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PubMedDate not supplied

[Multiple forms of tyrosine aminotransferase in rat liver cells and their role in cell homeostasis].

Properties and hormonal regulation of activity of multiple forms of tyrosine aminotransferase in rat liver cells are considered with particular reference to regulation of synthesis of the multiple forms of tyrosine aminotransferase by glucocorticoid hormones. A role of tyrosine aminotransferase multiple forms in the adaptive reactions of cells during induction and postinduction periods is discussed.

Animals↗

PubMed1982

Evidence for a dual effect of dibutyryl cyclic AMP on the synthesis of tyrosine aminotransferase in rat liver.

A single injection of dibutyryl cyclic AMP (Bt2cAMP) into adrenalectomized rats results in rapid and proportionate increases in hepatic tyrosine aminotransferase catalytic activity and in the amount of functional mRNA coding for this enzyme. This effect is transient in that mRNATAT peaks at 0.065% of total poly(A)+RNA activity at 1 h and is back to the basal level of 0.012% in 2.5 h. Enzyme activity peaks at 2.5 h and is back to the basal level by 5 h. If Bt2cAMP is repeatedly injected (0, 1, 2.5, and 4 h), enzyme activity remains at maximal levels for 4 to 5 h, whereas changes in mRNATAT activity are identical with those observed in the single injected rats. The rate of tyrosine aminotransferase synthesis at 5.5 h in the multiply injected rats, a time when mRNATAT has already returned to the basal level, is 3 to 4 times greater than that in either control or singly injected rats at the same time (0.3% of total protein versus 0.07%) and is equivalent to the maximal rate seen 1 h after the initial injection of the cyclic nucleotide. Since the rate of synthesis is increased in proportion to the increase in enzyme catalytic activity, stabilization of the enzyme against degradation is excluded as an induction mechanism at this late time point. These responses are not due to differences in the metabolism of Bt2cAMP, and the effect depends on the presence of metabolically active derivatives of this nucleotide. It thus appears that Bt2cAMP induces the synthesis of tyrosine aminotransferase in rat liver in two distinct ways. One is pretranslational and involves a transient and rapid increase in mRNATAT activity. The second appears to involve a delayed but sustained increase in translation of a basal level of mRNATAT.

Animals↗

PubMed1983

Catalytic and structural properties of trypsin-treated 4-aminobutyrate aminotransferase.

4-Aminobutyrate aminotransferase (EC 2.6.1.19, 4 aminobutyrate:2-oxoglutarate aminotransferase) is cleaved by trypsin, yielding an enzymatically active species which can be separated from the split peptides by gel filtration. The shortened enzyme derivative gives one band (Mr = 95,000) on polyacrylamide gradient gel electrophoresis. Changes in protein conformation induced by tryptic digestion were studied by fluorescence spectroscopy. The native enzyme tagged with the chromophore fluorescein yields a rotational relaxation time of 106 ns, whereas the trypsin-digested enzyme gives a rotational relaxation time of 33 ns. The decrease in rotational relaxation time is attributed to flexibility of the polypeptide chain with enhanced rotational freedom of the probe covalently linked to one thiol group. The reactivity of sulfhydryl groups toward 5,5'-dithiobis(2-nitrobenzoic acid) is also affected by trypsin cleavage. More--SH groups (2.6/dimer) become reactive toward 5,5'-dithiobis(2-nitrobenzoic acid) as a result of trypsin digestion. Local conformational fluctuations are induced as a result of tryptic cleavage, but the catalytic sites remain intact. The peptides released from 4-aminobutyrate aminotransferase were characterized by fingerprint analysis and their amino acid composition determined.

4-Aminobutyrate Transaminase↗

PubMed1982

4-Aminobutyrate aminotransferase, reaction of P'P2-bis(5'-pyridoxal) diphosphate with lysyl residues connected with catalytic activity.

4-Aminobutyrate aminotransferase is inactivated by preincubation with bispyridoxal-5-P (mixing molar ratio, 20:1) at pH 7.0. The reaction with bispyridoxal-5-P under pseudo-first order conditions proceeds at a slow rate (kobs = 0.03 min-1). The extent of chemical modification was determined by measuring the spectroscopic properties of P-pyridoxyl and P-pyridoxine chromophores formed after reduction of the enzyme reacted with P'P2-bis(5'-pyridoxal) diphosphate. Reduction with NaBH4 results in the incorporation of approximately 2.1 P-pyridoxyl residues/dimer. Thus, the blocking of 2 lysyl residues/dimer is needed for inactivation of the aminotransferase. The time course of inactivation is significantly affected by variations in the pH of the reaction mixtures. Plots of Kobs versus pH indicate the reaction of the bifunctional reagent with lysyl residues characterized by low pK values (pK = 7.3). The substrate alpha-ketoglutarate (10 mM) affords complete protection against inactivation, whereas pyridoxal-5-P failed to prevent the inactivation of the enzyme by bispyridoxal-5-P. It is postulated that binding of alpha-ketoglutarate to lysyl residues is the major factor contributing to stabilization of the catalytic site. Several lines of experimental evidence indicate that inactivation of the aminotransferase cannot be related to dissociation of the cofactor from the catalytic site. The bifunctional reagent bispyridoxal-5-P blocks lysyl residues other than those involved in the binding of the cofactor.

4-Aminobutyrate Transaminase↗

PubMed1982

[Properties of aromatic-amino-acid aminotransferases from two chloramphenicol-resistant Flavobacteria].

Two enzymes of chloramphenicol-resistant Flavobacterium strain CB 60 and strain CB 6 which catalyse the transamination of tyrosine, phenylalanine and tryptophan were enriched 43-fold and 31-fold. The molecular mass for the aromatic-amino-acid aminotransferases of both strains was found to be 120000 Da and the isoelectric point was at about pH 4.2-4.3. Both enzymes are not influenced by EDTA. A ping pong bi-bi-mechanism was obtained for the kinetic mechanism of the reaction. The aminotransferases of strain CB 60 and CB 6 differ in the pH optimum (pH 8.4-9.0 and 7.8-9.0), in the optimum of temperature (40.0 degrees C and 57.5 degrees C) and in a higher heat stability of the enzyme of strain CB 60 in comparison to that of strain CB 6. The catalytic activity of the enzyme of strain CB 60 is not influenced by cycloserine, contrary to the enzyme of CB 6 which is inhibited to 89%. Isonicotinohydrazide does not inhibit the enzyme of strain CB 6, but reduces the catalytic activity of the enzyme of strain CB 60 to 64%. Both enzymes are inhibited differently by phenylhydrazine [78% (CB 60) and 56% (CB 6)]. Data in percentages are related to 0.15 mM inhibitor. For the substrates tyrosine, phenylalanine, tryptophan and 2-oxoglutarate, the aminotransferase of strain CB 60 has Km values of 15.0, 16.6, 16.6 and 1.7mM, and for the enzyme of strain CB 6 Km values of 1.40, 1.33, 1.37 and 0.97mM were obtained.

Chloramphenicol↗

PubMed1981

4-Aminobutyrate aminotransferase. The presence of nonequivalent binding sites.

A homogeneous 4-aminobutyrate aminotransferase isolated from pig brain exhibits a kcat = 9.6 s-1 and contains only 1 mol of pyridoxal-5-P/mol of dimer. The equilibrium dissociation constant for pyridoxal-5-P tightly bound to the enzyme is 1 nM. Spectrophotometric titrations reveal that the enzyme binds a second molecule of pyridoxal-5-P with a KD = 3 microM. The reaction of enzyme containing 1 pyridoxal-5-P/dimer with the inhibitors NaBH4 and DL-gabaculine was studied by observing changes in the absorption spectrum of the bound coenzyme and by monitoring loss of catalytic activity. The native enzyme is inactivated by both inhibitors. Incubation of the resultant P-pyridoxyl aminotransferase and m-anthraniyl-PMP-aminotransferase with excess pyridoxal-5-P at 25 degrees C restores full catalytic activity. It is postulated that the dimeric enzyme contains two classes of catalytic binding sites, and that the binding site characterized by a weak affinity for pyridoxal-5-P (KD = 3 microM) becomes functional after specific chemical modification of the molecule of cofactor tightly bound to the protein (KD - 1 nM).

4-Aminobutyrate Transaminase↗

PubMed1993

Cloning of ornithine delta-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis.

Proline prototrophy was restored to an Escherichia coli proBA proline auxotroph by ornithine and a mothbean (Vigna aconitifolia) cDNA expression library. This novel strategy, "trans-complementation," allowed isolation of a cDNA encoding ornithine delta-aminotransferase (delta-OAT). This enzyme transaminates ornithine to glutamic-gamma-semialdehyde (GSA), thereby bypassing the block in GSA synthesis from glutamate in the proBA mutant. The identity of the mothbean enzyme was confirmed by its high sequence homology to mammalian and yeast delta-OATs as well as to a family of bacterial and fungal omega-aminotransferases and an absence of significant homology to various alpha-aminotransferases. The V. aconitifolia OAT cDNA encodes a polypeptide of 48.1 kDa. The native enzyme expressed in E. coli appears to be a monomer with Km of 2 mM for ornithine and 0.75 mM for alpha-ketoglutarate. Levels of mRNA in V. aconitifolia for delta 1-pyrroline-5-carboxylate synthetase (P5CS) and delta-OAT, the two key enzymes for proline synthesis, were monitored under different physiological conditions. Salt stress and nitrogen starvation induced P5CS mRNA levels and depressed OAT mRNA levels. Conversely, OAT mRNA level was elevated in plants supplied with excess nitrogen while the P5CS mRNA level was reduced. These data suggest that the glutamate pathway is the primary route for proline synthesis in plants during conditions of osmotic stress and nitrogen limitation whereas the ornithine pathway assumes prominence under high nitrogen input.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

PubMed1977

Activation of alanine aminotransferase in serum by pyridoxal phosphate.

Alanine aminotransferase activity in serum increases significantly when serum is incubated with pyridoxal phosphate. The increase depends on the L-alanine concentration in the final assay mixture, being greatest at 800 mmol/liter. Preincubation of 22 normal sera, in a 10:1 ratio with an 8.09 mmol/liter pyridoxal phosphate solution, resulted in an increase in the alanine aminotransferase activity from 10.5 +/- 4.9 U/liter (mean +/- SD) to 28.4 +/- 5.3 U/liter, an increase of 170%. The absolute amount of apoalanine aminotransferase is relatively constant over a wide range of enzyme activities.

Alanine↗

PubMed1993

Identification of the mitochondrial branched chain aminotransferase as a branched chain alpha-keto acid transport protein.

Conditions were developed which optimized reconstitution of branched chain alpha-keto acid transport activity, which was measured as alpha-ketoisocaproate (KIC) transport, and pyruvate transport activity. Reconstitutable KIC transport activity was about 40-fold higher in heart than in liver mitochondrial extracts and 40-fold higher than heart pyruvate transport activity but only about 7-fold higher than liver pyruvate transport activity. A purification procedure was developed for the branched chain alpha-keto acid and pyruvate transport proteins which resulted in partial purification of the proteins from rat heart mitochondria. Pyruvate transport activity appeared to be associated with a 32.5-kDa protein, whereas KIC transport activity appeared to be associated with two proteins around 41 kDa. As shown by immunoblotting and immunoaffinity chromatography, these proteins were recognized by an antiserum raised against purified rat heart mitochondrial branched chain aminotransferase (BCATm). Procedures used to extract BCATm from mitochondria effectively solubilized KIC transport activity, and both transaminase and transport activities in the sonicate supernatant were immunoprecipitated by BCATm antiserum. The tissue distribution of reconstitutable KIC transport activity was identical with the tissue distribution of BCATm. On the other hand, the distribution of reconstitutable pyruvate transport activity was distinct from that of KIC transport and branched chain aminotransferase activities, and pyruvate transport activity could not be immunoprecipitated by BCATm antiserum. When incorporated into phospholipid vesicles, purified BCATm exhibited branched chain alpha-keto acid transport activity but did not transport pyruvate. Transport was saturable, and Km values for KIC and alpha-ketoisovalerate uptake were 10 and 25 microM, respectively. Substrate competition experiments indicated that KIC transport could be inhibited substantially by branched chain alpha-keto acids and their derivatives but not by substrates for the pyruvate transporter. Transport was also inhibited by several aromatic carboxylic acid derivatives and alpha-ketoglutarate, but not by N-butylmalonate and succinate. Studies with covalent protein-modifying reagents indicated that transport was inhibited by sulfhydryl reagents, the histidine reagent diethyl pyrocarbonate, and the tyrosine reagent N-acetylimidazole. When BCATm was incorporated into phospholipid vesicles, pyridoxal 5-phosphate was an inhibitor of transport (75% inhibition at 10 mM) but had little effect on aminotransferase activity. The data indicate BCATm is a bifunctional protein catalyzing branched chain amino acid transamination and branched chain alpha-keto acid transport. The transport properties of BCATm suggest that this protein may be the branched chain alpha-keto acid transporter that was originally identified and characterized kinetically in isolated rat heart mitochondria (Hutson, S.M., and Rannels, S.L. (1985) J. Biol. Chem. 260, 14189-14193).

Animals↗

PubMed1997

Alfa interferon treatment of hepatitis C virus RNA-positive patients with normal or near-normal alanine aminotransferase levels.

OBJECTIVES: Alfa interferon therapy is conventionally offered only to chronic hepatitis C patients with abnormal serum alanine aminotransferase (ALT) values. Therapeutic response is traditionally gauged by normalization of liver enzymes. Treatment of patients with persistently normal serum aminotransferases is not routinely done. The purpose of this study was to determine whether standard therapy with alfa interferon can eradicate hepatitis C virus in viremic patients with persistently normal or near-normal serum aminotransferases. METHODS: Between 1990 and 1996 we evaluated 565 patients with chronic hepatitis C. Of these, 49 patients (8.7%) (15 men, 34 women) had normal or near-normal ALT levels (less than 1.5 times upper limit of normal) for at least 3 consecutive months. Of these, 15 patients were studied. Treatment consisted of interferon alfa-2b 3 million units thrice weekly for 6 months. RESULTS: Normal or near normal ALT levels are more common in women than men. All patients completed 6 months of therapy, and 12 patients completed 6 months of posttreatment follow-up. Only one patient lost hepatitis C virus RNA during treatment and viremia reappeared in this patient immediately after cessation of therapy. CONCLUSIONS: Standard antiviral therapy of patients with normal or near-normal ALT levels does not result in sustained viral eradication in most patients.

Alanine Transaminase↗

PubMedDate not supplied

[Aminotransferase levels in rheumatoid arthritis patients treated with methotrexate].

Increase of the aminotransferase levels in 53 patients treated with methotrexate (MTX) were analysed in a retrospective study. The mean dose of MTX was 7.46 mg/week (range 2.5-15 mg) during at least 30 weeks (mean time of MTX use 124 weeks). The aminotransferase levels were transitorily increased in 13 patients, always less than three times the upper limit of normal. Only in three patients the AST and ALT levels were persistently increased and lead to the drug discontinuation in two cases. These results showed that increase of aminotransferases was a frequent observation (24.5%) during the first two-three years of follow-up, without interference in the overall clinical management.

Adult↗

PubMed1997

Diagnosis of chronic camel trypanosomosis by detection of the antibody of trypanosome tyrosine aminotransferase.

Sera from animals with acute and chronic T. evansi infections were examined directly for trypanosome tyrosine aminotransferase activity and indirectly for the ability of these area from mice and camels with high parasitaemias contained significant levels of trypanosome tyrosine aminotransferase activity. In contrast the chronic sera from both mice and camels did not contain significant tyrosine aminotransferase activity but the chronic sera were able to neutralize the enzyme activity in trypanosome homogenates. In addition to the sera from other pathological conditions did not neutralize the enzyme activity. It is suggested that the inhibitory factor in the chronic sera is antibody. The potential use of the direct enzyme assay, and the indirect neutralization assay as diagnostic tools are discussed. Finally, the use of these assays to distinguish between early (acute) and late (chronic) infections are also suggested.

Animals↗

PubMedDate not supplied

[The dynamics of aminotransferase activity in the blood plasma as a reflection of the characteristics of the pathological process in patients with craniocerebral trauma].

The activity of alanin-aminotransferase (ALT) and aspartate-aminotransferase (AST) was measured in blood plasma of 103 patients with brain trauma. Sex-related differences were found. One day after the trauma ALT and AST activity rose significantly both in men and women with brain concussion, in males with mild and moderate brain contusion. In severe brain contusion the enzymes activity did not change. Eight days after the trauma AST activity significantly lowered in patients with brain concussion. No changes in aminotransferases activity one day after severe brain contusion evidence for biochemical maladaptation.

Adult↗

PubMed1965

INTERACTION OF ASPARTATE AMINOTRANSFERASE WITH AMINO ACIDS.

1. The capacity of various amino acids to convert the pyridoxal form of aspartate aminotransferase into the pyridoxamine form has been investigated. 2. Glutamate has the highest converting capacity; aspartate, alpha-aminopimelate, alpha-aminoadipate and other amino acids follow. 3. The converting capacity of the various amino acids assayed is connected with their structural features. 4. A possible role of amino acids as secondary substrates of aspartate aminotransferase is suggested.

Adipates↗

PubMed1998

Chemical anatomy of excitatory endings in the dorsal cochlear nucleus of the rat: differential synaptic distribution of aspartate aminotransferase, glutamate, and vesicular zinc.

In order to identify cytochemical traits relevant to understanding excitatory neurotransmission in brainstem auditory nuclei, we have analyzed in the dorsal cochlear nucleus the synaptic distribution of aspartate aminotransferase, glutamate, and vesicular zinc, three molecules probably involved in different steps of excitatory glutamatergic signaling. High levels of glutamate immunolabeling were found in three classes of synaptic endings in the dorsal cochlear nucleus, as determined by quantitation of immunogold labeling. The first type included auditory nerve endings, the second were granule cell endings in the molecular layer, and the third very large endings, better described as "mossy." This finding points to a neurotransmitter role for glutamate in at least three synaptic populations in the dorsal cochlear nucleus. The same three types of endings enriched in glutamate immunoreactivity also contained histochemically detectable levels of aspartate aminotransferase activity, suggesting that this enzyme may be involved in the synaptic handling of glutamate in excitatory endings in the dorsal cochlear nucleus. There was also extrasynaptic localization of the enzyme. Zinc ions were localized exclusively in granule cell endings, as determined by a Danscher-selenite method, suggesting that this ion is involved in the operation of granule cell synapses in the dorsal cochlear nucleus.

Animals↗

PubMed2005

Serum alanine aminotransferase in skeletal muscle diseases.

Although elevation of the levels of serum alanine aminotransferase (ALT) following liver injury is well known, confusion exists concerning skeletal muscle injury as the cause of this rise. We reviewed the records of 16 patients who had muscle necrosis without evidence of liver disease. The patients were divided into three groups: extreme exercise, polymyositis, and seizures. All patients exhibited markedly elevated creatine kinase and lactate dehydrogenase levels consistent with muscle injury. In acute cases, aspartate aminotransferase (AST) and ALT were both elevated, and the AST/ALT ratio was greater than 3, but this ratio approached 1 after a few days because of a faster decline in AST. In conclusion, this difference in half-life accounts for the comparable AST and ALT levels in our cases with chronic muscle injury.

Adult↗

PubMed1990

Optimal conditions for protease use in the assay of serum mitochondrial aspartate aminotransferase.

The optimal conditions for selective proteolytic inactivation of cytosolic aspartate aminotransferase (c-AST) to determine mitochondrial aspartate aminotransferase (m-AST) in serum were studied. Protease 401 was found to be effective over a pH range of 6.0-10.0. A pH of 9.5 with 0.5% albumin in the reagent mixture was determined to be optimal for inactivation of c-AST and preservation of m-AST, lactic dehydrogenase (LDH), and malic dehydrogenase (MDH) in the assay procedure. The presence of serum endogenous protein inhibitors such as alpha 1-antitrypsin and alpha 2-macroglobin did not inhibit protease 401.

Aspartate Aminotransferases↗

PubMed1992

User-defined serum aspartate and alanine aminotransferase, cholesterol, triglycerides, urea, and uric acid for the Beckman synchron CX 4/5 using Ames Sera-Pak reagents.

Beckman aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholesterol, triglycerides, urea, and uric acid Liquid Reagents for Synchron CX 4/5 (48, 48, 25, 60, 26, and 30 cents US/test, respectively) are expensive. We have established our own methods for serum AST, ALT, cholesterol, triglycerides, urea, and uric acid (6, 6, 5, 12, 13, and 6 cents US/test, respectively) using Ames Sera-Pak reagents. Linearity of our AST, ALT, cholesterol, triglycerides, urea, and uric acid methods were either similar to or higher than the Beckman methods. The within run and day-to-day run precisions were acceptable. Recovery of our AST, ALT, cholesterol, triglycerides, urea, and uric acid were excellent. Our results for AST, ALT, cholesterol, triglycerides, urea, and uric acid correlated well with the Beckman results. Bilirubin (340.8 mumol/L) did not significantly interfere on our AST, ALT, cholesterol, triglycerides, and urea, while its concentrations of 165.8 mumol/L started giving negative interference on uric acid. Turbidity (2+) did not interfere significantly on our AST and ALT but started giving positive interference on cholesterol, triglycerides, urea, and uric acid. Hemolysis (2+) gave positive interference on our cholesterol, triglycerides, urea, and uric acid. Stability of Ames Sera-Pak working reagents was at least 30 days for AST, ALT, urea, and uric acid and 40 days for cholesterol and triglycerides.

Alanine Transaminase↗