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PubMed1998

Kinetic properties and thermal stabilities of mutant forms of mitochondrial aspartate aminotransferase.

Kinetic properties and thermal stabilities of the precursor form of mitochondrial aspartate aminotransferase, the mature form lacking 9 amino acids from the N-terminus, and forms of the mature protein in which cysteine-166 had been mutated to serine or alanine were compared with those of the mature enzyme. The precursor and the cysteine mutants showed moderately impaired catalytic properties consistent with decreased ability to undergo transition from the open to the closed conformation which is an integral part of the mechanism of action of the enzyme. The deletion mutant had a kcat only 2% of that of the mature enzyme but also much reduced Km values for both substrates. In addition it showed enhanced reactivity of cysteine-166 with 5,5'-dithiobis(2-nitrobenzoate), which is characteristic of the closed form of the enzyme, with no enhancement of reactivity in the presence of substrates. This is taken to show that the deletion mutant adopts a conformation that is significantly different from that of the mature enzyme particularly in respect of the small domain. The deletion mutant was found to be more resistant to thermal inactivation over a range of temperatures than were the other forms of the enzyme consistent with its having a more tightly packed small domain.

Animals↗
PubMed1989

Increase in negative charge of cytosolic aspartate aminotransferase in vitamin B6 deficiency and during incubation.

Several subforms of cytosolic aspartate aminotransferase (AspATc) in the crude extracts of rat liver and kidney were separated by isoelectric focusing using immunoblotting and staining of activity to detect the enzyme protein and activity, respectively. Vitamin B6-deficiency resulted in decrease in the subforms with higher isoelectric points and increase in those with lower ones both in the liver and in the kidney. When pyridoxal phosphate was added to those preparations from vitamin B6-deficient rats, the isoelectric focusing pattern of kidney was recovered to the similar one to that of the controls. However, the liver preparation was affected only partially by the addition of PLP. The pattern of subforms was altered during in vitro incubation at 37 degrees C for 24 h in both liver and kidney preparations, and their patterns were very similar to that of liver preparation from VB6-deficient rats. The enzyme activity also decreased during this incubation, especially in preparations of the enzyme from the liver of vitamin B6-deficient rats. This loss of enzyme activity was not affected by addition of PLP alone, but was almost completely prevented by addition of substrate. The inactivation was recovered by addition of substrate and pyridoxal phosphate simultaneously. This finding suggests that the inactivation may be related with a conformational change around the catalytic site of the AspATc molecule.

Animals↗
PubMed1988

Coenzyme reorientations in the active sites of aspartate aminotransferase isoenzymes studied by linear dichroism method.

Cytosolic and mitochondrial pig heart aspartate aminotransferases (cAspAT and mAspAT) and chicken heart cAspAT have been oriented in a compressed slab of polyacrylamide gel and their linear dichroism LD spectra have been recorded. The coenzyme's tilt angles in the active sites of chicken cAspAT and pig mAspAT and their quasisubstrate complexes imitating catalytic intermediates have been computed. The computations are based on reduced linear dichroism values (delta A/A), the known directions of the transition dipole moments in the coenzyme ring and atomic coordinates of the coenzyme obtained by X-ray crystallography. It has been found that formation of the enzyme complex with glutarate and protonation of the internal pyridoxal-lysine aldimine induce reorientations of the coenzyme. As a result of protonation, the coenzyme ring tilts by 27 degrees in cAspAT and 13 degrees in mAspAT. Formation of the external aldimine with 2-methylaspartate is accompanied by tilting of the coenzyme ring by 44 degrees in cAspAT and 39 degrees in mAspAT. For the quinonoid complex with erythro-3-hydroxyaspartate, the tilt angles were found to be 63 degrees in cAspAT and 53 degrees in mAspAT. It is inferred that the basic features of the active site dynamics are similar in the three AspAT's studied. The differences in the coenzyme tilt angles between cAspAT and mAspAT may be linked to catalytic and structural peculiarities of the isoenzymes.

Animals↗
PubMed1988

The diagnostic accuracy of three recommended methods for serum aspartate aminotransferase assays in patients suspected of myocardial infarction and hepatobiliary diseases.

Serum aspartate aminotransferase (AST) activity was measured by the methods recommended by the Scandinavian Committee on Enzymes (SCE) and by the International Federation of Clinical Chemistry (IFCC) with pyridoxal phosphate (PLP) and without (-PLP) in one laboratory at 37 degrees C with the Abbott ABA-100 and in another at 30 degrees C with the IL Multistat III. Reference ranges were determined on 195 healthy hospital staff. Sera from 102 patients with suspected hepatobiliary disease (HBD) and 104 with suspected myocardial infarction (MI) were assayed at both laboratories by all three methods. Based on the above reference ranges, all assays with each method at both hospitals were abnormal in 59 of 67 cases with HBD and 53 of 55 with MI. In aggregate, all three methods yielded comparable rates of misclassification (20-23). The SCE method gave highest false negatives (18) and lowest false positives (5); the IFCC method gave lowest false negatives (1) and highest false positives (20); intermediate values of 8 false positives and 12 false negatives were given by the IFCC (-PLP) method. Using receiver operating characteristic (ROC) curves, the SCE method was clearly superior at 30 degrees C, and the IFCC (-PLP) method was marginally superior at 37 degrees C. However, when the decision threshold corresponded with a 2.5% false positive rate in the non-HBD, non-MI patients, the SCE method gave the lowest false negatives at both temperatures and, on the basis of the present data, must be considered to be the method of choice for AST activity determinations.

Aspartate Aminotransferases↗
PubMed2006

Aspartate aminotransferase to platelet ratio index in patients with alcoholic liver fibrosis.

OBJECTIVE: Aspartate aminotransferase (AST) to platelet ratio index (APRI) has been proposed as an easily determined and accurate noninvasive marker of liver fibrosis in chronic hepatitis C. To validate APRI in hepatitis C and to determine its usefulness in other liver diseases, we evaluated APRI in patients with liver fibrosis due to excessive alcohol consumption with or without viral hepatitis C. METHODS: A total of 1,308 subjects from two VA cooperative studies of alcoholic liver disease were evaluated. Liver biopsy was available from 781 noncirrhotic patients while a history of decompensation was present in 527. Alcohol intake was determined by self-report. Hepatitis C was confirmed by PCR. RESULTS: Ninety-eight percent were men with a mean age of 51.5 yr. Alcohol intake averaged 19 drinks/day for 20.6 yr. One hundred thirty-three (10.2%) were hepatitis C positive. In the HCV-positive subgroup, APRI had a sensitivity of 35.6% and a specificity of 29.7% for significant fibrosis. Of 64 patients classified as significant fibrosis, 21 (32.8%) were incorrectly classified. In the 507 HCV negative patients with biopsy confirmed fibrosis, the sensitivity of APRI for significant fibrosis was 13.2% and the specificity was 77.6%. Twenty percent were classified incorrectly. CONCLUSION: APRI has low sensitivity and specificity for the diagnosis of significant fibrosis in patients with alcoholic liver disease, including patients who have hepatitis C. Given the frequent history of alcohol use in patients with hepatitis C, APRI may be of limited usefulness in the diagnosis of fibrosis in many patients.

Aspartate Aminotransferases↗
PubMed1989

Health-based reference values of the Mini-Finland Health Survey: 1. Serum gamma-glutamyltransferase, aspartate aminotransferase and alkaline phosphatase.

The reference values for gamma-glutamyltransferase (GT), aspartate aminotransferase (ASAT) and alkaline phosphatase (AFOS) activities in serum have been produced on the basis of measurements done in the Mini-Finland Health Survey. A representative sample of all Finns aged 30 years or over comprised 8000 persons, of whom 99.2% participated in the actual health survey. Every effort was made to obtain reference values for the healthy ambulatory population. Three separate health-derived selection criteria were used to obtain such reference values for the above-mentioned enzymes: those based on the available literature, with minor modifications, the recommendations published by the Committee on Reference Values of the Scandinavian Society for Clinical Chemistry and Clinical Physiology, criteria that were obtained after subgroup comparisons of the obtained data, where all the factors affecting the enzyme levels were identified. The recommendations of the Expert Panel on Theory of Reference Values (1987) were strictly adhered to in the statistical analyses. The distribution of the serum activity of GT was very skewed. The overall intervals for men and women were 5.1-1460 and 4.7-748 U/l, respectively. The frequency distributions could be transformed to the normal ones logarithmically. The 95% inner reference intervals for GT in the three groups were 7-76, 7-65, and 8-57 U/l for men and 6-35, 6-30 and 6-32 U/l for women, respectively. For ASAT the full intervals of the enzyme levels in serum were 2.6-770 U/l for men and 8.3-172 U/l for women. After logarithmic transformation the respective reference intervals in the three selection groups were 14-42, 14-40 and 13-39 U/l for men and 13-33, 12-31 and 13-33 U/l for women. The full intervals of AFOS were 47.5-2755 and 5.4-816 U/l for men and women, respectively; after the logarithmic transformation the reference intervals of the three selection groups were 98-267, 97-254 and 97-264 U/l for men and 77-265, 75-231 and 75-250 U/l for women, respectively.

Adult↗
PubMed1995

Homologous proteins with different affinities for groEL. The refolding of the aspartate aminotransferase isozymes at varying temperatures.

The homologous cytosolic and mitochondrial isozymes of aspartate aminotransferase (c- and mAspAT, respectively) seem to follow very different folding pathways after synthesis in rabbit reticulocyte lysate, suggesting that the nascent proteins interact differently with molecular chaperones (Mattingly, J. R., Jr., Iriarte, A., and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 26320-26327). In an attempt to discern the structural basis for this phenomenon, we have begun to study the effect of temperature on the refolding of the guanidine hydrochloride-denatured, purified proteins and their interaction with the groEL/groES molecular chaperone system from Escherichia coli. In the absence of chaperones, temperature has a critical effect on the refolding of the two isozymes, with mAspAT being more susceptible than cAspAT to diminishing refolding yields at increasing temperatures. No refolding is observed for mAspAT at physiological temperatures. The molecular chaperones groEL and groES can extend the temperature range over which the AspAT isozymes successfully refold; however, cAspAT can still refold at higher temperatures than mAspAT. In the absence of groES and MgATP, the two isozymes interact differently with groEL, groEL arrests the refolding of mAspAT throughout the temperature range of 0-45 degrees C. Adding only MgATP releases very little mAspAT from groEL; both groES and MgATP are required for significant refolding of mAspAT in the presence of groEL. On the other hand, the extent to which groEL inhibits the refolding of cAspAT depends upon the temperature of the refolding reaction, only slowing the reaction at 0 degrees C but arresting it completely at 30 degrees C. MgATP alone is sufficient to effect the release of cAspAT from groEL at any temperature examined; inclusion of groES along with MgATP has no effect on the refolding yield but does increase the refolding rate at temperatures greater than 15 degrees C. These results demonstrate that groEL can have significantly different affinities for proteins with highly homologous final tertiary and quarternary structures and suggest that dissimilarities in the primary sequence of the protein substrates may control the structure of the folding intermediates captured by groEL and/or the composition of the surfaces through which the folding proteins interact with groEL.

Animals↗
PubMed1978

Evidence for the participation of aspartate aminotransferase in hepatic glucose synthesis in the suckling newborn rat.

Inhibition of liver aspartate aminotransferase by L-2-amino-4-methoxy-trans-3-butenoic acid in the suckling newborn rat causes a decrease in all gluconeogenic precursors from phosphoenolpyruvate to glucose and an accumulation of lactate but not of pyruvate. This suggests that the aspartate shuttle is operative and confirms the quantitative importance of lactate as a gluconeogenic precursor at this time during development.

Alanine↗
PubMed1981

Re-activation by glutamate or aspartate of amino-oxyacetate-inhibited aspartate aminotransferase in vitro and in isolated hepatocytes.

Experiments with isolated rat hepatocytes and with cell extracts indicate, in contrast with previous reports, that pyruvate does not block or reverse the inhibition of aspartate aminotransferase (EC 2.6.1.1) by amino-oxyacetate. That inhibition, however, is partially overcome by glutamate or aspartate either in cell extracts or in whole cells incubated with substrate combinations that cause accumulation of those amino acids.

Acetates↗
PubMed1985

Localization of aspartate aminotransferase and cytochrome oxidase in the cat retina.

Using immunohistochemical techniques, we demonstrate aspartate aminotransferase (AAT)-like immunoreactivity in cone pedicles and ganglion cells of the cat retina. An identical pattern was seen when we stained for cytochrome oxidase activity, a marker for neurons which have a high metabolic activity. Tetrodotoxin selectively blocked the cytochrome oxidase labeling of ganglion cells. AAT is a key enzyme in the metabolism of aspartate and glutamate and has been proposed as a marker for neurons which use aspartate/glutamate as a neurotransmitter. Due to the close correlation between AAT-like immunoreactivity and cytochrome oxidase activity, we suggest that, at least in the retina, AAT-like immunoreactivity in fact labels cells which have a high metabolic activity.

Animals↗
PubMed2003

Aspartate aminotransferase generates proagonists of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) binds planar aromatic compounds and up-regulates the transcription of a battery of xenobiotic-metabolizing enzymes. To identify proteins involved in the biosynthesis of endogenous AHR ligands, we screened extracts of various mouse tissues for AHR signaling activity. We found heart extract to activate AHR and identified the active component to be the enzyme aspartate aminotransferase (EC 2.6.1.1). We demonstrate that this transaminase can activate AHR signaling by converting l-tryptophan to indole-3-pyruvate. In turn, indole-3-pyruvate spontaneously reacts in aqueous solution to form a large number of compounds that act as agonists of AHR. Tyrosine and the serotonin-precursor 5-hydroxytryptophan also activate AHR signaling in combination with aspartate aminotransferase, suggesting that 4-hydroxyphenylpyruvate and 5-hydroxyindolepyruvate also act as proagonists of AHR. This study demonstrates that the known tryptophan metabolic-intermediate indole-3-pyruvate is a proagonist of AHR that reacts in aqueous solution to form a variety of AHR agonists.

Animals↗
PubMed2005

The promoter for C4-type mitochondrial aspartate aminotransferase does not direct bundle sheath-specific expression in transgenic rice plants.

For NAD-malic enzyme (NAD-ME)-type C4 photosynthesis, two types of aspartate aminotransferase (AAT) are involved. We examined the expression pattern of the Panicum miliaceum mitochondrial Aat gene (PmAat) and P. miliaceum cytosolic Aat gene (PcAat) in transgenic rice plants, which were specifically expressed in bundle sheath cells (BSCs) and mesophyll cells (MCs), respectively. Expression of a beta-glucuronidase (GUS) reporter gene under the control of the PcAat promoter was regulated in an organ-preferential and light-dependent manner in the transgenic rice plants. However, the PmAat promoter drove the GUS expression in all organs we tested without light dependency, and this non-preferential expression pattern was also observed in transgenic rice with introduction of the intact PmAat gene. The expression patterns of the rice counterpart Aat genes to PmAat or PcAat showed that the rice mitochondrial Aat (RmAat1) gene was expressed in all organs tested in a light-independent manner, while expression of the rice cytosolic Aat (RcAat1) gene showed an organ-preferential and light-dependent pattern. Taking these results together, we can generalize that the regulatory system of BSC-specific or light-dependent expression of mitochondrial Aat is not shared between P. miliaceum (C4) and rice (C3) and that the expression of the C4 genes introduced into rice mimics that of their counterpart genes in rice.

Aspartate Aminotransferase, Cytoplasmic↗
PubMed1990

Aspartate aminotransferase from Eleusine coracana, a C4 plant: purification, characterization, and preparation of antibody.

Aspartate aminotransferase (AspAT) isozymes from Eleusine coracana (an NAD-malic enzyme type C4 plant) were examined. Three groups of isoenzymes were identified (AspAT-1, AspAT-2, and AspAT-3). AspAT-1 (localized in the mesophyll cells) and AspAT-3 (localized in the bundle sheath cells), both of which are considered to function in the C4 acid pathway, were purified and their kinetic and physical properties studied. Both isoenzymes had a molecular mass of 80 kDa and were shown to consist of two identical 40-kDa monomers. Except for the higher affinity for aspartate and the lower activity for the forward direction (Asp----OAA) at lower pH exhibited by AspAT-3 compared with AspAT-1, the isozymes had similar kinetic properties. However they had quite different isoelectric points. Polyclonal antibodies raised against AspAT-3 preferentially cross-reacted with AspAT-3 but did show some cross-reactivity with AspAT-1.

Antibody Specificity↗
PubMed1985

Modification of the kinetic parameters of chicken liver cytoplasmic aspartate aminotransferase by lactate dehydrogenase.

A method for the purification of chicken liver soluble aspartate aminotransferase, lactate dehydrogenase free, is proposed. The preparation, which contained a mixture of the five molecular forms of the enzyme, showed a 120-fold increase in specific activity, with respect to the initial homogenates. Differences in Km(2-oxoglutarate) and saturating concentrations among solutions of purified enzyme and soluble fraction were due to the 2-oxoglutarate reductase activity of lactate dehydrogenase.

Animals↗
PubMed1994

Structure of genes that encode isozymes of aspartate aminotransferase in Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 photosynthetic cycle in NAD-malic enzyme-type C4 plants and are expressed at high levels in mesophyll cells and bundle sheath cells, respectively. We constructed a genomic library from Panicum miliaceum, a NAD-malic enzyme-type C4 plant, and cloned the genes for these isozymes. The sequence of the cloned gene for cytosolic AspAT spans 7800 bp and consists of 12 exons. The sequence of the cloned gene for mitochondrial AspAT spans 9000 bp and consists of 10 exons. The results of primer-extension analysis suggest that transcription may be initiated from multiple adjacent sites. Both genes have significant GC-rich regions around the site of initiation of transcription, and these regions showed no CpG suppression. The 5'- flanking regions of both genes include several short sequences similar to the regulatory elements found in other genes for components of the photosynthetic machinery. In particular, the cytosolic AspAT gene contains sequences similar to nuclear protein-binding sites in other mesophyll-expressed C4 photosynthetic genes and the mitochondrial AspAT gene contains elements for light-sensitive and constitutive expression of a bundle sheath-expressed gene. The results of Southern analysis indicated that there are at least two genes that encode each isozyme in the genome of P. miliaceum. A comparison of intron-insertion positions between AspAT genes of plants and animals revealed that several introns are located at identical positions. On the basis of a phylogenetic tree among AspATs and tyrosine aminotransferase, we have shown that the introns of aminotransferase genes antedate the divergence of eubacteria, archaebacteria, and eukaryotes.

Animals↗
PubMedDate not supplied

Effect of cycloheximide on increased aspartate aminotransferase in carbon tetrachloride hepatotoxicity.

The previously reported increases in liver and serum aspartate aminotransferase (ASAT) activities and liver protein content 24 hours after the administration of carbon tetrachloride (CCl4) were reduced by administering multiple doses of the protein synthesis inhibitor cycloheximide (CH). Liver ASAT and protein content were reduced to saline-injected control levels, and the serum ASAT increase was reduced by 45.0 percent in rats given CH. Although there are morphological features of severe hepatotoxicity in the cycloheximide-carbon tetrachloride-injected rats, cycloheximide does reduce the severity of these lesions and the regenerative response. These findings lend some support to the hypotheses that (1) the increase in liver ASAT activity and protein content after CCl4 is due to increased synthesis and (2) the increase in serum ASAT after CCl4 is most likely due to a combination of increased synthesis and leakage from necrotic and damaged cells.

Animals↗
PubMed2002

Macro-aspartate aminotransferase in a female with antibodies to hepatitis C virus.

Persistent elevation of aspartate aminotransferase (AST) activity in serum due to the presence of a macroenzyme form of AST (macro-AST) may lead to diagnostic confusion in many clinical conditions, particularly those associated with chronic liver disease. We describe a case of macro-AST arising in an adult female with a false-positive hepatitis C virus (HCV) RNA test result that was not accompanied by other biochemical or histologic evidence of liver disease. The presence of macro-AST in serum was confirmed utilizing size-exclusion, high performance liquid chromatography (HPLC) and Protein G-agarose beads to precipitate immune complexes of AST and immunoglobulin G followed by centrifugation and AST activity measurements in the supernatant. A brief review of the clinical enzymology of AST and methods used to quantify serum macro-AST activity is provided.

Adult↗