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PubMedDate not supplied

[Aspartate aminotransferase activity as dependent on the degree of serum dilution].

Dependence of the enzymatic reaction rate V. catalyzed by aspartate aminotransferase, on concentration of standard human blood serum Moni-trol I ("Dade", Switzerland) was studied. In the range of moderate dilutions, which did not exceed 1.3-times, the V value altered in accordance with the blood serum concentration C. Under conditions of extensive dilutions dependence of V on blood serum concentration became complicated. The non-linear type of V dependence on blood serum concentration appears to be due to deaggregation of the enzyme molecules with formation of more active fragments as a result of blood serum dilution. Two parameters should be used for characteristics of blood serum aspartate aminotransferase activity: the enzymatic activity of undiluted blood serum as well as the ratio between specific activities of the enzyme in an associated from and the products of its dissociation, i.e. when C-1 and C-0, respectively.

Aspartate Aminotransferases↗
PubMed2002

Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis.

Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.

Aspartate Aminotransferases↗
PubMed1987

Use of malate dehydrogenase immobilized on the dialyzer groove of the Autoanalyzer II for serum aspartate aminotransferase determination.

Malate dehydrogenase (EC 1.1.1.37) was immobilized on the lower groove of the dialyzer plate used for serum aspartate aminotransferase determination in the AutoAnalyzer II system. Immobilization was effected by covalently attaching malate dehydrogenase to the inner surface of the groove which was previously activated by treatment with glutaraldehyde at room temperature. The immobilized malate dehydrogenase catalyzed the reaction between oxaloacetate and NADH to form NAD in the coupled reaction originally proposed by Karmen. Results of the present method correlated well with those obtained by the Technicon SMA II system in which malate dehydrogenase is in solution (n = 99; r = 0.99; t = 0.30). The activity of immobilized malate dehydrogenase on the dialyzer groove was sufficient to measure serum aspartate aminotransferase for at least one month with continuous use. The stability of immobilized malate dehydrogenase was also dependent on the number of samples determined. The dialyzer plate is a reusable solid matrix for malate dehydrogenase immobilization. The expense of the present method is only half the cost of the method in which malate dehydrogenase is in solution.

Adolescent↗
PubMed1987

Stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate in aspartate aminotransferase. Activators and inhibitors.

Measurement of the stereospecific release of the pro-S proton from C-4' of enzyme-bound pyridoxamine 5'-phosphate provides an experimental means to probe parts of the active site of aspartate aminotransferase independently of substrate turnover (Tobler, H. P., Christen, P., and Gehring, H. (1986) J. Biol. Chem. 261, 7105-7108). The release of pro-S 3H from enzyme-bound [3H]pyridoxamine 5'-phosphate is 30,000 times faster than from free coenzyme. Enzyme-bound [3H]pyridoxine 5'-phosphate is not detritiated suggesting an essential role of the 4'-amino group. Formation of the unproductive complex of the [3H]pyridoxamine 5'-phosphate-enzyme with aspartate or glutamate results in a 400-fold acceleration of 3H release. In contrast, addition of borohydride or cyanoborohydride immediately stops 3H release. Experiments with a fluorescent reporter group and with differential chemical modifications indicate that the activating effect of aspartate on the release of 3H is accompanied by a shift of the so-called open/closed conformational equilibrium of the enzyme (Kirsch, J.F., Eichele, G., Ford, G. C., Vincent, M.G., Jansonius, J.N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525) toward the closed conformation; the inhibiting effect of borohydride and cyanoborohydride appears to be accompanied by a shift toward the open conformation. Apparently, at least part of the catalytic apparatus of aspartate aminotransferase becomes fully operative only in the closed conformation of the enzyme.

Animals↗
PubMed1977

Interspecies comparison of cytosolic and mitochondrial aspartate aminotransferases. Evidence for a more conservative evolution of the mitochondrial isoenzyme.

The degree of structural similarity between the mitochondrial isoenzymes of aspartate aminotransferase from pig heart and chicken heart was determined by means of their immunological cross-reactivity and compared with the degree of similarity between the cytosolic isoenzymes from the same two species. Quantitative microcomplement fixation revealed a remarkable similarity of the two mitochondrial isoenzymes corresponding to an immunological distance of 104. The structures of the two cytosolic isoenzymes, on the other hand, diverge with an immunological distance of 203. The apparent conservatism of mitochondrial aspartate aminotransferase indicates additional evolutionary constraints on the structure of this organelle-confined isoenzyme.

Animals↗
PubMed1994

[A new form of aspartate aminotransferase crystals].

A new crystal form of chicken cytosolic aspartate aminotransferase (EC 2.6.1.1) has been grown using a mixture of ammonium sulfate with ethanol as a precipitant. Crystals of the enzyme belong to the space group P 2(1)2(1)2(1) having the following unit cell dimensions: a = 62.38 A, b = 117.41 A, c = 124.34 A. There is one molecule of the enzyme in the asymmetric unit. The crystals diffract at 1.8 A resolution.

Animals↗
PubMed1995

[Aspartate aminotransferase of the human cerebellar cortex].

The distribution of aspartate aminotransferase (AST) was studied cytochemically in the cerebellar cortex of the man. Granular cell bodies were labeled, but staining was also found in mossy fibre glomerular synapses. In the molecular and Purkinje cell layers, intensive histochemical reaction was concentrated in the climbing fibres and the "basket" terminal plexus around the Purkinje cells pericaryon. Climbing and mossy fibres formed asymmetric synaptic contacts. Localization of AST in neuronal structures of the human cerebellar cortex suggests to consider these as aspartatergic.

Adolescent↗
PubMed1998

Crystallization and preliminary X-ray characterization of aspartate aminotransferase from an extreme thermophile, Thermus thermophilus HB8.

Recombinant aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8, has been crystallized in two different crystal forms. The crystals of both forms are orthorhombic and belong to space group P212121 with cell dimensions a = 124.3, b = 113.6 and c = 61.6 A for form I and a = 197.3, b = 109.7 and c = 80.3 A for form II. The crystals of form I and II diffract to 2.1 and 2.5 A resolution, respectively, on a conventional laboratory rotating-anode source. Two heavy-atom derivatives have been identified for form I.

Aspartate Aminotransferases↗
PubMedDate not supplied

[Alanine and aspartate aminotransferase activity in the grass carp oocytes, ovarian and perivitelline fluids].

The alanine aminotransferase activity in the ovarian fluid is much higher than in the mature oocytes and perivitelline fluid, the aspartate aminotransferase activity is higher in the perivitelline fluid than in the oocytes and ovarian fluid. The aspartate aminotransferase activity prevails in the mature oocytes and perivitelline fluid. The temperature optimum of the both activities is 38-44 degrees C, the pH optimum is between 7.5-7.6.

Alanine Transaminase↗
PubMed1988

Development of aspartate aminotransferase and glutaminase immunoreactivity in the rat auditory nerve.

Aspartate aminotransferase and glutaminase immunoreactive labeling of the auditory nerve has previously been reported. In the present study, the development of these immunoreactivities was examined in the auditory nerve of the rat, at ages ranging from 17 days gestation to four postnatal weeks. Cells and processes were examined in the cochlea, and fibers and terminals in the cochlear nucleus. In the cochlea, immunoreactive labeling with antisera to both enzymes was first seen at 20 gestational days, in spiral ganglion cells. It was not until two postnatal weeks, however, that this immunoreactive labeling was first seen in primary afferent terminals around spherical cells in the anteroventral cochlear nucleus. This correlates with the establishment of mature synaptic connections and function.

Aging↗
PubMed1981

Reactivity of sulphydryl groups of cytosolic and mitochondrial bovine aspartate aminotransferases.

Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs2 while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.

Animals↗
PubMed2001

Aspartate aminotransferase activity in human healthy and inflamed dental pulps.

Aspartate aminotransferase (AST) seems to be an important mediator of inflammatory processes. Its role in the progression and detection of inflammatory periodontal disease has been increasingly recognized in recent years. In the present study AST activity was analyzed in normal healthy human dental pulps, in reversible pulpitis, and in irreversible pulpitis. Enzymatic AST activity showed that the control values for the healthy pulps were 4.8 +/- 0.7 units/mg of pulp tissue. In reversible pulpitis specimens the AST activity increased to 7.98 +/- 2.1 units/mg of pulp tissue. In irreversible pulpitis specimens the values decreased to 2.28 +/- 1.7 units/mg of pulp tissue. Differences between the groups (control versus reversible pulpitis and reversible pulpitis versus irreversible pulpitis) were statistically significant (p = 0.0015). These results could point to a role of AST in the early events that lead to development of pulpal inflammation.

Acute Disease↗
PubMed1998

Directed evolution of an aspartate aminotransferase with new substrate specificities.

The substrate specificity of aspartate aminotransferase was successfully modified by directed molecular evolution using a combination of DNA shuffling and selection in an auxotrophic Escherichia coli strain. After five rounds of selection, one of the evolved mutants showed a 10(5)-fold increase in the catalytic efficiency (kcat/Km) for beta-branched amino and 2-oxo acids and a 30-fold decrease in that for the native substrates compared with the wild-type enzyme. The mutant had 13 amino acid substitutions, 6 of which contributed 80-90% to the total effect. Five of these six substitutions were conserved among the five mutants that showed the highest activity for beta-branched substrates. Interestingly, only one of the six functionally important residues is located within a distance of direct interaction with the substrate, supporting the idea that rational design of the substrate specificity of an enzyme is very difficult. The present results show that directed molecular evolution is a powerful technique for enzyme redesign if an adequate selection system is applied.

Amino Acid Sequence↗
PubMed1978

Aspartate aminotransferase activity and isoenzyme proportions in human liver tissues.

Aspartate aminotransferase (EC 2.6.1.1) activity and the distribution of its isoenzymes in human liver were examined. Rabbit antiserum against porcin soluble (i.e., non-mitochondrial) enzyme cross-reacted with the soluble enzyme of human origin and was used in an immunoprecipitation assay to quantitate the soluble and mitochondrial isoenzymes. These were separated by rapid, semiquantitative electrophoresis on cellulose acetate and by three other quantitative techniques: isoelectric focusing and anion-and cation-exchange chromatography. The mitochrondrial enzyme averaged 81% of the total activity in normal adult human liver (n = 4). Its contribution was dramatically reduced in single specimens of human fetal liver (56% of total activity) and hepatoblastoma tissue (38%). Total enzyme activities (mumol min-1 per gram of tissue) were: adult, 150; fetal, 38; tumor, 6. Total enzyme concentrations (micromoles of enzyme per kilogram of tissue) found were: adult, 10.8; fetal, 2.7; tumor, 0.4. The concentrations and isoenzyme distribution in human liver are compared to those in various animal model systems. Other methods for quantitative estimation of the isoenzymes and their adaptability for use in estimating concentrations in serum are reviewed.

Adolescent↗
PubMed1980

Uterine and kidney aspartate aminotransferase activity in ethinylestradiol- and norgestrel-treated rats.

Aspartate aminotransferase (AST) activity was measured in the kidneys and uteri of mature female rats to determine what effect, if any, treatment with ethinylestradiol (EE), norgestrel (NOR) or a combination of both hormones might have on the relationship of pyridoxal-PO4 to AST activity. Treatment with EE had no effect on the degree of saturation of kidney AST with pyridoxal-PO4, AST activity/mg kidney or total kidney AST activity compared to controls. Kidney weight was, however, significantly elevated (P < 0.05). The same AST parameters were elevated (P < 0.05) in rats treated with NOR or EE + NOR. Saturation of AST by pyridoxal-PO4 was not altered by hormone treatment in uteri, but AST activity/mg uterus and total uterine activity were lowered by NOR or EE + NOR treatment. Exposure to EE alone elevated these last two parameters. It is noteworthy that the same hormone can both stimulate or inhibit AST, depending on the organ in question. Since progestin-dominant contraceptives are becoming more commonplace, it is important to observe that the progestin was more effective in altering AST parameters than was the estrogen.

Animals↗
PubMed1994

Determination of aspartate aminotransferase activity by high-performance liquid chromatography.

A sensitive and reproducible assay of aspartate aminotransferase activity based on UV detection of the reaction products after their separation by HPLC is described. The main advantage is the direct measurement of the enzyme activity as micromoles of product (glutamate) formed within a known period of time without any coupled reaction. Further, with the chromatographic method, all components of the reaction mixture are identified, allowing the reaction course to be controlled and the possible presence of side-reactions to be monitored.

Animals↗
PubMed1987

Structural organization of the mouse mitochondrial aspartate aminotransferase gene.

Structural organization of the entire mouse mitochondrial aspartate aminotransferase (EC 2.6.1.1) gene was determined by analyzing the overlapping genomic clones obtained from a Charon 4A DNA library. The gene is 25 X 10(3) base-pairs long and contains ten exons interrupted by nine introns of various sizes. The 5' and 3'-flanking regions, the exact sizes and boundaries of the exon blocks including the transcription-initiation sites were determined. The 5' end of the gene lacks the prototypical 5' transcriptional regulatory sequence elements, such as TATA and CAAT boxes, but contains G + C-rich sequences, two putative binding sites for a cellular transcription factor, Sp1, and multiple transcription-initiation sites. Moreover, the sequences around the transcription-initiation sites are compatible with the formation of a number of potentially stable stem-loop structures. The leader sequence, which is essential for the transport of the protein into the mitochondria, is coded by the first exon and is separated from the mature protein by the first intron. The pyridoxal 5'-phosphate-binding domain, consisting of seven alternating beta-sheets and alpha-helical polypeptide strands, is separated by four introns present at the ends of alpha-helices. These genomic DNA structures suggest that the introns were not inserted into a previously uninterrupted coding sequence, but rather are products of evolution of the ancestral gene. However, a further correlation between the positions of introns relative to the well-defined structural domains of the mature protein was not obvious.

Animals↗