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)

Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that the active sites of glutamate aspartate transaminase function independently and a compulsory flip-flop mechanism is not involved.
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PMID:Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction. 117 14

Frontal and zonal analysis of the chromatography of aspartate aminotransferase (EC2.61.1), pig heart cytosolic enzyme, on Bio-Gel P150 shows that holo- and apoenzyme can dissociate at pH 8.3. Ultracentrifugation and fluorescence depolarization confirm this result. Kinetic analysis of the fluorescence depolarization experiments favors a biphasic phenomenon: a few minutes for the faster one and several hours for the slower one. The apparent dissociation constant is 0.8 muM for the apoenzyme and 0.18 muM for the pyridoxal 5'-phosphate form of the holoenzyme. In the presence of sucrose or 0.1 M L-aspartate or a mixture of 70 mM L-glutamate and 2 mM alpha-ketoglutarate, the holoenzyme is dimeric at concentrations higher than 5 nM. The addition of a mixture of the substrates L-glutamate and alpha-ketoglutarate to a monomeric holoenzyme leads to dimerization. The stability of the dimeric form is in the order: holoenzyme + substrates greater than apoenzyme.
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PMID:Dissociation of aspartate aminotransferase into subunits. Effect of ligands upon this dissociation. 119 65

Octanoate and L-palmitylcarnitine inhibited the synthesis of P-enolpyruvate from alpha-ketoglutarate and malate by isolated guinea pig liver mitochondria. A 50% reduction in P-enolpyruvate formation was obtained with 0.1 to 0.2 mM octanoate or with 0.06 to 0.10 mM L-palmitylcarnitine. At these concentrations, oxidative phosphorylation remained intact and only much higher concentrations of fatty acids altered this process. The addition of NH4Cl in the presence of malate and increasing concentrations of alpha-ketoglutarate (or vice versa) enhanced the formation of glutamate, aspartate, and P-enolpyruvate. The addition of increasing concentrations of NH4Cl in the presence of fixed amounts of malate and alpha-ketoglutarate had a similar effect. Furthermore, the inhibition of P-enolpyruvate synthesis by fatty acids and the reduction of the acetoacetate to beta-hydroxybutyrate ratio were reversed by the addition of NH4Cl. Cycloheximide, which blocks energy transfer at site 1 of the respiratory chain, decreased P-enolpyruvate formation. When cycloheximide and either octanoate or L-palmitylcarnitine were added together, there was an even greater reduction in P-enolpyruvate synthesis from either malate or alpha-ketoglutarate than was noted with either fatty acid alone. Since cycloheximide lowers the rate of ATP synthesis this may in turn reduce P-enolpyruvate formation by a mechanism independent of changes in the mitochondrial NAD+/NADH ratio caused by fatty acids. In the isolated perfused liver metabolizing lactate, the inhibitory effect of octanoate on gluconeogenesis was partially relieved by the addition of 1 mM NH4Cl, but remained unchanged in the presence of 2 mM NH4Cl, despite a highly oxidized NAD+/NADH ratio in the mitochondria. In contrast to glucose synthesis, urea formation was markedly increased during the infusion of 1 mM as well as 2 mM NH4Cl. After cessation of NH4Cl infusion, there was an increase in glucose production, to a rate as high as that observed in the absence of octanoate. This increase was accompanied by the disappearance of alanine, aspartate, and glutamate which had been stored in the liver during NH4Cl infusion. Urea synthesis also decreased progressively. These results indicate that gluconeogenesis in guinea pig liver is regulated, in part, by alterations in the mitochondrial oxidation-reduction state. However, the modulation of this effect by changing the concentrations of intermediates of the aspartate aminotransferase reaction indicates competition for oxalacetate between the aminotransferase reaction and P-enolpyruvate carboxykinase.
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PMID:Regulation of hepatic gluconeogenesis in the guinea pig by fatty acids and ammonia. 119 71

1. Isolated hepatocytes were used to establish the reasons for the accumulation of aspartate, previously observed when the isolated rat liver was perfused with ethanol in the presence of alanine or ammonium lactate. 2. The isolated cells did not form aspartate when incubated with alanine and ethanol, but much aspartate was formed on incubation with ammonium lactate and ethanol. 3. Urea was the main nitrogenous product on incubation with alanine, in contrast with the perfused liver, where major quantities of NH4+ are also formed. When the formation of urea was nullified by the addition of urease, alanine plus ethanol caused aspartate formation, indicating that aspartate formation depends on the presence of critical concentrations of NH4+. 4. The accumulated aspartate was present in the cytosol. Ethanol halved the content of 2-oxoglutarate in the cytosol and more than trebled that of glutamate in the mitochondria. 5. The findings support the assumption that 2-oxoglutarate formed by the mitochondrial aspartate aminotransferase is not translocated to the cytosol in the presence of ethanol and NH4+, because it is rapidly converted into glutamate, the dehydrogenation of ethanol providing the required NADH. Aspartate, however, is translocated to the cytosol and accumulates there because of the lack of stoicheiometric amounts of oxoglutarate.
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PMID:The accumulation of aspartate in the presence of ethanol in rat liver. 120 Oct 7

Chinese hamster ovary cells with a specific auxotrophy for proline were fused with human cells from a variety of sources and the resulting hybrids analyzed for human genetic markers. Of 63 hybrid clones examined, 27 possessed both proline and cytoplasmic glutamate oxaloacetate transaminase markers; 36 had neither; and no clones were found possessing one and not the other. These results constitute evidence that the proline and glutamate oxalocetate transaminase markers are syntenic. Evidence for absence of synteny between these and a variety of other human genes is presented. Biochemical tracer experiments established that the proline biosynthetic pathway through glutamate has been restored in the Pro+ hybrids.
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PMID:Synteny between the pro+ marker and human glutamate oxaloacetate transaminase. 123 10

Pulsed Fourier transform proton magnetic resonance spectroscopy was used to study the glutamate-alanine transaminase-catalyzed incorporation of deuterium from solvent deuterium oxide into the alpha and beta positions of L-alanine. It was found that the beta proton resonance signal initially disappears slightly faster than the signal due to the alpha proton, but whereas the alpha proton signal decays exponentially, that due to the beta proton signal does not. Eventually, the rate of decrease of the alpha proton signal becomes greater than that for the beta proton. This change in the relative rates is ascribed to a deuterium isotope effect upon substitution of an alpha proton by a deuteron. Furthermore, as deuterium begins to replace hydrogen, two classes of alanine become distinguishable, i.e. alanine which contains deuterium in the alpha position and hydrogen in the beta position, and alanine which contains hydrogen in the alpha position and deuterium in the beta position. Thus, removal of all 3 beta protons is not contingent upon loss of an alpha proton from the same molecule. The two classes of deuterated alanine may conceivably arise by a scrambling mechanism in which protons are transferred from the alpha to the beta position and vice versa. Present evidence excludes this scramblong mechanism and leads to the conclusion that deuterium incorporation into L-alanine involves, (a) the reversible enzymatic conversion of the classical ketimine enzymes intermediate to an enaminetype structure, and (b) considerable conservation of label during the prototropic shift from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate. It is also postulated that alanine binds at the active site in such a way as to bring the beta protons into close contact with a basic group on the enzyme surface. This group is distinct from that used in abstraction of an alpha proton. The beta protons of glutamate are not enzymatically removed; presumably glutamate binds in such a way that the beta protons cannot effectively interact with an enzyme base. Similar studies were carried out on soluble glutamate-aspartate transaminase; no evidence was found for significant enzyme-catalyzed deuterium incorporation into the beta position of L-glutamate, L-aspartate, and L-alanine.
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PMID:Proton magnetic resonance studies of glutamate-alanine transaminase-catalyzed deuterium exchange. Evidence for proton conservation during prototropic transfer from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate. 124 68

Feedback control between flux through the phosphorylating electron transport chain and the coordination of flux through individual steps of the citric acid cycle have been investigated under a number of different conditions of substrate availability and workloads in the isolated perfused rat heart. The transition from substrate-free perfusion to perfusion with glucose and insulin with no change of workload was associated with increases in the pool sizes of citric acid cycle intermediates except for oxaloacetate, but with an initial imbalance of flux through individual steps in the cycle and transport of anions of the malate-aspartate cycle across the mitochondrial membrane. Flux through citrate synthase initially increased while that through alpha-ketoglutarate dehydrogenase decreased. Of the components of the malate-aspartate cycle, flux through the malate-alpha-ketoglutarate exchange was increased prior to that through the glutamate-aspartate exchange and intramitochondrial aspartate aminotransferase. These changes can be accounted for on the basis of known kinetic controls of the enzyme and transport steps in response to increased pyruvate, acetyl-CoA, and NADH delivery at an approximately constant rate of ATP turnover.
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PMID:Coordination of citric acid cycle activity with electron transport flux. 126 91

Cytoplasmic aspartate aminotransferase from beef kidney loses 25% of its activity on nitration with tetranitromethane while the apoenzyme about 95%. In the holoenzyme 0.5 tyrosine residue and 1.0 tyrosine residue in the apoenzyme are nitrated per enzyme protomer. In addition 1 cysteine residue per protomer is oxidized in both. The presence of substrates, alpha-ketoglutarate and glutamate, both at ten times their Km values, does not change these results. Mercaptoethanol does not affect the residual activity of either the nitrated holo or apoenzyme. Dithionite abolishes the activity of the nitrated holoenzyme by reducing tha coenzyme moiety. It has no effect on the native holoenzyme or on either the native or nitroapoenzyme.
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PMID:Role of tyrosine residues in cytoplasmic aspartate aminotransferase from beef kidney. 127 58

The possible involvement of ionotropic and metabotropic quisqualate (QA) receptors in neuronal plasticity was studied in cultured glutamatergic cerebellar or hippocampal cells in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When cerebellar or hippocampal neurons were treated with QA, it elevated the specific activity of glutaminase in a dose-dependent manner. The half-maximal effect was obtained at about 0.1 microM, the maximum increase was at about 1 microM, but levels higher than 10 microM QA produced progressive reduction in glutaminase activity. In contrast, QA had little effects on the activities of lactate dehydrogenase and aspartate aminotransferase and the amount of protein, indicating that the increase in glutaminase was relatively specific. The QA-mediated increase in glutaminase was mimicked by the ionotropic QA receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; EC50, about 0.5 microM), but not by the metabotropic QA receptor agonist trans-(+-)-1-amino-cyclopentyl-1,3,dicarboxylate (t-ACPD; up to 0.5 mM). The specific ionotropic QA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited QA- and AMPA-mediated increases in glutaminase activity in a dose-dependent manner, whereas other glutamate receptor antagonists, D,L-2-amino-5-phosphonovalerate, gamma-D-glutamyl aminomethyl sulphonic acid and gamma-D-glutamyl diethyl ester were ineffective. The elevation of neurotransmitter enzyme was Ca(2+)-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of neurotransmitter enzyme by quisqualate subtype glutamate receptors in cultured cerebellar and hippocampal neurons. 133 Feb 9

Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.
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PMID:Glutamate metabolism in rat cortical astrocyte cultures. 134 25


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