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PubMed1996

An aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8.

The aspartate aminotransferase gene (AspAT, EC 2.6.1.1) of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced, and its gene product was overproduced. The purified T. thermophilus AspAT was stable up to about 80 degrees C at neutral pH. T. thermophilus AspAT was strictly specific for acidic amino acid substrates, such as aspartate, glutamate, and the respective keto acids. The gene coding for T. thermophilus AspAT showed that it comprised 1,155 bp with a high G+C content (70 mol%), and encoded a 385-residue protein with a molecular weight of 42,050. The amino acid sequence of T. thermophilus AspAT deduced from its gene showed about 15, 46, and 29% homology with those from Escherichia coli, Bacillus sp. YM-2, and Sulfolobus solfataricus, respectively. When the amino acid sequence of T. thermophilus AspAT was compared with that of E. coli AspAT, the number of Cys was found to have decreased from 5 to 1, that of Asn from 23 to 9, that of Gln from 16 to 8, and that of Asp from 20 to 13, all of which are known to be relatively labile at high temperatures. Conversely, the number of Pro was increased from 15 to 25, Arg from 22 to 32, and Glu 27 to 37. As shown by the E. coli AspAT structure, there was a marked tendency for the extra prolyl residues to be located around the surface of the molecule. This was quite different from that in the case of RecA protein, which shows an increased number of prolyl residues in the interior of its molecule. Different strategies of different proteins as to prolyl contribution to thermostability have been suggested. Despite the high degree of conservation of active-site residues, Arg292 in E. coli AspAT, which interacts with the distal carboxylate of the substrate, was not found in T. thermophilus AspAT. Arg89 may complement the function of Arg292.

Amino Acid Sequence↗

PubMed1985

The role of metal ions in the uptake of aspartate aminotransferase and malate dehydrogenase into isolated rat liver mitochondria in vitro.

To gain further insight into the mitochondrial receptor area which allows selective uptake of both purified aspartate aminotransferase and malate dehydrogenase into mitochondria, the inhibition of metal complexing agents such as bathophenanthroline and tiron on the uptake of both enzymes has been investigated. In view of the nature of the inhibition found, we propose the existence of metal ion(s) at or near the aspartate aminotransferase, but far from the malate dehydrogenase binding site.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed2002

Inhibition of aspartate aminotransferase by glycation in vitro under various conditions.

Incubation of 50 mM D-glucose with aspartate aminotransferase (AST, EC 2.6.1.1) preparations (purified pig heart enzyme or a rat liver 20,000 x g supernatant) at 25 degrees C had no effect on enzyme activity. 50 mM D-fructose or D-ribose gradually inhibited pig heart AST under the same conditions to zero activity after 14 days. 50 mM DL-glyceraldehyde decreased enzyme activity to zero after 6 days of incubation. The inhibition of pig heart AST by 50 mM D-fructose or D-ribose was marked even at a temperature of 4 degrees C but it was less pronounced than at 25 degrees C. There was no effect of 0.5 mM 2-oxoglutarate on AST activity during incubation, while the presence of 25 mM L-aspartate decreased it rapidly. 0.5 mM 2-oxoglutarate partly prevented inhibition of AST by D-ribose or D-fructose, while an analogous experiment with 25 mM aspartate resulted in a rapid decline similar to that in the absence of sugars.

Animals↗

PubMedDate not supplied

[Aspartate aminotransferase activity in the brain and spinal cord in acute and chronic alcoholic intoxication].

Resynthesis of aspartate via glutamate was decreased 6-8-fold in rat brain spinal cord tissues due to distinct inhibition of aspartate aminotransferase in acute alcohol intoxication. Consumption of glutamic acid was distinctly increased 1.5-2-fold in tissues of the central nervous system but its metabolism was altered. Normalization occurred within 12-24 hrs after the intake of alcohol. In the state of chronic alcohol intoxication the enzymatic activity was marked increased in brain and spinal cord; in this case the response to a single alcohol administration was altered as compared with acute alcohol intoxication.

Alcoholic Intoxication↗

PubMed1990

Aspartate aminotransferase: investigation of the active sites.

An investigation of the crystal structure of cytosolic pig-heart aspartate aminotransferase (AAT, E.C.2.6.1.1) was carried out to determine the structural requirements for ligand recognition by the active site. Structural differences were observed between the two active sites of the AAT dimer. The natural ligand, L-aspartate, was docked into both active sites using various methods. However, due to structural differences, the ligand was able to form all the necessary interactions for initial binding in only one of the active sites. The program GRID (P.J. Goodford, J. Med. Chem. 1985, 28, 849-857) was used to predict favorable binding sites for the functional groups of the aspartate ligand. These binding sites corresponded to the position of the docked aspartate ligand, indicating that substrate recognition takes place before any major conformational changes occur within the enzyme.

Animals↗

PubMed1981

Inhibition by cycloserine of mitochondrial and cytosolic aspartate aminotransferase in isolated rat hepatocytes.

Isolated hepatocytes were incubated with L-cycloserine and then treated with digitonin so that mitochondrial and cytosolic fractions were obtained in 5 s. Mitochondrial and total cellular aspartate aminotransferases (EC 2.6.1.1) were inactivated in parallel. The enzyme was also inhibited in isolated mitochondria incubated with L-cycloserine. These results, in contrast with previous reports, indicate that cycloserine reacts equally with mitochondrial and cytosolic aspartate aminotransferases.

Animals↗

PubMed1990

Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence.

The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm). The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme. However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1. Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization.

Animals↗

PubMed1998

Recombinant expression, purification, and characterization of three isoenzymes of aspartate aminotransferase from Arabidopsis thaliana.

Five different genes encoding isoenzymes of aspartate aminotransferase (AAT) have been identified in the plant Arabidopsis thaliana. cDNA sequences encoding three of these AAT isoenzymes, asp1 (mitochondrial), asp2 (cytosolic), and asp5 (plastid), were manipulated into bacterial expression vectors and the recombinant proteins expressed were purified from liquid culture using conventional methods. Yields of the purified isoenzymes varied from 11.5 mg/g wet wt cells (AAT5) to 0.95 mg/g wet wt cells (AAT2), an improvement of more than 1000-fold over typical yields of native isoenzymes obtained from plant tissues of other species. Analysis of the recombinant proteins on denaturing PAGE gels indicated subunit Mrs of between 44 and 45 K. Kinetic parameters (Km and kcat) obtained for all four substrates (aspartate, alpha-ketoglutarate, glutamate, and oxaloacetate) were consistent with values obtained for native AAT isoenzymes from other plant species. Further characterization of the purified recombinant enzymes alongside native enzymes from A. thaliana leaf tissue on AAT activity gels confirmed the identity of asp1 and asp2 as the mitochondrial and cytosolic AAT genes but indicated that asp5 may encode an amyloplastic rather than the chloroplastic enzyme.

Amino Acid Sequence↗

PubMed1987

Aspartate aminotransferase and alanine aminotransferase serum activities in small-for-date newborns.

The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum activities were evaluated in the first 12 hrs of life in 26 newborns small-for-gestational age, and correlated with birth weight and gestational age. We found a positive correlation between the ALT serum activity and both birth weight and gestational age of newborns small-for-gestational age. Therefore, the serum activity of neonatal ALT better reflects intrauterine fetal growth in relation to cellular enzymatic pool increase of small-for-date newborns.

Alanine Transaminase↗

PubMed1986

Effects of temperature on measurement of aspartate aminotransferase and alanine aminotransferase in commercial control sera.

The catalytic activity concentrations of aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2) in several commercial control sera were determined at three different temperatures. Although these sera were of various biological origins, the temperature-conversion factors calculated are nearly identical to those for native human sera. The proportional increase of the activity concentration caused by adding pyridoxal-5'-phosphate to the reaction mixture is independent of the reaction temperature. Arrhenius plots and some thermodynamic activation parameters of both enzymes are independent of the presence of the coenzyme pyridoxal-5'-phosphate.

Alanine Transaminase↗

PubMed1998

Arabidopsis mutants define an in vivo role for isoenzymes of aspartate aminotransferase in plant nitrogen assimilation.

Arabidopsis contains five isoenzymes of aspartate aminotransferase (AspAT) localized to the cytosol, chloroplast, mitochondria, or peroxisomes. To define the in vivo function of individual isoenzymes, we screened for Arabidopsis mutants deficient in either of the two major isoenzymes, cytosolic AAT2 or chloroplastic AAT3, using a native gel activity assay. In a screen of 8,000 M2 seedlings, three independent mutants deficient in cytosolic AAT2 (aat2) and two independent mutants deficient in chloroplastic AAT3 (aat3) were isolated. Mapping of aat2 and aat3 mutations and the five AspAT genes (ASP1-ASP5) established associations as follows: the mutation affecting aat2 maps with and cosegregates with ASP2, one of two expressed genes for cytosolic AspAT; the mutation affecting aat3 maps to the same location as the ASP5 gene encoding chloroplastic AspAT. Phenotypic analysis of the aat2 and aat3 mutants revealed a dramatic aspartate-related phenotype in one of the mutants deficient in cytosolic AAT2. The aat2-2 mutant displays an 80% reduction in levels of aspartate transported in the phloem of light-grown plants, and a 50% reduction in levels of asparagine transported in dark-adapted plants. These results indicate that cytosolic AAT2 is the major isoenzyme controlling aspartate synthesized for nitrogen transport in the light, and that this aspartate pool is converted to asparagine when plants are dark adapted.

Arabidopsis↗

PubMed1990

Substitution of S-(beta-aminoethyl)-cysteine for active-site lysine of thermostable aspartate aminotransferase.

The active site lysyl residue (K239) of the thermostable aspartate aminotransferase [EC 2.6.1.1] was replaced by cysteinyl residue by means of site-directed mutagenesis. The K239C mutant enzyme obtained was catalytically inactive. The reaction of the cysteinyl residue of the K239C mutant enzyme with ethylenimine led to the formation of S-(beta-aminoethylcysteinyl (SAEC) residue. The K239SAEC mutant enzyme obtained showed about 25% of the activity of wild-type enzyme, and absorbed at 375 nm, which suggested the internal Schiff base formation.

Amino Acids↗

PubMed1983

Immunoglobulin-complexed aspartate aminotransferase.

We report two cases of unexplained, isolated, persistently increased aspartate aminotransferase activity in serum. In the first patient, this lasted for 10 years and prompted multiple hospitalizations and medical/surgical consultations. For patient two, the abnormal enzyme activity resulted in hospitalization for further evaluation of possible heart disease. In spite of extensive investigation to determine the possible reason for the abnormality, no clear clinical cause was ever discovered in either patient. Electrophoretic and immunologic evidence indicates that the enzyme was complexed to IgG in the serum of both individuals. Recognition that this complex is probably benign may obviate other patients having to undergo unnecessary hospitalization, anxiety, and expense.

Aspartate Aminotransferases↗

PubMedDate not supplied

[Conformation of aspartate aminotransferase in crystals].

X-ray study of chicken cytosolic aspartate aminotransferase revealed conformational changes in the protein of two kinds: (1) a shift of the small domain adjacent to substrate-binding area due to interaction of the protein with two carboxyl groups of substrate and (2) a change in inclination of the coenzyme plane due to replacement of C = N bond of the coenzyme with Lys-258 by C = N bond with a substrate. An asymmetry in subunit behaviour is observed in both cases: the domain is shifted in one subunit and the coenzyme is rotated in other. Substrate-binding properties of each subunit are strictly dependent on the protein conformation in substrate-binding area.

Animals↗

PubMed1983

Serum mitochondrial aspartate aminotransferase in patients with polymyositis.

The clinical significance of serum aspartate aminotransferase (GOT) isozymes was studied in 18 patients with polymyositis. Abnormally high levels of mitochondrial GOT (mGOT) (6.2 +/- 1.2 IU/L, mean +/- SEM; normal, less than 2.0 IU/L) and cytosol GOT (sGOT) (95 +/- 21.6 IU/L; normal, less than 25 IU/L) were observed in sera. In polymyositic muscles, the sGOT level was significantly decreased but mGOT was not. The levels of serum sGOT and mGOT and the ratio of mGOT/tGOT before corticosteroid therapy correlated well with the severity of muscle weakness. Serial determination of CPK, sGOT, and mGOT during corticosteroid therapy revealed that mGOT most rapidly returned to normal. Exercise did not increase serum mGOT in polymyositis.

Adrenal Cortex Hormones↗

PubMed1983

Discrepancies in measurement of aspartate aminotransferase by continuous-flow analysis.

I compared results for aspartate aminotransferase (EC 2.6.1.1) obtained with a reaction-rate analyzer (LKB 2086 Mark Two), based on IFCC methodology, and a continuous-flow analyzer (the Technicon SMA 2) for 115 patients' sera and seven commercial quality-control sera. The data from the SMA 2 showed a clear positive bias in those sera with activities exceeding 40 U/L (the upper limit of the reference range). Independent data to support the bias of the SMA 2 and other continuous-flow analyzer systems are presented. Application of a correction factor to the SMA 2 data above the upper limit of the range significantly decreased this bias. Failure to apply such a factor to data obtained from continuous-flow analyzers could lead to serious clinical misinterpretation.

Aspartate Aminotransferases↗

PubMed1976

An assessment of some of the methods available for the determination of molecular weights of proteins as applied to aspartate aminotransferase from pig heart.

The isopotential specific volume of cytoplasmic aspartate aminotransferase from pig heart was found to be 0.763 ml g-1 whereas the value of the apparent specific volume obtained by summation of contributions from each type of amino acid in the protein is 0.735 ml g-1. Use of the experimentally determined isopotential specific volume largely abolishes the discrepancy between a previously reported value of the molecular weight of the native (dimeric) enzyme and that of the enzyme subunit obtained from its primary structure (46300). A new non-empirical method based on quantitative N-terminal analysis involving radioisotope dilution is described for the determination of subunit molecular weight of proteins. The method is capable of considerable accuracy and sensitivity. Some of the methods available for the determination of molecular weights ans subunit compositions of proteins are discussed.

Amino Acid Sequence↗

PubMed1981

Measurement of aspartate aminotransferase isoenzymes: six procedures compared.

Six procedures were evaluated for aspartate aminotransferase (EC 2.6.1.1) isoenzyme assay in human serum and tissue homogenates. Results of procedures based on immunochemical precipitation by use of antibodies directed against either the mitochondrial or (with greater precision) soluble isoenzyme correlated well with those by a differential kinetic assay involving both different pH conditions and adipate inhibition. Results with a DEAE-Sephadex ion-exchange chromatographic procedure correlated well with these techniques for specimens containing purified isoenzymes, but showed substantial positive bias for determination of the mitochondrial isoenzyme in human serum. An assay based on the differential effects of pH alone discriminated between the isoenzymes with less bias than did the chromatographic assay. Precision of the two differential pH assays was limited by significant reagent blank activity resulting from destruction of NADH at pH 6.0 or 6.2. An electrophoretic procedure in which diazonium salt is used to make oxalacetate visible was least accurate for measuring samples for which the isoenzyme composition was known.

Aspartate Aminotransferases↗