(L)H(+) and E(L), respectively) yields at least three forms of the Michaelis complex, whereas in the case of aspartate, only two species of this complex exist, E(L).SH(+) and E(L)H(+).S. The reaction of AspAT with 2-methylglutamate can be explained only when we consider all the protonation states of the Michaelis complex. Based on the previous crystallographic studies [Miyahara et al. (1994) J. Biochem. 116, 1001-1012], we consider that glutamate binds to the open form of AspAT and takes an extended conformation in the Michaelis complex, with the alpha-amino group of glutamate oriented in the opposite direction to the Schiff base. This is in contrast to the Michaelis complex of aspartate, in which a strong interaction of the alpha-amino group of aspartate and the Schiff base excludes the presence of the species E(L)H(+).SH(+). It is concluded that AspAT recognizes the two types of dicarboxylates with different chain lengths by changing the gross conformation of the enzyme protein.

Aspartate Aminotransferases↗

PubMed1996

Inactivation of brain and kidney aspartate aminotransferases by S-(1,2,-dichlorovinyl)-L-cysteine and by S-(1,1,2,2,-tetrafluoroethyl)-L-cysteine.

Long-term exposure to trichloroethylene can cause kidney cancer in experimental animals and humans. In addition, dichloroacetylene (a breakdown product of trichloroethylene) is nephrotoxic and neurotoxic. Both trichloroethylene and dichloroacetylene are metabolized in part to the corresponding cysteine S-conjugate (i.e. S-(1,2-dichlorovinyl)-L-cysteine) which is toxic. Cysteine S-conjugate beta-lyases convert S-(1,2,dichlorovinyl)-L-cysteine to pyruvate, ammonia and a reactive fragment that adds to macromolecules, depletes cellular thiols and causes lipid peroxidation. We now show that S-(1,2-dichlorovinyl)-L-cysteine and another nephrotoxic cysteine S-conjugate, S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, inactive purified cytosolic aspartate aminotransferase and purified alanine aminotransferase. These cysteine S-conjugates also inactive aspartate aminotransferase in cytosolic and mitochondrial fractions of rat brain and kidney. The present results suggest that some halogenated xenobiotics may be toxic in part through their conversion to the corresponding cysteine S-conjugate which inactivates key pyridoxal 5'-phosphate-containing enzyme.

Alanine Transaminase↗

PubMed1980

Translocation of mitochondrial aspartate aminotransferase through mitochondrial inner membrane. A cross-linking study with dimethyladipimidate.

Mitoplasts isolated from rat liver mitochondria were treated with dimethyladipimidate, a bifunctional alkylating agent. This agent causes, concurrently with modification of amino groups, loss of osmotic response. It was found that after cross-linking, the movement effector, succinate, was unable to induce the aspartate aminotransferase release from mitoplasts. In contrast, dimethyladipimidate-treated mitoplasts were still able to internalize 125I-labeled aspartate aminotransferase upon removal of exogenous succinate. The possible involvement of membrane asymmetry in the mechanism of translocation of porteins through the inner mitochondrial membrane is discussed.

Animals↗

PubMed1996

Aspartate aminotransferase complexed with erythro-beta-hydroxyaspartate: crystallographic and spectroscopic identification of the carbinolamine intermediate.

The crystal structure of mitochondrial aspartate aminotransferase (mAAT) of chicken complexed with erythro-beta-hydroxyaspartate has been determined at 2.4 A resolution. Pregrown crystals of mAAT complexed with the inhibitor maleate (closed enzyme conformation, orthorhombic space group C222(1)) were soaked in solutions of erythro-beta-hydroxyaspartate. The ligand exchange was monitored by microspectrophotometry. The active site turned out to be predominantly occupied by the carbinolamine intermediate. The carbinolamine is a true intermediate of the catalytic cycle forming the last covalently bound enzyme:substrate complex before release of the keto acid product. Occupancies of approximately 80% for the carbinolamine and of approximately 20% for the quinonoid intermediate were obtained. Two hydrogen bonds were identified that are potentially relevant for the accumulation of the carbinolamine intermediate: one to the hydroxyl group of Tyr 70* and the other to the epsilon-NH2 group of Lys 258.

Animals↗

PubMed1989

Beta-DL-methylene-aspartate, an inhibitor of aspartate aminotransferase, potently inhibits L-glutamate uptake into astrocytes.

[3H]Glutamate uptake into astrocytes in primary culture was potently inhibited by the aspartate analogues L- and D-aspartic acid, DL-threo-beta-hydroxy-aspartic acid-beta-hydroxymate (IC50's: 136, 259, 168, and 560 microM, respectively) and by beta-DL-methylene-aspartate, a suicide inhibitor of aspartate aminotransferase (IC50: 524 microM), and by the endogenous sulphur-containing amino acid L-cysteinesulfinic acid (IC50: 114 microM), [3H]Glutamate uptake was not significantly affected by either N-methyl-D-aspartate or DL-homocysteine thiolactone. These results demonstrate that other excitatory amino acids including aspartate and L-cysteinesulfinic acid (but excluding L-homocysteic acid) interact with the glutamate transport system of astrocytes. Inhibition of glutamate uptake may significantly increase the level of neuronal excitability.

Amino Acid Transport System X-AG↗

PubMed1995

Aspartate aminotransferase and glutamate dehydrogenase activities in the squid giant nerve.

The present study sought to investigate the presence and distribution of some enzymatic activities involved in the metabolism of glutamate in the giant nerve fiber of the tropical squid Sepioteuthis sepioidea. Specific activities of aspartate aminotransferase and glutamate dehydrogenase were evaluated in homogenates of the isolated giant fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. The activities of both enzymes were present in the tissue. The specific activity of aspartate aminotransferase was similar in axoplasm and sheaths. However, the specific activity of glutamate dehydrogenase was an order of magnitude higher in the sheaths. This finding is discussed in the framework of the hypothesis that proposes that a differential distribution of the enzymes of the glutamatergic system between the axonal and neuroglial compartments forms part of a system of communication between these cells whose neuronal signal may be glutamate.

Animals↗

PubMed1990

Purification and properties of L-aspartate aminotransferase of Chlamydomonas reinhardtii.

An enzyme which catalyzes the transamination of L-aspartate with 2-oxoglutarate has been purified 400-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. An apparent relative molecular mass of 138,000 was estimated by gel filtration. The enzyme is a dimer consisting of two identical subunits of Mr 65,000 each as deduced from PAGE/SDS studies. A stoichiometry of two molecules pyridoxal 5-phosphate/enzyme molecule was calculated. The enzyme has an isoelectric point of 8.48 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-aspartate. L-Aspartate or pyridoxal 5-phosphate, but not 2-oxoglutarate, protected the enzyme from heat inactivation. The purified enzyme was able to transaminate, although to a low extent, L-phenylalanine and L-tyrosine with 2-oxoglutarate, and L-serine, L-alanine and L-glutamine with oxaloacetate. L-Aspartate aminotransferase exhibited hyperbolic kinetics for 2-oxoglutarate and oxaloacetate, and nonhyperbolic behaviour for L-aspartate and L-glutamate. Apparent Km values were 0.55 mM for 2-oxoglutarate, 0.044 mM for oxaloacetate, 2.53 mM for L-aspartate and 3.88 mM for L-glutamate. Transamination of L-aspartate in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.

Aspartate Aminotransferases↗

PubMed1995

Reaction of aspartate aminotransferase with L-erythro-3-hydroxyaspartate: involvement of Tyr70 in stabilization of the catalytic intermediates.

The reaction of Escherichia coli aspartate aminotransferase (AspAT) with L-erythro-3-hydroxyaspartate (HOAsp) produces an intense absorption at 494 nm (epsilon = 13,650 M-1 cm-1), which is ascribed to the quinonoid intermediate. However, when Tyr70 of AspAT has been replaced by Phe, the enzyme shows only a faint absorption at 494 nm (epsilon = 522 M-1 cm-1) on the reaction with HOAsp. This indicates the involvement of the hydroxy group of Tyr70 in stabilizing the quinonoid intermediate formed from HOAsp and pyridoxal 5'-phosphate at the AspAT active site. Kinetic analysis of the absorption changes of the wild-type and Y70F mutant AspATs has shown that the reactions with HOAsp conform to the equation, EL + S<-->ES1<-->ES2<-->ES3<-->EM + P, in which there is a rapid formation of the quinonoid intermediate (ES2) from ES1, followed by a slow equilibrium between ES2 and ES3. ES3 absorbs primarily at 330 nm. The kinetic parameters for individual steps have been determined, and free energy profiles for the reactions of the two enzymes with HOAsp have been obtained. The stability of the quinonoid intermediates of the two enzymes in the normal catalytic reactions with aspartate has been assessed by static measurement of the spectra in the presence of both aspartate and oxalacetate, and the free energy profiles for the reactions have been similarly obtained. Comparison of the free energy levels in the profiles showed that the interaction of the beta-hydroxy group of HOAsp with the hydroxy group of Tyr70 accounts for 8.7 kJ mol-1 of the 18.5 kJ mol-1 stabilization of the quinonoid intermediate by the beta-hydroxy group. Model building of the active site of AspAT complexed with HOAsp suggests that the rest of the stabilization is mediated through the interaction of the beta-hydroxy group of HOAsp with the protonated epsilon-amino group of Lys258. This interaction is expected to strengthen the hydrogen-bonding network involving Tyr70, HOAsp, and the coenzyme phosphate. A similar network is possibly formed in the carbinolamine intermediate, suggesting ES3 to be the carbinolamine. A mechanism for the reaction of AspAT with HOAsp, which conforms to all the kinetic and spectroscopic data presented here, is proposed. This study provides a basis for subsequent spectroscopic characterization of the HOAsp-AspAT complex, which is a good model for the critical intermediate (quinonoid) structure of the AspAT-catalyzed reactions.

Amino Acid Sequence↗

PubMed1977

Intramitochondrial intermembranal reversible translocation of aspartate aminotransferase and malate dehydrogenase through the inner mitochondrial membrane.

The translocation of aspartate aminotransferase, malate dehydrogenase, and bulk protein from the rat liver inner mitochondrial membrane and matrix toward the intermembranal space induced by certain organic acids (movement effectors) has been studied. Experiments involving a two-stage dissolution of the mitochondrial membranes by the use of detergents strongly suggest that enzymes like aspartate amino-transferase can cross the inner mitochondrial membrane providing exogenous movement effector was present. Experiments which measured the changes in membranal distribution of malate dehydrogenase induced by the movement effectors also suggested the occurrence of a similar phenomenon for this enzyme in intact mitochondria. Control experiments revealed that under our experimental conditions, the inner mitochondrial membrane remained impermeable to small molecules, e.g., sucrose, and that the release of aspartate aminotransferease, malate dehydrogenase, insocitrate dehydrogenase, and bulk protein into the intermembranal space in the presence of succinate occurred at a much lower concentration of digitonin than that required to disrupt the inner mitochondrial membrane.

Animals↗

PubMed1984

Aspartate aminotransferase in Leishmania is a broad-spectrum transaminase.

The substrate specificity of aspartate aminotransferase (ASAT, E.C. 2.6.1.1.) from Leishmania was examined following observations of artefacts on gels stained for alanine aminotransferase (ALAT, E.C. 2.6.1.2.) after thin-layer starch-gel electrophoresis. Leishmanial ASAT acted on L-aspartate, L-alanine, L-tryptophan and L-tyrosine. Interpretation of ALAT zymograms must thus take into account the presence of interfering ASAT bands, and the need is emphasized for rigorous controls in isoenzyme electrophoresis.

Alanine Transaminase↗