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PubMed1979

Determination of serum aminotransferases: activation by pyridoxal-5'-phosphate in relation to substrate concentration.

To investigate the activation of aspartate- and alanine aminotransferases by pyridoxal-5'-phosphate, we determined the enzymatic activity in serum in two different ways: (a) Preincubation of the serum alone or the serum with pyridoxal-5'-phosphate and starting the reaction by the addition of the serum sample or the serum sample + coenzyme, respectively. (b) Preincubation of the serum or the serum with pyridoxal-5'-phosphate in the reaction medium and starting the reaction by adding 2-oxoglutarate. There are only small differences in activities of both aminotransferases determined according to these two different methods. The stimulation by pyridoxal-5'-phosphate is also of the same order, when both methods are compared. Further, these enzymatic activities were measured with use of various concentrations of substrates. From our experiments we conclude that the degree of stimulation of the apoenzyme of the two enzymes is independent of which way the enzymatic reaction is carried out or the substrate concentration, except that aspartate aminotransferase activity is more stimulated by the coenzyme at higher 2-oxoglutarate concentrations.

Alanine Transaminase↗

PubMed1994

The roles of myoglobin, MB iso-enzyme of creatine phosphokinase and aspartate aminotransferase in serum in the acute phase of myocardial infarction.

The diagnostic value of serum myoglobin as compared to MB iso-enzyme of creatine phosphokinase and aspartate aminotransferase was investigated in 25 patients admitted on suspicion of acute myocardial infarction with a duration of symptoms less than 6 hours. In group 1 (acute myocardial infarction group), the first blood sample, obtained at a mean time of 3.27 hours after onset of infarction, invariably showed increased myoglobin (mean 2.6-fold normal) whereas MB iso-enzyme of creatine phosphokinase and aspartate aminotransferase were often normal. Peak myoglobin values occurred earlier than peak serum MB iso-enzyme of creatine phosphokinase values. The highest peak values of serum myoglobin were found in patients with extensive myocardial infarction. In group 2 (non-acute myocardial infarction or control group) serial determinations of serum myoglobin, serum MB iso-enzyme of creatine phosphokinase and aspartate aminotransferase were within normal limits. Hence the importance lies with the early detection of serum myoglobin in acute myocardial infarction.

Adult↗

PubMed1977

[Interaction of aspartate aminotransferase with aminooxyalkylcelluloses].

Interaction of aspartate aminotransferase with various aminooxycelluloses capable of reacting with carbonyl compounds to form oximes has been studied with respect to the distance of the H2NO-groups from the polymer matrix. Aminotransferase does not react with aminooxycelluloses, when the amono-oxygroups are located at small distances from the matrix. When these celluloses interact with the enzyme in the presence of the substrate amino acid, we manage to obtain the amino-form of amino-transferase containing no pyridoxylidine form quantitatively. The cellulose with the H2NO-groups located at considerable distances from the polysaccharide matrix, form the "aminotransferase oxime--aminooxycellulose" complex, which is subsequently split either to a choloenzyme or apoenzyme of PLP and aminooxycellulose, depending on the experimental conditions used. The adsorbents under study may present some interest both in terms of isolation and purfication of pyridoxal enzymes and their effects on enzymatic systems, which contain carbonylic compounds.

Adsorption↗

PubMedDate not supplied

[Aspartate and alanine aminotransferase activity in the adrenal cortex of human embryos and fetuses].

Aspartate aminotransferase was detected in the adrenals of 6-7-week human embryos, the activity being the highest at the 7-9th and 12-14th weeks. Alanine aminotransferase was found in 15-week foetuses. During all the period investigated (6-28 weeks), the activity of aspartate aminotransferase was found to be higher than that of the second enzyme. The activity of both enzymes is higher in female foetuses than in male ones. However, age changes in the activity of both enzymes in male embryos and foetuses were of the same sign as in female ones.

Adrenal Cortex↗

PubMedDate not supplied

Elevated serum aminotransferase induced by isoniazid in relation to isoniazid acetylator phenotype.

Of 143 patients with pulmonary tuberculosis receiving isoniazid therapy, 52 (36%) had a transient elevation in serum aminotransferases. Among 74 patients taking isoniazid, rifampicin and streptomycin, 35 (47%) had such an increase, while of 69 patients taking isoniazid, amino-salicylic acid and streptomycin, 17 (24%) did; this difference was significant. Isoniazid therapy could be continued in all patients with the abnormal test results. In 36 of the patients receiving isoniazid, rifampicin and streptomycin, isoniazid and its metabolites were studied in the serum and urine using high-performance liquid chromatography after the oral administration of 10 mg per kg of isoniazid. We had chosen for this test 18 patients with normal aminotransferase levels and 18 with abnormal levels. There were 14 rapid acetylators in the patients with abnormal aminotransferase levels and 7 rapid acetylators in the patients with normal levels; this difference was significant. These results indicate that liver dysfunction is more often caused by an isoniazid/rifampicin regimen, and patients who are rapid acetylators are more susceptible.

Acetylation↗

PubMed2004

Murine alanine aminotransferase: cDNA cloning, functional expression, and differential gene regulation in mouse fatty liver.

Alanine aminotransferase (ALT) is a widely used index of liver integrity or hepatocellular damage in clinics as well as a key enzyme in intermediatary metabolism. In this study, we have cloned the complementary DNAs of murine homologues of human alanine aminotransferase 1 and 2 (ALT1 and ALT2). The deduced peptides of murine ALT1 (mALT1) and ALT2 (mALT2) share 87% and 93% identity, respectively, with their human counterparts at the amino acid level. Murine ALT genes localize to separate chromosomes, with mALT1 gene (gpt1) on chromosome 15 and mALT2 gene (gpt2) on chromosome 8. The murine gpt1 and gpt2 differ in messenger RNA expression: gpt1 is mainly expressed in liver, bowel, and white adipose tissue and gpt2 is highly expressed in muscle, liver, and white adipose tissue. Expression of recombinant mALT1 and mALT2 proteins in Escherichia coli (E. coli) produced functional enzymes that catalyze alanine transamination. The potential diagnostic value of ALT isoenzymes in liver disease was evaluated in an obese animal model. In fatty livers of obese mice, ALT2 gene expression is induced 2-fold, but ALT1 remains the same. Furthermore, in fatty liver, total hepatic ALT activity is elevated significantly by 30% whereas aspartate aminotransferase (AST) activity remains unchanged. In conclusion, these results indicate that ALT2 may be responsible for the increased ALT activity in hepatic steatosis and provide evidence that an ALT isoenzyme-specific assay may have more diagnostic value than the total ALT activity assay currently in clinical use.

Alanine Transaminase↗

PubMed2005

Value of two noninvasive methods to detect progression of fibrosis among HCV carriers with normal aminotransferases.

The course of hepatitis C virus (HCV) infection carriers with normal/near-normal aminotransferases (NALT) is usually mild; however, in a few, fibrosis progression occurs. We aimed to verify whether monitoring by liver biopsy might be replaced by noninvasive methods and to identify factors associated with fibrosis progression in patients with persistently normal alanine aminotransferases. We studied 40 untreated HCV-RNA-positive subjects (22 male; median age, 44 years), who underwent two liver biopsies, with a median interval of 78.5 months, during which alanine aminotransferase concentrations (median number of determinations: 12) never exceeded 1.2 times the upper normal limit. Within 9 months from the second biopsy, they were tested by the shear elasticity probe (Fibroscan) and the artificial intelligence algorithm FibroTest. METAVIR fibrosis scores were analyzed in relationship to demographic, clinical, and viral parameters. Weighted kappa analysis was used to verify whether the results of noninvasive methods agreed with histology. Significant fibrosis (> or = F2), present at the first biopsy in only one patient (2.5%), was observed at the second biopsy in 14 patients (35%). At multivariate analysis, excess alcohol consumption in the past (>20 g/d; P = .017) and viral load (>8.0 x 10(6) copies/mL; P = .021) were independent predictors of progression. In identifying patients with significant fibrosis, inter-rater agreement was excellent for Fibroscan (weighted kappa = 1.0), and poor for FibroTest (weighted kappa = -0.041). In conclusion, among HCV carriers with NALT, Fibroscan is superior to the FibroTest in the noninvasive identification of fibrosis, for which excess alcohol consumption in the past and high viral load represent risk factors.

Adult↗

PubMed1977

The influence of culture conditions on the induction of tyrosine aminotransferase by cyclic nucleotides in rat hepatoma cells.

The increase in tyrosine aminotransferase activity which occurs in rat hepatoma tissue culture (HTC) cells in response to cyclic AMP analogs has been shown to be an enzyme induction, similar to the larger response observed in certain other hepatoma cells and in liver. A specific antibody to tyrosine aminotransferase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by N6,O2'-dibutyryl cyclic AMP in HTC cells. The effect on tyrosine aminotransferase is also produced by various 8-substituted derivatives of cyclic AMP and occurs whether or not the enzyme has been preinduced with a glucocorticoid. The response of the enzyme is greater when HTC cells are maintained in monolayer than in suspension cultures. Neither cell growth nor serum is required for the response.

Antibodies↗

PubMed1975

Effect of uridine diphospho-N-acetylglucosamine and sodium salicylate on L-glutamine-D-fructose-6-phosphate aminotransferase activity from rat gastric mucosa.

Mechanisms of inhibitory action of sodium salicylate on L-glutamine-D-fructose-6-phosphate aminotransferase, prepared from rat gastric mucosa, were studied. Sodium salicylate at lower concentrations (10-20 mM) inhibited reversibly aminotransferase activity by competing with fructose-6-phosphate. At higher concentrations, sodium salicylate inactivated the enzyme irreversibly, with an inactivation rate following first-order kinetics with respect to the enzyme concentration. Uridine-5'-diphospho-N-acetylglucosamine is an endogenous feedback inhibitor. It inhibited the aminotransferase-catalyzed reaction also by competing with fructose-6-phosphate but with an inhibiting activity 1000 times that of sodium salicylate. Uridine-5'-diphospho-N-acetylglucosamine reduced the salicylate inhibition of the enzymic reaction and protected the enzyme from salicylate-induced irreversible inactivation. At a fixed concentration of uridine-5'-diphospho-N-acetylglucosamine (7x10(-6)M), an increase of salicylate concentration produced an increase in enzyme activity as compared with the control.

Animals↗

PubMed2006

Effect of pH on the structure and stability of Bacillus circulans ssp. alkalophilus phosphoserine aminotransferase: thermodynamic and crystallographic studies.

pH is one of the key parameters that affect the stability and function of proteins. We have studied the effect of pH on the pyridoxal-5'-phosphate-dependent enzyme phosphoserine aminotransferase produced by the facultative alkaliphile Bacillus circulans ssp. alkalophilus using thermodynamic and crystallographic analysis. Enzymatic activity assay showed that the enzyme has maximum activity at pH 9.0 and relative activity less than 10% at pH 7.0. Differential scanning calorimetry and circular dichroism experiments revealed variations in the stability and denaturation profiles of the enzyme at different pHs. Most importantly, release of pyridoxal-5'-phosphate and protein thermal denaturation were found to occur simultaneously at pH 6.0 in contrast to pH 8.5 where denaturation preceded cofactor's release by approximately 3 degrees C. To correlate the observed differences in thermal denaturation with structural features, the crystal structure of phosphoserine aminotransferase was determined at 1.2 and 1.5 A resolution at two different pHs (8.5 and 4.6, respectively). Analysis of the two structures revealed changes in the vicinity of the active site and in surface residues. A conformational change in a loop involved in substrate binding at the entrance of the active site has been identified upon pH change. Moreover, the number of intramolecular ion pairs was found reduced in the pH 4.6 structure. Taken together, the presented kinetics, thermal denaturation, and crystallographic data demonstrate a potential role of the active site in unfolding and suggest that subtle but structurally significant conformational rearrangements are involved in the stability and integrity of phosphoserine aminotransferase in response to pH changes.

Alkalies↗

PubMed1996

Coupled enzymatic assay for estimation of branched-chain L-amino acid aminotransferase activity with 2-Oxo acid substrates.

A convenient continuous spectrophotometric assay for estimation of branched-chain L-amino acid aminotransferase activity was established: Branched-chain 2-oxo acid-dependent transamination of L-glutamate was coupled-via 2-oxoglutarate-to L-aspartate aminotransferase plus L-malate dehydrogenase or to L-alanine aminotransferase plus L-lactate dehydrogenase as indicator systems. The rate of transamination can be monitored specifically by measuring the decrease in NADH absorbance at 334 nm over time. The method was applied, e.g., for evaluation of some kinetic properties of the rat heart (iso)enzyme.

Animals↗

PubMed1996

In vitro association with peroxisomes and conformational change of peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase in rat and human livers.

To understand the targeting mechanisms of peroxisomal serine:pyruvate aminotransferase, in vitro import experiments were carried out using this 43 kDa peroxisomal enzyme, which was synthesized in a coupled transcription/translation system. Being different from other peroxisomal enzymes, such as acyl-CoA oxidase and urate oxidase used in previous in vitro import experiment, the soluble form of peroxisomal serine:pyruvate aminotransferase was fairly resistant to proteinase K digestion. However, the enzyme recovered in the peroxisomal fraction was proteinase K sensitive. This indicates that the association of peroxisomal serine:pyruvate aminotransferase with the peroxisomal membrane causes a marked conformational change in the structure of this protein.

Alanine Transaminase↗

PubMed1993

Isolation and characterization of an ornithine aminotransferase-related sequence (OATL3) mapping to 10q26.

We used a near full-length human ornithine delta-amino-transferase cDNA, huOAT6, as a probe under low stringency hybridization conditions to identify a new autosomal ornithine delta-aminotransferase-related sequence (OATL3). Cloning and characterization of this sequence reveal it to be a partial nonprocessed pseudogene corresponding to exon 3 and flanking intronic sequences of the ornithine delta-aminotransferase structural gene. Using somatic cell hybrids and fluorescence in situ hybridization, we mapped OATL3 to 10q26, adjacent to the ornithine delta-aminotransferase structural gene locus.

Base Sequence↗

PubMed1999

Crystal structure of human ornithine aminotransferase complexed with the highly specific and potent inhibitor 5-fluoromethylornithine.

Ornithine aminotransferase (l-ornithine:2-oxoacid delta-aminotransferase; EC 2.6.1.13), a pyridoxal-5'-phosphate-dependent mitochondrial enzyme controls the l-ornithine level in tissues by catalyzing the transfer of the delta-amino group of l-ornithine to 2-oxoglutarate, producing l-glutamate- gamma-semialdehyde and l-glutamate. (2S, 5S)-5-Fluoromethylornithine is the only inhibitor exclusively specific for ornithine aminotransferase known to date. Both in vitro and in vivo, it blocks the enzyme by a suicide reaction leading to a covalent adduct with the cofactor. The crystal structure of the enzyme-inhibitor complex was solved at a resolution of 1.95 A. No significant conformational changes compared with the native enzyme structure were observed. The structure reveals the atomic details of the cofactor-inhibitor adduct and its interactions with the active site of the enzyme. The main residues responsible for specific binding of the inhibitor are Arg180, which forms a strong salt bridge with the alpha-carboxylate and Tyr55, which is involved in a short hydrogen bond with the alpha-amino group. The experimental observation that in the racemic mixture, (2S, 5S)-5-fluoromethylornithine is exclusively responsible for the enzyme inhibition can be explained on the basis of the active site topology. Model building studies strongly suggest that the natural substrate l-ornithine, in its external aldimine adduct with the enzyme, makes use of the same recognition site as the inhibitor. It is proposed that the neutralization of the active site Arg413 by a salt bridge with Glu235 also plays an important role in productive binding of both 5-fluoromethylornithine and l-ornithine. Arg180 and Arg413 are believed to be instrumental in recognition of l-glutamate, by binding its gamma and alpha-carboxylate groups, respectively. This requires a different side-chain conformation of Glu235. Lys292 is the only obvious candidate for catalyzing the rate-limiting proton transfer steps in the transamination reaction.

Amino Acid Sequence↗

PubMed1999

Crystal structure of phosphoserine aminotransferase from Escherichia coli at 2.3 A resolution: comparison of the unligated enzyme and a complex with alpha-methyl-l-glutamate.

Phosphoserine aminotransferase (PSAT; EC 2.6.1.52), a member of subgroup IV of the aminotransferases, catalyses the conversion of 3-phosphohydroxypyruvate to l-phosphoserine. The crystal structure of PSAT from Escherichia coli has been solved in space group P212121 using MIRAS phases in combination with density modification and was refined to an R-factor of 17.5% (Rfree=20.1 %) at 2.3 A resolution. In addition, the structure of PSAT in complex with alpha-methyl-l-glutamate (AMG) has been refined to an R-factor of 18.5% (Rfree=25.1%) at 2.8 A resolution. Each subunit (361 residues) of the PSAT homodimer is composed of a large pyridoxal-5'-phosphate binding domain (residues 16-268), consisting of a seven-stranded mainly parallel beta-sheet, two additional beta-strands and seven alpha-helices, and a small C-terminal domain, which incorporates a five-stranded beta-sheet and two alpha-helices. A three-dimensional structural comparison to four other vitamin B6-dependent enzymes reveals that three alpha-helices of the large domain, as well as an N-terminal domain (subgroup II) or subdomain (subgroup I) are absent in PSAT. Its only 15 N-terminal residues form a single beta-strand, which participates in the beta-sheet of the C-terminal domain. The cofactor is bound through an aldimine linkage to Lys198 in the active site. In the PSAT-AMG complex Ser9 and Arg335 bind the AMG alpha-carboxylate group while His41, Arg42 and His328 are involved in binding the AMG side-chain. Arg77 binds the AMG side-chain indirectly through a solvent molecule and is expected to position itself during catalysis between the PLP phosphate group and the substrate side-chain. Comparison of the active sites of PSAT and aspartate aminotransferase suggests a similar catalytic mechanism, except for the transaldimination step, since in PSAT the Schiff base is protonated. Correlation of the PSAT crystal structure to a published profile sequence analysis of all subgroup IV members allows active site modelling of nifs and the proposal of a likely molecular reaction mechanism.

Alanine Transaminase↗

PubMed2001

Crystal structure of histidinol phosphate aminotransferase (HisC) from Escherichia coli, and its covalent complex with pyridoxal-5'-phosphate and l-histidinol phosphate.

The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants. The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5'-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9). Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5'-phosphate (PMP) at 1.5 A resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 A resolution. This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine.HisC is a dimeric enzyme with a mass of approximately 80 kDa. Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an alpha/beta/alpha topology, and a smaller domain. An N-terminal arm contributes to the dimerization of the two monomers. The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes. Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases. The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis. Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure. Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups. We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site.

Amino Acid Sequence↗

PubMed1983

Regulation of the synthesis of tyrosine aminotransferase: the relationship to mRNATAT.

The activity of the hepatic enzyme tyrosine aminotransferase (TAT) is the sum of many diverse regulatory factors. These include the developmental stage of the animal, the hormonal and nutritional environment of the animal (or tissue culture cell), other extrinsic and intrinsic regulatory cycles and factors (including cytoplasmic substances), and chromatin structure. Although TAT is subject to a number of post-translational modifications, alterations in catalytic activity always parallel changes in enzyme amount. In a few instances this is due to a selective change in TAT degradation, but most are due to changes in the rate of aminotransferase synthesis. Recent studies have shown that TAT synthesis is generally directly correlated with the activity, and presumably amount, of the mRNA that codes for tyrosine aminotransferase.

Adrenalectomy↗

PubMed1991

Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli.

In bacteria 5-aminolevulinate, the universal precursor in the biosynthesis of the porphyrin nucleus of hemes, chlorophylls and bilins is synthesised by two different pathways: in non-sulphur purple bacteria (Rhodobacter) or Rhizobium 5-aminolevulinate synthase condenses glycine and succinyl-CoA into 5-aminolevulinate as is the case in mammalian cells and yeast. In cyanobacteria, green and purple sulphur bacteria, as in chloroplasts of higher plants and algae a three step pathway converts glutamate into 5-aminolevulinate. The last step is the conversion of glutamate 1-semialdehyde into 5-aminolevulinate. Using a cDNA clone encoding glutamate 1-semialdehyde aminotransferase from barley, genes for this enzyme were cloned from Synechococcus PCC6301 and Escherichia coli and sequenced. The popC gene of E. coli, previously considered to encode 5-aminolevulinate synthase, appears to be a structural gene for glutamate 1-semialdehyde aminotransferase. Domains with identical amino acid sequences comprise 48% of the primary structure of the barley, cyanobacterial and putative E. coli glutamate 1-semialdehyde aminotransferases. The cyanobacterial and barley enzymes share 72% identical residues. The peptide containing a likely pyridoxamine phosphate binding lysine is conserved in all three protein sequences.

Amino Acid Sequence↗