Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.6.1.2 (alanine aminotransferase)
 26,722 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes.
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PMID:Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase. 730 49

The amphistomes Gigantocotyle explanatum and Gastrothylax crumenifer utilize leucine, alanine, proline and methionine during in vitro incubations. Autoradiography on sections of these flukes reveal a time-dependent differential incorporation of tritium-labelled amino acids in various tissues. The tegument appears to be the primary surface through which amino acids are absorbed. Following absorption, the reappearance of [3H]-leucine and [3H]-alanine on the tegumental surface during late chase periods indicates their possible involvement in tegumental secretion. A combination of diffusion and carrier-mediated uptake, possibly involving gamma-glutamyl transpeptidase, is indicated. The transport loci show differences in carrier-affinity (Kt) and maximum uptake velocities (Vmax) for amino acids under study, which suggest multiple transport molecules. Metabolic studies reveal that aspartate, alanine, ornithine, proline, leucine and methionine undergo transamination through 2-oxoglutarate-linked transaminases, distributed in the cytosolic and mitochondrial fractions of G. explanatum and G. crumenifer. With the exception of alanine transaminase, the enzyme levels in the cytosolic fraction were higher than the mitochondrial fraction of the two amphistomes. Predominantly cytosolic glutamate dehydrogenase which was comparatively higher in G. explanatum, catalyse amination of alpha-ketoglutarate. A high level of cytosolic arginase alone does not indicate a functional urea cycle. A tentative pathway of amino acid metabolism in these amphistomes is proposed.
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PMID:[3H]-amino acid uptake and metabolic studies on Gigantocotyle explanatum and Gastrothylax crumenifer (Digenea: Paramphistomidae). 763 32

The assay of oxaloacetate and alpha-ketoglutarate in biological samples is complicated by their chemical instability and low concentrations. We present a quantitative assay for physiological concentrations of these metabolites by isotope dilution gas chromatography-mass spectrometry. Samples are spiked with the corresponding internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate prior to their treatment with hydroxylamine. After ethyl acetate extraction and evaporation of the organic phases, the oximes are converted to t-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry of the [M-57]+ ion in electron impact. Although the internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate are not commercially available, they can easily be synthesized in 30 min by reacting [1,2,3,6-13C4]citrate with citrate lyase, and L-[U-13C5]glutamate with pyruvate and glutamate-pyruvate transaminase, respectively. Because of their chemical instability, the internal standards are prepared on the day of the analysis. A stock solution of [1,2,3,6-13C4]citrate is prepared from L-[U-13C4]aspartate using citrate synthase and glutamate-oxaloacetate transaminase and then purified and kept frozen until required. The detection limit of the method is 0.05 nmol in a given sample. The method was applied to measurements of oxaloacetate and alpha-ketoglutarate in human blood and rat liver.
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PMID:Assay of blood and tissue oxaloacetate and alpha-ketoglutarate by isotope dilution gas chromatography-mass spectrometry. 773 61

Experimental studies have demonstrated preferential injury to the sinusoidal endothelium during liver preservation with University of Wisconsin (UW) or Euro-Collins solution. This endothelial cell injury has an unclear pathogenesis, and it has not yet been studied in the human liver. Therefore, we analyzed the effluent of 21 human liver allografts after cold storage. Markers of hepatocellular and nonparenchymal cell injury were assessed. After preservation with UW solution, early effluent samples contained 1823 +/- 1494 U/l lactate dehydrogenase (LDH), 493 +/- 516 U/l alanine aminotransferase (ALT) and 132 +/- 97 U/l creatine kinase (CK; 92 +/- 92 U/l CK-BB). The effluent of livers preserved in histidine-tryptophan-ketoglutarate (HTK) solution contained 3681 +/- 2009 U/l LDH, 1139 +/- 599 U/l ALT and 282 +/- 120 U/l CK (165 +/- 91 U/l CK-BB). Comparison of effluent enzyme activities with liver tissue enzyme activities indicates that the release of the endothelial cell/nonparenchymal cell marker creatine kinase was higher, by a factor of 7-8, than the release of hepatocellular enzymes. Effluent thrombomodulin concentrations were 123 +/- 248 ng/ml (UW) and 604 +/- 299 ng/ml (HTK), and effluent glucose concentrations, 40.3 +/- 27.0 mM (726 +/- 486 mg/dl; UW) and 10.4 +/- 4.5 mM (187 +/- 81 mg/dl; HTK). We conclude that prominent endothelial cell injury also occurs in human liver grafts after preservation with UW solution or HTK solution. This endothelial cell injury is unlikely to be caused by hypoxia-induced energy deficiency, as it affects a cell type with a high glycolytic capacity in the presence of high glucose levels.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nonparenchymal cell and hepatocellular injury to human liver grafts assessed by enzyme-release into the perfusate. 793 84

Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.
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PMID:Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi. 810 Apr 16

The retina of honeybee drone is a nervous tissue with a crystal-like structure in which glial cells and photoreceptor neurons constitute two distinct metabolic compartments. The phosphorylation of glucose and its subsequent incorporation into glycogen occur in glia, whereas O2 consumption (QO2) occurs in the photoreceptors. Experimental evidence showed that glia phosphorylate glucose and supply the photoreceptors with metabolic substrates. We aimed to identify these transferred substrates. Using ion-exchange and reversed-phase HPLC and gas chromatography-mass spectrometry, we demonstrated that more than 50% of 14C(U)-glucose entering the glia is transformed to alanine by transamination of pyruvate with glutamate. In the absence of extracellular glucose, glycogen is used to make alanine; thus, its pool size in isolated retinas is maintained stable or even increased. Our model proposes that the formation of alanine occurs in the glia, thereby maintaining the redox potential of this cell and contributing to NH3 homeostasis. Alanine is released into the extracellular space and is then transported into photoreceptors using an Na(+)-dependent transport system. Purified suspensions of photoreceptors have similar alanine aminotransferase activity as glial cells and transform 14C-alanine to glutamate, aspartate, and CO2. Therefore, the alanine entering photoreceptors is transaminated to pyruvate, which in turn enters the Krebs cycle. Proline also supplies the Krebs cycle by making glutamate and, in turn, the intermediate alpha-ketoglutarate. Light stimulation caused a 200% increase of QO2 and a 50% decrease of proline and of glutamate. Also, the production of 14CO2 from 14C-proline was increased. The use of these amino acids would sustain about half of the light-induced delta QO2, the other half being sustained by glycogen via alanine formation. The use of proline meets a necessary anaplerotic function in the Krebs cycle, but implies high NH3 production. The results showed that alanine formation fixes NH3 at a rate exceeding glutamine formation. This is consistent with the rise of a glial pool of alanine upon photostimulation. In conclusion, the results strongly support a nutritive function for glia.
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PMID:Glial cells transform glucose to alanine, which fuels the neurons in the honeybee retina. 812 Jun 29

Male weanling rats were maintained on magnesium-deficient diet for 30 d and compared with pair-fed control rats fed magnesium-supplemented diet. Magnesium deficiency led to slow growth and finally to a significant decrease in body weight (P < 0.001) accompanied by a significant hypomagnesaemia, hypomagnesuria and hyperoxaluria (P < 0.001 in each case) in experimental rats as compared to the control rats. Magnesium deficiency altered the glyoxylate metabolism in the liver and kidney mitochondria by significantly decreasing glyoxylate oxidation (by 26 per cent in liver and 17 per cent in kidney) and activity of alpha-ketoglutarate:glyoxylate carboligase enzyme (by 35 per cent in liver and 27 per cent in kidney) in the experimental animals. A significant increase in the specific activities of glycolic acid oxidase (P < 0.001) and glycolic acid dehydrogenase (P < 0.01) and a significant decrease in alanine transaminase (P < 0.01) was also observed in magnesium-deficient rats. No change in liver and kidney lactate dehydrogenase was observed. Thus magnesium deficiency in rats leads to accumulation of glyoxylate in the tissues, a part of which is converted into oxalate, thereby promoting hyperoxaluria.
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PMID:Oxalate metabolism in magnesium-deficient rats. 827 58

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.
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PMID:Coupled enzymatic assay for estimation of branched-chain L-amino acid aminotransferase activity with 2-Oxo acid substrates. 866 May 88

Incubation of pig heart cytosolic aspartate aminotransferase (pyridoxal 5'-phosphate form) with 10 mM 2-oxoglutaconic acid dimethyl ester for 2 h at 25 degrees C (pH 7.0) results in slight inactivation (approximately 15%). However, incubation of the enzyme with glutamate, or prior conversion of the enzyme to the pyridoxamine 5'-phosphate form, results in more extensive inactivation. The inactivation of the enzyme by 2-oxoglutaconic acid dimethyl ester is most pronounced in the presence of both glutamate and alpha-ketoglutarate. N-Ethylmaleimide was previously shown to alkylate two surface cysteine residues (I and II) and to react syncatalytically with a third cysteine residue (III) of cytosolic pig heart aspartate aminotransferase [Birchmeier et al. (1973) J. Biol. Chem. 248, 1751-1759]. Alkylation of cysteine III results in inactivation of the enzyme, despite the fact that this residue is not essential for catalysis. The present results suggest that 2-oxoglutaconic acid dimethyl ester reacts with the enzyme in a similar fashion to that exhibited by N-ethylmaleimide. Some inactivation by alkylation of a susceptible group at the active site cannot be ruled out. However, the rate of inactivation of cytosolic pig heart aspartate aminotransferase is proportional to the concentration of 2-oxoglutaconic acid dimethyl ester up to a concentration of at least 40 mM, suggesting that the compound binds very poorly to the active site or that alkylation at the active site is slow compared with syncatalytic alkylation of cysteine III. The t 1/2 for inactivation of pig heart cytosolic aspartate aminotransferase by 40 mM 2-oxoglutaconic acid dimethyl ester (in the presence of 10 mM L-glutamate, pH 7.2, 25 degrees C) is 9 min. Incubation of cytosolic pig heart aspartate aminotransferase with 10 mM 2-oxoglutaconate for 2 h (25 degrees C, pH 7.2) results in significant inactivation (approximately 30%). The enzyme is protected against inactivation by the presence of alpha-ketoglutarate, but glutamate enhances the inactivation. These findings suggest that 2-oxoglutaconate is an active site-directed inhibitor. The binding of 2-oxoglutaconate to the enzyme exhibits saturation kinetics (K1 approximately 2 mM), but the rate of inactivation is slow (limiting rate constant for inactivation in the presence of L-glutamate approximately 0.01 min-1; pH 6.0, 25 degrees C; t 1/2 max approximately 70 min). This finding suggests that 2-oxoglutaconate does not readily react in a syncatalytic fashion with cysteine III. Possibly, the two negative charges of 2-oxoglutaconate do not allow ready approach to cysteine III. Rather, the findings suggest that 2-oxoglutaconate binds at the active site of the pyridoxal 5'-phosphate form of the enzyme as an affinity labeling reagent. However, the increased rate of 2-oxoglutaconate-induced inactivation in the presence of glutamate suggests that this unsaturated alpha-keto acid also exhibits the properties of a kcat inhibitor. 2-Oxoglutaconate inactivates aspartate aminotransferase in cytosolic and mitochondrial fractions of rat kidney and purified pig heart alanine aminotransferase. Injection of 2-oxoglutaconate into mice results in inhibition of kidney aspartate aminotransferase. 2-Oxoglutaconate is a substrate of glutamate dehydrogenase. The kinetic constants are similar to those obtained with alpha-ketoglutarate. The results suggest that unsaturated alpha-keto acids and their esters may be useful probes for the study of alpha-keto acid-utilizing enzymes.
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PMID:Irreversible inactivation of aspartate aminotransferase by 2-oxoglutaconic acid and its dimethyl ester. 890 17

Transamination reaction is the first step in the catabolism of most of the L-amino acids. Alanine is an important molecule in the inter-organ nitrogen transport, conveying them from muscle to the liver. Amino groups from this amino acid are generally first transferred to alpha-ketoglutarate in the cytosol of liver cells to form glutamate and leaving behind the corresponding alpha-keto acid analog. Measurements of the alanine aminotransferase (EC2.6.1.2.) activity were compared in liver, mammary gland and skeletal muscle in virgin, lactating and weaning dam rats. In this study liver was the principal tissue involved in alanine transamination, while muscle showed a reduction in the enzyme activity during lactation. Results indicate an increase in alanine amino-transferase activity in the mammary gland during lactation and weaning when compared with virgin rats. This suggests that mammary gland during lactation is an important extra-hepatic tissue involved in the metabolism of alanine and probably shunted into the pathways for amino group metabolism in terms of nitrogen economy.
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PMID:Alanine aminotransferase activity in mammary tissue, muscle and liver of dam rat during lactation and weaning. 898 75


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