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PubMed1978

Purification and characterisation of D-amino acid aminotransferase from Rhizobium japonicum.

Rhizobium japonicum has D-amino acid aminotransferase and alanine racemase activities. The D-amino-acid aminotransferase has been partially purified and characterized. This enzyme has a broad specificity and is very active with D-alpha-aminobutyrate and D-aspartate as well as D-alanine and D-glutamate. The stereospecificity of the enzyme for D-amino acids was apparently absolute with respect to product inhibition, pyridoxamine formation as well as catalytic activity. The apparent molecular weight was 58,000 and the pH optimum was 7.8-7.9. The equilibrium constant in the direction of D-glutamate formation was 1.9. Initial-velocity kinetic studies indicate the enzyme acts by a ping-pong mechanism. The dissociation constant for pyridoxal phosphate and the Michaelis constants (+/- standard errors) for D-alanine and 2-oxoglutarate were determined to be 0.51 +/- 0.06 micrometer, and 2.13 +/- 0.18 and 0.058 +/- 0.005 mM respectively. The enzyme is moderately inhibited (30%) by 4 mM p-chloromercuribenzoate.

Alanine Transaminase↗

PubMed1983

Inhibition of coenzyme activation of aspartate aminotransferase.

Forty compounds were surveyed for their effect on the activation of pig heart apoaspartate aminotransferase by pyridoxamine 5'-phosphate. Most of the nucleotides, sugar phosphates, coenzymes, phospholipid precursors and inorganic oxyanions tested were found to be inhibitory. With few exceptions, the only requirement for a substance to be inhibitory is the presence of a di- or polyanionic moiety analogous to the 5'-phosphate group of the cofactor. In spite of the lack of overall structural similarity to pyridoxamine 5'-phosphate, inorganic pyrophospate and apparently other inhibitors are characterized by dissociation constants comparable in magnitude to that previously reported for the natural cofactor. The physiological significance of the inhibition of coenzyme activation of apoaspartate aminotransferase by these common biological compounds is not known.

Animals↗

PubMed1987

Molecular cloning and in vivo expression of a precursor to rat mitochondrial aspartate aminotransferase.

A 2.4 kilobase cDNA for rat mitochondrial aspartate aminotransferase (E.C. 2.6.1.1.) was isolated and sequenced. The predicted presequence is 93% homologous to the presequences of the enzyme from pig and mouse. The predicted amino acid sequence of the mature enzyme differs from that determined directly by amino acid sequencing (Huynh, Q.K., Sakakibara, R., Watanabe, T., and Wada, H. (1981) J. Biochem. (Tokyo) 90, 863-875) at 13 amino acids residues. The most important difference is at position 140 where the cDNA encodes a tryptophanyl residue rather than the previously reported glycine. This critical residue is now seen to be conserved in all aspartate aminotransferases. The coding region of this cDNA was inserted into the plasmid cloning vector pKK233-2 and used to stably express an unfused precursor in Escherichia coli JM105.

Amino Acid Sequence↗

PubMed1985

Immunocytochemical localization of glutaminase-like and aspartate aminotransferase-like immunoreactivities in the rat and guinea pig hippocampus.

There is considerable evidence that pathways of the hippocampus use an excitatory amino acid as transmitter. We have attempted to immunocytochemically identify excitatory amino acid neurons in the hippocampus of the rat and guinea pig using antiserum to glutaminase and antiserum to aspartate aminotransferase, which have been proposed as markers for aspartergic/glutamergic neurons. Glutaminase-like immunoreactivity was seen in granule cells in the dentate gyrus and fibers and puncta associated with the mossy fiber pathway in the hilus and stratum lucidum of the hippocampus. At the ultrastructural level, glutaminase-like immunoreactivity was observed in mossy fiber terminals in the stratum lucidum. Glutaminase-like immunoreactivity was also seen in pyramidal cells in regio inferior and regio superior and in cells in layer two of the entorhinal cortex. Schaffer collateral terminals, commissural fiber terminals and perforant pathway terminals were not seen at the light microscopic level. Glutaminase-like immunoreactivity is thus found in the cell bodies of proposed excitatory amino acid neurons of hippocampal pathways, but does not appear to label all terminals. Aspartate aminotransferase-like immunoreactivity was not seen in any cells, fibers or terminals in the rat or guinea pig hippocampus.

Animals↗

PubMed1986

Immunocytochemical localization of aspartate aminotransferase and glutaminase immunoreactivities in the cerebellum.

The distributions of glutaminase and aspartate aminotransferase were studied immunocytochemically in the cerebellum of the guinea pig and the rat. In the granule cell layer, both antibodies gave a similar staining pattern. Granule cell bodies were labeled, but staining was also found to lie outside the cell body, associated with what appear to be synaptic processes. In the molecular and Purkinje cell layers, aspartate aminotransferase was concentrated in stellate and basket cell bodies and in terminal baskets beneath Purkinje cells. Glutaminase, however, was not concentrated in these structures.

Animals↗

PubMed1976

Reactivation of the apoenzyme of aspartate aminotransferase in serum.

Apoenzyme of aspartate aminotransferase in serum can be reactivated conveniently by addition of 100 mumoles/l pyridoxal phosphate to the reaction mixture, without extending the usual minimum pre-incubation period in the operation of the LKB 8600 reaction rate analyzer. Normal sera contain some apoenzyme, the amount of which, as well as that of holoenzyme, is greatly increased by damage to skeletal muscle. This may be due to direct injury or to the indirect effects of anoxia; e.g., following surgery with extracorporeal circulation. Myocardial infarction also increases the levels of both apo- and holoenzymes, but changes in the two levels follow similar time courses and apo- and holo-aminotransferases disappear from the circulation at similar rates.

Apoenzymes↗

PubMed1978

The changes in activation of intracellular aspartate aminotransferase by pyridoxal 5-phosphate after cell death.

Aspartate aminotransferase activity was measured in plasma, in liver and in heart mitochondrial and cytoplasmic preparations from rats immediately after death and after a post-mortem interval of 15 h. No significant stimulation of activity on addition of pyridoxal 5-phosphate to the assay medium could be demonstrated in any preparations obtained immediately after death. Significant stimulation occurred in both cytoplasmic and mitochondrial preparations of liver and myocardium after a 15-h post-mortem interval, but not in plasma stored for the same period. It appears, therefore, that variations in the intracellular saturation of apoenzyme with coenzyme cannot account for the observed differences in activation of aspartate aminotransferase by pyridoxal 5-phosphate in sera from patients with myocardial infarction and liver disease. Changes in degree of saturation of apoenzyme seem to occur intracellularly after cell death or injury and before release into the circulation.

Animals↗

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↗

PubMed1981

Multiple molecular forms of human cytoplasmic aspartate aminotransferase.

Human liver cytoplasmic aspartate aminotransferase was found to exhibit five subforms with isoelectric points of 5.15, 5.30, 5.45, 5.60, and 5.80. Treatment with neuraminidase did not affect their electrophoretic mobility. The immunochemical and steady-state kinetic properties of the subforms were identical. Heat treatment increased the proportion of acidic subforms, but all forms were present in fresh tissue. 2-Mercaptoethanol or inhibitors of proteolysis failed to protect against the formation of the subforms with lower isoelectric points. Multiple molecular forms with similar properties were found for the enzyme of human erythrocytes. This evidence is consistent with deamidation of asparaginyl or glutaminyl residues as the origin of the multiple forms. Human mitochondrial aspartate aminotransferase presented as a single molecular form with an isoelectric point of 9.7.

Aspartate Aminotransferases↗

PubMed1984

Mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in sera of patients after myocardial infarction.

Mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase (AST) were studied in the sera of 42 patients following acute myocardial infarction and compared to creatine kinase (CK), lactate dehydrogenase (LDH) and alanine aminotransferase (ALT). Mitochondrial AST( ASTm ) was detected in 93% (39/42) of patients. Maximum recorded ASTm activity was 59.5 +/- 8.8 U/l and was found 39.4 +/- 3.5 hours after the onset of symptoms (chest pain) of myocardial infarction. In contrast the maximum recorded cytoplasmic AST ( ASTc ) activity was greater (327 +/- 23 U/l) and it occurred earlier (33.5 +/- 2.2 hours) after onset of infarction compared to ASTm . ASTm correlated significantly (p less than 0.05) with ASTc , LDH and ALT but not with total CK or CK-MB. ASTc correlated significantly (p less than 0.05) with total CK, CK-MB and LDH but not ALT. Maximum recorded ASTm activity was significantly associated with the clinical assessment of left ventricular failure ( Killip classification) but not with ventricular arrhythmias. In a subset of 15 patients evaluated with invasive hemodynamic measurements of cardiac output and pulmonary capillary wedge pressure. ASTm correlated significantly (p less than 0.05) and better than CK-MB with the hemodynamic assessment of left ventricular dysfunction. Thus ASTSm can be readily identified in sera of patients after acute myocardial infarction and may be of value in the evaluation of patients with acute myocardial infarction.

Adult↗

PubMed1986

Mitochondrial aspartate aminotransferase linked to immunoglobulin G of the kappa-lambda type: report of a case.

Macromolecular aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase EC 2.6.1.1, AST) was found in the serum of a patient with benign hypertension. The serum total AST and mitochondrial AST (mAST) activities were proportionately higher. The abnormal AST was found to be a macromolecular complex composed of mAST and immunoglobulin G of the kappa-lambda type. The dissociated IgG from the complex was shown to combine with human and rat mAST, but not with cytosolic AST of both species. Molecular mass of the macromolecular AST was estimated to be 360,000 Da. These results indicate that the complex may consist of one IgG molecule associated with two mAST molecules. By the method of papain digestion the binding site of immunoglobulin in the complex appeared to be located in the Fab portion of the IgG molecule. This finding strongly suggests that the AST-immunoglobulin complex is a specific antigen-antibody complex.

Adult↗

PubMed1984

Cooperative effects in the binding of pyridoxal 5'-phosphate to mitochondrial apo-aspartate aminotransferase.

Titrations of mitochondrial apo-aspartate aminotransferase with pyridoxal 5'-phosphate in the presence of AMP, contrary to what has been observed in the case of the cytosolic isoenzyme [(1983) FEBS Lett. 153, 98-102], show sigmoidal isotherms, with Hill coefficients ranging from nH = 1.4, in the absence of AMP, to nH = 1.8, in the presence of 5.9 mM AMP. The experimental data were successfully fitted by the Monod-Wyman- Changeaux model. The best fit, in the absence of AMP, was obtained with L = 30, KR = 4.72 X 10(-7) M and KT = 1.18 X 10(-5) M. Binding curves in the presence of AMP fit the model by keeping KR as a constant. This implies that AMP could bind to the apoenzyme only in the T state. In contrast, binding curves in the presence of phosphate ion (Pi) showed a less pronounced cooperativity, the Hill coefficient dropping to nH = 1.0 in the presence of 0.1 mM Pi. The above results suggest a regulatory role of AMP and Pi in the reconstitution of aspartate aminotransferase.

Adenosine Monophosphate↗

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 ↗

PubMed1993

Cloning and sequence analysis of A cDNA encoding bovine cytosolic aspartate aminotransferase.

1. Complementary DNA encoding cytosolic aspartate aminotransferase was isolated from an adult bovine heart library. 2. The amino acid sequence deduced for the protein (412 amino acids) is extremely similar (> 94% identity) to that of porcine cytosolic aspartate aminotransferase but interesting differences were noticed comparing the position of cysteine residues.

Amino Acid Sequence↗

PubMed1994

The regulation of aminotransferase activity by carbamoyl-phosphate.

We studied the effect of carbamoylphosphate (CP) on L-aspartate aminotransferase (GOT) and L-alanine aminotransferase (GPT), compared to its effect on L-threonine deaminase (TD). GPT and GOT were slightly inhibited by CP, while TD was strongly inhibited. GPT and TD, but not GOT, were inactivated when preincubated with CP. Only GOT was enhanced by pyridoxal 5'-phosphate (PLP), but not when the coenzyme was preincubated with CP. When the enzymes were resolved by p-chloromercuribenzoate (PCMB) treatment to apoenzymes, only GOT retained 47% of the original activity. Reconstitution of the apoenzymes with PLP also followed different course; activities of GPT and TD were completely restored while GOT remained partially inactivated. Treatment of apoenzymes with CP resulted in impairment of their reconstitution except GPT, activity of which could be completely restored. When PLP was pre-treated with CP before reconstitution, however, even GPT was only partially restored. The data indicated that CP affect activities of these enzymes at different levels, holoenzymes, PLP and probably apoenzymes. Under a concentration of PLP, activity of GOT would be most enhanced, followed by TD then GPT. In the presence of CP, this effect would be eliminated.

Alanine Transaminase↗

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↗

PubMed1985

Slow- and tight-binding inhibition of aspartate aminotransferase by L-hydrazinosuccinate.

The inhibition of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) by L-hydrazinosuccinate has been studied. The velocity of the enzyme reaction decreased with time when the reaction was initiated by the addition of enzyme to a mixture of the assay components and L-hydrazinosuccinate, while it increased slowly from a low level when a preincubated mixture of the enzyme and the inhibitor was added to the reaction mixture to initiate the reaction. Nearly 50% decrease in the initial reaction velocity was produced by a prolonged preincubation of the enzyme with the inhibitor, both at low concentrations of about 2 nM. These findings indicate that the inhibition is of the slow- and tight-binding type. The time-course of the reaction of the enzyme and the inhibitor, examined by the change in activity, was not in accord with single-step mechanisms, but rather appeared to follow biphasic kinetics. The inhibition could be fully reversed only in the presence of L-cysteine sulfinate or large excess of L-aspartate to convert the regenerated enzyme to its pyridoxamine form. The time-course of the reversal followed pseudo-first-order kinetics. Quantitative analysis of the experimental data has shown that the results are consistent with a mechanism of enzyme-inhibitor interaction which involves a reaction of two consecutive, reversible steps. The overall inhibition constant for L-hydrazinosuccinate was calculated to be approx. 0.2 nM.

Animals↗

PubMed1985

The primary structure of mitochondrial aspartate aminotransferase from human heart.

The complete amino acid sequence of the mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from human heart has been determined based mainly on analysis of peptides obtained by digestion with trypsin and by chemical cleavage with cyanogen bromide. Comparison of the sequence with those of the isotopic isoenzymes from pig, rat and chicken showed 27, 29 and 55 differences, respectively, out of a total of 401 amino acid residues. Evidence for structural microheterogeneity at position 317 has also been obtained.

Amino Acid Sequence↗