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PubMed1981

Direct immunological determination of aspartate aminotransferase isoenzymes.

We examined the suitability of a rapid immunological technique to determine the amount of aspartate aminotransferase (EC 2.6.1.1) isoenzymes in human serum or tissue extracts. Purified isoenzymes were absorbed as a monolayer to the surface of an indium metal film on glass. The enzyme retains immunological reactivity, allowing the specific binding of aspartate aminotransferase antibodies at the surface. The amount of isoenzyme in a specimen is estimated from the competition for the antibody between the free isoenzyme in the specimen and that at the surface. The surface is further incubated with goat antibodies to rabbit IgG, and the extent of antibody binding is determined by densitometry. There is no cross reactivity between the cytoplasmic and mitochondrial forms, so these two isoenzymes can be determined simultaneously. The minimum detectable concentration by this technique is about 50 micrograms of enzyme protein per liter. The within-day coefficient of variation for determination of either isoenzyme was about 20%. Our results suggest that normal and patients' sera contain considerably more immunologically active than catalytically active isoenzymes.

Aspartate Aminotransferases↗
PubMed1986

Inhibition of aspartate aminotransferase by D-hydrazinosuccinate: comparison with L-hydrazinosuccinate.

L-Hydrazinosuccinate has been reported to be a slow- and tight-binding inhibitor of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) and to interact with the enzyme via a reaction of two consecutive steps. The present work examined the effects of D-hydrazinosuccinate on the same enzyme for comparison. D-Hydrazinosuccinate showed a potent inhibition in a slow-binding manner: transamination became slower with time when the reaction was initiated by the addition of enzyme to a mixture of the assay components and D-hydrazinosuccinate, while the reaction was initially very slow and became faster with time when the enzyme was preincubated with the inhibitor before the initiation of reaction. Analysis of the time-course of interaction of the enzyme with D-hydrazinosuccinate suggested a reversible single-step reaction mechanism and gave an inhibition constant of approx. 3 nM, in contrast to the two-step mechanism, and a much lower inhibition constant of 0.2 nM for L-hydrazinosuccinate. Comparison of the rate constants for the reaction steps in the interaction of the enzyme with D- and L-enantiomers confirmed that the difference in the reaction mechanism was mainly responsible for the stronger inhibition by the L-enantiomer. Spectral studies showed that D- and L-hydrazinosuccinate both produced complexes with the enzyme probably in the form of aldimine, and thereafter only the complex with L-hydrazinosuccinate further changed to another species more slowly, consistent with the two-step mechanism. The configuration of the hydrazino group is therefore crucial for the conversion of aldimine complexes to more tightly bound complexes.

Animals↗
PubMed1994

Isolation and properties of mitochondrial aspartate aminotransferase from red muscle of grey mullet, Mugil auratus Risso.

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.

Animals↗
PubMed1979

[Kinetics of the aspartate-aminotransferase reaction catalyzed by free and immobilized cells of E. coli].

The kinetics of the aspartate-aminotransferase reaction were studied, using free and immobilized cells of E. coli, strain 85 as an enzyme source. It was shown that the reaction is limited by mass transport of the reagents through the bacterial cell membrane even at high concentrations of the substrates in the surrounding solution. The polyacrylamide gel-incorporated cells of E. coli, strain 85 catalyze the aspartate-aminotransferase reaction more effectively as compared to free or destroyed cells. In the latter case the reaction is characterized by the following kinetic parameters: the effective values of the stationary rate of the product accumulation and its stationary efflux from the cell are equal to (15,37 +/- 0.4) . 10(-6) mole/s/mg of protein and (3,01 +/- 0,8) . 10(-20) mole/s per 1 cell. respectively. The steady-state constant for glutamate synthesis from aspartic acid is equal to 0,22--0,23.

Aspartate Aminotransferases↗
PubMed1975

The binding of 8-anilinonaphthalene-1-sulphonate to cytoplasmic aspartate aminotransferase from pig heart.

Anilinonaphthalenesulphonate binds to cytoplasmic aspartate aminotransferase with high affinity (Kd about 10 muM) and with a stoicheiometry of one molecule per dimer. It is not displaced by aliphatic or aromatic dicarboxylate substrate analogues. The enzyme is believed to be a symmetrical dimer with identical subunits; it can evidently function asymmetrically in binding anilinonaphthalenesulphonate.

Anilino Naphthalenesulfonates↗
PubMed1989

Evidence that 31P NMR is a sensitive indicator of small conformational changes in the coenzyme of aspartate aminotransferase.

The pH dependence of 31P-NMR spectra of pig cytosolic aspartate aminotransferase, containing either N-(5'-phosphopyridoxyl)-L-aspartate or pyridoxal 5'-deoxymethylenephosphonate in place of the normal coenzyme pyridoxal 5'-phosphate, has been analysed. The chemical shifts of phosphopyridoxylaspartate and of pyridoxal 5'-deoxymethylenephosphonate model Schiff base in free solution show pK values of 6.3 and 7.4, attributable to the second deprotonation step of phosphate and phosphonate, respectively. However, these compounds behave very differently when bound to apoaspartate aminotransferase. 31P-NMR spectra of these enzyme derivatives indicate that the phosph(on)ate group remains dianionic throughout the pH range 4-8.5. A clear correlation between apparent pK values obtained from spectrophotometric titration of the coenzyme chromophore and those obtained by 31P NMR indicates that the same ionisation is being reported by both methods. The data are interpreted, on the basis of available crystallographic structures of chicken mitochondrial aspartate aminotransferase, to indicate that in each case the alteration in 31P chemical shift results from a conformational change in the coenzyme 5' side chain, in which one of the structures involves a near-eclipsed pair of bonds. Such a stressed conformation produces slight alterations in bond angles around the phosphorus atom, which in turn cause the observed change in 31P chemical shift. The evidence is taken to indicate that in this case 31P NMR is a sensitive reporter of stress in enzyme-bound pyridoxal 5'-phosphate and its derivatives.

Animals↗
PubMed1975

Aspartate aminotransferase activity during early development of chicken embryo.

Aspartate aminotransferase (AAT) activity is studied, employing two different procedures, during early development stages of chicken embryo. ATT activity is steady from pre-streak to the definitive primitive streak stage after which it suddenly increases as growth proceeds. INH or IIH administration in this embryonic system led to almost instantaneous and complete disappearance of AAT activity which could be reversed to 80 percent by treatment with equimolar pyridoxal phosphate. Histochemical studies from the literature support a view that the period of intense differentiation coincides with an increased RNA content. The present study shows more AAT activity per mg wet embryo during the same development stages. Whether this is due to availability of more aspartic acid for pyrimidine is not clear due to possible presence of two AAT activities, the many competing reactions that can use aspartic acid, and in situ conditions during differentiation.

Animals↗
PubMed1986

Immunohistochemical localization of glutamate, glutaminase and aspartate aminotransferase in neurons of the pontine nuclei of the rat.

The pontine nuclei form the key relay nuclei in the cerebropontocerebellar pathway. Although a great deal of information is available regarding the anatomy of this region, the identity of the neurotransmitter(s) contained in the neurons of the pontine gray are not known. The aim of the present investigation is to utilize immunohistochemical techniques to determine whether glutamate, a putative excitatory transmitter, and the enzymes responsible for its metabolism, are found in pontine neurons. Both glutaminase, an enzyme which converts glutamine to glutamate, and aspartate aminotransferase, an enzyme which is involved in the interconversion between glutamate and aspartate, have been proposed to be markers of neurons which use excitatory amino acids as neurotransmitters. The present study utilizes a monoclonal antibody against carbodiimide-fixed glutamate and polyclonal antisera against glutaminase and aspartate aminotransferase in conjunction with the indirect peroxidase technique or the peroxidase-labeled biotin-avidin procedure to localize glutamatergic neurons in the pontine nuclei of the rat. Numerous neurons in all subdivisions of the pontine nuclei were found to contain carbodiimide-fixed glutamate-like immunoreactivity, glutaminase-like immunoreactivity or aspartate aminotransferase-like immunoreactivity. Horseradish peroxidase was injected into the cerebellum of four rats for use with a combined retrograde transport-immunohistochemical procedure. Double-labeled neurons were observed in all subdivisions of the pontine nuclei, indicating that pontine neurons which contain glutamate-like immunoreactivity project to the cerebellum. Based on the hypothesis that increased levels of glutamate, glutaminase and aspartate aminotransferase reflect a transmitter role for glutamate, the present data raise the possibility that glutamate may be a major neurotransmitter of pontocerebellar fibers.

Animals↗
PubMed1989

The unfolding and refolding of cytoplasmic aspartate aminotransferase from pig heart.

The unfolding of cytoplasmic aspartate aminotransferase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from protein fluorescence, c.d. and thiol-group reactivity indicated that the enzyme was unfolded in 6 M-GdnHCl. Spectroscopic studies showed that this unfolding was accompanied by dissociation of the pyridoxal 5'-phosphate cofactor. On dilution of the GdnHCl, re-activation of the enzyme occurred in reasonable yield, provided that dithiothreitol and pyridoxal 5'-phosphate were present. The regain of activity obeyed second-order kinetics. In the absence of added dithiothreitol and pyridoxal 5'-phosphate, substantial formation of high-Mr aggregates occurred.

Adenosine Diphosphate↗
PubMed1979

Generation of aspartate aminotransferase multiple forms by deamidation.

The development of aspartate aminotransferase subforms in vitro was followed by densitometry after thin-film isoelectric focusing. At the same time ammonia production was measured. Each reaction can be expressed in terms of a first-order process in which 2 mol of glutamine or asparagine/mol of dimer are deamidated with a half time of 22 days. The more negatively charged subforms developed in vitro were almost fully active. Another process occurred leading to inactivation by coenzyme modification, and this was independent of deamidation. Although the enzyme formed absorbed maximally at 340nm, it was different from the naturally occurring inactive enzyme that absorbs at this wavelength.

Ammonia↗
PubMed1982

Increase of inactive form of aspartate aminotransferase in pyridoxine-deficient rat liver.

Cytosolic aspartate aminotransferase from rat liver was separated into at least 3 subforms, focused at pH 6.2, 5.9, and 5.7, by isoelectric focusing. Increase of subforms with low pI values was observed in pyridoxine-deficient rat liver. These subforms with low pI values showed low catalytic activities relative to their antigenic activities. The Km values for substrate and optimal pH values of the two main subforms were not significantly different in pyridoxine-deficient and control rat livers. Some conformational change of enzyme in the cytosol of pyridoxine-deficient rat liver was suggested by circular dichroic and fluorescent spectra. The N and C terminals of the enzyme from both pyridoxine-deficient rats and controls were shown to be alanine and glutamine, respectively.

Animals↗
PubMed1991

The K258R mutant of aspartate aminotransferase stabilizes the quinonoid intermediate.

Lys-258 of aspartate aminotransferase forms a Schiff base with pyridoxal phosphate and is responsible for catalysis of the 1,3-prototropic shift central to the transamination reaction sequence. Substitution of arginine for Lys-258 stabilizes the otherwise elusive quinonoid intermediate, as assessed by the long wavelength absorption bands observed in the reactions of this mutant with several amino acid substrates. The external aldimine intermediate is not detectable during reactions of this mutant with amino acids, although the inhibitor alpha-methylaspartate does slowly and stably form this species. These results suggest that external aldimine formation is one of the rate-determining steps of the reaction. The pyridoxamine-5'-phosphate-like enzyme form (330-nm absorption maximum) is unreactive toward keto acid substrates, and the coenzyme bound to this species is not dissociable from the protein.

Amino Acid Sequence↗
PubMed1978

Effect of some--SH and other reagents on aspartate aminotransferase and L-alanine aminotransferase of Paramphistomum explanatum Fischoeder, 1901.

Studies on aspartate aminotransferase (GOT) and L-alanine aminotransferase (GPT) of Paramphistomum explanatum have shown that GPT activity has more than twice the activity of GOT. The effect os some--SH reagents like cadmium, mercury, silver and iodoacetamide revealed that both enzymes were inhibited except that GOT was insensitive to cadmium ions. GPT was found to be much more sensitive to--SH reagents than GOT. There was unusual reaction to the two thiols used, cysteine and mercaptoethanol. Cysteine inhibited both the enzymes and mercaptoethanol activated GPT and inhibited GOT. Thiols in combination with iodoacetamide showed that the strong inhibitory effect of cysteine on both enzymes was reduced by iodoacetamide, but with mercaptoethanol the inhibitory effect on GOT was greater than when either of them was used alone, while GPT the effect of either counteracted each other. EDTA activated both enzymes and partially protected mercury inhibition of both enzymes and silver inhibition GOT only. It provided no protection against silver inhibition of GPT but complete protection of GPT against total inhibition by cadmium ions.

Alanine Transaminase↗
PubMed1989

Immobilization of aspartate aminotransferase on agarose.

Various methods for immobilization of aspartate aminotransferase (AspAT; from cytosolic fraction of pig heart) on agarose were tested. Aldehyde-, thiol-, and CNBr-activated agaroses were studied in detail. The capacity of the aldehyde support to firmly bind protein was less than 0.2 mg/ml, whereas the apparent remaining specific activity of the bound AspAT was high (50-63% of soluble AspAT). The maximum capacity of SH-agarose to bind enzymatic protein was 3 mg/ml; the apparent remaining activity was 30-40%, and the specific activity determined by Vmax was 51%. Chemical coupling on to thiol-agarose did not denature the enzyme, as 93% of protein and 83% of the activity were recovered after release of the enzyme from the support. Enzyme protein was quantitatively bound to CNBr-activated agarose (up to 10 mg/ml of the gel). The apparent specific activities were 27-35%, while the value calculated from Vmax was 46%. Active site-protecting agents within the CNBr-coupling were tested. Bromphenol blue increased the apparent specific activity to 60% and Vmax to 80% at 3-fold molar concentration at the active sites. Kinetic constants for immobilized preparations were determined.

Aldehydes↗
PubMedDate not supplied

[Linear dichroism of chicken cytosol aspartate aminotransferase oriented in polyacrylamide gel].

Cytosolic chicken heart aspartate aminotransferase (EC 2.6.1.1) was incorporated in polyacrylamide gel and partially oriented by compressing the gel block in two mutually perpendicular directions. The linear dichroism (LD) was recorded in a dichrograph equipped with a quarter-wavelength device which transforms circularly polarized light into linearly polarized. Spectra were resolved with lognormal distribution curves. A marked difference has been found between reduced linear dichroism values (LD/A) in the absorption bands of the protonated (430 nm) and nonprotonated (360 nm) forms of the internal pyridoxal phosphate--lysine aldimine. This finding indicates that protonation of the internal aldimine bond induces a change in direction of the transition dipole moment within the coenzyme ring or reorientation of the ring. Formation of the external aldimine with 2-methylaspartate is accompanied by a decrease of the reduced LD value in the 430 nm band. On the other hand, binding of the dicarboxylate anions, which imitates formation of the noncovalent adsorption Michaelis complex, results in a marked increase of the reduced LD value in the 430 nm band. These data suggest that the coenzyme ring tilts in opposite directions upon noncovalent substrate binding and upon subsequent formation of the external aldimine.

Animals↗
PubMed1994

Genomic structure, expression and evolution of the alfalfa aspartate aminotransferase genes.

Genomic clones encoding two isozymes of aspartate aminotransferase (AAT) were isolated from an alfalfa genomic library and their DNA sequences were determined. The AAT1 gene contains 12 exons that encode a cytosolic protein expressed at similar levels in roots, stems and nodules. In nodules, the amount of AAT1 mRNA was similar at all stages of development, and was slightly reduced in nodules incapable of fixing nitrogen. The AAT1 mRNA is polyadenylated at multiple sites differing by more than 250 bp. The AAT2 gene contains 11 exons, with 5 introns located in positions identical to those found in animal AAT genes, and encodes a plastid-localized isozyme. The AAT2 mRNA is polyadenylated at a very limited range of sites. The transit peptide of AAT2 is encoded by the first two and part of the third exon. AAT2 mRNA is much more abundant in nodules than in other organs, and increases dramatically during the course of nodule development. Unlike AAT1, expression of AAT2 is significantly reduced in nodules incapable of fixing nitrogen. Phylogenetic analysis of deduced AAT proteins revealed 4 separate but related groups of AAT proteins; the animal cytosolic AATs, the plant cytosolic AATs, the plant plastid AATs, and the mitochondrial AATs.

Amino Acid Sequence↗