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PubMed1987

Intramitochondrial location and some characteristics of chicken liver aspartate aminotransferase.

Chicken liver mitochondrial aspartate aminotransferase was found to be located in the intermembrane space and bound to the inner mitochondrial membrane. Purification of two mitochondrial fractions containing aspartate aminotransferase activity was performed. Both fractions showed similar chromatographic behaviour and identical isoelectric point and molecular weight values. There were no significant differences in the general kinetic mechanism, Km values, substrates inhibition and effect of various anions on the activity of mitochondrial aspartate aminotransferase purified from both fractions.

Animals↗
PubMed1993

Escherichia coli aromatic amino acid aminotransferase: characterization and comparison with aspartate aminotransferase.

Aromatic amino acid aminotransferase (ArAT) from Escherichia coli was overexpressed in E. coli cells, purified, and characterized. The enzyme was similar to aspartate aminotransferase (AspAT) of E. coli in many aspects, such as gross protein structure and spectroscopic properties. The reactions of pyridoxal 5'-phosphate-form ArAT with amino acids and pyridoxamine 5'-phosphate-form ArAT with oxo acids were investigated using stopped-flow spectrophotometric techniques. The kinetic parameters for these "half" reactions could excellently explain the ArAT-catalyzed overall transamination reactions at pH 8.0. Reactions of ArAT with aspartate and tryptophan which had been deuterated at position 2 showed isotope effects of 2.5 and 6.0 in the kcat values of the half-reactions, showing that the proton-transfer step is at least partially rate-limiting for these reactions. ArAT and AspAT showed overlapping substrate specificity. Both ArAT and AspAT were active toward dicarboxylic substrates. ArAT showed, however, 10(3)-fold higher activity toward aromatic substrates than AspAT. This high activity toward aromatic substrates was in part ascribed to the active site hydrophobicity of ArAT, which was suggested to be about 1.4 times as large as that of AspAT. In addition to dicarboxylic substrate analogs, aromatic substrate analogs such as carboxylic acids, 2-methyl amino acids, and 3-hydroxy amino acids caused characteristic changes in the absorption spectra of ArAT, while these aromatic analogs did not significantly change the spectra of AspAT. In particular, the erythro-3-hydroxy analogs of phenylalanine and aspartate caused a prominent absorption of ArAT at around 500 nm, which is generally ascribed to the accumulation of quinonoid intermediates. The threo forms of these 3-hydroxy analogs acted as substrates for ArAT. The erythro and threo forms of 3-hydroxyaspartate reacted with AspAT similarly as they reacted with ArAT; however, both forms of 3-phenylserine were poor substrates for AspAT, although phenylalanine was a fairly good substrate for AspAT. The observations on the two erythro-3-hydroxy amino acids show the similar orientation of these analogs in the active site of ArAT, probably through a hydrogen-bonding network involving the hydroxy groups of the analogs and Tyr70, and suggest that the aromatic binding pocket is near or even overlaps the side-chain-carboxylate-binding site for dicarboxylic substrates.

Amino Acid Sequence↗
PubMed1997

Stability of aspartate aminotransferase from Sulfolobus solfataricus.

Aspartate aminotransferase from Sulfolobus solfataricus (SsAspAT) is an extremely thermophilic and thermostable dimeric enzyme which retains its structure and reaches maximal activity at 100 degrees C. The structural stability of this protein was investigated by coupling isothermally and thermally induced denaturation studies to molecular modeling. Gel filtration analysis indicated that SsAspAT unfolds with an N2 reversible 2D mechanism. In the molecular model, a cluster of hydrophobic residues was shown at the interface between the subunits of SsAspAT and suggested this cluster as a structural feature stabilizing the enzyme quaternary structure. At 25 degrees C, SsAspAT is less resistant to guanidinium chloride-induced denaturation than the cytosolic aspartate aminotransferase from pig heart (cpAspAT), which was chosen as a mesophilic counterpart in the thermodynamic analysis since it shares with SsAspAT the two-state unfolding mechanism. Therefore, in the case of aspartate aminotransferases, thermal stability does not correlate with the stability against chemical denaturants. Isothermal denaturation curves at 25 degrees C and melting profiles recorded in the presence of guanidinium chloride showed that the delta G degrees (H2O) at 25 degrees C of SsAspAT exceeds that of cpAspAT by roughly 15 kJ/mol; the parameter delta n, related to the number of binding sites for the denaturant differentially exposed in unfolded and folded states, is higher for SsAspAT than for cpAspAT; and delta Cp is lower for the thermophilic enzyme than for the mesophilic one by 8 kJ/K.mol. These results are indicative of a less hydrophobic core for SsAspAT than cpAspAT. In agreement with this, the molecular model predicts that some charged side chains are buried in SsAspAT and interact to form an H-bond/ion-pair network.

Amino Acid Sequence↗
PubMed1994

The regulation of alanine and aspartate aminotransferase by different aminothiols and by vitamin B-6 derivatives.

We examined the effects on alanine aminotransferase and aspartate aminotransferase of different aminothiols (L-cysteine, D-cysteine, cysteamine, L-cysteine ethyl ester, L-cysteine methyl ester) and several vitamin B-6 derivatives (pyridoxal, pyridoxamine, pyridoxol, pyridoxol 5'-phosphate), before and after treatment with KOCN, which transforms these molecules into the corresponding carbamoyl derivatives. Only GPT, and not GOT, was specifically inhibited by L-cysteine and, to a lesser extent, by D-cysteine. The association reaction: PLP + apo GPT<-->holo GPT was inhibited by the vitamin B-6 derivatives, and this inhibition was prevented by pretreatment of the vitamin B-6 derivatives with KOCN. All the observed effects occurred at pH 7, 37 degrees C, at mM and even lower concentrations of reagents. Hence, they all potentially play a physiological role, in the regulation of the PLP dependent enzymes and of the vitamin B-6 levels in the cell.

Alanine Transaminase↗
PubMed1990

New automated measurement of mitochondrial aspartate aminotransferase with use of protease 401.

Total mitochondrial aspartate aminotransferase (EC 2.6.1.1), the sum of apo- and holo-mitochondrial aspartate aminotransferase activity in human serum, was measured by using a proteolytic method: inactivation of cytosolic aspartate aminotransferase with cytosolic aspartate aminotransferase-inactivating protease 401 from Streptomyces violaceochromogenes. Cytosolic aspartate aminotransferase is completely inactivated, and apo-mitochondrial aspartate aminotransferase is completely activated by pyridoxal 5'-phosphate within 5 min. Results by the proposed method correlated well with those by an immunochemical method (r = 0.994, n = 145) and showed excellent inhibitory activity of the protease for holo- and apo-cytosolic aspartate aminotransferase up to 5000 U/L and activation of mitochondrial apo-aspartate aminotransferase up to 2000 U/L in the presence of 100 mumol of pyridoxal 5'-phosphate per liter. Within-run Cvs were good (1.13-7.49%). Mean values for total mitochondrial aspartate aminotransferase and apo-mitochondrial aspartate aminotransferase activities in serum of the healthy subjects were 4.8 (SD 0.9) and 1.8 (SD 0.8) U/L, respectively (n = 154). Various common interferents tested did not affect this assay.

Aspartate Aminotransferases↗
PubMed2003

Release of pyridoxal 5'-phosphate upon unfolding of mitochondrial aspartate aminotransferase.

Dimeric mitochondrial aspartate aminotransferase (mAAT) contains a molecule of pyridoxal 5'-phosphate (PLP) tightly attached to each of its two identical active sites. The presence of this natural reporter allows us to study separately local perturbations in the architecture of this critical region of the molecule during unfolding. Upon unfolding of the enzyme with guanidine hydrochloride (GdnHCl), the coenzyme is completely released from the active site. The transition midpoint for the dissociation of PLP is 1.4+/-0.02 M when determined by size-exclusion chromatography (SEC) and 1.6+/-0.02 M when the protein-bound PLP is estimated by electrospray mass spectrometry (ESI-MS). In both cases the transition midpoint is higher than that of inactivation (1.3+/-0.01 M). On the other hand, the midpoint of the unfolding transition obtained by monitoring changes in ellipticity at 356 nm, which reflects the asymmetric environment of the PLP cofactor at the active site, is 1.19+/-0.011 M guanidine. These results indicate that the unfolding of mAAT is a multi-step process which includes an intermediate containing bound PLP but lacking catalytic activity.

Animals↗
PubMed1998

Identification of an adipocyte-specific negative glucose response region in the cytosolic aspartate aminotransferase gene.

Cytosolic aspartate aminotransferase (cAspAT) participates in gluconeogenesis in the liver and is expected to exert a glyceroneogenic function in the adipose tissue when the supply of glucose is limited. Here we demonstrate that adipose cAspAT messenger RNA (mRNA) is increased when rats are fed a low carbohydrate diet. In the 3T3-F442A, BFC-1 adipocyte cell lines and differentiated adipocytes in primary culture, a 24 h glucose deprivation induces approximately a 4-fold increase in cytosolic AspAT (cAspAT) mRNA, whereas mitochondrial AspAT mRNA remains unchanged. cAspAT activity is also increased in a weaker but reproducible manner. Addition of glucose within a physiological range of concentrations reverses the increase of cAspAT mRNA in 8 h (EC50 = 1.25 g/liter). Such a regulation requires protein synthesis and is specific for adipocytes differentiated in culture. It does not occur in Fao or H4IIE hepatoma cells, in C2 muscle cells, or in 293 kidney cells. 2-deoxyglucose mimicks glucose, while 3-orthomethyl-glucose has no effect, suggesting that glucose-6-phosphate is the effector. cAspAT mRNA stability is not affected by glucose deprivation. To ascertain the transcriptional nature of the glucose effect, we have stably transfected 3T3-F442A adipoblasts with constructs containing the chloramphenicol acetyltransferase reporter gene under the control of either 5'-deletions of the cAspAT gene promoter or internal fragments in an heterologous context. We demonstrate that a glucose response element(s) is present in the region between -1838 and -1702 bp relative to the translation start site. In this region, three DNA sequences bind nuclear proteins from adipocytes as shown by footprinting experiments. Our results indicate that cAspAT gene transcription is repressed by glucose selectively in adipocytes.

Adipocytes↗
PubMed1985

Effect of phosphate and other inorganic anions on the activity of chicken liver cytosolic aspartate aminotransferase.

Chicken liver aspartate aminotransferase was inhibited by several inorganic anions. The inhibitory effect of the anions was related to their chaotropic character. Apparent Km (2-oxoglutarate) and Km (L-aspartate) values depended on the molarity of the buffer. The profile of the curves obtained did not depend on the nature of the enzyme sample assayed. Phosphate slightly inhibited the holoaspartate aminotransferase and was a strong inhibitor of apoaspartate aminotransferase with respect to pyridoxal phosphate.

Animals↗
PubMed2002

Molecular analysis of the role of two aromatic aminotransferases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism of Pyrococcus furiosus.

The genes encoding aromatic aminotransferase II (AroAT II) and aspartate aminotransferase (AspAT) from Pyrococcus furiosus have been identified, expressed in Escherichia coli and the recombinant proteins characterized. The AroAT II enzyme was specific for the transamination reaction of the aromatic amino acids, and uses a-ketoglutarate as the amino acceptor. Like the previously characterized AroAT I, AroAT II has highest efficiency for phenylalanine (k(cat)/Km = 923 s(-1) mM(-1)). Northern blot analyses revealed that AroAT I was mainly expressed when tryptone was the primary carbon and energy source. Although the expression was significantly lower, a similar trend was observed for AroAT II. These observations suggest that both AroATs are involved in amino acid degradation. Although AspAT exhibited highest activity with aspartate and alpha-ketoglutarate (k(cat) approximately 105 s(-1)), it also showed significant activity with alanine, glutamate and the aromatic amino acids. With aspartate as the amino donor, AspAT catalyzed the amination of alpha-ketoglutarate, pyruvate and phenyl-pyruvate. No activity was detected with either branched-chain amino acids or alpha-keto acids. The AspAT gene (aspC) was expressed as a polycistronic message as part of the aro operon, with expression observed only when the aromatic amino acids were absent from the growth medium, indicating a role in the biosynthesis of the aromatic amino acids.

Amino Acid Sequence↗
PubMed1986

[Aspartate aminotransferase activity high affinity (3H) glutamate/(3H) aspartate uptake in rat nervous tissue in postnatal development].

The regional distribution and cellular localization of aspartate aminotransferase (AspAT) as a glutamate and aspartate metabolizing enzyme was studied in the hippocampal formation, in dorsal root ganglia, and in superior cervical ganglia during the postnatal development of the rat. At birth, in all neuronal tissues studied the enzyme activity was rather low. Whereas in the dendritic layers of the hippocampal formation enzyme levels rose strikingly during the first weeks of postnatal life, those of peripheral ganglia remained remarkably stable. The high affinity uptake of [3H]labelled L-glutamate and D-aspartate were studied in surface autoradiograms of incubated slices of hippocampal formation during postnatal development of the rat. Our data indicate that the postnatal development of high affinity uptake capacity is parallel to the increase of histochemically demonstrable AspAT activity in the hippocampal formation suggesting that the enzyme may be involved in glutamate/aspartate neurotransmitter metabolism.

Aging↗
PubMed1981

Multiple molecular forms of human cytoplasmic aspartate aminotransferase.

Human liver cytoplasmic aspartate aminotransferase was found to exhibit five subforms with isoelectric points of 5.15, 5.30, 5.45, 5.60, and 5.80. Treatment with neuraminidase did not affect their electrophoretic mobility. The immunochemical and steady-state kinetic properties of the subforms were identical. Heat treatment increased the proportion of acidic subforms, but all forms were present in fresh tissue. 2-Mercaptoethanol or inhibitors of proteolysis failed to protect against the formation of the subforms with lower isoelectric points. Multiple molecular forms with similar properties were found for the enzyme of human erythrocytes. This evidence is consistent with deamidation of asparaginyl or glutaminyl residues as the origin of the multiple forms. Human mitochondrial aspartate aminotransferase presented as a single molecular form with an isoelectric point of 9.7.

Aspartate Aminotransferases↗
PubMed1996

Crystal structures and solution studies of oxime adducts of mitochondrial aspartate aminotransferase.

The interaction of mitochondrial aspartate aminotransferase with hydroxylamine and five derivatives (in which the hydroxyl hydrogen is replaced by the side chain of naturally occurring amino acids) was investigated by X-ray diffraction as well as by kinetic and spectral measurements with the enzyme in solution. The inhibitors react with pyridoxal 5'-phosphate in the enzyme active site, both in solution and in the crystalline state, in a reversible single-step reaction forming spectrally distinct oxime adducts. Dissociation constants determined in solution range from 10(-8) M to 10(-6) M depending on the nature of the side-chain group. The crystal structures of the adducts of mitochondrial aspartate aminotransferase with the monocarboxylic analogue of L-aspartate in the open and closed enzyme conformation were determined at 0.23-nm and 0.25-nm resolution, respectively. This inhibitor binds to both the open and closed crystal forms of the enzyme without disturbing the crystalline order. Small differences in the conformation of the cofactor pyridoxal phosphate were detected between the omega-carboxylate of the inhibitor and Arg292 of the neighbouring subunit is mainly responsible for the attainment of near-coplanarity of the aldimine bond with the pyridine ring in the oxime adducts. Studies with a fluorescent probe aimed to detect shifts in the open/closed conformational equilibrium of the enzyme in oxime complexes showed that the hydroxylamine-derived inhibitors, even those containing a carboxylate group, do not induce the 'domain closure' in solution. This is probably due to the absence of the alpha-carboxylate group in the monocarboxylic hydroxylamine-derived inhibitors, emphasizing that both carboxylates of the substrates L-Asp and L-Glu are essential for stabilizing the closed form of aspartate aminotransferase.

Amino Acid Sequence↗
PubMed1983

Use of beta-methylene-D,L-aspartate to assess the role of aspartate aminotransferase in cerebral oxidative metabolism.

Several inhibitors of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle, were investigated for their effects on cerebral oxidative metabolism in vitro. beta-Methylene-D,L-aspartate (2 mM), aminooxyacetate (0.1 mM), and D,L-vinylglycine (20 mM) all significantly reduced the activity of aspartate aminotransferase and the rate of oxygen consumption of rat cerebral cortex slices respiring on glucose. In the presence of beta-methyleneaspartate, a one-to-one correlation was found between the degree of inhibition of tissue respiration and the degree of inhibition of transaminase activity. Slices of rat liver incubated in the presence of glucose and beta-methyleneaspartate showed a similar one-to-one relationship between inhibition of oxygen comsumption and inhibition of aspartate aminotransferase activity, whereas with rat kidney cortex slices, the inhibition of aspartate aminotransferase activity was greater than the inhibition of oxygen consumption. Structural analogs of beta-methyleneaspartate (D,L-beta-methyl-D,L-aspartate, gamma-methyl-D,L-glutamate, and alpha-methyl-D,L-didehydroglutamate) that did not inhibit the activity of aspartate aminotransferase similarly did not inhibit the rate of oxygen consumption by cerebral cortex slices. In the presence of beta-methyleneaspartate, pyruvate oxidation by cerebral cortex slices was inhibited to almost the same extent as was glucose oxidation, and the oxidation of succinate was decreased by approximately 20%. The artificial electron acceptor phenazine methosulfate (0.1 mM) only partially overcame the beta-methyleneaspartate-mediated inhibition of respiration with glucose as substrate. The content of ATP and phosphocreatine declined steadily in slices incubated with glucose and beta-methyleneaspartate. At 1 h the concentration of lactate and the lactate/pyruvate ratio, an indicator of the cytoplasmic redox state, increased threefold, whereas the concentrations of malate, citrate, and aspartate decreased. The findings are interpreted in the context of the hypothesis that enzymes common to the malate-aspartate shuttle and the tricarboxylic acid cycle are physically complexed in brain, so that inhibition of aspartate aminotransferase, a component of the complex, impedes the flow of carbon through both metabolic pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminooxyacetic Acid↗
PubMed1984

Partial amino-acid sequence and cysteine reactivities of cytosolic aspartate aminotransferase from horse heart.

Cytosolic aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from horse heart has five cysteine residues, two of which can be titrated with 5,5'-dithiobis(2-nitrobenzoid acid) in the native enzyme with no impairment of catalytic activity. The rate of modification is unaffected by the presence of substrates. Reaction with N-ethylmaleimide leads to loss of catalytic activity, the rate of inactivation being increased by the presence of substrates. Peptides containing 361 amino-acid residues (about 88% of the total number in the protein) have been isolated and aligned by comparison with the known sequence of the isotopic isoenzyme from pig heart. In the regions compared, 342 of the residues are identical. Hence, assuming that those regions are representative of the whole, then the cytosolic isoenzymes from horse and from pig have about 95% identity of structure. Uniquely among the mammalian cytosolic aspartate aminotransferases so far examined, the enzyme from horse heart is acetylated at the N-terminus.

Amino Acid Sequence↗
PubMed1990

Proteolytic measurement of mitochondrial aspartate aminotransferase in human serum.

A new proteolytic measurement of serum mitochondrial aspartate aminotransferase was evaluated using cytosolic aspartate aminotransferase inactivating protease. Some of the proteases, such as, alpha-chymotrypsin, subtilisin and cytosolic aspartate aminotransferase inactivating protease 401 from Streptomyces species, also specifically inactivated cytosolic aspartate aminotransferase, but not mitochondrial, aspartate aminotransferase. The protease 401 was the most heat stable for storage and showed a higher inactivation rate for cytosolic aspartate aminotransferase--up to 7000 IU/L--more than 200-fold the upper limit. The coefficient of variation of the proteolytic method was less than 10%. Results by the present method correlated with those by the immunochemical method (r = 0.970) and the regression curve was Y = 0.95X + 1.60 (Y: immunochemical method; X: proteolytic method). In the present assay system, reference values for mitochondrial aspartate aminotransferase activity in 500 healthy people ranged from 2.0-7.2 U/L (mean 3.8 U/L).

Aspartate Aminotransferases↗
PubMed1989

[Serum activity of mitochondrial aspartate aminotransferase and extrahepatic cholestasis].

Serum mitochondrial aspartate aminotransferase (mAST) level and the mitochondrial aspartate aminotransferase/total aspartate aminotransferase ratio (mAST/AST) have been proposed as sensitive markers of chronic alcoholism. Their specificity, however, remains poorly defined. The purpose of this study was to compare these markers in three groups of hospitalized patients: group I, 80 patients with chronic alcoholic liver disease; group II, 51 patients with chronic liver disease without alcoholism; group III, 44 patients with extrahepatic cholestasis (due to choledocholithiasis in 21 and malignant in 23). mAST was measured after immuno-precipitation of cytoplasmic aspartate aminotransferase. The normal values of mAST (less than or equal to 2 mu/l) and mAST/AST (less than or equal to 6 p. 100) were defined in a group of 59 non alcoholic subjects without liver disease (controls). mAST was increased as compared with controls in 91 p. 100 of the patients of group I, 20 p. 100 of group II, 61 p. 100 of group III. mAST was comparable in groups I (mean +/- SD: 10 +/- 10.8) and III (10.3 +/- 12.9), and higher than in group II (1.8 +/- 2.4). m/AST was increased in 59 p. 100 of the patients of group I, 6 p. 100 of group II and 36 p. 100 of group III. It was higher in group I (8 +/- 4 p. 100) than in group III (6 +/- 4 p. 100, p less than 0.02), and particularly higher in both these groups than in group II (2 +/- 1 p. 100, p less than 0.00001). mAST was correlated to AST in each of these three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcoholism↗