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At least 667 records · Page 37Linked to original sources
PubMed1989

Glutamate dehydrogenase and aspartate aminotransferase in leukocytes of patients with motor neuron disease.

We report the results of glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) activities in leukocytes of 24 patients with motor neuron disease (MND) and 20 matched control subjects. In 62.5% of patients suffering from MND, we detected a leukocyte GDH deficiency (+/- 2 SD) as compared with the mean value obtained in controls. By contrast, there was no difference in leukocyte AAT activities in affected and nonaffected subjects. Abnormal cellular glutamate metabolism might be involved in the pathogenesis of MND.

Adult↗
PubMed1991

Structural and functional role of the amino-terminal region of porcine cytosolic aspartate aminotransferase. Catalytic and structural properties of enzyme derivatives truncated on the amino-terminal side.

In porcine cytosolic aspartate aminotransferase, a dimeric enzyme, the amino-terminal region anchoring onto the neighboring subunit is linked to the adjoining floppy peptide segment (residues 12-47), an integral part of the small domain whose facile movement upon substrate binding is a striking "induced fit" feature of this enzyme. To assess the contribution by the amino-terminal region to small domain movement and protein stability, a series of enzyme derivatives truncated on the amino-terminal side (residues 1-9) was prepared by using oligonucleotide-directed in vitro mutagenesis. Deletion of residues 1-3 showed no effect on catalytic activity and heat stability. Del 1-5 mutant enzyme with an extra methionine at position 5 showed only 43% of the kappa cat value (in the overall transamination) of the wild-type enzyme. Further deletion up to residue 9 resulted in a slight decrease in kappa cat values. Del 1-9 mutant enzyme still retained a kappa cat value of 33% that of wild-type enzyme. Km values for aspartate and 2-oxoglutarate increased sharply upon deletion of residues 1-9. Accordingly, Del 1-9 mutant enzyme showed a striking decrease in the kappa cat/Km value, to only 2% of that for the wild-type enzyme. Deletion of amino-terminal residues 1-9 resulted also in a large decrease in thermostability and in an enhanced susceptibility to limited proteolysis by protease 401, which is known to cleave at Leu20 of the wild-type enzyme. These findings indicate that an increase in the conformational freedom of the floppy segment (residues 12-47) would occur upon the loss of most of the anchorage region, thereby presenting an entropic barrier to conformational changes that facilitate substrate binding with high affinity.

Amino Acid Sequence↗
PubMed1995

Multienzyme control serum (Seraclear-HE) containing human enzymes from established cell lines and other sources. 2: Evaluation as candidate working enzyme Reference Material for alanine and aspartate aminotransferases.

Seraclear-HE, containing seven enzyme analytes from human sources, was evaluated as an intermethod calibrator for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) to transfer Reference Method values to seven routine methods, including one based on hydrogen peroxide detection for possible unification of values (interlaboratory comparability of data). The commutabilities of AST from erythrocytes and ALT from a hepatoma cell line were studied between the consensus methods of Japan Society of Clinical Chemistry (chosen as the Reference Methods) and each of the automated routine methods at reaction temperatures of 30 degrees C and 37 degrees C. For AST, calibration of patients' sera with Seraclear-HE decreased average intermethod variation (CV) from 12% to 2%; for ALT, the decrease was from 20% to 3%. For both enzymes, Seraclear-HE was judged to be commutable between the Reference Methods and each of the methods investigated. The limitations for such use are discussed.

Alanine Transaminase↗
PubMed2000

Aspartate aminotransferase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125. Cloning, expression, properties, and molecular modelling.

The gene encoding aspartate aminotransferase from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 was cloned, sequenced and overexpressed in Escherichia coli. The recombinant protein (PhAspAT) was characterized both at the structural and functional level in comparison with the E. coli enzyme (EcAspAT), which is the most closely related (52% sequence identity) bacterial counterpart. PhAspAT is rapidly inactivated at 50 degrees C (half-life = 6.8 min), whereas at this temperature EcAspAT is stable for at least 3 h. The optimal temperature for PhAspAT activity is approximately 64 degrees C, which is some 11 degrees C below that of EcAspAT. The protein thermal stability was investigated by following changes in both tryptophan fluorescence and amide ellipticity; this clearly suggested that a first structural transition occurs at approximately 50 degrees C for PhAspAT. These results agree with the expected thermolability of a psychrophilic enzyme, although the observed stability is much higher than generally found for enzymes isolated from cold-loving organisms. Furthermore, in contrast with the higher efficiency exhibited by several extracellular psychrophilic enzymes, both kcat and kcat/Km of PhAspAT are significantly lower than those of EcAspAT over the whole temperature range. This behaviour possibly suggests that the adaptation of this class of endocellular enzymes to a cold environment may have only made them less stable and not more efficient. The affinity of PhAspAT for both amino-acid and 2-oxo-acid substrates decreases with increasing temperature. However, binding of maleate and 2-methyl-L-aspartate, which both inhibit the initial steps of catalysis, does not change over the temperature range tested. Therefore, the observed temperature effect may occur at any of the steps of the catalytic mechanism after the formation of the external aldimine. A molecular model of PhAspAT was constructed on the basis of sequence homology with other AspATs. Interestingly, it shows no insertion or extension of loops, but some cavities and a decrease in side chain packing can be observed.

Amino Acid Sequence↗
PubMed1984

Gamma-glutamyltranspeptidase, aspartate aminotransferase, and erythrocyte mean corpuscular volume as indicators of alcohol consumption in liver disease.

Gamma-glutamyltransferase (GGT), aspartate aminotransferase (ASAT), and erythrocyte mean corpuscular volume (MCV) were used as indicators of continued alcohol abuse in 178 patients with known liver disease studied for 5-49 months after diagnosis. Abstension was checked by interviews and blood alcohol determinations. Persistent alcoholics had significantly higher values for all three tests than abstainers. At values above upper normal limits, GGT was the most sensitive test, but the least specific. A positive predictive value of 80% for continued alcohol consumption was chosen, and the corresponding discriminating levels of the tests were compared. With this specificity GGT was the least sensitive test, and it did not add to the efficiency of ASAT or MCV when the tests were combined. Abstinence could not be predicted by low levels of the tests. It is concluded that these biochemical tests are valuable as indicators of alcohol consumption in patients with liver disease and that ASAT and MCV are equally efficient, whereas GGT is inferior to these.

Adult↗
PubMed1992

Prolactin stimulates transcription of aspartate aminotransferase in prostate cells.

Prolactin (PRL) has been reported to stimulate citrate production and the activity of mitochondrial aspartate aminotransferase (mAAT) and its precursor form pmAAT in prostate epithelial cells. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused the same result as PRL, which suggests that the PRL effect on mAAT activity might be mediated by protein kinase C (PKC) stimulation of pmAAT gene transcription. Both PRL and TPA increased the level of pmAAT mRNA by 2.5- to 3-fold in pig prostate cells. The PKC inhibitor gossypol completely inhibited the PRL and TPA induced increases. In addition, the effects of both PRL and TPA were inhibited by down-regulation of prostate PKC. Nuclear run-off assays indicated that PRL and TPA induction of pmAAT occurred primarily at the transcriptional level. The stimulation of pmAAT transcription by TPA suggests that the pmAAT gene contains a TPA response element. Thus, these results are consistent with our previous observation that PRL directly induces pmAAT and that the mechanism of this PRL effect might involve stimulation of PKC.

Animals↗
PubMed1988

Kinetic studies on the binding of gostatin, a suicide substrate for aspartate aminotransferase, with the isoenzymes from porcine heart mitochondria and cytosol.

The reaction of pig heart mitochondrial and cytosolic aspartate aminotransferases (abbreviated to mAspAT and cAspAT, respectively) with an enzyme-suicide substrate (mechanism-based inhibitor), gostatin (5-amino-2-carboxyl-4-oxo-1,4,5,6-tetrahydropyridine-3-acetic acid) was studied kinetically, by following the spectral change with a micro-stopped-flow apparatus, as well as the inactivation of the enzyme activity. No significant difference in kinetic behavior was observed between mAspAT and cAspAT. From the analysis of time-dependent spectral change, no positive evidence for the existence of spectrophotometrically distinguishable intermediates was obtained. Both the spectral change and the inactivation followed, at least in appearance, simple bimolecular association kinetics, under the conditions studied. However, the second-order rate constant of the spectral change was found to be 1.5 to 2 times as large as that of the inactivation. The effects of pH and temperature on k(on) (the second-order rate constant of the spectral change) were also studied.

Amino Acids, Dicarboxylic↗
PubMedDate not supplied

Serum activity of mitochondrial aspartate aminotransferase: a sensitive marker of alcoholism with or without alcoholic hepatitis.

Serum activity of the mitochondrial isoenzyme of aspartate aminotransferase (mAST) was measured with an immunological method in 74 subjects. Fourty-six were chronic alcoholics with (30) or without (16) obvious alcoholic liver disease; 28 were nonalcoholic controls among whom 14 had acute or chronic viral hepatitis, the remaining 14 being healthy individuals. Mean mAST activity was much higher in all the alcoholic subjects, with or without liver disease, 10.4 and 1.95 units per liter, respectively, than in the healthy controls (0.43, p less than 0.001). The mean mAST to total AST ratio was similar in the healthy controls and in the patients with viral hepatitis (2.98 and 3.19%, NS), whereas it was about 4 times higher in the alcoholics with a sensitivity which reached 93% in the patients with alcoholic liver disease and 100% in those without. Both gamma-glutamyl transpeptidase and glutamate dehydrogenase serum activities were far less sensitive and specific. As almost all chronic alcoholics had similar abnormal values of mAST/total AST ratio, this leads to question whether "normal" liver may really exist in any of such subjects.

Adult↗
PubMed2003

Molecular cloning of the cDNA encoding aspartate aminotransferase from bean root nodules and determination of its role in nodule nitrogen metabolism.

A cDNA clone encoding aspartate aminotransferase (PVAAT-2) (EC 2.6.1.1) was isolated from the common bean Phaseolus vulgaris nodule cDNA library. The nucleotide sequence analysis of the full-length cDNA allowed its identification by comparison with sequence databases. The amino acid sequence of the bean PvAAT-2 showed high similarity with the AAT-2 isoforms described in other leguminous plants. The amino-terminal region of the PvAAT-2 contains a sequence, which shares common features of plastid transit peptides. Southern blot analysis showed that the PvAAT-2 clone is encoded by a single gene in the P. vulgaris genome. Analysis of the PvAAT-2 mRNA levels suggests that the expression of this gene is nodule enhanced. The PvAAT-2 transcript is more abundant in nodules with increased synthesis of amides and is down-regulated in conditions where ureides accumulate. When plants were supplemented with ureides or with amides, PvAAT-2 expression was reduced, while it was not affected when plants were treated with allopurinol, an inhibitor of ureide synthesis. On the other hand, the expression of asparagine synthetase (another enzyme involved in the synthesis of amides) is not affected either by ureides or amides. These data suggest a role for AAT-2 in the mechanism involved in the synthesis of nitrogen compounds in bean nodules.

Acetylene↗
PubMed1977

Active-site labeling of aspartate aminotransferases by the beta,gamma-unsaturated amino acid vinylglycine.

The pyridoxal form of both cytosolic and mitochondrial aspartate aminotransferase is irreversibly inactivated consequent to its interaction with the beta,gamma-unsaturated substrate analogue vinylglycine. Per catalytic cycle, 90% of the enzyme molecules are inactivated while 10% escape inactivation by transamination to the pyridoxamine form. In the presence of vinylglycine plus 2-oxoglutarate, inactivation is complete because of retransamination of the pyridoxamine form to the susceptible pyridoxal form. Peptide analyses after inactivation with [1-14C]vinylglycine showed that vinylglycine alkylates the active-site lysine residue 258 which forms the internal aldimine with the coenzyme pyridoxal 5'-phosphate. The coenzyme itself is left intact; resolution of the inactivated enzyme by base or trichloroacetic acid yields pyridoxal-5'-P. The absorption spectrum of the inactivated enzyme (lambdamax 335 nm) suggests that the cofactor is bound as a substituted aldimine. The proposed pathway of alkylation of Lys-258 involves abstraction of the alpha proton from vinylglycine, isomerization to the alpha,beta-unsaturated enamine, and subsequent nucleophilic attack of the epsilon-amino group of the lysyl residue at the beta carbon of the inhibitor. The determination of the amino acid sequence around the coenzyme-binding lysyl residue in the mitochondrial isoenzyme from chicken gave Ala-(epsilon-Pxy)Lys-Asn-Met-(Gly,Leu,Tyr) which is identical with the other mitochondrial transaminases examined so far.

Amino Acid Sequence↗
PubMed2003

[Effect of smoking on the periodontium and the level of aspartate aminotransferase in gingival cervical fluid].

OBJECTIVE: To study the effect of smoking on the periodontium and level of aspartate aminotransferase in gingival cervical fluid (GCF-AST). METHODS: A proportion matched case-control filtration was performed in examined population, college students, outpatients from 1999 May to 2001 March 115 smokers aged from 23 to 65 years (102 men and 13 women) and 90 non-smokers aged from 25 to 70 years (80 men and 10 women) were chosen. Debris index (DI), calculus index (CI), periodontal disease index (PDI), GCF-AST were measured. RESULTS: No obvious differences were observed in DI in smokers (0 degrees, 27.2%; 5 degrees, 5.0%) and non-smokers (0 degrees, 27.8%; 5 degrees, 4.2%),whereas obvious differences were found in CI in smokers and non-smokers. The percentages of patients without calculus were lower in smokers (9.8%) than in non-smokers (20.0%). The percentages of patients with weighty calculus were higher in smokers (25.4%) than in non-smokers (12.8%). The PDI values in smokers were higher than in non-smokers. The percentages of their normal periodontium were lower in smokers (9.6%) than in non-smokers (20.8%). The percentages of their periodontitis were higher in smokers (38.5%) than in non-smokers (25.8%). The smoking quantity were positively related to periodontitis (P < 0.001). No obvious differences were found in the level of GCF-AST with same PDI (P > 0.05). CONCLUSION: Smoking is considered as one of the risk factors in the prevalence of periodontal disease, but may not have any direct effect on GCF-AST.

Adult↗
PubMed1986

Cryoenzymological study of aspartate aminotransferase. Detection of intermediates by monitoring single turnovers with a true substrate.

The mechanism of action of mitochondrial aspartate aminotransferase has been investigated by cryoenzymological methods. For the first time a single half-reaction of enzymic transamination with a fast-reacting natural substrate could be monitored. The cryosolvent (50% methanol) did not affect the kinetic parameters for the overall reaction at 4 degrees C with cysteine sulfinate and oxaloacetate as substrates. The Km value for cysteine sulfinate at -44 degrees C, as determined from single-turnover experiments, was only slightly higher than that at 4 degrees C with and without cryosolvent. The kcat values obtained from analysis of the overall reaction at 4 degrees C to -33 degrees C give a linear Arrhenius plot (Ea = 87 kJ mol-1), which extrapolates to the kcat value estimated from single-turnover experiments at -44 degrees C. Apparently no change in the reaction path occurs over this large temperature range. On mixing pyridoxal enzyme and cysteine sulfinate at -44 degrees C, an intermediate absorbing at 430 nm was observed, which decayed in a biphasic process and most probably reflects the external aldimine. Under all conditions tested a build-up of a quninonoid intermediate was not observed, indicating that the protonation at C4' of the coenzyme is far from being rate-limiting and/or the equilibrium favors strongly the aldimine. The initial decay rate of the 430-nm intermediate indicates that this step might be partly rate-determining. However, the slower turnover rate as well as the shapes of intermediate spectra suggests another step, most likely the hydrolysis of the ketimine, to be actually rate-limiting.

Aspartate Aminotransferases↗
PubMed1984

Inhibition of aspartate aminotransferase by hydrazinosuccinate.

DL-Hydrazinosuccinic acid was synthesized by the reaction of DL-bromosuccinic acid with hydrazine. The compound strongly inhibited aspartate aminotransferase and gave 50% inhibition at 1.3 microM when added simultaneously with L-aspartate to an assay mixture containing enzyme. Incubation of the enzyme with the compound prior to assay resulted in a much stronger inhibition, which proceeded time-dependently. The inhibition was protectable with L-aspartate and was substantially reversed by dialysis.

Animals↗
PubMed1998

Conformation of aspartate aminotransferase isozymes folding under different conditions probed by limited proteolysis.

The partially homologous mitochondrial (mAAT) and cytosolic (cAAT) aspartate aminotransferase have nearly identical three-dimensional structures but differ in their folding rates in cell-free extracts and in their affinity for binding to molecular chaperones. In its native state, each isozyme is protease-resistant. Using limited proteolysis as an index of their conformational states, we have characterized these proteins (a) during the early stages of spontaneous refolding; (b) as species trapped in stable complexes with the chaperonin GroEL; or (c) as newly translated polypeptides in cell-free extracts. Treatment of the refolding proteins with trypsin generates reproducible patterns of large proteolytic fragments that are consistent with the formation of defined folding domains soon after initiating refolding. Binding to GroEL affords considerable protection to both isozymes against proteolysis. The tryptic fragments are similar in size for both isozymes, suggesting a common distribution of compact and flexible regions in their folding intermediates. cAAT synthesized in cell-free extracts becomes protease-resistant almost instantaneously, whereas trypsin digestion of the mAAT translation product produces a pattern of fragments qualitatively akin to that observed with the protein refolding in buffer. Analysis of the large tryptic peptides obtained with the GroEL-bound proteins reveals that the cleavage sites are located in analogous regions of the N-terminal portion of each isozyme. These results suggest that (a) binding to GroEL does not cause unfolding of AAT, at least to an extent detectable by proteolysis; (b) the compact folding domains identified in AAT bound to GroEL (or in mAAT fresh translation product) are already present at the early stages of refolding of the proteins in buffer alone; and (c) the two isozymes seem to bind in a similar fashion to GroEL, with the more compact C-terminal portion completely protected and the more flexible N-terminal first 100 residues still partially accessible to proteolysis.

Aspartate Aminotransferases↗
PubMed1990

Light microscope study of the enzymatic activities of aspartate aminotransferase, GABA transaminase and acetylcholinesterase in the mesencephalic trigeminal nucleus neural complex of Mauremys caspica.

The enzymatic activities of aspartate aminotransferase, GABA-transaminase and acetylcholinesterase were studied by means of histochemical methods in the mesencephalic trigeminal nucleus (MTN) neural complex of the turtle Mauremys caspica. Light microscope observations have demonstrated that MTN neurons have a positive reaction for these enzymes.

4-Aminobutyrate Transaminase↗
PubMed1987

Distributions of the activities of aspartate aminotransferase isoenzymes in rat retinal layers.

Distributions of the activities of the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase were determined in rat retinal layers. cAAT was highest in the photoreceptor inner segments, inner nuclear layer and inner plexiform layer; mAAT was highest in the inner segments. The high activity in the inner segments indicates that both isoenzymes are involved in energy metabolism in addition to a possible role in neurotransmission.

Animals↗
PubMed1986

Inactivation of cytosolic aspartate aminotransferase accompanying modification of Trp 48 by N-bromosuccinimide.

Reaction of N-bromosuccinimide with pig heart cytosolic aspartate aminotransferase led to loss of the enzymatic activity. Chemical analysis indicated the modification of two tryptophan residues. At a low ratio of N-bromosuccinimide to enzyme, oxidation of Trp 122 occurred without affecting the enzymatic activity. Increase in the ratio resulted in the oxidation of Trp 48 with a concomitant decrease in enzyme activity. The modified enzyme did not react with substrates and their analogs. Trp 48 is not within the active site but in the hinge region linking the large domain of the enzyme to the small domain that shows dynamic movement upon binding substrates. The present result suggests that oxidation of Trp 48 may impair the structural integrity of the interdomain interface.

Animals↗
PubMed1990

Purification of rat liver mitochondrial aspartate aminotransferase and separation of its isoforms utilizing high-performance liquid chromatography.

A rapid method for purification of mitochondrial aspartate aminotransferase from rat liver employing high-performance liquid chromatography is reported. The product is purified 80-fold with a recovery greater than or equal to 50% in a single day. The amino acid composition, N-terminal amino acid sequence, specific activity, and spectral characteristics of the isolated enzyme are similar to those previously reported for this protein. The protein is homogeneous by standard electrophoretic and chromatographic criteria, but can be resolved into at least five isoforms by a carboxymethylated resin column using high-performance liquid chromatography. The principal isoform initially isolated is converted into two additional isoforms with lower specific activity upon storage at 4 degrees C.

Amino Acid Sequence↗