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)

A previously described digitonin-perfusion technique [Quistorff, Grunnet & Cornell (1985) Biochem. J. 226, 289-297], by which intracellular material of rat liver could be liberated, has been refined, now allowing release of cytosol of high purity from both periportal and perivenous parts of the same liver. The cytosolic fractions are obtained by perfusing the liver for short intervals (10-20 s) with digitonin (4-5 mg/ml), first in the normal perfusion direction and then, after an interval of 1-2 min, in the retrograde direction, the eluate being collected during and after both intervals. The technique is termed 'dual-digitonin-pulse perfusion'. The eluate fractions showed a peak specific activity of the cytosolic enzymes alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and pyruvate kinase (PK) of 3-5-fold higher than obtained in a biopsy from the same liver. For glutamine synthetase (GS) a 10-fold higher specific activity was obtained. Zonation, defined as the ratio of the specific activities in periportal and perivenous eluates, of ALAT, LDH and PK was 10, 1.7 and 0.70 respectively. Zonation of GS was less than 0.01. These factors may be modified by a slight zonation of cytosolic protein of 1.2-1.3. Peak concentrations in the eluate of ATP, ADP, Pi, NAD+ and glycerol 3-phosphate were 32.5 +/- 11.4, 19.9 +/- 4.3, 71.9 +/- 25.4, 2.41 +/- 0.83 and 6.84 +/- 2.74 nmol/mg of protein for periportal eluates. There was no difference between periportal and perivenous eluates except for glycerol 3-phosphate, which was significantly higher in perivenous eluates, 12.8 +/- 4.5 nmol/mg of protein.
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PMID:Dual-digitonin-pulse perfusion. Concurrent sampling of periportal and perivenous cytosol of rat liver for determination of metabolites and enzyme activities. 360 84

The RS-isomers of beta-mercapto-alpha-ketoglutarate, beta-methylmercapto-alpha-ketoglutarate and beta-methylmercapto-alpha-hydroxyglutarate have been synthesized. Beta-Mercapto-alpha-ketoglutarate was a potent inhibitor, competitive with isocitrate and noncompetitive with NADP+, of the mitochondrial NADP-specific isozyme from pig heart (Ki = 5 nM; Km (DL-isocitrate)/Ki(RS-beta-mercapto-alpha-ketoglutarate) = 650) and pig liver, the cytosolic isozyme from pig liver (I0.5 = 23 nM), and the NADP-linked enzymes from yeast (Ki = 58 nM) and Escherichia coli (Ki = 58 nM) at pH 7.4 and with Mg2+ as activator. beta-Mercapto-alpha-ketoglutarate was also an effective inhibitor of NADP-isocitrate-dehydrogenase activity in intact liver mitochondria. beta-Mercapto-alpha-ketoglutarate was a much less potent inhibitor for heart NAD-isocitrate dehydrogenase (Ki = 520 nM) than for the NADP-specific enzyme. beta-Methylmercapto-alpha-ketoglutarate (I0.5 = 10 microM) was a much less effective inhibitor than the beta-mercapto derivative for heart NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarates were substrates for the oxidation of NADPH by heart NADP-isocitrate dehydrogenase without requiring CO2. beta-Methylmercapto-alpha-hydroxyglutarate, the expected product of reduction of beta-methylmercapto-alpha-ketoglutarate, did not cause reduction of NADP+ but it was an inhibitor competitive with isocitrate for NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarate derivatives were alternate substrates for alpha-ketoglutarate dehydrogenase and the cytosolic and mitochondrial isozymes of heart aspartate aminotransferase but had no effect on glutamate dehydrogenase or alanine aminotransferase.
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PMID:beta-Sulfur substituted alpha-ketoglutarates as inhibitors and alternate substrates for isocitrate dehydrogenases and certain other enzymes. 394 94

The effects of a homologous series of fatty acids with a chain length of two to eight on the rate of pyruvate oxidation and covalent interconversions of the pyruvate dehydrogenase complex (PDH) were studied in isolated perfused rat hearts. In the Langendorff-perfused heart beating at 5 Hz against an aortic pressure of 59 mmHg (7.85 kPa), a positive linear correlation was found between the fraction of PDH existing in the active non-phosphorylated form of pyruvate dehydrogenase complex (PDHa) and the pyruvate oxidation rate until the PDHa fraction increased to 48%. This value resulted in a saturation of the citric acid cycle and further activation did not increase the metabolic flux. The PDHa content of the tissue was higher during infusion of odd carbon number fatty acids than during infusion of even carbon number fatty acids. Propionate caused an almost maximal (93%) activation of PDH. A negative correlation was found between the mitochondrial NADH/NAD+ ratio and the PDHa content. A negative correlation was also found between the acetyl-CoA/CoA ratio and the tissue PDHa content. The rate of labelled CO2 production, the specific radioactivity of tissue alanine and the metabolic balance sheet demonstrated that the alanine aminotransferase reaction in the total tissue does not reach equilibrium with the mitochondrial pyruvate pool during propionate oxidation, but the equilibrium is reached during the oxidation of even-number carbon fatty acids. This suggests that pyruvate is formed from propionate-derived metabolites also in the cytosol, although the primary metabolism of propionate occurs in the mitochondria. The results indicate that the rate of pyruvate oxidation in the myocardium is mainly regulated by covalent interconversion of PDH. During propionate oxidation the PDHa content in the tissue can increase beyond the point of saturation of the citric acid cycle and this indicates that feedback inhibition of the enzyme is rate-determining under these conditions.
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PMID:Regulation of pyruvate dehydrogenase during infusion of fatty acids of varying chain lengths in the perfused rat heart. 408 5

1. Changes in the concentrations of ammonia, glutamine, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, alanine, aspartate, malate, lactate, pyruvate, NAD(+), NADH and adenine nucleotides were measured in freeze-clamped rat liver during ischaemia. 2. Although the concentrations of most of the metabolites changed rapidly during ischaemia the ratios [glutamate]/[2-oxoglutarate][NH(4) (+)] and [3-hydroxybutyrate]/[acetoacetate] changed equally and the value of the expression [3-hydroxybutyrate][2-oxoglutarate][NH(4) (+)]/[acetoacetate][glutamate] remained approximately constant, indicating that the 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase systems were at near-equilibrium with the mitochondrial NAD(+) couple. 3. The value of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] was about 0.7 in vivo and remained fairly constant during the ischaemic period of 5min, although the concentrations of alanine and oxoglutarate changed substantially. No explanation can be offered why the value of the ratio differed from that of the equilibrium constant of the alanine aminotransferase reaction, which is 1.48. 4. Injection of l-cycloserine 60min before the rats were killed increased the concentration of alanine in the liver fourfold and decreased the concentration of the other metabolites measured, except that of pyruvate. During ischaemia the concentration of alanine did not change but that of aspartate almost doubled. 5. After treatment with l-cycloserine the value in vivo of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] rose from 0.7 to 2.4. During ischaemia the value returned to 0.8. 6. The effects of l-cycloserine are consistent with the assumption that it specifically inhibits alanine aminotransferase. 7. Most of the alanine formed during ischaemia is probably derived from pyruvate and from ammonia released by the deamination of adenine nucleotides and glutamine. The alanine is presumably formed by the combined action of glutamate dehydrogenase and alanine aminotransferase. 8. The rate of anaerobic glycolysis, calculated from the increase in the lactate concentration, was 1.3mumol/min per g fresh wt. 9. Although the concentrations of the adenine nucleotides changed rapidly during ischaemia, the ratio [ATP][AMP]/[ADP](2) remained constant at 0.54, indicating that adenylate kinase established near-equilibrium under these conditions.
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PMID:Effects of ischaemia on metabolite concentrations in rat liver. 431 90

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.
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PMID:The intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm. 439 82

We describe an improved method for determination of alcohol dehydrogenase (EC 1.1.1.1) activity in 60 microL of human serum, based on conversion of ethanol to acetaldehyde with simultaneous reduction of NAD+ in glycine NaOH buffer (pH 9.0) at 37 degrees C in a centrifugal analyzer. The final concentration of NAD+ was 10 mmol/L and ethanol was 20 mmol/L. The dilution curve was linear with enzyme activity up to 200 U/L, and results by this method correlated well with those by a manual method (N Engl J Med 279: 241-248, 1968). Within-run precision (CV) was 0.9 to 8.2% over the range of 4.5 to 88.1 U/L, and day-to-day precision was 5.4 to 5.6%. In sera from 198 healthy individuals, mean alcohol dehydrogenase activity was 1.6 (SD 1.2, range 0-5) U/L. To evaluate the clinical utility of determining alcohol dehydrogenase, we measured the activity of alanine aminotransferase and alcohol dehydrogenase in sera from 470 patients with various diseases in our hospital, and found that results for the two enzymes did not correlate well.
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PMID:Improved assay for alcohol dehydrogenase activity in serum by centrifugal analysis. 638 26

The activity of certain key enzymes involved in glutamic acid metabolism was studied in purified brain mitochondria and in mitochondrial subfractions separated in a discontinuous 1.2--1.6 mol/l sucrose gradient. Alanine aminotransferase and glutamate dehydrogenase were found to be matrix enzymes and aspartate aminotransferase to be associated with the inner mitochondrial membranes. After the purified mitochondria had been separated into 5 subfractions, aspartate aminotransferase and NAD+-dependent isocitrate dehydrogenase were found to be bound to the lighter mitochondrial subfractions settling at the 1.4--1.5 mol/l sucrose boundary while alanine aminotransferase, 4-aminobutyrate transaminase and glutamate dehydrogenase were associated with the heavier subfractions settling below 2.4 mol/l sucrose. The highest specific activity of the given enzymes was found in the subfraction settling at the 1.4--1.5 mol/l sucrose boundary, the only exception being alanine aminotransferase activity, whose maximum was found in the subfractions settling in 1.5 and 1.6 mol/l sucrose. It was concluded that alanine aminotransferase, in conjunction with glutamate dehydrogenase, is linked to NH3 binding and to the oxidation of reduced adenine nucleotides; in addition, alanine aminotransferase is presumed to have the function of transporting glutamate from the mitochondria to the extramitochondrial space.
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PMID:Alanine aminotransferase and some other enzymes in different populations of free brain cortex mitochondria. 645 52

The possibility that the glycosomes present in the bloodstream form of Trypanosoma brucei [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360--364] constitute a separate pool of glycolytic intermediates within the cell was investigated. In titrations of intact cells with digitonin, a differential activation of glycolytic enzymes was observed. Enolase, pyruvate kinase and the cell-sap marker alanine aminotransferase were activated at 0.05 mg digitonin per mg protein. The nine glycosomal enzymes involved in the conversion of glucose and glycerol into 3-phosphoglycerate were activated only at digitonin concentrations between 0.7 and 9.8 mg/mg protein. In subcellular fractions the activities of the latter enzymes were all latent between 70 and 92%. Latency was abolished by addition of 0.1% Triton X-100 or partly by five cycles of freezing and thawing. We conclude that the glycosomal enzymes are surrounded by a membrane, which forms a permeability barrier to intermediates and co-factors of glycolysis. The concentrations of glycolytic intermediates and of adenine nucleotides were measured under aerobic conditions as well as in the presence of 1 mM salicylhydroxamic acid, a respiratory inhibitor. Addition of salicylhydroxamic acid caused the following changes: (a) The levels of almost all glycolytic intermediates measured decreased. Glycerol-3-phosphate, however, increased fourfold. (b) The phosphate potential was drastically lowered from 2900 to 450 M-1. (c) The trypanosomes became more reduced, as monitored by a change in the apparent redox state of the NADH/NAD+ courple from E'h = -189 to E'h = -219 mV. From the high levels of metabolite concentrations found and from comparison of the apparent mass-action ratios calculated for the separate glycolytic reactions with those for other organisms, we conclude that in bloodstream form T. brucei the glycolytic intermediates are present in the glycosomes as well as in the cytosol and that the two pools of intermediates equilibrate with each other, despite the presence of the glycosomal membrane.
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PMID:Glycolysis in Trypanosoma brucei. 676 64

Measurements have been made of the hepatic soluble and mitochondrial GOT and GPT and mitochondrial NAD+ glutamate dehydrogenase activities in thioacetamide-treated rats for 30 days. There is a significant fall in the GOT and GPT soluble activities from the effect of chronic thioacetamide administration while the mitochondrial activities become markedly increased in both cases. Glutamate dehydrogenase also increased from the effect of this hepatotoxic substance. Protein determined in the soluble and mitochondrial fractions, showed decreased levels in the cytosolic extracts and increased levels in the mitochondrial ones. Morphological aspects of liver cells showed hypertrophic mitochondria located around the likewise hypertrophic nucleus. The existence of functionally very active mitochondria in the generating liver, induced by thioacetamide, as well as metabolic mechanisms for the regulating control under pathological circumstances, can be a consequence of the increased ammonia concentration.
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PMID:[Changes caused by thioacetamide in GOT and GPT aminotransferases and glutamate dehydrogenase in rat liver. Ultrastructural study]. 714 64

Fed and fasted rats were injected with L-tryptophan (12.5 mg/100 g body weight) and the specific activities of L-glutamic: NAD oxidoreductase (deaminating) (EC 1.4.1.2) (GDH), L-aspartic-2-ketoglutaric aminotransferase (EC 2.6.1.1) (GOT) and L-alanine-2-ketoglutaric aminotransferase (EC 2.6.1.2) (GPT) from hepatic mitochondria and cytosol were compared. L-tryptophan results in a decrease of mitochondrial GDH activity by 22% and of cytosolic GPT and GOT by 42% and 38% respectively in the liver of fasted rats. Xanthurenate is a potent inhibitor of purified extramitochondrial GPT, whereas anthranilate and quinolinate are less potent inhibitors. L-tryptophan, 5-OH-tryptophan and indole exert a slight inhibition. Kynurenine, 5-OH-tryptamine, tryptamine, picolinic acid, nicotinic acid and indoloacetic acid do not show any inhibition of GPT. It is suggested that L-tryptophan injection inhibits extramitochondrial GPT by its transformation to xanthurenate and anthranilate.
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PMID:Effect of L-tryptophan injection in rats on some enzymes of amino acid metabolism in liver. I. In vitro studies of the effect of L-tryptophan and its metabolites on the extramitochondrial L-alanine: 2-ketoglutaric aminotransferase. 722 74


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