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PubMed1975

Kinetics of steroid induction and deinduction of tyrosine aminotransferase synthesis in cultured hepatoma cells.

The specific rate of synthesis of tyrosine aminotransferase (EC 2.6.1.5; L-tyrosine:2-oxoglutarate aminotransferase) is used as a measure of the level of functional, cytoplasmic, tyrosine aminotransferase-specific mRNA in cultured rat hepatoma cells. An analysis of the kinetics of change in this rate after the addition or withdrawal of glucocorticosteroids sets an upper limit on the half-life of the enzyme-specific mRNA of 1-1.5 hr, whether or not steroid is present. The inactivation rate of the enzyme mRNA is independent of the growth condition of the cells, occuring equally rapidly in the presence or absence of serum or insulin, both of which induce tyrosine aminotransferase in these cells. The implications of these results for the mechanism of steroid induction are discussed.

Animals↗

PubMed1982

Differences in rates of tyrosine aminotransferase deinduction with cyclic AMP and glucocorticoids.

As an indirect test of the possibility that cyclic AMP produces its stimulatory effects on the synthesis of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) by stabilizing its mRNA, the kinetics of decline in the rate of synthesis of this enzyme was measured in rat hepatoma cells following inducer removal or addition of RNA synthesis inhibitors. In all cases in which cells were previously exposed to cyclic AMP, the decline in enzyme synthesis after removal of inducer or addition of inhibitors of RNA synthesis was 4 to 5 times more rapid than in cells exposed to a glucocorticoid (dexamethasone) which also induces the aminotransferase. Thus, it seems unlikely that cyclic AMP had been acting by stabilizing the mRNA that directs the synthesis of the aminotransferase. Possible explanations for these paradoxical results are suggested. A more rapid decline in enzyme synthesis was also seen in cells induced with both cyclic AMP and glucocorticoid, suggesting direct interaction between the two inducers.

Animals↗

PubMed1982

Induction of tyrosine aminotransferase mRNA by glucocorticoids and cAMP in fetal rat liver.

Tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) enzyme and mRNA activity were not detectable in day 20 fetal rat liver. Precocious induction of catalytic activity by in utero injection of dibutyryl cAMP was a direct consequence of the de novo appearance of translatable tyrosine aminotransferase mRNA. In contrast, in utero injection of hydrocortisone acetate failed to elicit fetal liver enzyme activity. This failure was due to the inability of the steroid hormone to induce the appearance of tyrosine aminotransferase mRNA activity. In fetal rat liver explants, either compound was capable of stimulating the synthesis of adult levels of enzyme and mRNA. However, catalytic and mRNA activity comparable with that seen in vivo 24 hr after birth required the concerted action of both inducers.

Animals↗

PubMed1995

Cloning and expression of the mammalian cytosolic branched chain aminotransferase isoenzyme.

The cDNA for the rat cytosolic branched chain aminotransferase (BCATc) has been cloned. The BCATc cDNA encodes a polypeptide of 410 amino acids with a calculated molecular mass of 46.0 kDa. By Northern blot analysis, BCATc message of approximately 2.7 kilobases was readily detected in rat brain, but was absent from liver, a rat hepatoma cell line, kidney, and skeletal muscle. When expressed in COS-1 cells, the enzyme is immunologically indistinguishable from the native enzyme found in rat brain cytosol. Comparison of the rat BCATc sequence with available data bases identified the Escherichia coli (and Salmonella typhimurium) branched chain aminotransferase (BCAT) and revealed a Haemophilus influenzae BCAT, a yeast BCAT, which is hypothesized to be a mitochondrial form of the enzyme, and the murine BCATc (protein ECA39). Calculated molecular masses for the complete proteins are 33.9 kDa, 37.9 kDa, 42.9 kDa, and 43.6 kDa, respectively. The rat BCATc sequence was 84% identical with murine BCATc, 45% identical with yeast, 33% identical with H. influenzae, 27% identical with the E. coli and S. typhimurium BCAT, and 22% identical with the evolutionary related D-amino acid aminotransferase (D-AAT) (Tanizawa, K., Asano, S., Masu, Y., Kuramitsu, S., Kagamiyama, H., Tanaka, H., and Soda, K. (1989) J. Biol. Chem. 264, 2450-2454). Amino acid sequence alignment of BCATc with D-AAT suggests that the folding pattern of the overlapping mammalian BCATc sequence is similar to that of D-AAT and indicates that orientation of the pyridoxal phosphate cofactor in the active site of the eukaryotic BCAT is the same as in D-AAT. Thus, BCAT are the only eukaryotic aminotransferases to abstract and replace the proton on the re face of the pyridoxal phosphate cofactor. Finally, requirements for recognition of substrate L-amino acid and alpha-carboxylate binding are discussed.

Amino Acid Sequence↗

PubMed2004

Crystal structures of glutamine:phenylpyruvate aminotransferase from Thermus thermophilus HB8: induced fit and substrate recognition.

The following three-dimensional structures of three forms of glutamine:phenylpyruvate aminotransferase from Thermus thermophilus HB8 have been determined and represent the first x-ray analysis of the enzyme: the unliganded pyridoxal 5'-phosphate form at 1.9 A resolution and two complexes with 3-phenylpropionate and alpha-keto-gamma-methylthiobutyrate at 2.35 and 2.6 A resolution, respectively. The enzyme shows high activity toward phenylalanine, tyrosine, tryptophan, kynurenine, methionine, and glutamine. The enzyme is a homodimer, and each subunit is divided into an N-terminal arm and small and large domains. Based on its folding, the enzyme belongs to fold type I, aminotransferase subclass Ib. The subclass I aminotransferases whose structures have so far been determined exhibit a large movement of the small domain region upon binding of a substrate. Similarly, the glutamine:phenylpyruvate aminotransferase undergoes a large movement in part of the small domain to close the active site. The active-site pocket has a shape and size suitable to enclose the side chain of an aromatic amino acid or that of methionine. The inner side of the pocket is mostly hydrophobic, but also has polar sites. The kynurenine complex generated by computer modeling fits the pocket of the enzyme and its hydrophilic groups interact with the polar sites of the pocket.

Protein Conformation↗

PubMed2004

Structural studies of the catalytic reaction pathway of a hyperthermophilic histidinol-phosphate aminotransferase.

In histidine biosynthesis, histidinol-phosphate aminotransferase catalyzes the transfer of the amino group from glutamate to imidazole acetol-phosphate producing 2-oxoglutarate and histidinol phosphate. In some organisms such as the hyperthermophile Thermotoga maritima, specific tyrosine and aromatic amino acid transaminases have not been identified to date, suggesting an additional role for histidinol-phosphate aminotransferase in other transamination reactions generating aromatic amino acids. To gain insight into the specific function of this transaminase, we have determined its crystal structure in the absence of any ligand except phosphate, in the presence of covalently bound pyridoxal 5'-phosphate, of the coenzyme histidinol phosphate adduct, and of pyridoxamine 5'-phosphate. The enzyme accepts histidinol phosphate, tyrosine, tryptophan, and phenylalanine, but not histidine, as substrates. The structures provide a model of how these different substrates could be accommodated by histidinol-phosphate aminotransferase. Some of the structural features of the enzyme are more preserved between the T. maritima enzyme and a related threonine-phosphate decarboxylase from S. typhimurium than with histidinol-phosphate aminotransferases from different organisms.

Amino Acid Sequence↗

PubMedDate not supplied

Effect of cefazolin on aminotransferase activity in the rat.

Cefazolin given sc to male rats in daily doses of 0.5-2 g per kilogram of body weight significantly decreased alanine aminotranferase activity in serum, liver, kidney, heart, and brain 2-4 wk from the beginning of the treatment. Serum aspartate aminotransferase was also reduced, but serum alkaline phosphatase and tissue pyruvate decarboxylase activities remained unaltered. In female rats, daily sc administration of cefazolin at 0.1-1 g/kg also brought about a dose-related reduction of alanine and aspartate aminotransferase activities, which reached statistical significance at high dose levels. The effect of cefazolin at low concentrations was partly reversed by administration of pyridoxal in vivo. Paradoxically, at higher dose levels pyridoxal potentiated the action of cefazolin on serum aminotranferases. The low enzyme activities were elevated by subsequent addition of pyridoxal 5'-phosphate in vitro. Similar results were obtained when rats were treated with isoniazid at daily oral doses of 200 mg/kg; administration of pyridoxal completely restored alanine aminotransferase activity to the normal level within 2 wk. Cefazolin was metabolized in vivo, resulting in some metabolites that probably possessed a hydrazine group, since positive reactions were obtained with p-dimethylaminobenzaldehyde and Fast Blue B salt. The potentiation of decreased aminotransferase activity by pyridoxal indicated, however, some dissimilarity in the effect between isoniazid and cefazolin.

Alanine Transaminase↗

PubMed1997

Separate effects of the coffee diterpenes cafestol and kahweol on serum lipids and liver aminotransferases.

The coffee diterpene cafestol occurs in both robusta and arabica beans. It is present in unfiltered coffee brews and raises serum concentrations of cholesterol, triacylglycerols, and alanine aminotransferase in humans. The effects are linear with the cafestol dose. Unfiltered coffee also contains the related compound kahweol, which occurs only in the major coffee strain arabica. The activity of kahweol is unknown. In a randomized, double-blind crossover study, we gave 10 healthy male volunteers either pure cafestol (61-64 mg/d) or a mixture of cafestol (60 mg/d) and kahweol (48-54 mg/d) for 28 d. Relative to baseline values, cafestol raised mean (+/-SEM) total serum cholesterol concentrations by 0.79 +/- 0.14 mmol/L (31 +/- 5 mg/dL), low-density-lipoprotein (LDL) cholesterol by 0.57 +/- 0.13 mmol/L (22 +/- 5 mg/dL), fasting triacy-glycerols by 0.65 +/- 0.12 mmol/L (58 +/- 11 mg/dL), and alanine aminotransferase by 18 +/- 2 U/L (all P < 0.01). Relative to cafestol alone, the mixture of cafestol plus kahweol increased total cholesterol by another 0.23 +/- 0.16 mmol/L (9 +/- 6 mg/dL) (P = 0.08), LDL cholesterol by 0.23 +/- 0.16 mmol/L (9 +/- 6 mg/dL) (P = 0.09), triacylglycerols by 0.09 +/- 0.10 mmol/L (8 +/- 9 mg/dL) (P = 0.20), and alanine aminotransferase by 35 +/- 11 U/L (P = 0.004). Thus, the effect of cafestol on serum lipid concentrations was much larger than the additional effect of kahweol, and the hyperlipidemic potential of unfiltered coffee mainly depends on its cafestol content. Both cafestol and kahweol raised alanine aminotransferase concentrations, and their hyperlipidemic effect thus seems not to be coupled with their effect on liver cells.

Administration, Oral↗

PubMed1978

Loss of in vitro inactivation of rat liver tyrosine aminotransferase with dietary vitamin B6 restriction.

Tyrosine aminotransferase, in the presence of 8 mM L-cysteine, is inactivated in incubated liver homogenates prepared from normal rats, but not in those from rats deprived of vitamin B6. In this study we fed rats a diet deficient in vitamin B6 to determine the length of time required for in vitro inactivating activity to be lost from liver homogenates. After 2 weeks, the half-life of tyrosine aminotransferase in liver homogenates from vitamin B6-deficient rats was 5.9 hours, and from control rats, 1.8 hours. After 3 weeks, tyrosine aminotransferase was no longer inactivated in homogenates prepared from livers of deficient rats. The pyridoxal phosphate (PLP) concentration of plasma from rats fed the vitamin B6-deficient diet dropped from 89 ng/ml to 14 ng/ml after 1 week and to 7 ng/ml after 2 weeks. In 5 weeks the PLP concentration of liver from vitamin B6-adequate rats increased from 2.9 microgram/g to 6.6 microgram/g while in deficient rats it dropped to 2 microgram/g. The loss of tyrosine aminotransferase inactivating activity in the livers of vitamin B6-deficient rats occurred at approximately the same time that the concentration of PLP in the livers of rats fed the two diets began to show marked differences.

Animals↗

PubMed1986

Purification and characterization of yeast L-kynurenine aminotransferase with broad substrate specificity.

L-Kynurenine aminotransferase [L-kynurenine:2-oxoglutarate aminotransferase (cyclizing), EC 2.6.1.7] has been purified to homogeneity and crystallized from cell-free extracts of a yeast, Hansenula schneggii, grown in a medium containing L-tryptophan as an inducer. The enzyme has a molecular weight of about 100,000 and consists of two subunits identical in molecular weight (52,000). The enzyme exhibits absorption maxima at 280, 335, and 430 nm, and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. The enzyme-bound pyridoxal 5'-phosphate shows negative circular dichroic extrema, in contrast with other pyridoxal 5'-phosphate acting on L-amino acids. In addition to L-kynurenine and alpha-ketoglutarate, which are the most preferred substrates, a large number of L-amino acids and alpha-keto acids can serve as substrates; the extremely broad substrate specificity is the most characteristic feature of this yeast enzyme. The enzyme activity is significantly affected by both carbonyl and sulfhydryl reagents. Certain dicarboxylic acids such as adipate and pimelate act as competitive inhibitors. Addition of various substrate amino acids to the culture medium results in the inductive formation of aminotransferases which are immunochemically indistinguishable from L-kynurenine aminotransferase.

Amino Acids↗

PubMed2003

The spectrum of fatty liver in obese children and the relationship of serum aminotransferases to severity of steatosis.

OBJECTIVES: Non-alcoholic fatty liver disease is an emerging diagnosis in the pediatric population. Previously, ultrasonography and serum aminotransferases have been used to estimate prevalence of the disorder. A lack of concordance has been noted between these two diagnostic tests. To better understand the spectrum of fatty liver in obese children and the relationship of serum aminotransferases to the severity of steatosis, hepatic MRI was used to quantitate fat content. METHODS: Twenty-two children, ages 6 to 18 years, with obesity (BMI > 95th percentile for age) and hepatomegaly (liver edge more than 2 cm below the right costal margin) underwent hepatic MRI for fat quantitation. Hepatic MRI was performed using a modification of the Dixon method that used a fast gradient echo sequence rather than traditional spin echo. Scan times were sufficiently brief to allow completion within a single breath hold. Serum aminotransferases were obtained within one month of MRI. RESULTS: Twenty-one of 22 subjects had an elevated hepatic fat fraction. Seven of 7 subjects with a fat fraction of < or =18% had a normal serum ALT. Twelve of 13 subjects with fat fraction of >18% had an elevated serum ALT. Hepatic fat fraction correlated with serum ALT but did not correlate with age, BMI, or serum AST. CONCLUSION: The spectrum of fatty liver is larger than detected by screening for abnormal serum aminotransferases alone. Abnormalities in serum ALT occur exclusively in more severe cases of fatty liver.

Adolescent↗

PubMed2006

Glutamate:glyoxylate aminotransferase modulates amino acid content during photorespiration.

In photorespiration, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine. Previous studies demonstrated that alanine aminotransferase-like protein functions as a photorespiratory GGAT. Photorespiratory transamination to glyoxylate, which is mediated by GGAT and serine glyoxylate aminotransferase (SGAT), is believed to play an important role in the biosynthesis and metabolism of major amino acids. To better understand its role in the regulation of amino acid levels, we produced 42 GGAT1 overexpression lines that express different levels of GGAT1 mRNA. The levels of free serine, glycine, and citrulline increased markedly in GGAT1 overexpression lines compared with levels in the wild type, and levels of these amino acids were strongly correlated with levels of GGAT1 mRNA and GGAT activity in the leaves. This accumulation began soon after exposure to light and was repressed under high levels of CO(2). Light and nutrient conditions both affected the amino acid profiles; supplementation with NH(4)NO(3) increased the levels of some amino acids compared with the controls. The results suggest that the photorespiratory aminotransferase reactions catalyzed by GGAT and SGAT are both important regulators of amino acid content.

Amino Acids↗

PubMed1990

Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P(2) from Lupin Root Nodules.

Twenty-one monoclonal antibodies were raised against the aspartate aminotransferase-P(2) isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. Induction of this isoenzyme is positively correlated with the onset of N(2) fixation in effective root nodules and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. The monoclonal antibodies produced were all of the IgG class, recognized five different epitopes on the protein, and represented greater than 90% of the available epitopes. These epitopes were not unique to lupin nodule aspartate aminotransferase-P(2) but were shown to be present on the enzyme from tobacco leaves and potato. Four of the epitopes were conformational with a fifth epitope recognized by the appropriate monoclonals in both its native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium Iupini NZP2257 extracts. Antibodies against two epitopes showed some cross-reaction with the constitutive aspartate aminotransferase-P(1) isoenzyme also found in lupin root nodules. However, affinity of these monoclonals for AAT-P(1) was three orders of magnitude lower than for AAT-P(2). Monoclonals against the other epitopes appeared to be specific for aspartate aminotransferase-P(2).

Journal Article↗

PubMed1999

Crystallization and preliminary crystallographic analysis of the Escherichia coli tyrosine aminotransferase.

Tyrosine aminotransferase catalyzes transamination for both dicarboxylic and aromatic amino-acid substrates. The substrate-free Escherichia coli tyrosine aminotransferase (eTAT) bound with the cofactor pyridoxal 5'-phosphate (PLP) was crystallized in the trigonal space group P3(2). A low-resolution crystal structure of eTAT was determined by molecular-replacement methods. The overall folding of eTAT resembles that of the aspartate aminotransferases, with the two identical subunits forming a dimer in which each monomer binds a PLP molecule via a covalent bond linked to the epsilon-NH(2) group of Lys258. Comparison of the structure of eTAT with those of the open, half-open or closed form of chicken or E. coli aspartate aminotransferases shows the eTAT structure to be in the open conformation.

Animals↗

PubMed2005

Liver alanine aminotransferase, insulin resistance and endothelial dysfunction in normotriglyceridaemic subjects with type 2 diabetes mellitus.

BACKGROUND: Plasma levels of liver transaminases, including alanine aminotransferase (ALT), are elevated in most cases of nonalcoholic fatty liver disease (NAFLD). Elevated ALT levels are associated with insulin resistance, and subjects with NAFLD have features of the metabolic syndrome that confer high-risk cardiovascular disease. Alanine aminotransferase predicts the development of type 2 diabetes (DM2) in subjects with the metabolic syndrome. However, the role of elevated ALT levels in subjects with overt DM2 has yet not been explored. MATERIALS AND METHODS: In a cross-sectional study, 64 normotriglyceridaemic subjects with DM2 were studied with regard to the relation between liver transaminases with whole-body insulin sensitivity, measured with the euglycaemic hyperinsulinaemic clamp and with brachial artery flow-mediated dilation (FMD) as a marker of endothelial dysfunction. RESULTS: On average, patients were normotriglyceridaemic (plasma triglycerides 1.3 +/- 0.4 mmol L-1) and had good glycaemic control (HbA1c 6.2 +/- 0.8%). The mean ALT level was 15.0 +/- 7.5 U L-1, and the mean aspartate aminotransferase concentration equalled 10.6 +/- 2.6 U L-1. Alanine aminotransferase levels were negatively associated with whole-body insulin sensitivity as well as with FMD (both P = 0.03, in multivariate analyses; regression coefficients beta [95%CI]: -0.76 [-1.4 to -0.08] and -0.31 [-0.58 to -0.03] respectively). CONCLUSIONS: In metabolically well-controlled patients with DM2, ALT levels are related to decreased insulin-sensitivity and an impaired conduit vessel vascular function.

Adult↗

PubMed1977

4-amino-hex-5-enoic acid, a selective catalytic inhibitor of 4-aminobutyric-acid aminotransferase in mammalian brain.

Incubation of rat brain 4-aminobutyrate aminotransferase with 4-amino-hex-5-enoic acid, a substrate analog of 4-aminobutyric acid, results in a time-dependent irreversible loss of enzymatic activity. In the presence of 0.1 mM inhibitor the half-life of the inactivation process is approximately 6 min. Low concentrations of L-glutamic acid or 4-aminobutyric acid protect against this inactivation, while 2-oxoglutarate prevents this protection, suggesting that only the pyridoxal form of the enzyme is susceptible to inhibition by 4-amino-hex-5-enoic acid. The irreversible inhibition of mammalian 4-aminobutyrate aminotransferase by 4-amino-hex-5-enoic acid is selective. There is no inhibition of this enzyme from Pseudomonas fluorescens with the inhibitor at mM concentrations. Even at 10 mM there is no irreversible inhibition of mammalian glutamate decarboxylase or of aspartate aminotransferase, while alanine aminotransferase is inhibited over 500 times more slowly than rat brain 4-aminobutyrate transaminase.

4-Aminobutyrate Transaminase↗

PubMed1978

4-Aminobutyrate aminotransferase fluorescence studies.

The kinetics of resolution of the pyridoxamine phosphate form of the enzyme 4-aminobutyrate aminotransferase were monitored by fluorescence spectroscopy. 2 mol pyridoxamine phosphate are released/mol enzyme, indicating that two molecules of cofactor are involved in catalysis. The apoprotein is reconstituted by addition of pyridoxal phosphate; the apparent rate constant corresponding to the formation of active species is not a linear function of the concentration of cofactor. A multistep mechanism is proposed for the reconstitution of 4-aminobutyrate aminotransferase. A slow phase of reactivation of the aminotransferase is observed when the apoprotein is allowed to reconstitute in the presence of pyridoxal kinase, ATP and pyridoxal. The enzyme 4-aminobutyrate aminotransferase is a dimeric protein made up of subunits of identical molecular weight. It is characterized by a rotational relaxation time of 110 ns. The dimeric structure does not dissociate into subunits over a wide range of protein concentration (4--0.2 micrometer) at neutral pH.

4-Aminobutyrate Transaminase↗

PubMed1980

Regulation of rat-liver tyrosine-aminotransferase mRNA by hydrocortisone and by N6,O2'-dibutyryladenosine 3',5'-phosphate.

The intraperitoneal injection of either hydrocortisone of N6,O2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP) results in a specific increase in functional tyrosine aminotransferase mRNA (mRNATAT) activity in rat liver that is proportional to the degree of enzyme induction. Both require continuous RNA synthesis. There are several differences in the response to these inducers: (a) the magnitude of the increase is greater following hydrocortisone injection than after Bt2cAMP; (b) the peak response is seen within 1 h following the injection of Bt2cAMP as compared to the 5 h required for the maximal response following hydrocortisone injection; (c) finally, although both responses are rapid, the lag period which precedes the accumulation of functional tyrosine aminotransferase mRNA activity following the injection of hydrocortisone is at least 20 min whereas following Bt2cAMP it is 5-10 min. The administration of actinomycin D to rats 5 h after they were treated with hydrocortisone causes an additional twofold increase in tyrosine aminotransferase enzymatic activity, a phenomenon known as superinduction, but does not prevent the normal decrease in its mRNA seen at this time. This dissociation of enzyme and mRNA activities indicates that superinduction of tyrosine aminotransferase is not due to a selective stabilization of the mRNA which codes for this protein.

Animals↗