Cysteine sulfinate aminotransferase and aspartate aminotransferase isoenzymes of rat brain. Purification, characterization, and further evidence for identity.
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Animals↗
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Results for “ASPARTATE AMINOTRANSFERASE”
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[Alanine aminotransferase and aspartate aminotransferase activity in cultured chicken fibroblasts in the process of growing and after infection by vaccinia virus].
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Alanine Transaminase↗
[Measurement of alanine aminotransferase and aspartate aminotransferase activity with a 2-channel automatic flow analyser].
A flow automaton for the estimation of the enzyme activities of the ALAT and ASAT is described: 1. In precision and exactness the automaton achieves results comparable to the manual UV-test. The VK are clearly below 5%. 2. Sera with enzyme activities greater than 200 U/l are diluted with human albumin solution (20 g/l) 1 : 5 or 1 : 10. 3. The speed of the analysis with simultaneous estimation of the activity of the ALAT and ASAT from one test is 40 tests/h. 4. The technological disadvantages of the colour test regarding the kinetic measurement are vastly evaded by the selected device parameters and conditions.
Alanine Transaminase↗
[Alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), alkaline phosphatase (AP) and gamma-glutamyltransferase (GGP) in intestinal diseases of dogs].
331 dogs, suffering from different intestinal diseases with diarrhea, were classified into the groups of "acute noninfectious", "infectious", "chronic" and "secondary enteropathies". The serum enzymes ALT, AST, GLDH, AP and GGT were determined. In all groups increases of enzyme activities were to be found. The highest and most frequent increases have been observed in acute noninfectious and in secondary enteropathies. The enzyme pattern in acute noninfectious enteropathies indicate a secondary liver disturbance in consequence of the intestinal disease, whereas the liver participation in secondary enteropathies is the effect of the primary disease other than intestinal disturbances. In comparison to this the height and number of increases of liver enzyme activities were low in acute infectious and in chronic enteropathies.
Alanine Transaminase↗
Serum versus heparinized plasma for alanine aminotransferase and aspartate aminotransferase of normal individuals.
The influence of heparin on the estimation of aminotransferases was investigated in a reaction medium containing phosphate or Tris buffer. Heparin scarcely affects the ASAT determination, but the ALAT activity in plasma is about 70% of the activity in serum, when the enzyme determination is carried out in Tris buffer. There exists a lag phase due to which the decrease in absorbance with the incubation time is nonlinear. When the Tris buffer is omitted, the same lag phase is observed in the ALAT determination. This lag phase is not caused by the indicator enzyme LDH. Addition of phosphate to the incubation medium without any buffer abolishes this lag phase. The possibility exists that heparin influences the structure of the proteins present in plasma and that these proteins, in their turn, decrease the enzymatic activity of ALAT.
Alanine Transaminase↗
Aspartate aminotransferase from a thermophilic formate-utilizing methanogen, Methanobacterium thermoformicicum strain SF-4: relation to serine and phosphoserine aminotransferases, but not to the aspartate aminotransferase family.
The primary structure of the aspartate aminotransferase (AspAT) of an archaebacterium, Methanobacterium thermoformicicum strain SF-4, has been determined by cloning and sequencing of the gene for the enzyme. The gene had a consensus promoter and a ribosome binding sequence of methanogens in the 5' untranslated region, followed by an open reading frame starting with ATG and terminating with TGA. The deduced amino acid sequence was identical with the partial amino acid sequences of the enzyme including the N-terminal sequence, and the deduced molecular weight of 41,684 was virtually identical to that reported earlier for this enzyme [Tanaka, T., Yamamoto, S., Taniguchi, M., Hayashi, H., Kuramitsu, S., Kagamiyama, H., & Oi, S. (1992) J. Biochem. 112, 811-815]. The gene was expressed in Escherichia coli by inserting it into an expression vector just downstream of the lacZ promoter, and this verified that the cloned gene really encodes the Methanobacterium AspAT. The primary structure of the Methanobacterium AspAT showed extremely low homology, 5%, with AspATs of eubacteria, eukaryotes, and a thermoacidophilic arachaebacterium, Sulfolobus solfataricus. On the other hand, the Methanobacterium AspAT showed remarkable amino acid sequence homology, 31.5%, with rat serine:pyruvate aminotransferase and, 13.5%, with E. coli phosphoserine aminotransferase. Thus, the Methanobacterium AspAT apparently belongs to subgroup IV of the aminotransferases [Mehta, P.K., Hale, T.I., & Christen, P. (1993) Eur. J. Biochem. 214, 549-561], but not to subgroup I, in which all the AspATs known so far are included.
Amino Acid Sequence↗
Proteinase K inactivation of cytosolic aspartate aminotransferase isoenzyme for measurement of human serum mitochondrial aspartate aminotransferase.
We studied a new proteinase K assay method for human serum mitochondrial aspartate aminotransferase. We found that proteinase K showed no inactivation of human mitochondrial aspartate aminotransferase isoenzyme and complete inactivation of cytosolic aspartate aminotransferase. Previous studies have shown that selective proteolytic measurement for mitochondrial aspartate aminotransferase in serum using the protease 401 cleaved peptide bond at Leu 20 from the amino-terminal bond shows complete inactivation of cytosolic aspartate aminotransferase and slight inactivation of mitochondrial aspartate aminotransferase isoenzyme, depending on protease concentration. In this investigation, we found that the proteinase K method does not depend on protease concentration. The proteinase K enzyme inactivation of cytosolic aspartate aminotransferase is caused by the cleavage of the peptide bond at Ileu 21 from the aminoterminal bond. In studies with various animal cytosolic aspartate aminotransferase isoenzymes, proteinase K almost completely inactivated cytosolic aspartate aminotransferase. Precision and correlation using proteinase K for measurement of serum mitochondrial aspartate aminotransferase in human showed a good coefficient of variation (within-run < 4.45%) and a coefficient of correlation of r = 0.985 (N = 125).
Adult↗
Application of immobilized aspartate aminotransferase and immobilized aspartate aminotransferase-malate dehydrogenase coupled system to micro-assay of L-aspartic acid.
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Animals↗
Measurement of aminotransferases: Part 1. Aspartate aminotransferase.
Aminotransferases are ubiquitous enzymes of mammalian cells and several are of important diagnostic use. The application of aspartate aminotransferase activity measurements in serum from individuals suffering from myocardial infarction brought about a new dimension in clinical laboratory testing in the 1950s. This review focuses on measurement techniques for aspartate aminotransferase and their application (a subsequent article will review other aminotransferases). Assay techniques measuring enzyme activity are direct spectrophotometric measurements, manometric techniques, assays using dye substances, coupled enzyme techniques, and radiometric procedures. Of these procedures, the one employing malate dehydrogenase and NADH is the most important and is covered in particular detail. The estimation of the mitochondrial isoenzyme of aspartate aminotransferase is also of clinical interest, in particular for estimating severity of disease or in specific applications (e.g., chronic alcoholism). Methods reviewed for estimation of this enzyme are electrophoresis, chromatography, differential kinetic behavior, and immunochemical separation. Determination of the enzyme protein by techniques independent of its catalytic activity are also reviewed.
Animals↗
The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes.
In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage.
Animals↗
Biosynthesis of aspartate aminotransferases. Both the higher molecular weight precursor of mitochondrial aspartate aminotransferase and the cytosolic isoenzyme are synthesized on free polysomes.
The site of synthesis of the higher molecular weight precursor of mitochondrial aspartate aminotransferase (Sonderegger, P., Jaussi, R., and Christen, P. (1980) Biochem. Biophys. Res. Commun. 94, 1256-1260) has been determined by separation of free and membrane-bound polysomes under ionic conditions imitating the intracellular milieu and in vitro read-out translation of the two polysome fractions in a rabbit reticulocyte lysate. The amounts of the precursor of mitochondrial aspartate aminotransferase synthesized by free and membrane-bound polysomes were compared with the relative extent of the synthesized of cytosolic aspartate aminotransferase in the same fractions. Only a small (less than 10% of total) and for both isoenzymes quantitatively equivalent fraction was found to be produced by the membrane-bound polysome fraction; very likely, it has to be attributed to contaminating free polysomes. Apparently, the import of mitochondrial aspartate aminotransferase into the mitochondria does not involve an association of polysomes with intracellular membranes.
Animals↗
Cloning and nucleotide sequencing of Rhizobium meliloti aminotransferase genes: an aspartate aminotransferase required for symbiotic nitrogen fixation is atypical.
In Rhizobium meliloti, an aspartate aminotransferase (AspAT) encoded within a 7.3-kb HindIII fragment was previously shown to be required for symbiotic nitrogen fixation and aspartate catabolism (V. K. Rastogi and R.J. Watson, J. Bacteriol. 173:2879-2887, 1991). A gene coding for an aromatic aminotransferase located within an 11-kb HindIII fragment was found to complement the AspAT deficiency when overexpressed. The genes encoding these two aminotransferases, designated aatA and tatA, respectively, have been localized by subcloning and transposon Tn5 mutagenesis. Sequencing of the tatA gene revealed that it encodes a protein homologous to an Escherichia coli aromatic aminotransferase and most of the known AspAT enzymes. However, sequencing of the aatA gene region revealed two overlapping open reading frames, neither of which encoded an enzyme with homology to the typical AspATs. Polymerase chain reaction was used to selectively generate one of the candidate sequences for subcloning. The cloned fragment complemented the original nitrogen fixation and aspartate catabolism defects and was shown to encode an AspAT with the expected properties. Sequence analysis showed that the aatA protein has homology to AspATs from two thermophilic bacteria and the eukaryotic tyrosine aminotransferases. These aminotransferases form a distinct class in which only 13 amino acids are conserved in comparison with the well-known AspAT family. DNA homologous to the aatA gene was found to be present in Agrobacterium tumefaciens and other rhizobia but not in Klebsiella pneumoniae or E. coli.
Amino Acid Sequence↗
International Federation of Clinical Chemistry (IFCC) Scientific Committee, Analytical Section: approved recommendation (1985) on IFCC methods for the measurement of catalytic concentration of enzymes. Part 2. IFCC method for aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1).
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Aspartate Aminotransferases↗
Effects of substrate structural analogues on the enzymatic activities of aspartate aminotransferase isoenzymes.
Aspartate aminotransferase (AAT, EC 2.6.1.1) catalyses the transamination of L-asparate to oxaloacetate. It has been reported that AAT from different plant sources can catalyse the transamination of other compounds structurally similar to the natural substrates. Specificity and kinetic studies were performed with two aspartate aminotransferase isoenzymes (AAT-1 and AAT-2) from leaves of Lupinus albus L. cv Estoril using different amino donors and acceptors. Both isoenzymes showed residual activity for some of the substrates tested. Competitive inhibition was found with most of the structural analogues which is typical of a ping-pong bi-bi kinetic mechanism. It was found that both isoenzymes can use 2-amino-4-methoxy-4-oxobutanoic acid as amino donor. AAT-2 uses 2-amino-4-methoxy-4-oxobutanoic acid at a similar rate as L-aspartate but AAT-1 uses this substrate at a slower rate. The use of this amino donor by AAT isoenzymes has not been reported previously, and our results indicate structural differences between both isoenzymes.
Amino Acids↗
Tyrosine 70 fine-tunes the catalytic efficiency of aspartate aminotransferase.
The aspartate aminotransferase mutant Y70F exhibits kcat = 8% and kcat/KM = 2% of the wild type values for the transamination of aspartate and alpha-ketoglutarate. The affinity of the enzyme for the noncovalently bound inhibitor maleate is reduced 17-fold by the mutation, while only a 2.5-fold reduction is observed for alpha-methylaspartate, which forms a stable, covalent external aldimine. The high population of the quinonoid intermediate formed in the reaction of the wild type with beta-hydroxyaspartate is more than 75% diminished by the mutation. The values of the Y70F C alpha-H kinetic isotope effects for the aspartate reaction are larger than those of wild type (DV = 2.4 vs 1.52; D(V/K) = 2.5 vs 1.7). Conversely, the Y70F value of D(V/K) for the glutamate reaction is decreased compared to wild type (1.75 vs 2.5). These results, combined with previous studies of Lys258 mutants, eliminate Tyr70 as an essential component of the catalytic apparatus, with the caveat that the functionally of the deleted hydroxyl group is possibly replaced by a water molecule.
Aspartate Aminotransferases↗
Aspartate aminotransferase activity is required for aspartate catabolism and symbiotic nitrogen fixation in Rhizobium meliloti.
A mutant of Rhizobium meliloti, 4R3, which is unable to grow on aspartate has been isolated. The defect is specific to aspartate utilization, since 4R3 is not an auxotroph and grows as well as its parent strain on other carbon and nitrogen sources. The defect was correlated with an inability to fix nitrogen within nodules formed on alfalfa. Transport of aspartate into the mutant cells was found to be normal. Analysis of enzymes involved in aspartate catabolism showed a significantly lower level of aspartate aminotransferase activity in cell extracts of 4R3 than in the wild type. Two unrelated regions identified from a genomic cosmid bank each complemented the aspartate catabolism and symbiotic defects in 4R3. One of the cosmids was found to encode an aspartate aminotransferase enzyme and resulted in restoration of aspartate aminotransferase activity in the mutant. Analysis of the region cloned in this cosmid by transposon mutagenesis showed that mutations within this region generate the original mutant phenotypes. The second type of cosmid was found to encode an aromatic aminotransferase enzyme and resulted in highly elevated levels of aromatic aminotransferase activity. This enzyme apparently compensated for the mutation by its ability to partially utilize aspartate as a substrate. These findings demonstrate that R. meliloti contains an aspartate aminotransferase activity required for symbiotic nitrogen fixation and implicate aspartate as an essential substrate for bacteria in the nodule.
Aspartate Aminotransferases↗
The role of His143 in the catalytic mechanism of Escherichia coli aspartate aminotransferase.
In aspartate aminotransferase (AspAT), His143 is located within a hydrogen-bonding distance to Asp222 that forms a strong ion pair with the ring nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). His143 of Escherichia coli AspAT was replaced by Ala or Asn. The mutant enzyme H143A showed a slight increase in the maximum velocity of the overall transamination reaction between aspartate and 2-oxoglutarate, while H143N AspAT showed a decrease to 60% in the maximum rate of the overall reactions in both directions. In all of the half-transamination reactions with four substrates, aspartate, glutamate, oxalacetate, and 2-oxoglutarate, the catalytic competence as defined by kmax/Kd decreased by 3-18-fold upon replacing His143 by either Ala or Asn. The extent of the decrease varied from one substrate to another; it was largely contributed to by the decrease in affinities for all substrates. The equilibrium constants, [PMP-form] [keto acid]/[( PLP-form] [amino acid]), decreased by over 10-fold upon the mutations at position 143. Both H143A and H143N AspATs exhibited a considerably decreased affinity for 2-methylaspartate, an external-aldimine-forming substrate analogue, yet without appreciable alteration in the affinity for succinate and glutarate, which are non-aldimine-forming analogues. All these findings suggest that, although His143 is not essential for catalysis, it might assist the formation of enzyme-substrate complex.
Aspartate Aminotransferases↗
Aspartate Aminotransferase in Alfalfa Root Nodules : III. Genotypic and Tissue Expression of Aspartate Aminotransferase in Alfalfa and Other Species.
Aspartate aminotransferase (AAT) plays an important role in nitrogen metabolism in all plants and is particularly important in the assimilation of fixed N derived from the legume-Rhizoblum symbiosis. Two isozymes of AAT (AAT-1 and AAT-2) occur in alfalfa (Medicago sativa L.). Antibodies against alfalfa nodule AAT-2 do not recognize AAT-1, and these antibodies were used to study AAT-2 expression in different tissues and genotypes of alfalfa and also in other legume and nonlegume species. Rocket immunoelectrophoresis indicated that nodules of 38-day-old alfalfa plants contained about eight times more AAT-2 than did nodules of 7-day-old plants, confirming the nodule-enhanced nature of this isozyme. AAT-2 was estimated to make up 16, 15, 5, and 8 milligrams per gram of total soluble protein in mature nodules, roots, stems, and leaves, respectively, of effective N(2)-fixing alfalfa. The concentration of AAT-2 in nodules of ineffective non-N(2)-fixing alafalfa genotypes was about 70% less than that of effective nodules. Western blots of soluble protein from nodules of nine legume species indicated that a 40-kilodalton polypeptide that reacts strongly with AAT-2 antibodies is conserved in legumes. Nodule AAT-2 immunoprecipitation data suggested that amide- and ureide-type legumes may differ in expression and regulation of the enzyme. In addition, Western blotting and immunoprecipitations of AAT activity demonstrated that antibodies against alfalfa AAT-2 are highly cross-reactive with AAT enzyme protein in leaves of soybean (Glycine max L.), wheat (Triticum aestivum L.), and maize (Zea mays L.) and in roots of maize, but not with AAT in soybean and wheat roots. Results from this study indicate that AAT-2 is structurally conserved and localized in similar tissues among diverse species.
Journal Article↗