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)

Sensitive flow-injection analyses of aspartate, glutamate, 2-oxoglutarate, and oxaloacetate were developed. The analytes were enzymatically coupled with NADH which was monitored by light emission from immobilized bacterial bioluminescence enzymes. Aspartate (or oxaloacetate) was assayed on the basis of NADH consumption by introducing the sample through a coimmobilized aspartate aminotransferase-malate dehydrogenase column. The assay responded linearly from 100 pmoles to 5 nmoles per assay. Glutamate (2-oxoglutarate) was determined by formation of NADH in the glutamate dehydrogenase reaction. The measuring range for glutamate was from 10 pmoles to 100 nmoles per assay. The precision of the flow-injection method was generally excellent, and the sensitivities of the described assays were 100-1000-fold higher than with spectrophotometric methods. The immobilized enzyme preparations were stable for several months in storage, and the enzyme columns could be used for 600-800 analyses. Flow-injection analyses of amino acids and related compounds by NADH/bioluminescence-coupled reactions provide a sensitive, fast, and inexpensive assay method for a wide variety of purposes.
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PMID:Flow-injection analysis of amino acids and their metabolites by immobilized vitamin B6-dependent enzymes. Sensitive determination of L-aspartate, L-glutamate, 2-oxoglutarate, and oxaloacetate. 197 15

In vivo studies with L-[13N]glutamate in the Walker 256 carcinosarcoma implanted under the renal capsule of female Sprague-Dawley rats demonstrate that uptake of glutamate and the rate of incorporation of the nitrogen label from this amino acid into metabolites is slower in the tumor than in nontumorous kidney tissue. Glutamate dehydrogenase, glutaminase, and alanine aminotransferase activities are significantly lower within the tumor than within the adjoining kidney. However, the tumor expresses high levels of aspartate aminotransferase, attesting to the importance of this enzyme in the metabolism of glutamate. Indeed, high performance liquid chromatographic analysis showed that the principal metabolic fate of label derived from L-[13N]glutamate in the tumor is incorporation into aspartate. Measurement of specific activity ratios of glutamate to aspartate shows that the transfer of nitrogen from glutamate to aspartate is rapid and that equilibration of label among components of the aspartate aminotransferase reaction is attained within minutes after tumor uptake. Analyses of the nontumorous portion of the implanted kidney also showed that aspartate is the major recipient of glutamate nitrogen. However, high performance liquid chromatographic analyses of deproteinized tissue revealed that glutamine and ammonia are also significant 13N-labeled metabolites formed from L-[13N]glutamate within the kidney. Proportionately lower amounts of these labeled metabolites were found in the tumor.
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PMID:Short-term metabolic fate of L-[13N]glutamate in the Walker 256 carcinosarcoma in vivo. 197 67

In studies on the uptake and metabolism of [14C]glutamate by Bradyrhizobium japonicum bacteroids we found that, in the presence of unlabeled malate, succinate or alpha-ketoglutarate, substantial label was recovered in alpha-ketoglutarate in the reaction mixtures. As much as 30% of the total 14C supplied could be found in alpha-ketoglutarate in the reaction mixtures after 30 min and this occurred in the absence of detectable labeling of alpha-ketoglutarate in the cells. The labeling of alpha-ketoglutarate was almost completely inhibited by aminooxyacetate (aminotransferase inhibitor). Direct assay of aspartate aminotransferase in intact bacteroids was possible in the presence of very dilute Triton X-100 (less than or equal to 0.02%, w/v). The response of the aminotransferase to detergent was similar to the response of phosphodiesterase, a periplasmic marker, and different from malate dehydrogenase and beta-hydroxybutyrate dehydrogenase, cytoplasmic markers. Comparison of maximum enzyme activity assayable with intact bacteroids and maximum activity in sonicated bacteroids indicated that about half of the total cellular aminotransferase activity was accessible to the external medium. The combined labeling and enzyme assay results indicated that B. japonicum bacteroids have a capability for transamination in the periplasmic space. Although this may not be important in the transfer of reducing equivalents from host cytoplasm to bacteroids in nodules, the transamination capability may facilitate the acquisition of metabolites by free-living bacteria.
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PMID:Periplasmic metabolism of glutamate and aspartate by intact Bradyrhizobium japonicum bacteroids. 197 84

In aspartate aminotransferase (AspAT), His143 is located within a hydrogen-bonding distance to Asp222 that forms a strong ion pair with the ring nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). His143 of Escherichia coli AspAT was replaced by Ala or Asn. The mutant enzyme H143A showed a slight increase in the maximum velocity of the overall transamination reaction between aspartate and 2-oxoglutarate, while H143N AspAT showed a decrease to 60% in the maximum rate of the overall reactions in both directions. In all of the half-transamination reactions with four substrates, aspartate, glutamate, oxalacetate, and 2-oxoglutarate, the catalytic competence as defined by kmax/Kd decreased by 3-18-fold upon replacing His143 by either Ala or Asn. The extent of the decrease varied from one substrate to another; it was largely contributed to by the decrease in affinities for all substrates. The equilibrium constants, [PMP-form] [keto acid]/[( PLP-form] [amino acid]), decreased by over 10-fold upon the mutations at position 143. Both H143A and H143N AspATs exhibited a considerably decreased affinity for 2-methylaspartate, an external-aldimine-forming substrate analogue, yet without appreciable alteration in the affinity for succinate and glutarate, which are non-aldimine-forming analogues. All these findings suggest that, although His143 is not essential for catalysis, it might assist the formation of enzyme-substrate complex.
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PMID:The role of His143 in the catalytic mechanism of Escherichia coli aspartate aminotransferase. 200 66

The mitochondrial (m-AAT) and the cytoplasmic (c-AAT) isoenzyme activities of the glutamate synthesizing enzyme aspartate aminotransferase have been localized in the rat retina on the ultrastructural level using enzyme histochemistry. Reaction product of c-AAT was found selectively in cone pedicles, in presynaptic terminals of a subpopulation of amacrine cells and of horizontal cell processes, which are connected to rods. Rod spherules, terminals of cone-related horizontal cells and of bipolar cells reacted negatively, as well as ganglion cells, nerve fibre layer and optic nerve, m-AAT reaction product was found in all neuronal structures, most densely in the photoreceptor inner segments. The localization of c-AAT activity is in accordance with its presumed meaning in the production of releasable glutamate.
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PMID:Cytochemical demonstration of aspartate aminotransferase activity in the rat retina. 205 2

Liver necrosis was produced in rats by administering 3 doses of a mixture of carbon tetrachloride + olive oil, 2 ml/kg, ip. The liver damage was evidenced by the elevated levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transpeptidase (gamma-GT) and by histopathological observations of liver sections. Aspartate and glutamate administration (100 mg/kg, ip) significantly reduced these elevated levels of AST, ALT, and gamma-GT. Carbon tetrachloride induced liver necrosis was also found to be significantly reduced in aspartate and glutamate pretreated animals as observed macroscopically and histologically.
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PMID:Effect of aspartate and glutamate on carbon tetrachloride induced liver damage in rats. 209 35

The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm). The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme. However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1. Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization.
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PMID:Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence. 210 17

In situ hybridization histochemistry (ISHH) using synthetic oligonucleotide probes has been used to identify cells containing the mRNAs coding for glutaminase (GluT), aspartate aminotransferase (AspT) and glutamic acid decarboxylase (GAD). The distribution of GAD mRNA confirms previous descriptions and matches the distribution of GAD detected using specific antibodies. AspT mRNA is widely distributed in the brain, but is present at high levels in GABAergic neuronal populations, some that may be glutamatergic, and in a subset of neurons which do not contain significant levels of either GAD or GluT mRNA. Particularly prominent are the neurons of the magnocellular division of the red nucleus, the large cells in the deep cerebellar nuclei and the vestibular nuclei and neurons of the lateral superior olivary nucleus. GluT mRNA does not appear to be present at high levels in all GAD-containing neurons, but is seen prominently in many neuronal populations that may use glutamate as a neurotransmitter, such as neocortical and hippocampal pyramidal cells, the granule cells of the cerebellum and neurons of the dentate gyrus of the hippocampus. The heaviest labelling of GluT mRNA is seen in the lateral reticular nucleus of the medulla. ISHH using probes directed against the mRNAs encoding these enzymes may be an important technique for identifying glutamate and aspartate using neuronal populations and for examining their regulation in a variety of experimental and pathological circumstances.
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PMID:Distribution of messenger RNAs encoding the enzymes glutaminase, aspartate aminotransferase and glutamic acid decarboxylase in rat brain. 216 7

The four half-transamination reactions [the pyridoxal form of Escherichia coli aspartate aminotransferase (AspAT) with aspartate or glutamate and the pyridoxamine form of the enzyme with oxalacetate or 2-oxoglutarate] were followed in a stopped-flow spectrometer by monitoring the absorbance change at either 333 or 358 nm. The reaction progress curves in all cases gave fits to a monophasic exponential process. Kinetic analyses of these reactions showed that each half-reaction is composed of the following three processes: (1) the rapid binding of an amino acid substrate to the pyridoxal form of the enzyme; (2) the rapid binding of the corresponding keto acid to the pyridoxamine form of the enzyme; (3) the rate-determining interconversion between the two complexes. This mechanism was supported by the findings that the equilibrium constants for half- and overall-transamination reactions and the steady-state kinetic constants (Km and kcat) agreed well with the predicted values on the basis of the above mechanism using pre-steady-state kinetic parameters. The significant primary kinetic isotope effect observed in the reaction with deuterated amino acid suggests that the withdrawal of the alpha-proton of the substrates is rate determining. The pyridoxal form of E. coli AspAT reacted with a variety of amino acids as substrates. The Gibbs free energy difference between the transition state and the unbound state (unbound enzyme plus free substrate), as calculated from the pre-steady-state kinetic parameters, showed a linear relationship with the accessible surface area of amino acid substrate bearing an uncharged side chain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pre-steady-state kinetics of Escherichia coli aspartate aminotransferase catalyzed reactions and thermodynamic aspects of its substrate specificity. 220 6

The enzyme aspartate aminotransferase was demonstrated cytochemically in the rat hippocampus 4, 7, and 14 days after unilateral entorhinal cortex lesion. At the light microscopic level the enzyme showed a significant activity decrease in the ipsilateral entorhinal terminal field which was similar at all postlesion times investigated. Non-denervated areas, i.e. the inner one-third of the dentate gyrus molecular layer and the radiatum layer of CA2/3, showed an increase of aminotransferase activities. At the electron microscopic level in the entorhinal terminal field of the control (unoperated) side aspartate aminotransferase was localized preferentially in a great number of boutons, containing the cytoplasmic and mitochondrial isoenzymes. Following entorhinal lesion a significant loss of these positively reacting boutons was seen. Most of the degenerating boutons contained reaction product but a small number was negative for aspartate aminotransferase. From 4 to 14 postlesion days the positively reacting boutons of the non-denervated supragranular zone expanded outward into the denervated area according to the known terminal proliferation of the commissural and associational systems. The remaining denervated entorhinal terminal field was reinnervated predominantly by negatively reacting boutons (probably terminal proliferations of septal afferents) and by a small number of positively reacting boutons (probably terminal proliferations of the crossed temporo-dentate pathway). The presence of cytoplasmic aspartate aminotransferase in the terminals of a well-known glutamatergic system is discussed in relation to the possible importance of this enzyme for the production of releasable glutamate.
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PMID:Glutamate producing aspartate aminotransferase in glutamatergic perforant path terminals of the rat hippocampus. Cytochemical and lesion studies. 222 38


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