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PubMedDate not supplied

Serum aminotransferase levels and histological disease in chronic hepatitis C.

The reliability of serum aminotransferase (ASAT and ALAT) levels, currently used in deciding on performing liver biopsy and to assess interferon therapy in chronic hepatitis C has been questioned. In Belgium, interferon therapy is actually only reimbursed for treatment of chronic hepatitis C when serum aminotransferase levels are more than twice the upper limit of normal. The aim of the present study was to assess the relationship between serum aminotransferase levels and histological severity of chronic hepatitis C. Sixty-seven liver biopsies from 51 different patients with chronic hepatitis C and presenting with elevated ASAT and/or ALAT levels, were retrospectively evaluated using the original terminology (minimal hepatitis, chronic persistent hepatitis, chronic active hepatitis, cirrhosis), the Knodell score and the components of the Bianchi-Gudat score, where grading (portal inflammation, piecemeal necrosis, intra-acinar necrosis and inflammation) and staging components (fibrosis/ cirrhosis) are quantitated separately. The correlation between amino-transferase levels measured at or near to the biopsy date and histological criteria were evaluated using Spearman's rank correlation. About one third of the patients, including patients with chronic active hepatitis and cirrhosis, presented with ASAT and ALAT levels less than twice the upper limit of normal. ASAT levels correlated with originally determined histological severity, the numerical Knodell score and the numerical scores for piecemeal necrosis, for intra-acinar necrosis and inflammation and for fibrosis in the Bianchi-Gudat score. ALAT levels correlated only with intra-acinar necrosis and inflammation. It is concluded that limiting interferon therapy to patients with aminotransferase levels over twice the upper limit of normal excludes a large proportion of patients from potentially curative treatment. ASAT levels are more useful than ALAT to assess the histological severity of the disease, probably because this mitochondrial enzyme is present in higher quantities in the liver as compared to the cytosolic ALAT, and is more released when tissue damage is more severe.

Adult↗

PubMed1983

Development of tyrosine aminotransferase in perinatal rat liver: changes in functional messenger RNA and the role of inducing hormones.

Expression of the hepatic enzyme tyrosine aminotransferase was analyzed in the perinatal period of development in the rat, when this expression undergoes significant changes associated with hepatocyte differentiation. In late prenatal liver both enzyme and functional mRNA gene products are present at levels 10- to 15-fold below those in the fully differentiated adult liver. This low level of expression in fetal liver is refractory to induction by glucocorticoids, but both gene products are increased to a limited extent by cyclic AMP. This induction by cyclic AMP (cAMP) does not confer glucocorticoid-responsiveness on expression. By 3 hr after birth both functional mRNA and enzyme levels are significantly increased, an increase which continues until a peak is reached at 12 hr that is appreciably above the adult levels. Both gene products then decline until adult levels are reached by 24 hr. The postnatal shift in aminotransferase expression is accompanied by acquisition of the capacity to respond to glucocorticoids. Treatment of newborns with an antiglucocorticoid steroid or with glucose suppresses the postnatal overshoot of expression, but neither treatment affects the increase from fetal to adult levels of expression. The results indicate that prior to birth, expression of the aminotransferase gene is partially repressed, a repression that is lifted essentially immediately upon birth. The hormones capable of inducing aminotransferase synthesis have no apparent necessary role in this process.

Animals↗

PubMed2006

Crystal structures of the PLP- and PMP-bound forms of BtrR, a dual functional aminotransferase involved in butirosin biosynthesis.

The aminotransferase (BtrR), which is involved in the biosynthesis of butirosin, a 2-deoxystreptamine (2-DOS)-containing aminoglycoside antibiotic produced by Bacillus circulans, catalyses the pyridoxal phosphate (PLP)-dependent transamination reaction both of 2-deoxy-scyllo-inosose to 2-deoxy-scyllo-inosamine and of amino-dideoxy-scyllo-inosose to 2-DOS. The high-resolution crystal structures of the PLP- and PMP-bound forms of BtrR aminotransferase from B. circulans were solved at resolutions of 2.1 A and 1.7 A with R(factor)/R(free) values of 17.4/20.6 and 19.9/21.9, respectively. BtrR has a fold characteristic of the aspartate aminotransferase family, and sequence and structure analysis categorises it as a member of SMAT (secondary metabolite aminotransferases) subfamily. It exists as a homodimer with two active sites per dimer. The active site of the BtrR protomer is located in a cleft between an alpha helical N-terminus, a central alphabetaalpha sandwich domain and an alphabeta C-terminal domain. The structures of the PLP- and PMP-bound enzymes are very similar; however BtrR-PMP lacks the covalent bond to Lys192. Furthermore, the two forms differ in the side-chain conformations of Trp92, Asp163, and Tyr342 that are likely to be important in substrate selectivity and substrate binding. This is the first three-dimensional structure of an enzyme from the butirosin biosynthesis gene cluster.

Amino Acid Sequence↗

PubMed1998

Crystal structure of human recombinant ornithine aminotransferase.

Ornithine aminotransferase (OAT), a pyridoxal-5'-phosphate dependent enzyme, catalyses the transfer of the delta-amino group of L-ornithine to 2-oxoglutarate, producing L-glutamate-gamma-semialdehyde, which spontaneously cyclizes to pyrroline-5-carboxylate, and L-glutamate. The crystal structure determination of human recombinant OAT is described in this paper. As a first step, the structure was determined at low resolution (6 A) by molecular replacement using the refined structure of dialkylglycine decarboxylase as a search model. Crystallographic phases were then refined and extended in a step-wise fashion to 2.5 A by cyclic averaging of the electron density corresponding to the three monomers within the asymmetric unit. Interpretation of the resulting map was straightforward and refinement of the model resulted in an R-factor of 17.1% (Rfree=24.3%). The success of the procedure demonstrates the power of real-space molecular averaging even with only threefold redundancy. The alpha6-hexameric molecule is a trimer of intimate dimers with a monomer-monomer interface of 5500 A2 per subunit. The three dimers are related by an approximate 3-fold screw axis with a translational component of 18 A. The monomer fold is that of a typical representative of subgroup 2 aminotransferases and very similar to those described for dialkylglycine decarboxylase from Pseudomonas cepacia and glutamate-1-semialdehyde aminomutase from Synechococcus. It consists of a large domain that contributes most to the subunit interface, a C-terminal small domain most distant to the 2-fold axis and an N-terminal region that contains a helix, a loop and a three stranded beta-meander embracing a protrusion in the large domain of the second subunit of the dimer. The large domain contains the characteristic central seven-stranded beta-sheet (agfedbc) covered by eight helices in a typical alpha/beta fold. The cofactor pyridoxal-5'-phosphate is bound through a Schiff base to Lys292, located in the loop between strands f and g. The C-terminal domain includes a four-stranded antiparallel beta-sheet in contact with the large domain and three further helices at the far end of the subunit. The active sites of the dimer lie, about 25 A apart, at the subunit and domain interfaces. The conical entrances are on opposite sides of the dimer. In the active site, R180, E235 and R413 are probable substrate binding residues. Structure-based sequence comparisons with related transaminases in this work support that view. In patients suffering from gyrate atrophy, a recessive hereditary genetic disorder that can cause blindness in humans, ornithine aminotransferase activity is lacking. A large number of frameshift and point mutations in the ornithine aminotransferase gene have been identified in such patients. Possible effects of the various point mutations on the structural stability or the catalytic competence of the enzyme are discussed in light of the three-dimensional structure.

Binding Sites↗

PubMed1979

Branched chain amino acid aminotransferase isoenzymes of Pseudomonas cepacia.

Pseudomonas cepacia grew rapidly using a mixture of all three branched chain amino acids as carbon source, but failed to use individual branched chain amino acids as sole carbon source. Extracts of bacteria grown on branched chain amino acids had between 2- and 3-fold higher levels of alpha-ketoglutarate-dependent branched chain amino acid aminotransferase activity than extracts of glucose-grown bacteria. The increase in enzyme activity was due to the presence of a second aminotransferase not detected in extracts of glucose-grown bacteria. The enzyme, which presumably plays a role in branched chain amino acid degradation, had an apparent molecular weight (mol.wt.) of 75,000. The other aminotransferase was formed constitutively and apparently functions in synthesis of branched chain amino acids. It was more stable than the 75,000 mol.wt. enzyme, and was purified to homogeneity and found to be a 180,000 mol.wt. oligomer containing 6 subunits of approximately 30,000 mol.wt. Antiserum prepared against the purified enzyme inhibited its activity but failed to influence the activity of the 75,000 mol.wt. aminotransferase, suggesting that the two isoenzymes are encoded by different genes.

Humans↗

PubMed1983

L-glutamine D-fructose-6-P aminotransferase regulation by glucose-6-P and UDP-N-acetylglucosamine.

L-Glutamine D-Fructose-6-P aminotransferase regulates hexosamine synthesis. An affinity purified human fibroblast aminotransferase and specific radioisotope assays developed by us were used to show an independent inhibition of the aminotransferase by Glucose-6-P. More interestingly, at concentration of UDP-N-Acetylglucosamine and glucose-6-P where either sugar has no independent inhibitory effect, there is an allosteric and significant inhibition of the aminotransferase.

Allosteric Regulation↗

PubMed1989

Activities of pyruvate dehydrogenase, enzymes of citric acid cycle, and aminotransferases in the subcellular fractions of cerebral cortex in normal and hyperammonemic rats.

Activity levels of pyruvate dehydrogenase, enzymes of citric acid cycle, aspartate and alanine aminotransferases were estimated in mitochondria, synaptosomes and cytosol isolated from brains of normal rats and those injected with acute and subacute doses of ammonium acetate. In mitochondria isolated from animals treated with acute dose of ammonium acetate, there was an elevation in the activities of pyruvate, isocitrate and succinate dehydrogenases while the activities of malate dehydrogenase (malate----oxaloacetate), aspartate and alanine aminotransferases were suppressed. In subacute conditions a similar profile of change was noticed excepting that there was an elevation in the activity of alpha-ketoglutarate dehydrogenase in mitochondria. In the synaptosomes isolated from animals administered with acute dose of ammonium acetate, there was an increase in the activities of pyruvate, isocitrate, alpha-ketoglutarate and succinate dehydrogenases while the changes in the activities of malate dehydrogenase, aspartate and alanine amino transferases were suppressed. In the subacute toxicity similar changes were observed in this fraction except that the activity of malate dehydrogenase (oxaloacetate----malate) was enhanced. In the cytosol, pyruvate dehydrogenase and other enzymes of citric acid cycle except malate dehydrogenase were enhanced in both acute and subacute ammonia toxicity though their activities are lesser than that of mitochondria. In this fraction malate dehydrogenase (oxaloacetate----malate) was enhanced while activities of malate dehydrogenase (malate----oxaloacetate), aspartate and alanine aminotransferases were suppressed in both the conditions. Based on these results it is concluded that the decreased activities of malate dehydrogenase (malate----oxaloacetate) in mitochondria and of aspartate aminotransferase in mitochondria and cytosol may be responsible for the disruption of malate-aspartate shuttle in hyperammonemic state.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗

PubMed2000

Overexpression of the lat gene in Nocardia lactamdurans from strong heterologous promoters results in very high levels of lysine-6-aminotransferase and up to two-fold increase in cephamycin C production.

The level of lysine-6-aminotransferase (encoded by the lat gene), an enzyme that commits lysine to the cephamycin biosynthesis pathway, is very low in wild type Nocardia lactamdurans. Two lat overexpression systems (pAMEXlat and pSAFlat) were constructed to express the promoterless lat gene of N. lactamdurans from the strong promoters amyP (of the alpha-amylase gene) and safP (of the secretion activating factor gene) of Streptomyces griseus. Both constructions led to very high levels of lysine-6-aminotransferase (between 8- and 15-fold) in the cells. Expression of lat from the amy promoter was optimal in glycerol-containing medium and was negatively regulated by glucose. The high levels of lysine-6-aminotransferase resulted in a 50-200% increase in cephamycin C production in the standard fermentation conditions. Onset of cephamycin C biosynthesis occurred at the same time in control and in lat-overexpressing strains, but the cephamycin production rate was clearly higher in transformants overexpressing the lat gene. Furthermore, HPLC analysis of cephamycin C in the culture broths revealed an early depletion of biosynthetic intermediates and an accumulation of cephamycin C when the lat gene was overexpressed. These results indicate that lysine-6-aminotransferase activity is limiting for cephamycin C biosynthesis under some culture conditions.

Cephamycins↗

PubMed1988

Purification of ornithine aminotransferase by immunoadsorption.

Ornithine aminotransferase was purified by conventional biochemical methods from rat kidney, rat liver, and human liver. Affinity-purified antibodies raised to the rat kidney enzyme were used to produce an immunoadsorbent enabling a one-step purification of ornithine aminotransferase to be made from crude human liver extracts. The harsh chemical conditions often required to desorb immunoadsorbents were avoided by isolating antibodies with low functional affinity and employing an electrophoretic desorption method which allowed the enzyme activity to be retained. The close structural similarity between human and rat ornithine aminotransferase was demonstrated by immunodiffusion reactions. It was therefore possible to purify the enzyme from human liver using immobilized antibodies raised against rat kidney ornithine aminotransferase. Furthermore, desorption was more readily achieved due to the lower affinity for the human enzyme.

Animals↗

PubMed1986

Kinetic properties and characteristics of mouse liver mitochondrial asparagine aminotransferase.

Mouse liver asparagine aminotransferase has been found to be a mixture of enzyme forms having a cytosolic component and a mitochondrial component. The molecular weight of the mitochondrial enzyme is 70,800. The mitochondrial asparagine aminotransferase is strongly inhibited by aminooxyacetate. It is less affected by D-cycloserine but a small amount of inhibition is observed. Cysteine strongly inhibits the enzyme as do several sulfhydryl modifying reagents. The activities of the cytosolic and mitochondrial aminotransferases have been separated, and the kinetic properties of the mitochondrial form determined. The mouse liver mitochondrial asparagine aminotransferase is fairly specific for asparagine, utilizing very few amino acids as alternate amino donors and none to a great extent. The keto acid specificity is very broad, but glyoxylate is one of the most active amino group acceptors. The kinetic properties of the mitochondrial enzyme are also reported here and the data indicate strong substrate and product inhibition. Abortive complex formation may account for the deviation of the double reciprocal plots from the expected pattern.

Amino Acids↗

PubMed1988

Significance of the alanine aminotransferase reaction in the formation of alpha-ketoglutarate in rat liver mitochondria.

The total production of alpha-ketoglutarate from glutamate and isocitrate was estimated in isolated rat liver mitochondria. Mitochondrial alanine aminotransferase converts glutamate to alpha-ketoglutarate [A.K. Groen et al. (1982) Eur. J. Biochem. 122, 87-93], thus participating in the net formation of the tricarboxylic acid cycle intermediates from glutamate. The present investigation indicates a significant contribution of the alanine aminotransferase reaction to glutamate oxidation by isolated rat liver mitochondria in the presence of bicarbonate. It amounted to 41-74 and 7-31% of the total utilization of glutamate in States 4 and 3, respectively, in various conditions in vitro, at pyruvate concentrations in the range of 0.1-10 mM. The participation of glutamate in the total production of alpha-ketoglutarate at physiological concentrations of glutamate, citrate, and isocitrate varied in the range of 72-82%. It was calculated that alpha-ketoglutarate formation by the reaction of alanine aminotransferase amounted to 30 and 5% of the total mitochondrial alpha-ketoglutarate production in States 4 and 3, respectively, at physiological concentrations of its precursors and in the presence of 0.5 mM malate and 0.1 mM pyruvate. It constituted 77-97% of the net production of the tricarboxylic acid cycle intermediates from glutamate in rat liver mitochondria. The importance of alpha-ketoglutarate production via the alanine aminotransferase reaction under various physiological conditions is discussed.

Alanine↗

PubMed1980

Reactivity of one thiol group in the dimeric protein, 4-aminobutyrate aminotransferase.

The enzyme 4-aminobutyrate aminotransferase (4-aminobutyrate:2-oxoglutarate aminotransferase, EC 2.6.1.19) from pig brain is inactivated by incubation with 5-5'-dithiobis-2-nitrobenzoic acid at pH 7.4. The reaction of one SH group per dimer brings about 95% loss of aminotransferase activity. The reaction with 5-5'-dithiobis-2-nitrobenzoic acid under pseudo-first-order conditions proceeds at a slow rate (Kobs = 0.05 min-1) and the substrate 4-aminobutyrate has no effect on the rate of inactivation. The inactivation of the enzyme cannot be related to dissociation of the cofactor (pyridoxal-P and pyridoxamine-P) from the catalytic site. One thiol group of the enzyme reacts with N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonic acid at pH 7.4. The reaction of approximately one SH group per dimer causes 95% loss of aminotransferase activity. The absorption and fluorescence properties of the enzyme tagged with the chromophore were used to gain information on the microenvironment of the reactive SH group critically connected with catalytic activity. The fluorescent properties, emission maximum (470 nm), fluorescence lifetime (19 ns) and degree of exposure to the collisional quencher acrylamide (Kq = 1.8 . 10(8) M-1 . s-1) indicate that the chromophore is shielded by the protein matrix.

4-Aminobutyrate Transaminase↗

PubMed1980

Translation of poly-A RNA from rat liver in vitro. Evidence for a high molecular weight subunit of tyrosine aminotransferase.

Poly-A RNA extracted from the rat liver was translated in a cell-free wheat germ system and a rabbit reticulocyte lysate. The subunit of tryptophan pyrrolase precipitated by specific antiserum after synthesis in vitro has the same molecular weight as the corresponding subunit derived from the rat liver. With specific antiserum prepared against tyrosine aminotransferase, however, a radioactive protein from both the in vitro assays was precipitated with an about 5% higher molecular weight than the tyrosine aminotransferase subunit precipitated from rat liver. The immunological evidence and the comparison of the specific peptide patterns prepared by cyanogen bromide treatment showed that the in vitro product corresponds to tyrosine aminotransferase. Various concentrations of potassium or spermidine used in the wheat germ translation system did not alter the size of the enzyme subunit synthesized. The run of the tyrosine aminotransferase purified form the rat liver in the SDS-polyacrylamide gel electrophoresis was not influenced by treatment with Escherichia coli alkaline phosphatase. The possibility is discussed that the larger enzyme synthesized in vitro represents a precursor molecule which is cleaved proteolytically in vivo.

Animals↗

PubMed1980

Enhancement by streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine of the tyrosine aminotransferase activity in cultured rat liver cells: role of dexamethasone and NAD.

The activity of tyrosine aminotransferase (TAT) (EC 2.6.1.5) was enhanced 3-fold after a 5-h exposure of cultured rat liver cells (RLC) to streptozotocin (SZ) at concentrations higher than 100 microgram/ml (0.38 mM) in the presence of 10 nM dexamethasone, a potent glucocorticoid inducer for the enzyme. The structurally related carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) also enhanced the aminotransferase in the presence of the glucocorticoid, but its optimal concentration was at 100 ng/ml (0.68 microM). While the cellular NAD (NAD+ + NADH) concentration was reduced to 60% of the control levels, the rate of poly(ADP-ribose) formation in the isolated cell nuclei was unaffected by treating the cells with SZ. The enhancement of tyrosine aminotransferase by SZ and MNNG was effectively prevented by nicotinamide. Using nicotinamide and its derivatives such as 1-methyl-, N'-methyl- or 6-amino-derivatives it was found that the degree of enzyme induction is almost inversely proportional to the cellular NAD content, though the activity of nuclear poly(ADP-ribose)polymerase remains unchanged. The results indicate that SZ or MNNG, in combination with dexamethasone, stimulate the induction of tyrosine aminotransferase through their NAD lowering action.

Animals↗

PubMed1986

Influence of streptozotocin upon the induction of tyrosine aminotransferase, the content of NAD and of 5-methyl cytosine in rat liver.

The antibiotic, streptozotocin, has carcinostatic, carcinogenic, and diabetogenic properties. Moreover, it is capable of inducing the enzyme tyrosine aminotransferase in a permanent line of rat liver cells. In the present publication, the effects of streptozotocin upon the induction of tyrosine aminotransferase, NAD synthesis, and methylation of DNA in different organs were analyzed in vivo. If administered alone, streptozotocin slightly induced tyrosine aminotransferase. The induction of tyrosine aminotransferase caused by tryptophan or nicotinamide was inhibited by streptozotocin. Streptozotocin reduced the NAD content of the liver. NAD synthesis induced by tryptophan was reduced by streptozotocin, while that induced by nicotinamide was enhanced. DNA methylation in the form of 5-methyl cytosine was not influenced by streptozotocin.

5-Methylcytosine↗

PubMed1991

Studies on alanine aminotransferase in nematodes.

L-alanine aminotransferase was demonstrated in a range of gastrointestinal, free-living and entomophagous nematodes. As in mammals, nematode L-alanine aminotransferase was found to exist in the form of mitochondrial and cytosolic isoenzymes. Whilst the majority of nematode enzymes exhibited a greater overall capacity for L-alanine synthesis than for L-alanine catabolism in vitro, the opposite was true for rat liver L-alanine aminotransferase. In contrast with rat liver, certain gastrointestinal nematodes were apparently able to transaminate D-alanine at low rates. H. contortus cytosolic L-alanine aminotransferase differed significantly from the mammalian enzyme with respect to both thermal stability and response to potential protective reagents.

Alanine Transaminase↗

PubMed1985

Isolation, characterization and chromosomal mapping of the mouse tyrosine aminotransferase gene.

The tyrosine aminotransferase (TAT) gene is expressed in a tissue and developmental-specific manner. In addition, this gene is regulated by glucocorticoid and polypeptide hormones and its expression is affected when a regulatory region near the albino locus of the mouse is deleted. In order to allow studies of the molecular effects of these deletion mutations we have isolated and characterized the mouse TAT gene. The gene is 9.2 x 10(3) bases in length and consists of 12 exons which give rise to a 2.3 x 10(3) base long messenger RNA. The DNA sequence at the 5' end of the gene was determined and compared with the corresponding sequence of the rat tyrosine aminotransferase gene. The sequence comparison showed extensive homology over the entire region sequenced. In addition, DNA: DNA heteroduplex studies between the mouse and rat tyrosine aminotransferase genes revealed that this homology extends over the entire gene and its flanking sequences. The mouse tyrosine aminotransferase gene has been mapped distal to the serum esterase-1 locus on mouse chromosome 8, using a restriction fragment length polymorphism between two mouse species. Since the albino deletions are located on mouse chromosome 7, the assignment of the TAT gene to chromosome 8 suggests that a regulatory factor(s) affecting TAT gene expression acts in trans.

Animals↗

PubMed1985

4-Aminobutyrate aminotransferase. Conformational changes induced by reduction of pyridoxal 5-phosphate.

Conformational changes induced in 4-aminobutyrate aminotransferase (4-aminobutyrate:2-oxoglutarate aminotransferase, EC 2.6.1.19) by conversion of pyridoxal-5-P to pyridoxyl-5-P were examined by two independent methods. The reactivity of the SH groups of the reduced enzyme is increased by chemical modification of the cofactor. 1.8 SH per dimer of modified enzyme react with DTNB, whereas 1.2 SH per dimer of the native enzyme react with the attacking reagent under identical experimental conditions. The modified and native forms of the enzyme bind the fluorescent probe ANS, but the number of binding sites for ANS is increased as result of conversion of P-pyridoxal to P-pyridoxyl. After the conformational changes onset by reduction of the cofactor, the modified enzyme binds one molecule of pyridoxal-5-P with a Kd of 0.1 microM to become catalytically competent. The catalytic site of the reduce enzyme was probed with P-pyridoxal analogs. Like resolved 4-aminobutyrate aminotransferase, the reduced species recognize the phosphorothioate analog and regain 40% of the total enzymatic activity. Since the catalytic parameters of reduced and native 4-aminobutyrate aminotransferase are indistinguishable, it is concluded that the additional catalytic site of the reduced enzyme is functionally identical to that of the native enzyme.

4-Aminobutyrate Transaminase↗