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PubMed1975

Interlaboratory proficiency, intermethod comparison, and calibrator suitability in assay of serum aspartate aminotransferase activity.

Sources of variation in assays of aspartate aminotransferase (EC 2.6.1.1) activity were examined in an interlaboratory survey and through an examination of materials used as calibration materials in these assays. Four highly stable lyophilized specimens containing human cytoplasmic enzyme, with activities of 0, 22, 46, and 96 U/liter at 30 degrees C and optimal substrate concentrations, were assayed by 319 laboratories. Mean values obtained on these specimens by laboratories using 2,4-dinitrophenylhydrazine kits varied among manufacturers and deviated from values expected from this procedure. The average coefficient of variation (CV) with these kits was greater than 20%. Automated continuous-flow procedures with use of diazonium salt showed the best precision (av CV, less than 10%). However, the automated continuous-flow malate dehydrogenase/NADH coupled method produced an average CV greater than 20%. Results from each of the automated methods were related to a reference malate dehydrogenase/NADH coupled continuous kinetic assay method by temperature relationships alone. Mean values from manual diazonium salt procedures were 1.7-fold greater than similar reference values (av CV was 18%). The higher results were attributed to the use of poorly-defined units and to an artifact caused by chromophore stabilizers in this procedure when aqueous samples are used. The average CV in continuous kinetic methods varied among kit manufacturers, ranging from 6 to 28% for the specimen of highest activity. Variations in results were much larger at 366 nm than at 340 nm than at 340ity. Variations in results were much larger at 366 nm than at 340 nm. Interassay relationships of these methods are presented. Concentrations of pyruvate in commercially available calibration materials differed between manufacturers, varied in stability, and deviated from the expected concentration. For some colorimetric assays the precision attained on reported absorbance values for the enzyme specimens was of the same order of magnitude as that for pyruvate standards. Other sources of error are revealed by the interlaboratory survey. The value of commercially available sources of enzyme activity as calibration or control materials was assessed by evaluating the following properties: activity at suboptimal concentrations of L-aspartate or 2-oxoglutarate, temperature effects, preincubation lability owing to aspartate and phosphate, pyridoxal phosphate saturation, contamination with glutamate dehydrogenase, and manufacturer's rated activity. These properties are compared to those of human cytoplasmic enzyme in a human serum matrix.

Aspartate Aminotransferases↗
PubMed1980

Increased rat liver homogenate, mitochondrial, and cytosolic aspartate aminotransferase activity in acute carbon tetrachloride poisoning.

Rat liver homogenate, subcellular fractions, and sera were assayed for aspartate aminotransferase 24 h after the administration of carbon tetrachloride. Enzyme activity per total liver per gram initial body weight was significantly increased in the homogenate, and in the mitochondrial and cytosolic fractions. Protein content per total liver per gram initial body weight was also increased so that the specific activity of the enzyme was unchanged. Enzyme activity per gram liver wet weight was decreased consistent with hepatomegaly, edema, and dilution of enzyme. Enzyme and isoenzyme activities of serum were increased. These findings support the hypothesis that increased synthesis may be a source of increased serum aspartate aminotransferase and its isoenzymes as a response to hepatocellular injury.

Animals↗
PubMed1987

Regulation of aspartate aminotransferase isoenzymes by hydrocortisone in the liver of aging rats.

The activities and induction patterns of the isoenzymes of aspartate aminotransferase of the liver of male rats of various ages were studied. The activity of cytoplasmic aspartate aminotransferase increases gradually till adulthood and remains constant thereafter with increasing age of the rat. However, the activity of mitochondrial isoenzyme remains constant throughout the life span of the rat. Adrenalectomy decreases and hydrocortisone increases the activity of cytoplasmic isoenzyme in rats of all ages. In contrast, the above treatments do not shown any affect on the activity of mitochondrial isoenzyme in the liver of rats of all ages.

Adrenalectomy↗
PubMed1982

Mitochondrial aspartate aminotransferase and the effect of testosterone on citrate production in rat ventral prostate.

Mitochondrial aspartate transamination was investigated as a major source of oxalacetate for citrate synthesis in rat ventral prostate. Citrate accumulation was measured in isolated mitochondria incubated with acetyl coenzyme A and various combinations of amino acids. Aspartate plus alpha ketoglutarate in the presence of acetyl coenzyme A resulted in significant citrate accumulation. Neither aspartate nor alpha ketoglutarate alone resulted in any significant citrate accumulation. Aspartate and alpha ketoglutarate use was comparable to glutamate and citrate production. The results indicated the presence of a mitochondrial aspartate aminotransferase. Castration (3 days) caused a significant decrease in citrate production from aspartate plus alpha ketoglutarate as well as a decrease in mitochondrial AAT activity in prostate although no effect on kidney activity occurred. A single injection of 1 mg. testosterone propionate to castrate rats significantly increased prostate mitochondrial AAT activity within 24 hours while MDH activity was unaltered. A double reciprocal plot indicated that testosterone might regulate the level of mitochondrial AAT in prostate. Ventral prostate also contain a uniquely high level of endogenous aspartate. These studies indicate that aspartate might be the major 4-carbon source of oxalacetate for citrate synthesis. Also testosterone possibly regulated prostate citrate production by its effect on the level of mitochondrial AAT activity.

Animals↗
PubMed1988

Evidence that aspartate aminotransferase activity and ketodicarboxylate carrier function are essential for biosynthesis of transmitter glutamate.

Based on the selective inhibition of glutamate release in cerebellar granule cells in primary cultures by the aspartate aminotransferase inhibitor, aminooxyacetic acid, and by the ketodicarboxylate carrier inhibitor, phenylsuccinate, a novel model for synthesis of transmitter glutamate is suggested: Glutamate is formed from glutamine in the mitochondrial intramembrane space by phosphate-activated glutaminase, transported across the inner membrane in exchange with aspartate, transaminated in the matrix to alpha-ketoglutarate, which via the ketodicarboxylate carrier is transferred to the cytoplasm, and transaminated to form transmitter glutamate. Such a mechanism would explain the functional role of aspartate aminotransferase in glutamatergic neurons.

Aminooxyacetic Acid↗
PubMed1977

Biological variability in aspartate aminotransferase activity in serum of healthy persons, and effect of in vitro supplemantation with pyridoxal 5-phosphate.

Aspartate aminotransferase (EC 2.6.1.1) activities in sera from nine healthy individuals were monitored during two weeks, both with and without first supplementing the serum with pyridoxal phosphate. Pyridoxal phosphate supplementation caused a mean increase of 39% (range, 33-55%) in measured activity. The biological variability during the two-week period was independent of pyridoxal phosphate supplemantation. The intra-individual variability (CV) was 5.3% and 5.1% with and without pyridoxal phosphate supplementation, respectively; the corresponding inter-individual variability was 13.2% and 13.6%. We conclude that the reference interval will be insensitive to intra-individual fluctuations in aspartate aminotransferase activity in serum, whether or not the serum is supplemented with pyridoxal phosphate.

Adult↗
PubMed1992

Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate.

Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.

Aspartate Aminotransferases↗
PubMed1983

Primary structure of aspartate aminotransferase from horse heart and comparison with that of other homotopic and heterotopic isoenzymes.

Sulphydryl groups of mitochondrial aspartate aminotransferase from horse heart were titrated with 5,5'-dithiobis (2-nitrobenzoic acid). From analysis of peptic peptides, 378 amino acid residues (94.3% of the total) in the protein were identified. The results of amino acid sequence analysis are compared with those of cytosolic and mitochondrial aspartate aminotransferases from other sources.

Amino Acid Sequence↗
PubMed1980

Three-dimensional structure of a pyridoxal-phosphate-dependent enzyme, mitochondrial aspartate aminotransferase.

X-ray diffraction studies to 2.8-A resolution have yielded the three-dimensional structure of mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), an isologous alpha 2 dimer (Mr = 2 x 45,000). The subunits are rich in secondary structure and contain two domains, one of which anchors the coenzyme, pyridoxal 5'-phosphate. Each active site lies between the subunits and is composed of residues from both of them.

Animals↗
PubMed1994

Three-dimensional structure of a mutant E. coli aspartate aminotransferase with increased enzymic activity.

The aspartate and tyrosine aminotransferases from Escherichia coli have 43% sequence identity and nearly identical active sites. Both are equally good enzymes for dicarboxylate substrates, but the latter transaminates aromatic amino acids 1000 times faster. In an attempt to discover the critical residues for this differential substrate specificity, the aspartate aminotransferase mutant V39L has recently been prepared. It showed improved Kcat/Km values for aspartate, glutamate and tyrosine and the corresponding oxo acids, mainly due to two to ten times lower Km values. For example, the Km values of V39L (wild type) for Asp and Glu are 0.12 (1.0) and 0.85 (2.7) mM respectively. The mutant was co-crystallized with 30 mM maleate from both polyethylene glycol and ammonium sulfate. Both structures were solved and refined to R-factors of 0.22 and 0.20 at 2.85 and 2.5 A resolution respectively. They bear strong resemblance to the closed structure of the wild type enzyme complexed with maleate. The unexpected feature is that, for the first time, the closed form was produced in crystals grown from ammonium sulfate. It is concluded that the mutation has shifted the conformational equilibrium towards the closed form, which leads to generally reduced substrate Kms.

Aspartate Aminotransferases↗
PubMed1984

Effect of administration of triiodothyronine on aspartate aminotransferase in pyridoxine-deficient rats.

The state of thyroid function and the effect of triiodothyronine (T3) administration on aspartate aminotransferase isozymes in the livers of rats fed on a diet with or without pyridoxine were examined. Decreased thyroid function in pyridoxine-deficient rats was demonstrated using malic enzyme as a marker of thyroid function in the liver. Administration of T3 increased cytosolic aspartate aminotransferase in the liver of pyridoxine-deficient rats, but not of control rats.

Animals↗
PubMed1994

X-ray crystallographic study of pyridoxamine 5'-phosphate-type aspartate aminotransferases from Escherichia coli in three forms.

The three-dimensional structures of pyridoxamine 5'-phosphate-type aspartate aminotransferase from Escherichia coli and its complexes with maleate and glutarate have been determined by X-ray crystallography at 2.2, 2.1, and 2.7 A resolution, respectively. The enzyme is a dimeric form comprising two identical subunits, each of which is divided into one large and one small domain. The complex with maleate showed that substrate (or inhibitor) binding induced a large conformational change from the "open" to the "closed" form, resulting in closure of the active site by the small domain movement, as was observed in the pyridoxal 5'-phosphate-type enzyme. In the open form, three hydrophobic residues (hydrophobic plug) at the entrance of the active site are exposed to solvent. Maleate binding make the active site more hydrophobic by charge compensation and release of water molecules, facilitating the movement of the hydrophobic plug into the active site pocket to induce a large conformational change in the enzyme. Maleate is fixed rigidly in the active site pocket by extensive salt bridges and a hydrogen bonding network, guaranteeing the stereo-specificity of the catalysis and giving a Michaelis complex model. Contrary to our expectation, the glutarate complex was in the open form, suggesting that the equilibrium between the open and closed forms lies far toward the open form in solution. The water molecules located in the active site pocket were almost completely conserved between Escherichia coli and chicken mitochondrial aspartate aminotransferase with the same type of cofactor and the same conformation.

Aspartate Aminotransferases↗
PubMed1988

Rat cytosolic aspartate aminotransferase: molecular cloning of cDNA and expression in Escherichia coli.

cDNA clones for rat cytosolic aspartate aminotransferase (cAspAT, L-aspartate:2-oxoglutarate aminotransferase) [EC 2.6.1.1] were isolated from a rat cDNA library, and the primary structure of the gene for cAspAT was deduced from its cDNA sequence. Rat cAspAT consists of 412 amino acids and its molecular weight is 46,295. The deduced amino acid sequence of rat cAspAT was compared with the sequences of AspATs from other species. The degree of sequence identities of rat/mouse cAspAT, rat/pig cAspAT, rat/chicken cAspAT, rat/pig mAspAT, and rat/Escherichia coli AspAT were 97.1, 89.6, 81.7, 48.1, and 41.2%, respectively. A coding region of rat cAspAT cDNA was inserted into E. coli expression vector pUC9, and enzymatically active cAspAT was expressed as a beta-galactosidase-cAspAT hybrid protein. This hybrid protein represented about 18% of the soluble proteins in E. coli and its kinetic properties were comparable with those of cAspAT preparations purified from rat liver.

Amino Acid Sequence↗
PubMedDate not supplied

The effect of X-irradiation on the alanine--and aspartate aminotransferase activity in the liver, kidney and spleen of mouse.

The effect of X-irradiation on the alanine- and aspartate aminotransferase activity in the liver, kidney and spleen of mouse. Acta Physiol. Pol. 1975, 26 (1): 95-101. The alanine- and aspartate aminotransferase (GOT and GPT) activities and the protein content were measured in the liver, kidney and spleen homogenates of mice exposed to a single whole body X-irradiation with a 900 r dose. The assays were performed in 6 h intervals during the first day and 24 h intervals from the 2nd until the 6th day after the exposure. Significant differences in the enzymatic activity were found in the course of 24 h in control animals and a marked increase of this activity was found after irradiation. This may be explained by changes in the permeability of the mitochondrial membrane for enzyme molecules.

Alanine Transaminase↗
PubMed2002

Racial and ethnic differences in alcohol-associated aspartate aminotransferase and gamma-glutamyltransferase elevation.

BACKGROUND: Recent analyses have confirmed that Hispanic and black non-Hispanic Americans are at an increased risk for death from liver cirrhosis. The reasons for this are unknown. As a common cause of cirrhosis, differing sensitivities to alcohol-related hepatocellular injury may play a role. This study compared racial and ethnic aspartate aminotransferase and gamma-glutamyltransferase level elevations within alcohol-drinking categories. METHODS: A cross-sectional analysis of adult subjects from the Third National Health and Nutrition Examination Survey. Logistic regression models were used to estimate the risk for elevation of aspartate aminotransferase and gamma-glutamyltransferase levels among Mexican American and black non-Hispanic subjects compared with white non-Hispanic subjects within categories of alcohol use. Adjustment was made for age, sex, exposure to hepatitis C and B, and body mass index. RESULTS: Among current drinkers, black non-Hispanic and Mexican Americans were more likely to have a 2-fold elevation in aspartate aminotransferase levels when compared with white non-Hispanic Americans. This was most pronounced in the highest-frequency drinkers (Mexican Americans: odds ratio, 9.1 [95% confidence interval, 3.9-21.0]; and black non-Hispanic Americans: odds ratio, 3.1 [95% confidence interval, 1.4-6.8]). No racial and ethnic differences were apparent among current abstainers. A similar pattern was found for 2-fold gamma-glutamyltransferase level elevations. CONCLUSIONS: Among current drinkers, Mexican and black non-Hispanic Americans may have an increased risk for hepatocellular injury. These results require confirmation in other study populations for whom validated measures of quantity and pattern of drinking exist.

Adult↗
PubMed1985

Effect of pyridoxine administration on the induction of cytosolic aspartate aminotransferase in the liver of rats treated with hydrocortisone.

The effects of pyridoxine and hydrocortisone administrations on the rate of synthesis of cytosolic aspartate aminotransferase in adrenalectomized pyridoxine-deficient rat livers were examined. Induction of cytosolic aspartate aminotransferase by hydrocortisone was observed 6 to 10 h after the injection. Treatment with pyridoxine daily for 6 days but not for the 2 days, resulted in suppression of the enzyme synthesis. Similar suppression of enzyme synthesis was observed in rats without hydrocortisone treatment. The activity of tryptophan oxygenase, which is known to be induced by glucocorticoid and does not contain pyridoxal phosphates, was higher in the livers of pyridoxine-deficient rats than in that of controls. The possible effects of pyridoxal phosphate on the action of glucocorticoid are discussed based on the results.

Adrenalectomy↗