Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P17174 (aspartate aminotransferase)
 14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The response of guinea pig macrophages to migration inhibitory factor (MIF) is altered by several chemical treatments. Treatment of macrophages with the diazonium salt of sulfanilic acid (5 x 10(-6) to 4 x 10(-4) M) significantly increases the response of these cells to MIF. Treatment with acetic anhydride also augments the response of these cells to MIF. The latter finding suggests that alteration of amino, hydroxyl, or sulfhydryl groups is involved in this phenomenon. Treatment of macrophages with sodium periodate (2 x 10(-5) to 10(-3) M) which is known to oxidize cis-glycols and with hydroxylamine (2 x 10(-5) to 2 x 10(-3) M), which reacts with carbonyl groups also increases response to MIF. The following experiments suggest that the significant alteration occurs at the level of the cell surface. Incubation of macrophages with the diazonium salt of sulfanilic acid at 4 degrees C, at which temperature pinocytosis is largely inhibited, is sufficient to increase the MIF response. The activity of the cytoplasmic enzyme aspartate aminotransferase, which in homogenates is susceptible to inactivation by low concentrations of the diazonium salt of sulfanilic acid, is not decreased when intact macrophages are incubated with high concentrations of the diazonium salt of sulfanilic acid. Cumulatively, these findings suggest that modification of different functional groups on the macrophage surface causes the same physiologic effect.
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PMID:Chemical treatment of macrophages increases their responsiveness to migration inhibitory factor (MIF). 18 95

The mechanism of inhibition of ornithine aminotransferase [EC 2.6.1.13] by L-canaline (alpha-amino-gamma-amino-oxybutyric acid) was investigated. Spectral changes of pyridoxal 5'-phosphate in ornithine aminotransferase on addition of L-canaline showed that L-canaline formed an oxime-type compound with pyridoxal 5'-phosphate that had the same spectra as the compound formed on addition of hydroxylamine to the holoenzyme. Kinetic studies indicated that hydroxylamine was a reversible noncompetitive inhibitor, whereas L-canaline was an irreversible inhibitor of ornithine aminotransferase. Other analogs, such as delta-aminovaleric acid and alpha-N-acetyl-L-ornithine, also reacted with the pyridoxal 5'-phosphate of the enzyme, but these compounds were competitive inhibitors with respect to L-ornithine. L-Canaline and hydroxylamine also reacted with pyridoxal 5'-phosphate in pig heart aspartate aminotransferase [EC 2.6.1.1] to produce an oxime, but both of them were reversible and noncompetitive inhibitors of the enzyme. The Ki value of hydroxylamine for ornithine aminotransferase was 4.3 X 10(-7) M and those of L-canaline and hydroxylamine for aspartate aminotransferase were 1.7 X 10(-4) M and 2.2 X 10(-5) M, respectively.
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PMID:Mode of inhibition of ornithine aminotransferase by L-canaline. 62 4

Hydroxylamine and its derivatives of general formula H2NOR react with aldehydes and aldimines to produce oximes. If R corresponds to the side chain of a natural amino acid, such compounds can be thought of as analogs of the corresponding amino acids, lacking the alpha-carboxylate group. Oximes formed between such compounds and pyridoxal phosphate in the active site of aspartate amino-transferase mimic external aldimine intermediates that occur during catalysis by this enzyme. The properties of oxime derivatives of mitochondrial aspartate aminotransferase with hydroxylamine and 6 compounds H2NOR were studied by absorption spectroscopy and circular dichroism in solution and by linear dichroism in crystals. Stable oximes, absorbing at lambda max congruent to 380 nm and exhibiting a negative Cotton effect, were obtained with the carboxylate-containing compounds. The oximes formed with carboxylate-free compounds showed somewhat different properties and stability. With H-Tyr a stable complex absorbing at lambda max congruent to 370 nm rather than at 380 nm, was obtained, H-Ala and H-Phe produced unstable oximes with the initial absorption band at lambda max congruent to 380 nm that was gradually replaced by a band at lambda max congruent to 340 nm. The species absorbing at 340 nm were shown to be coenzyme-inhibitor complexes which were gradually released from the enzyme. A similar 330-340 nm absorption band was observed upon reaction of the free coenzyme with all hydroxylamine inhibitors at neutral pH-values. The results of the circular dichroism experiments in solution and the linear dichroism studies in microcrystals of mAspAT indicate that the coenzyme conformation in these inhibitor/enzyme complexes is similar to that occurring in an external aldimine analogue, the 2-MeAsp/mAspAT complex. Co-crystallizations of the enzyme with the H2NOR compounds were also carried out. Triclinic crystals were obtained in all cases, suggesting that the "closed" structure cannot be stabilized by a single carboxylate group.
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PMID:Complexes of aspartate aminotransferase with hydroxylamine derivatives: spectral studies in solution and in the crystalline state. 250 50

A rapid, sensitive and specific procedure has been developed for the determination of p-tyrosine aminotransaminase activity. The assay is based on high-performance liquid chromatography (HPLC) separation and electrochemical detection of the pyruvate product, which has been derivatized with hydroxylamine to form a stable oxime. Using this method the product at the low pmol level can be measured. A comparison of the kinetic parameters of the rat liver tyrosine aminotransferase and rat brain non-specific aspartate aminotransferase towards p-tyrosine has been made.
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PMID:New sensitive high-performance liquid chromatographic method for p-tyrosine aminotransferase assay. 287 80

In bovine platelets aspartate aminotransferase has a high activity. The enzyme in vitro is inhibited in a dose dependent manner by aminooxyacetate (IC50 = 10(-4) M), hydroxylamine (IC50 = 10(-4) M), and cycloserine (IC50 = 5 X 10(-3). The inhibitory effect of all the three compounds is strongest at low substrate (aspartate) concentration. Blocking of aspartate aminotransferase activity by these compounds in intact platelets is accompanied by the inhibition of ADP and collagen-induced aggregation. Among the three compounds the strongest inhibitor of platelet aggregation was hydroxylamine, which was also the most effective inhibitor of aspartate aminotransferase. Other metabolic blockers, i.e. dinitrophenol (DNP), rotenone and antimycin also inhibited the aggregation of platelets, and a synergism has been demonstrated between DNP, rotenone and antimycin A action on platelet aggregation and blockade of aspartate aminotransferase activity. The results are interpreted to mean that transamination is of importance in the energy production in the activated platelet, probably through its participation in reducing equivalents transport from the cytosol to the mitosol via the malate: oxaloacetate: aspartate shuttle.
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PMID:The importance of aspartate aminotransferase for platelet aggregation. 375 38

1. An improved procedure is reported for purification of the amine dehydrogenase from methylamine-grown Pseudomonas AM1 which yielded a product homogeneous by sedimentation and disc-electrophoretic analysis, with molecular weight of 133000. 2. The purified enzyme had absorption maxima at 280 and 430nm. On aging, a third peak appeared at 325nm, and the 430nm peak decreased in intensity. This spectrum was independent of pH. 3. Addition of 2.5mm-semicarbazide, phenylhydrazine, hydrazine or hydroxylamine produced modified spectra with maxima respectively at 400, 440, 395 and 425nm. 4. Aerobic addition of methylamine resulted in a bleaching of the 430nm peak and the appearance of a new one at 325nm. This spectral change was retained after removal of the methylamine by dialysis. The original spectrum could be restored on addition of phenazine methosulphate. 5. Addition of borohydride partially inactivated the enzyme and produced spectral changes similar to those observed with methylamine. Pre-treatment with methylamine prevented the inactivation by borohydride. The degree of inactivation could be increased by alternate phenazine methosulphate and borohydride treatments. 6. The addition of methylamine or borohydride each caused shifts in the fluorescence emission maximum from 348 to 380nm. 7. Lineweaver-Burk plots of reciprocal activity against reciprocal concentration of either of the substrates n-butylamine or phenazine methosulphate were consistent with a mechanism that involves interconversion of two free forms of the enzyme by the two substrates. 8. The enzyme, although spectrally modified, was not inactivated by dialysis against diethyldithiocarbamate, and contained about 0.27 g-atom of copper/mol, with small traces of cobalt, iron and zinc. 9. Conventional methods of resolution did not release the prosthetic group. Heat denaturation after treatment of the enzyme with methylamine liberated a yellow chromophore which did not reactivate resolved aspartate aminotransferase, and whose spectral, electrophoretic and fluorescence properties did not agree with any recognizable pyridoxal derivatives. 10. Despite the inconclusive results with the isolated chromophore, the observations on the enzyme suggest that it may contain a pyridoxal derivative bound as a Schiff's base which is converted into the pyridoxamine form on aerobic treatment with methylamine and reconverted into the pyridoxal form with phenazine methosulphate. 11. The copper detected is probably not involved in the enzyme mechanism, since most copper-chelating agents are not inhibitory, and since the enzyme does not react with oxygen.
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PMID:Microbial oxidation of amines. Spectral and kinetic properties of the primary amine dehydrogenase of Pseudomonas AM1. 512 84

The bimolecular association and first-order dissociation rate constants for the reactions of aminooxyacetate and hydroxylamine with the cytosolic aspartate aminotransferase (EC 2.6.1.1) of pig heart were estimated from pH 4.8 to pH 9.5. The acidic form of the enzyme (pK = 6.3) was more reactive than the unprotonated enzyme, but the rates of breakdown of the complexes were not affected by pH. The equilibrium dissociation constants, which were of the order of magnitude of 10(-7) M, were thus lowest at acidic pH values. Aminooxyacetate and hydroxylamine reacted similarly, with the former being more reactive. Glutarate and acetate inhibited the rates of formation and dissociation but had no effect on the overall equilibrium constants between enzyme and inhibitor. On the other hand, the substrate analog erythro-beta-hydroxy-L-aspartate, which forms a complex with the enzyme prosthetic group, acted competitively by preventing the inhibitors from reacting. The rate constants for formation of complexes with free pyridoxal phosphate were much less than those for the enzyme and, unlike the enzyme, hydroxylamine was more reactive than aminooxyacetate.
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PMID:Effects of ligands and pH on the reactions of aspartate aminotransferase with aminooxyacetate and hydroxylamine. 646 3

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

Following the chromatographic separation of the grey mullet (Mugil auratus Risso) red muscle extract, two fractions with aspartate aminotransferase activity were detected. One of the anticipated enzymes was purified to homogeneity. The isolated enzyme was a dimeric protein composed of identical subunits with the overall M(r) of about 65,000. It consisted of three electrophoretically distinct subforms with isoelectric points at pH 8.50, 8.70 and 8.85, respectively. The Michaelis-Menten constants of the substrates L-aspartate and 2-oxoglutarate were estimated to be 0.29 +/- 0.012 mM and 0.45 +/- 0.016 mM, respectively. For the reverse reaction, the Km for L-glutamate was 8.57 +/- 2.1 mM and for oxaloacetate it was 0.13 +/- 0.035 mM. The inhibition of the isolated enzyme by hydroxylamine was of a mixed linear noncompetitive type for L-aspartate as a substrate, whereas with 2-oxoglutarate hyperbolic uncompetitive inhibition was observed. The inhibition by aminooxyacetic acid and D,L-glyceraldehyde 3-phosphate was of a mixed linear noncompetitive type with respect to L-aspartate and 2-oxoglutarate. The isolated enzyme was slightly affected by maleate and succinate and no effects were produced by adipate. According to its subcellular distribution, susceptibility to inhibitors molecular and catalytic properties the isolated enzyme belonged to the mitochondrial form of aspartate aminotransferase.
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PMID:Isolation and properties of mitochondrial aspartate aminotransferase from red muscle of grey mullet, Mugil auratus Risso. 791 42

The interaction of mitochondrial aspartate aminotransferase with hydroxylamine and five derivatives (in which the hydroxyl hydrogen is replaced by the side chain of naturally occurring amino acids) was investigated by X-ray diffraction as well as by kinetic and spectral measurements with the enzyme in solution. The inhibitors react with pyridoxal 5'-phosphate in the enzyme active site, both in solution and in the crystalline state, in a reversible single-step reaction forming spectrally distinct oxime adducts. Dissociation constants determined in solution range from 10(-8) M to 10(-6) M depending on the nature of the side-chain group. The crystal structures of the adducts of mitochondrial aspartate aminotransferase with the monocarboxylic analogue of L-aspartate in the open and closed enzyme conformation were determined at 0.23-nm and 0.25-nm resolution, respectively. This inhibitor binds to both the open and closed crystal forms of the enzyme without disturbing the crystalline order. Small differences in the conformation of the cofactor pyridoxal phosphate were detected between the omega-carboxylate of the inhibitor and Arg292 of the neighbouring subunit is mainly responsible for the attainment of near-coplanarity of the aldimine bond with the pyridine ring in the oxime adducts. Studies with a fluorescent probe aimed to detect shifts in the open/closed conformational equilibrium of the enzyme in oxime complexes showed that the hydroxylamine-derived inhibitors, even those containing a carboxylate group, do not induce the 'domain closure' in solution. This is probably due to the absence of the alpha-carboxylate group in the monocarboxylic hydroxylamine-derived inhibitors, emphasizing that both carboxylates of the substrates L-Asp and L-Glu are essential for stabilizing the closed form of aspartate aminotransferase.
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PMID:Crystal structures and solution studies of oxime adducts of mitochondrial aspartate aminotransferase. 866 90


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