Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.6.1.1 (aspartate aminotransferase)
21,665 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

hisH encodes imidazole acetol phosphate (IAP) aminotransferase in Zymomonas mobilis and is located immediately upstream of tyrC, a gene which codes for cyclohexadienyl dehydrogenase. A plasmid containing hisH was able to complement an Escherichia coli histidine auxotroph which lacked the homologous aminotransferase. DNA sequencing of hisH revealed an open reading frame of 1,110 bp, encoding a protein of 40,631 Da. The cloned hisH product was purified from E. coli and estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular mass of 40,000 Da. Since the native enzyme had a molecular mass of 85,000 Da as determined by gel filtration, the active enzyme species must be a homodimer. The purified enzyme was able to transaminate aromatic amino acids and histidine in addition to histidinol phosphate. The existence of a single protein having broad substrate specificity was consistent with the constant ratio of activities obtained with different substrates following a variety of physical treatments (such as freeze-thaw, temperature inactivation, and manipulation of pyridoxal 5'-phosphate content). The purified enzyme did not require addition of pyridoxal 5'-phosphate, but dependence upon this cofactor was demonstrated following resolution of the enzyme and cofactor by hydroxylamine treatment. Kinetic data showed the classic ping-pong mechanism expected for aminotransferases. Km values of 0.17, 3.39, and 43.48 mM for histidinol phosphate, tyrosine, and phenylalanine were obtained. The gene structure around hisH-tyrC suggested an operon organization. The hisH-tyrC cluster in Z. mobilis is reminiscent of the hisH-tyrA component of a complex operon in Bacillus subtilis, which includes the tryptophan operon and aroE. Multiple alignment of all aminotransferase sequences available in the database showed that within the class I superfamily of aminotransferases, IAP aminotransferases (family I beta) are closer to the I gamma family (e.g., rat tyrosine aminotransferase) than to the I alpha family (e.g., rat aspartate aminotransferase or E. coli AspC). Signature motifs which distinguish the IAP aminotransferase family were identified in the region of the active-site lysine and in the region of the interdomain interface.
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PMID:Imidazole acetol phosphate aminotransferase in Zymomonas mobilis: molecular genetic, biochemical, and evolutionary analyses. 788 15

We have recorded 500-MHz 1H NMR spectra in the 10-18-ppm range for aspartate aminotransferase from Escherichia coli and for three specific mutant forms. Histidine 143 has been replaced by either alanine or asparagine. In the third mutant, tryptophan 140 has been replaced by phenylalanine. The NMR spectrum of the native enzyme is very similar to that of porcine cytosolic aspartate aminotransferase in the most downfield region. However, the resonances of the proton on the ring nitrogen of the pyridoxal 5'-phosphate (peak A) and on the His-143 imidazole ring (peak B) of the E. coli enzyme are broader and more readily lost at low pH or higher temperatures than those of the porcine enzyme. The possible role of tautomerism in promoting such broadening is discussed. In the histidine mutant proteins, peak A of the pyridoxal 5'-phosphate form is too broad to see under most conditions but is clearly present in the pyridoxamine phosphate form. Peak B is missing in the 2 histidine mutants. Observation of nuclear Overhauser effects further confirms the identity of B as the resonance of HN epsilon 2 of His-143 and that of peak D at approximately 11.8 ppm as HN epsilon 2 of His-189. The mutant spectra also provide insight into electronic interactions between groups in and near the active site which confirm and supplement conclusions drawn from spectra of porcine cAspAT. While no clear loss of a peak was observed for the Trp-140 mutant in its free form, the spectrum of the succinate complex lacked a strong band at 11.26 ppm. This may represent the Trp-140 indole NH proton which has been shifted downfield by binding to a succinate carboxylate group. While our results confirm the basic similarity of cytosolic aspartate aminotransferase and E. coli aspartate aminotransferase 1H NMR spectra, they also point out differences that may be useful in identifying resonances. A large number of mutant proteins have been prepared for the E. coli enzyme. The present results provide essential information for future study of these mutants and for study of NMR spectra of isotopically labeled enzyme.
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PMID:NMR studies of 1H resonances in the 10-18-ppm range for aspartate aminotransferase from Escherichia coli. 796 37

Serum amino acid (AA) profiles are altered in epilepsy. It is not clear whether this is due to the disease process itself or to other variables such as seizure type, seizure frequency, duration of illness, medication, or altered liver function. We investigated serum AA profiles and liver enzymes in 73 epileptic patients and 90 healthy subjects and evaluated the data by analysis of variance to discriminate between age, sex, seizure type, duration of illness, seizure frequency, antiepileptic drug (AED) and increased serum liver enzyme levels, and their putative interaction with the serum AA profile. There was no correlation between the changes in the AA profile and age, duration of illness, seizure frequency, and seizure type. Seventy-two percent of the AED-treated patients and 33% of the unmedicated patients showed an increase in one or several serum liver enzymes [alanine aminotransferase (ALT), aspartate aminotransferase (AST), and/or gamma-glutamyl transferase (gamma-GT)]; particularly gamma-GT. We observed a significant increase in serum concentrations of glutamine and glycine and decreased levels of taurine, threonine, serine, valine, methionine, isoleucine, leucine, phenylalanine, histidine, tryptophan, and arginine in AED-treated patients but not in unmedicated patients. These results show that the changes in the serum AA profiles of epileptic patients treated with AEDs occur in patients with alteration of serum liver enzymes; whether this implies a causal relation is still uncertain.
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PMID:Serum amino acids, liver status, and antiepileptic drug therapy in epilepsy. 809 92

The Schiff base formed between Lys258 of Escherichia coli aromatic amino acid aminotransferase (ArAT) and the coenzyme pyridoxal 5'-phosphate (PLP) has a pKa value of 6.65. The pH dependency of the kinetic parameters was consistent with a mechanism by which the enzymatic form with the nonprotonated Schiff base productively binds aspartate, phenylalanine, and tryptophan. The Schiff base pKa value rose by 1.7-2.1 unit on binding of substrate analogs, and this strongly suggested protonation of the Schiff base upon formation of the Michaelis complex with substrates. The protonated "internal" Schiff base in the Michaelis complex is supposed to be attacked by the deprotonated substrate amino group, and this explains excellently the mechanism of transaldimination to form the PLP-substrate Schiff base. Phenylpropionate and indolepropionate caused similar increases in the pKa value to maleate. [Arg292-->Ala] ArAT showed the same pKa value as the wild-type enzyme. Therefore, neutralization of Arg292 by omega-carboxylate of dicarboxylic ligands, which had been well documented in aspartate aminotransferase to increase the Schiff base pKa, has little effect on the protonation of the Schiff base in ArAT. Thus the structure of ArAT is deliberately organized so that the Schiff base pKa is effectively modulated by substrates having only one carboxylate group.
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PMID:Protonation state of the active-site Schiff base of aromatic amino acid aminotransferase: modulation by binding of ligands and implications for its role in catalysis. 818 25

Aromatic amino acid aminotransferase (ArAT) from Escherichia coli was overexpressed in E. coli cells, purified, and characterized. The enzyme was similar to aspartate aminotransferase (AspAT) of E. coli in many aspects, such as gross protein structure and spectroscopic properties. The reactions of pyridoxal 5'-phosphate-form ArAT with amino acids and pyridoxamine 5'-phosphate-form ArAT with oxo acids were investigated using stopped-flow spectrophotometric techniques. The kinetic parameters for these "half" reactions could excellently explain the ArAT-catalyzed overall transamination reactions at pH 8.0. Reactions of ArAT with aspartate and tryptophan which had been deuterated at position 2 showed isotope effects of 2.5 and 6.0 in the kcat values of the half-reactions, showing that the proton-transfer step is at least partially rate-limiting for these reactions. ArAT and AspAT showed overlapping substrate specificity. Both ArAT and AspAT were active toward dicarboxylic substrates. ArAT showed, however, 10(3)-fold higher activity toward aromatic substrates than AspAT. This high activity toward aromatic substrates was in part ascribed to the active site hydrophobicity of ArAT, which was suggested to be about 1.4 times as large as that of AspAT. In addition to dicarboxylic substrate analogs, aromatic substrate analogs such as carboxylic acids, 2-methyl amino acids, and 3-hydroxy amino acids caused characteristic changes in the absorption spectra of ArAT, while these aromatic analogs did not significantly change the spectra of AspAT. In particular, the erythro-3-hydroxy analogs of phenylalanine and aspartate caused a prominent absorption of ArAT at around 500 nm, which is generally ascribed to the accumulation of quinonoid intermediates. The threo forms of these 3-hydroxy analogs acted as substrates for ArAT. The erythro and threo forms of 3-hydroxyaspartate reacted with AspAT similarly as they reacted with ArAT; however, both forms of 3-phenylserine were poor substrates for AspAT, although phenylalanine was a fairly good substrate for AspAT. The observations on the two erythro-3-hydroxy amino acids show the similar orientation of these analogs in the active site of ArAT, probably through a hydrogen-bonding network involving the hydroxy groups of the analogs and Tyr70, and suggest that the aromatic binding pocket is near or even overlaps the side-chain-carboxylate-binding site for dicarboxylic substrates.
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PMID:Escherichia coli aromatic amino acid aminotransferase: characterization and comparison with aspartate aminotransferase. 821

The University of Wisconsin (UW) solution consists of a relatively complex mixture of agents. In this study we compared simpler preservation solutions, namely, histidine-tryptophan-ketoglutarate (HTK) and phosphate-buffered sucrose (PBS) with different compositions of UW solution in the isolated perfused rabbit liver model. Livers were stored cold for 24 and 48 h. After 24 h of preservation, the amount of bile produced in UW-preserved livers was significantly greater (P < 0.05) than that in HTK-preserved livers. Also, there was less LDH released into the perfusate in UW-preserved livers. There was more edema and lower K +/Na+rations in HTK-preserved livers than in UW-preserved livers (all data P < 0.05). After 48 h of preservation, the differences between livers preserved in UW or HTK solution were less noticeable than at 24 h and bile production was similar. LDH and AST release were greater in HTK-preserved livers than in UW livers, but these differences were not statistically significant. Preservation in PBS for 48 h was worse than in either UW or HTK solution. Substitution of polyethylene glycol (PEG) for hydroxyethyl starch (HES) in 48-h UW-preserved livers was not effective. We conclude that solutions simpler in composition than UW solution may be effective in kidney transplantation but do not appear suitable for successful liver preservation.
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PMID:Comparison of solutions for preservation of the rabbit liver as tested by isolated perfusion. 857 38

Indian River male broiler chickens growing from 7 to 28 d of age were fed on diets containing 120, 210 and 300 g crude protein/kg diet and 0, 1.67 or 16.7 g added tryptophan (TRP)/kg diet. The hypothesis tested was that crude protein levels and TRP would affect both growth and neurotransmitter metabolism. Heart, brain and pancreatic neurotransmitter (noradrenaline (NA), dopamine (DA), serotonin (5-HT) and 5-hydroxy-indole-3-acetic acid (5-HIAA)) concentrations were determined by HPLC separation and electrochemical detection. Malate dehydrogenase (2-oxoglutarate decarboxylating) (NADP+) (MDH(NADP+); EC 1.1.1.40), isocitrate dehydrogenase (NADP+) (ICD(NADP+); EC 1.1.1.42) and aspartate aminotransferase (AAT; EC 2.6.1.1) activities were also measured. Supplemental TRP decreased growth and feed intake. Increasing dietary crude protein decreased MDH(NADP+), but increased (ICD(NADP+) and AAT activities. Additional dietary TRP decreased MDH(NADP+) activity, but had no effect on other enzyme activities. Cardiac NA concentrations were directly related to dietary crude protein levels while pancreatic levels were inversely related. An increase in dietary crude protein decreased both brain NA and DA. Supplemental dietary TRP increased both 5-HIAA and 5-HT. Changes in feed intake caused by different levels of both dietary crude protein and TRP are accompanied by altered levels of neurotransmitters. The present study indicates that much larger amounts of TRP are required to make simultaneous changes in feed intake and neurotransmitters.
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PMID:Crude protein and supplemental dietary tryptophan effects on growth and tissue neurotransmitter levels in the broiler chicken. 877 19

In the end-stage renal disease patient, certain uremic compounds could influence the cellular accumulation of aluminum (Al). In this study, we examined the effect of 15 uremic ultrafiltrate fractions obtained by HPLC on the uptake and toxicity of Al in mouse hepatocytes (MH) in culture, a model system in which Al is taken up bound to transferrin (Tf). Uremic fractions 4 to 8, 12, 14, and 15 increased cellular Al uptake and aspartate aminotransferase release and decreased cell growth when Tf-Al, not Al citrate, was added to culture media. Compounds that have been extracted previously from these ultrafiltrate fractions (p-cresol, xanthine, tryptophan, hippuric acid, and o-hydroxyhippuric acid) were then tested for their effect on Al uptake and toxicity in MH at concentrations found in uremic serum. Significant Al uptake by MH was observed only when p-cresol was added together with Tf-Al. Time-response curves showed increased Al uptake and toxicity at p-cresol concentrations of 3 mg/dl in culture media. Dose-response curves confirmed that Al uptake and cell toxicity were proportional to p-cresol from 1.5 mg/dl to 3 mg/dl in culture media. p-Cresol was not toxic to MH in the absence of Tf-Al in media. p-Cresol increased Tf-associated Al uptake only because there was no effect on Al uptake when Al citrate was substituted, and studies with Tf-I125-Al in the presence of this compound showed increased Tf-I125 taken up by MH. p-Cresol did not increase Tf saturation with Al. p-Cresol also increased Tf-Al uptake in Friend erythroleukemia and neuroblastoma cells in culture. Our studies suggest that p-cresol and uremic fractions 4 to 8, 12, 14, and 15 increase the uptake and toxicity of Al in cultured MH. These compounds may play a role in the accumulation and toxicity of Al in the liver of end-stage renal disease patients and possibly in all cells that express Tf receptors.
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PMID:P-Cresol, a uremic compound, enhances the uptake of aluminum in hepatocytes. 918 61

1. Living-related liver transplantation has some advantages in the evaluation of novel clinical protocols, since many complicated factors affecting initial graft function are almost uniform in grafts obtained from healthy donors. 2. To compare histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin (UW) solution in terms of tissue oxygenation in living-related liver transplantation, oxygen saturation of haemoglobin (SO2) in hepatic tissue and its heterogeneity (CV, coefficient of variation) were measured by near-infrared spectroscopy. The HTK and UW groups consisted of 15 and 49 successful transplants respectively, in which no statistical differences in background were observed. 3. In the HTK group, hepatic SO2 after portal vein reflow was higher (P < 0.01) than that in the UW group, as was that after hepatic artery reflow (P < 0.05). In the UW group, hepatic SO2 remained at the lower level at the end of the operation. 4. Furthermore, the increase in CV after portal vein reflow was normalized after hepatic artery reflow in the HTK group. However, the CV remained at a high level at the end of the operation in the UW group. 5. Postoperative peak aspartate aminotransferase level in the HTK group was lower than that in the UW group (P < 0.05). 6. In cadaveric liver transplantation, higher hepatic SO2 and lower CV of hepatic SO2 in the early phase after reperfusion compared with the UW group (n = 18) were also observed in the HTK group (n = 30) (P < 0.05). 7. In conclusion, recovery of tissue oxygenation and its heterogeneity after reperfusion in HTK-preserved livers were more rapid and homogeneous than in UW-preserved livers in living-related liver transplantation. Accordingly, HTK solution may be a potential alternative to UW solution.
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PMID:Hepatic preservation with histidine-tryptophan-ketoglutarate solution in living-related and cadaveric liver transplantation. 927 7

The aim of these investigations was to examine the influence of "natural fibrous feedstuffs" as wheat bran and alfalfa meal on criteria of vitamin B6 metabolism of adult sows subjected to a low vitamin B6 supply. Two experiments were conducted in two periods with 12 sows (180 kg BW) and 3 groups each. The supplements were in the first experiment 0 g, 225 g and 675 g wheat bran, and in the second experiment 0 g, 575 g and 1150 g alfalfa meal to a compound feed, low in vitamin B6 content. The criteria were fecal and urinary vitamin B6 concentration and excretion, vitamin B6 concentration in blood, hematological criteria, activity of aspartate aminotransferase in erythrocytes (EAST) and xanthurenic acid excretion in the tryptophan load test. Vitamin B6 concentration in feces amounted 10-12 micrograms/g DM and was neither influenced by quality or amount of the fibrous products. Vitamin B6 excretion was increased by each supplement and 60-70% of vitamin B6 was excreted via feces. Fecal vitamin B6 excretion was enlarged linearly by increasing fibrous supplementation. Bacterially fermentable substrates from wheat bran induced a higher bacterial vitamin B6 synthesis compared to cellulose.
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PMID:[The effect of wheat bran and green meal supplements on vitamin B6 metabolism of sows fed suboptimal vitamin B6 supply]. 932 21


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