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PubMed1999

A kinetic model of mitochondrial aspartate aminotransferase transmigration in hepatobiliary disorders.

A hypothetical kinetic model of transmigration of mitochondrial aspartate aminotransferase (m-AST) from liver to blood and its elimination from blood was constructed and assessed for prediction by computer simulation. The elimination of m-AST from plasma in healthy rats followed first-order kinetics. Depletion of m-AST from the liver after induction of hepatobiliary disorders in rats with alpha-naphthylisothiocyanate (ANIT) also followed first-order kinetics. In contrast, the changes in metabolic rate in the liver after ANIT administration, as estimated from the plasma indocyanine green clearance, approximated to a second-order time function. Based on these data, the time-dependent change in plasma m-AST activity after ANIT administration was simulated by computer according to a hypothetical kinetic model. The result of computer simulation of the change in plasma m-AST coincided well with that obtained experimentally, suggesting that continuous measurements of m-AST may be helpful to the clinical diagnosis of liver injury.

1-Naphthylisothiocyanate↗

PubMed1984

A unifying reference system for clinical enzymology: aspartate aminotransferase and the International Clinical Enzyme Scale.

A review of methodology for determining aspartate aminotransferase (ASAT; EC 2.6.1.1), including recent national and international recommendations, indicates that standardization of methodology alone will not bring interlaboratory compatibility of ASAT results. We propose that an additional component to standardization is needed, namely, enzyme reference materials. Furthermore, we suggest that stable, well-defined ASAT materials from human sources are currently available. These primary reference materials and the state-of-the-art IFCC Reference Method for ASAT provide the basis for a unifying reference system for ASAT. Given such a reference system, we propose a practical way to promote compatibility of currently incompatible numerical results for ASAT through the use of one ASAT scale of units, the "International Clinical Enzyme Scale." This scale-unification concept would permit all current methods, instruments, and temperature choices to be used for ASAT determinations in the daily working laboratory. We present illustrative examples and demonstrate the unique ability of this concept to promote compatibility of the ASAT results from numerous laboratories using many different ASAT methods.

Aspartate Aminotransferases↗

PubMed1983

Immunochemical quantitation of isoenzymes of aspartate aminotransferase and lactate dehydrogenase.

The applicability of immunochemical techniques to the determination of aspartate aminotransferase (AspAT, EC 2.6.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) isoenzymes in human serum are reviewed. In the case of AspAT, the human enzymes of mitochondrial (m-AspAT) and cytosolic (s-AspAT) origin were purified to homogeneity from liver and erythrocytes respectively and used to prepare isoenzyme-specific anti-sera in rabbits. Immunoprecipitation and immunoinhibition assays using partially purified antibodies or monovalent Fab fragments were found to provide better accuracy and precision than column chromatographic, electrophoretic, or differential kinetic techniques. A variety of immunochemical techniques were examined for the determination of enzyme protein including radioimmunoassay, turbidimetric procedures, and an assay using the indium slide technique. In the last, purified isoenzyme was absorbed as a monolayer to the surface of an indium metal film upon glass. The enzyme retains immunological reactivity, allowing the specific binding of antibody at the surface. The minimum detectable concentration by this technique is greater than 50 micrograms/L of enzyme protein; results suggest that normal and patient sera contain considerably more immunologically reactive s- and m-AspAT than catalytically active enzyme.

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↗

PubMed1974

The nature of the multiple forms of cytoplasmic aspartate aminotransferase from pig and sheep heart.

Starch-gel electrophoresis of sheep heart aspartate aminotransferase was carried out over the range pH7.0-8.5. The enzyme separates into three subforms in the same way as the pig heart enzyme. As the pH was increased the distance migrated by each subform increased by the same amount, so that they remained the same distance apart. Titration of the enzyme over the appropriate pH range was used to calculate the difference in charge between the subforms and it was concluded that they differ by one charged group per dimer from their nearest neighbour on the electrophoretogram over the whole pH range studied. It was also shown that the pig-heart alpha and beta subforms differ by almost one charged group per dimer in the range pH5.5-5.7 and that the spacing between the subforms on starch-gel electrophoresis at pH8.0 is the same as that for the sheep enzyme. Since the charge difference between the subforms is maintained over such a wide range of pH, it is concluded that they probably differ from each other in covalent structure, because of the improbability that conformational differences can give rise to such behaviour. The relationship between the subforms and inactive binding of the coenzyme is also examined.

Animals↗

PubMed1996

Characterization and mapping of cDNA encoding aspartate aminotransferase in rice, Oryza sativa L.

Fifteen cDNA clones, putatively identified as encoding aspartate aminotransferase (AST, EC 2.6.1.1.), were isolated and partially sequenced. Together with six previously isolated clones putatively identified to encode ASTs (Sasaki, et al. 1994, Plant Journal 6, 615-624), their sequences were characterized and classified into 4 cDNA species. Two of the isolated clones, C60213 and C2079, were full-length cDNAs, and their complete nucleotide sequences were determined. C60213 was 1612 bp long and its deduced amino acid sequence showed 88% homology with that of Panicum miliaceum L. mitochondrial AST. The C60213-encoded protein had an N-terminal amino acid sequence that was characteristic of a mitochondrial transit peptide. On the other hand, C2079 was 1546 bp long and had 91% amino acid sequence homology with P. miliaceum L. cytosolic AST but lacked in the transit peptide sequence. The homologies of nucleotide sequences and deduced amino acid sequences of C2079 and C60213 were 54% and 52%, respectively. C2079 and C60213 were mapped on chromosomes 1 and 6, respectively, by restriction fragment length polymorphism linkage analysis. Northern blot analysis using C2079 as a probe revealed much higher transcript levels in callus and root than in green and etiolated shoots, suggesting tissue-specific variations of AST gene expression.

Amino Acid Sequence↗

PubMed1993

Enzyme variation at the aspartate aminotransferase locus in members of the Anopheles gambiae complex (Diptera: Culicidae).

The enzyme aspartate aminotransferase (AAT) currently is used to identify Anopheles quadriannulatus Theobald, the animal-biting, nonmalaria vector species of the Anopheles gambiae complex. Samples of An. quadriannulatus from South Africa and An. gambiae Giles s.str. from the island of Grand Comoros and the People's Republic of Congo have shown variation in electromorph frequencies that indicate that AAT has five alleles. The three slowest alleles are found in An. quadriannulatus and the three fastest in An. gambiae, An. arabiensis Patton and An. merus Dönitz. One of these is common to both An. quadriannulatus and An. gambiae. This overlap indicates a potential misidentification of 0.3% of unknown females with a further 2.1% being unidentifiable. However, all of the specimens in the overlap area were classified correctly using octanol dehydrogenase (ODH). Variation at the ODH locus in An. quadriannulatus is recorded for the first time, with four of 157 specimens being heterozygous for the fast allele. The probability of both AAT and ODH giving an incorrect or indecisive identification is 0.0005. The slowest AAT alleles were present in samples from a single locality, indicating the lack of gene flow between subpopulations of An. quadriannulatus in close geographic proximity in the Shingwedzi area, South Africa. A modified method for multiple gel casting is given.

Alleles↗

PubMed1995

Pyridoxal 5'-phosphate modulates expression of cytosolic aspartate aminotransferase gene by inactivation of glucocorticoid receptor.

The level of mRNA for cytosolic aspartate aminotransferase (cAST) in the liver of vitamin B6-deficient rats was found to be 7-fold higher than that of the control rats. The administration of hydrocortisone to adrenalectomized vitamin B6-deficient rats induced expression of hepatic cAST mRNA and the induction was suppressed by the simultaneous administration of pyridoxine. Since the 5' regulatory region of the rat cAST gene contains several sequences showing homology to glucocorticoid-responsive elements, we synthesized an oligonucleotide probe of glucocorticoid-responsive element sequence and assayed the binding activity of liver nuclear extract to the oligonucleotide by gel mobility shift analysis. We found that the binding activity of nuclear extract prepared from the liver of vitamin B6-deficient rats was far greater than that of the control rats, indicating that the DNA-binding activity of glucocorticoid receptor was enhanced by vitamin B6 deficiency. We further found that preincubation of the nuclear extract from the vitamin-deficient liver with pyridoxal 5'-phosphate brought about a rapid and extensive decrease in the binding of the extract to the glucocorticoid-responsive element. Congeners of pyridoxal phosphate, such as pyridoxamine 5'-phosphate, pyridoxal, pyridoxamine and pyridoxine, did not show an inhibitory effect. These observations suggest that pyridoxal 5'-phosphate modulates cAST gene expression by inactivating the binding activity of glucocorticoid receptor to glucocorticoid-responsive elements.

Adrenalectomy↗

PubMed1992

X-ray structure refinement and comparison of three forms of mitochondrial aspartate aminotransferase.

The X-ray crystal structures of three forms of the enzyme aspartate aminotransferase (EC 2.6.1.1) from chicken heart mitochondria have been refined by least-squares methods: holoenzyme with the co-factor pyridoxal-5'-phosphate bound at pH 7.5 (1.9 A resolution), holoenzyme with pyridoxal-5'-phosphate bound at pH 5.1 (2.3 A resolution) and holoenzyme with the co-factor pyridoxamine-5'-phosphate bound at pH 7.5 (2.2 A resolution). The crystallographic agreement factors [formula: see text] for the structures are 0.166, 0.130 and 0.131, respectively, for all data in the resolution range from 10.0 A to the limit of diffraction for each structure. The secondary, super-secondary and domain structures of the pyridoxal-phosphate holoenzyme at pH 7.5 are described in detail. The surface area of the interface between the monomer subunits of this dimeric alpha 2 protein is unusually large, indicating a very stable dimer. This is consistent with biochemical data. Both subunit and domain interfaces are relatively smooth compared with other proteins. The interactions of the protein with its co-factor are described and compared among the three structures. Observed changes in co-factor conformation may be related to spectral changes and the energetics of the catalytic reaction. Small but significant adjustments of the protein to changes in co-factor conformation are seen. These adjustments may be accommodated by small rigid-body shifts of secondary structural elements, and by packing defects in the protein core.

Aspartate Aminotransferases↗

PubMed1999

A prospective study of the causes of notably raised aspartate aminotransferase of liver origin.

BACKGROUND AND AIMS: To ascertain the causes of raised aspartate aminotransferase (AST) presumed to be of hepatic origin in two hospitals and the local community served by a centralised biochemistry laboratory. METHODS: From June 1996 to February 1997 all patients with AST greater than 400 U/l were identified by the biochemistry laboratory; the patients' clinical records were studied to determine the diagnosis, the clinical outcome, and whether the raised AST and its significance had been noted. RESULTS: A total of 137 patients with a hepatic cause for the raised AST were found. The cause of the raised AST was hepatic ischaemia/hypoxia in 68, pancreatobiliary disease in 33, primary hepatocellular disease in 23, hepatic malignancy in five, and hepatic haematoma in one. In seven patients the diagnosis was unclear. The overall mortality was high (22%) with the highest mortality in the hepatic ischaemia group (37%). The recording and interpretation of the causes of raised AST was poor with only 48% having the correct diagnosis. In 38% the raised AST was apparently not noticed by the attending clinicians. CONCLUSIONS: The commonest cause of a hepatitis like biochemical picture was hepatic hypoxia (50%) followed by pancreatobiliary disease (24%). Drug induced hepatic necrosis (8.8%) was uncommon and viral hepatitis was rare (3.6%). AST concentrations returned towards normal most rapidly in patients with hepatic hypoxia and calculous biliary obstruction. Hepatitis, viral or otherwise, is an uncommon cause of a typical hepatitic biochemical result in this community.

Adult↗

PubMed1986

Cloning and sequence analysis of mRNA for mouse aspartate aminotransferase isoenzymes.

The nucleotide sequences of mRNAs for the mouse mitochondrial and cytosolic aspartate aminotransferase isoenzymes (mAspAT and cAspAT) (EC 2.6.1.1) were determined from complementary DNAs. The mAspAT mRNA comprises minimally 2460 nucleotides and codes for a polypeptide of 430 amino acid residues corresponding to the precursor form of the mAspAT (pre-mAspAT). The cAspAT mRNA comprises minimally 2086 nucleotides and codes for a polypeptide of 413 amino acid residues. The region coding for the mature mAspAT and that for the cAspAT show about 53% overall homology. The former shares 49% and the latter 48% of homology, respectively, with that of the Escherichia coli aspC gene, which has been shown to code for the E. coli AspAT (Kuramitsu, S., Okuno, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 1259-1262). When the deduced amino acid sequence of the mouse pre-mAspAT was compared with that of the pig pre-mAspAT polypeptide, we found that they share a 94% homology and that the mouse pre-mAspAT yields a presequence consisting of 29 amino acid residues and a mature mAspAT, consisting of 401 amino acid residues. These numbers and the amino acid residues present at the putative cleavage site are all in complete agreement in these two species. The deduced amino acid sequence of the mouse cAspAT shares 91% homology with that of the pig cAspAT. Comparisons of the nucleotide and deduced amino acid sequences between the mouse and E. coli AspATs suggest that the mammalian mAspAT gene is more closely related to the E. coli aspC gene than is the mammalian cAspAT gene.

Amino Acid Sequence↗

PubMed1997

Refolding intermediates of acid-unfolded mitochondrial aspartate aminotransferase bind to hsp70.

The cytosolic (cAAT) and mitochondrial (mAAT) isozymes of eukaryotic aspartate aminotransferase share a high degree of sequence identity and almost identical three-dimensional structure. The rat liver proteins can be refolded and reassembled into active dimers after unfolding at low pH. However, refolding of the mitochondrial form after unfolding at pH 2.0 is arrested in the presence of hsp70, whereas this chaperone does not affect the refolding of the cytosolic isozyme unfolded under similar conditions. To elucidate the nature of the differential interaction between hsp70 and the two transaminase forms, we have characterized their refolding from their acid-unfolded states. The recovery of activity of the cytosolic enzyme is monophasic and can be adequately described by a single first-order reaction. By contrast, two sequential first-order rate-limiting steps can be detected for the refolding and reactivation of the mitochondrial protein. The overall refolding pathway of mAAT includes a very fast collapse to an intermediate with 80% of the secondary structure of the active dimer. This is followed by a slow isomerization to form assembly-competent monomers that rapidly associate to form an inactive dimer and a final structural rearrangement of the dimer to the native conformation. Analysis of the interaction of hsp70 with intermediates along the folding pathway of mAAT shows that the polypeptide loses its ability to bind to the chaperone after it has proceeded through the first isomerization/fast dimerization steps. Thus it appears that only the first collapsed intermediate states in the folding of mAAT bind hsp70. By contrast a faster refolding of cAAT from this collapsed state could explain, at least in part, the inability of hsp70 to bind this isozyme.

Animals↗

PubMed2002

Immunogold localization of mitochondrial aspartate aminotransferase in mitochondria and on the cell surface in normal rat tissues.

Mitochondrial aspartate aminotransferase (mAspAT) (E.C. 2.6.1.1), an important enzyme in amino acid metabolism, is identical to a fatty acid-binding protein (FABPpm) isolated from plasma membranes of several cell types. Employing a monospecific polyclonal antibody to rat mAspAT, we have used immunogold electron microscopy to study the subcellular distribution of mAspAT in various mammalian tissues. Immunogold labeling of rat tissue sections embedded in LR Gold resin showed strong labeling of mitochondria in all tissues examined (viz. liver, pancreas, pituitary, spleen, heart, kidney, submandibular gland). In addition, strong and specific labeling was also observed at a number of non-mitochondrial sites including various locations in kidney, such as on cell surface in distal tubules and cortical collecting ducts, in condensing vacuoles, along cell boundaries between adjoining cells, and in endothelial cells lining capillaries in the glomerulus. Surface labeling due to mAspAT was also seen in arteriolar endothelial cells and in lymphocytes. These findings support the previous identification of mAspAT as both a mitochondrial enzyme and a plasma membrane protein. It is suggested that in accordance with its established role in other cells and tissues, the surface-located mAspAT in kidney and endothelial cells is involved in the fatty acid transport process. The dual-localization of mAspAT, as well as a large number of other mitochondrial proteins (viz. Hsp60, Hsp10, Cytochrome c, TRAP-1 and P32 (gC1q-R)) in recent studies, within both mitochondria and at various specific extramitochondrial sites raises fundamental questions about the role of mitochondria in cell structure and function, and about the mechanisms that exist in normal cells for protein translocation from mitochondria to other compartments. These results have implications for the role of mitochondria in apoptosis and different diseases.

Animals↗

PubMed2001

Strain is more important than electrostatic interaction in controlling the pKa of the catalytic group in aspartate aminotransferase.

Systematic single and multiple replacement studies have been applied to Escherichia coli aspartate aminotransferase to probe the electrostatic effect of the two substrate-binding arginine residues, Arg292 and Arg386, and the structural effect of the pyridoxal 5'-phosphate-Asn194-Arg386 hydrogen-bond linkage system (PLP-N-R) on the pK(a) value of the Schiff base formed between pyridoxal 5'-phosphate (PLP) and Lys258. The electrostatic effects of the two arginine residues cannot be assessed by simple mutational studies of the residues. PLP-N-R lowers the pK(a) value of the PLP-Lys258 Schiff base by keeping it in the distorted conformation, which is unfavorable for protonation. Mutation of Arg386 eliminates its hydrogen bond with Asn194 and partially disrupts PLP-N-R, thereby relaxing the strain of the Schiff base. On the other hand, mutation of Arg292, the large domain residue that interacts with the small domain residue Asp15, makes the domain opening easier. Because PLP-N-R lies between the two domains, the domain opening increases the strain of the Schiff base. Therefore, the true electrostatic effects of Arg292 and Arg386 could be derived from mutational analysis of the enzyme in which PLP-N-R had been completely disrupted by the Asn194Ala mutation. Through the analyses, we could dissect the electrostatic and structural effects of the arginine mutations on the Schiff base pK(a). The positive charges of the two arginine residues and the PLP-N-R-mediated strain of the Schiff base lower the Schiff base pK(a) by 0.7 and 1.7, respectively. Thus, the electrostatic effect of the arginine residues is not as strong as has historically been thought, and this finding substantiates our recent finding that the imine-pyridine torsion of the Schiff base is the primary determinant (2.8 unit decrease) of the extremely low pK(a) value of the Schiff base [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-15085].

Arginine↗

PubMed2002

Molecular and physiological analysis of Arabidopsis mutants defective in cytosolic or chloroplastic aspartate aminotransferase.

Arabidopsis mutants deficient in cytosolic (AAT2) or chloroplastic (AAT3) aspartate (Asp) aminotransferase were characterized at the molecular and physiological levels. All of the ethyl methane sulfonate- or nitrosomethylurea-generated mutants are missense mutations, as determined by sequencing of the ASP2 gene from the cytosolic aat2 mutants (aat2-1, aat2-2, aat2-4, and aat2-5) and the ASP5 gene from the chloroplastic aat3 mutants (aat3-1, aat3-2, and aat3-4). A T-DNA insertion mutant in cytosolic AAT2 (aat2-T) was also identified. All the cytosolic aat2 and chloroplastic aat3 mutants have less than 6% AAT2 and less than 3% AAT3 activity, respectively, as determined by the native gel assay; however, none are nulls. The metabolic and physiological affect of these mutations in AAT isoenzymes was determined by measuring growth and amino acid levels in the aat mutants. Two aat2 mutants (aat2-2 and aat2-T) show reduced root length on Murashige and Skoog medium. For aat2-2, this growth defect is exaggerated by Asp supplementation, suggesting a defect in Asp metabolism. Amino acid analysis of the aat mutants showed alterations in levels of Asp and/or Asp-derived amino acids in several aat2 alleles. Two aat2 mutants show dramatic decreases in Asp and asparagine levels in leaves and/or siliques. As such, the cytosolic AAT2 isoenzyme appears to serve a nonredundant function in plant nitrogen metabolism of Asp and Asp-derived amino acids.

Amino Acid Substitution↗

PubMed1981

[Modification of blood serum mineral levels and aspartate aminotransferase, leucine aminopeptidase and alkaline phosphatase activity and plasma in glucose levels in healthy cattle by Ca-Mg-containing solutions].

Three high-performance cows in lactation were tested each for the effects of two kinds of infusion each, 500 ml of Tetamag-2 solution (60 g of calcium gluconate and 60 g of magnesium adipate) and 500 ml of Calcimag (100 g of calcium gluconate and 20 g of magnesium adipate), upon the levels of calcium, magnesium, Pa, potassium, and sodium as well as upon the activities of aspartate-aminotransferase, leucine-aminopeptidase, and alkaline phosphatase in blood serum and upon glucose levels in blood plasma. Migration of calcium out of blood plasma was found to take place at much higher rate than that of magnesium. Infusion of Pa and glucose was followed soon by some temporary drop of values. No directional effects were recordable from potassium and sodium. Aspartate-aminotransferase activity did not change in response to transfusion. Intravenous infusion of Tetamag-2 solution caused strong temporary rise in magnesium concentration and, consequently, activation of leucine-aminopeptidase.

Alkaline Phosphatase↗

PubMed1976

The phosphopyridoxyl peptide from the mitochondrial aspartate aminotransferase of beef kidney.

The sequence around the coenzyme-binding lysine of mitochondrial aspartate amino-transferase from beef kidney was determined. The holoenzyme was treated with NaB3H4 and digested with thermolysin; the labelled peptide was isolated and its sequence proved to be identical with sequences around the coenzyme site from the same isoenzyme of different organs and animals (pig heart and sheep liver). The sequences of the phosphopyridoxyl peptides of the mitochondrial aspartate aminotransferases appear to be closely related to the corresponding peptides from the cytoplasmic isoenzymes.

Amino Acids↗

PubMed1984

Histochemical localization of aspartate aminotransferase activity in the hippocampal formation and in peripheral ganglia of the rat with special reference to the glutamate transmitter metabolism.

The regional distribution and cellular localization of aspartate aminotransferase (AspAT), an glutamate/aspartate metabolizing enzyme, was studied in the hippocampal formation, in dorsal root ganglia, and in superior cervical ganglia during the postnatal development of the rat. In the dendritic layers of the hippocampal formation the enzyme activity was found to be very low in newborns, increased markedly from the second week of life and reached adult levels during the postweaning period. These results coincide with parameters of the maturation of glutamatergic structures in this brain region. The staining intensity of AspAT in the peripheral ganglia, in which glutamate/aspartate inputs do not obviously occur, did not display any evident differences during the postnatal development. The increase of AspAT activity parallel to the maturation of glutamatergic/aspartatergic mechanisms suggest that the enzyme studied is involved in glutamate/aspartate neurotransmitter metabolism.

Age Factors↗