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PubMed1975

[The influence of pyridoxal 5'-phosphate on the temperature relationships of aspartate aminotransferase isoenzymes].

The relationship between temperature and the behaviour of aspartate aminotransferase was investigated in the presence of pyridoxal 5'-phosphate. The addition in vitro of pyridoxal 5'-phosphate caused an increase in the activity and altered the thermal behaviour of aspartate aminotransferase. In choosing the temperature for the determination of enzymic activity, the concentration of the coenzyme must therefore also be considered.

Aspartate Aminotransferases↗

PubMed1976

Aspartate aminotransferase activity and glutamic dehydrogenase in the cerebellar cortex in several species of animals. A histochemical study.

The histochemical study of the consumption of glutamic acid by way of the aspartate aminotransferase and the glutamic dehydrogenase in the cerebellar cortex of several species of animals have demonstrated that in that nerve centre exists some structures in which the mentioned consumption is specially or exclusively realized by means of one way and not for other different one. Is observed, as well, that in the rats, chicken and lizard, the baskets that surround the Purkinje cells are constituted by basket cells axons and by recurrent collaterals of Purkinje axons and that those structures have an intense aspartate aminotransferase activity, but not glutamic dehydrogenase. The aspartate aminotransferase activity was not observed on the other side, in the perikarya of the Purkinje cells of the related animals. However, there exists intense glutamic dehydrogenase activity. On the other hand, in the toad was not observed baskets with aspartate aminotransferase activity but this enzyme was presented on the other side in the perikarya of the Purkinje cells. All these observations have suggested the possibility that this special utilization of the glutamic acid is in some way concerned with the transmission phenomenons of the nerve impulse.

Animals↗

PubMed1990

Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species.

Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp. strain YM-2. The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pIs of 4.1 and 4.5 upon isoelectric focusing. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra. The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent. The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and alpha-ketoglutarate as an amino acceptor; the KmS were determined to be 3.0 mM for L-aspartate and 2.6 mM for alpha-ketoglutarate. The enzyme was most active at 70 degrees C and had a higher thermostability than the enzyme from Escherichia coli. The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E. coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.

Amino Acid Sequence↗

PubMed1981

Response of rat liver aspartate aminotransferase to carbon tetrachloride.

Rat liver aspartate aminotransferase activity per total liver per gram initial body weight was increased 24 hours after carbon tetrachloride injection suggesting that increased synthesis may be a source of increased serum enzyme activity as a response to hepatocellular injury. Protein content per total liver per gram initial body weight was also increased so that the specific activity of the enzyme was unchanged. Enzyme activity per gram liver wet weight was decreased consistent with hepatomegaly, edema, and dilution of enzyme. Total liver per gram initial body weight is suggested as the optimal reference standard.

Animals↗

PubMed1975

[Ultrastructural location of aspartate aminotransferase in the cerebellum of mice].

The histochemical location of aspartate aminotransferase by ultrastructural observation in the mouse cerebellum has been investigated. The enzymatic activity was observed specifically in the fibre endings of basket cells and in the probable terminations of golgi cells axons. This observation suggests the intervention of aspartate aminotransferase in the specific function of the cerebellum inhibitor axons.

Animals↗

PubMed1990

The development of computer programs to predict and minimise the effect of sample pyruvate on aspartate aminotransferase assays.

Some NADH-linked aspartate aminotransferase (AST) assay systems may produce significant errors due to endogenous pyruvate because the pre-incubation time employed is too short and/or because the reagent lactate dehydrogenase (LD) activity is inadequate. Such errors in patient specimens may go unnoticed if quality control materials themselves contain a low pyruvate concentration. A theory of pyruvate interference in AST assay is formulated and calculation programs are described which will allow a laboratory to assess whether their particular AST methods are likely to be subject to such interference and also to predict a way of removing any error thus identified.

Aspartate Aminotransferases↗

PubMed1989

Crystallization and preliminary X-ray studies of an aspartate aminotransferase mutant from Escherichia coli.

Mutant aspartate aminotransferase V39L (Val39 replaced by Leu) from Escherichia coli has been crystallized into a monoclinic cell from a polyethylene glycol solution (pH 7.5) by vapor diffusion. The space group and the unit cell dimensions have been determined using a precession camera, a CAD4 diffractometer and a Nicolet Xentronics area detector to be P2(1) with a = 86.8 A, b = 79.9 A, c = 89.4 A, beta = 118.74 degrees. The crystals diffract to better than 2.3 A and are suitable for X-ray structure analysis.

Aspartate Aminotransferases↗

PubMed1982

Kinetics of coupled reactions catalyzed by aspartate aminotransferase and glutamate dehydrogenase.

Liver mitochondrial aspartate aminotransferase and glutamate dehydrogenase catalyze following sequence of reactions: see formula in text. In the presence of a slight excess of dehydrogenase, the time course of NADPH oxidation resulting from the overall reaction goes through a lag phase and reaches a linear phase. The slopes of the linear part of this curve is a linear function of transaminase concentration. At high concentration (approximately or equal to 10 microM) of both enzymes the lag phase, as observed after rapid mixing of the two enzymes in a Durrum stopped-flow spectrophotometer, is shorter, than that predicted from the kinetic parameters determined for the separate reactions catalyzed by each enzyme.

Animals↗

PubMed2002

Aspartate aminotransferase isotope exchange reactions: implications for glutamate/glutamine shuttle hypothesis.

Aspartate aminotransferase (AAT) catalyzes amino group transfer from glutamate (Glu) or aspartate (Asp) to a keto acid acceptor-oxaloacetate (OA) or alpha-ketoglutarate (KG), respectively. Data presented here show that AAT catalyzes two partial reactions resulting in isotope exchange between 3H-labeled Glu or 3H-labeled Asp and the cognate keto acid in the absence of the keto acid acceptor required for the net reaction. Tritiated keto acid product was detected by release of 3H2O from C-3 during base-induced enolization. Tritium released directly from C-2 (or C-3) by the enzyme was also evaluated and is a small fraction of that released because of exchange to the keto acid pool. Exchange is dependent on AAT concentration, time-dependent, proportional to the amino-to-keto acid ratio, and blocked by aminooxyacetate (AOA), an AAT inhibitor. Enzymatic conversion of [3H]KG to Glu by glutamic dehydrogenase (GDH) or of [3H]OA to malate by malic dehydrogenase (MDH) "protects" the label from release by base, showing that base-induced isotope release is from keto acid rather than a result of release during the exchange process. AAT isotope exchange is discussed in the context of the glutamate/glutamine shuttle hypothesis for astrocyte/neuron carbon cycling.

Alkalies↗

PubMed2003

Crystallization and preliminary X-ray analysis of the Mj0684 gene product, a putative aspartate aminotransferase, from Methanococcus jannaschii.

A putative aspartate aminotransferase from the hyperthermophilic archaeon Methanococcus jannaschii encoded by the Mj0684 gene has been overexpressed in Escherichia coli and crystallized at 296 K using the sitting-drop vapour-diffusion method. The crystals belong to space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 111.87, c = 60.86 A. They diffract to 2.2 A resolution using Cu Kalpha X-rays. The asymmetric unit contains a single subunit of the recombinant Mj0684 gene product, giving a corresponding V(M) of 2.25 A(3) Da(-1) and a solvent content of 45.3% by Volume. An X-ray diffraction data set has been collected to 2.2 A at 295 K.

Aspartate Aminotransferases↗

PubMed1994

Testis-specific transcription start site in the aspartate aminotransferase housekeeping gene promoter.

We have studied the expression and regulation of the rat testis cytosolic aspartate aminotransferase gene. The cytosolic aspartate aminotransferase activity was 5-fold lower in the testis than in the liver and kidney. A 1.9-kilobase mRNA form was detected in the rat testis in contrast to the 2.1- and 1.8-kilobase forms present in other organs. Using Northern blot and S1 mapping analyses, we found that the proximal polyadenylation site was almost exclusively used in the testis as opposed to other organs where the distal site was preferentially used. RNase protection and primer extension analysis showed that transcription was initiated at multiple sites in all organs, but the pattern of those start sites was different in the testis; in particular, a novel transcription start site was specifically detected in this organ (at position -115 from the translation start site). This site was first observed in 29-day-old rats and was maximally utilized in the adult testis. DNase I footprinting using testis nuclear extracts revealed the presence of three sites of DNA-protein interaction in the 250-base pair proximal promoter, a pattern similar to the one found using liver nuclear extracts. However, the proteins bound had different properties as shown by gel retardation experiments. We conclude that the pattern of transcription initiation and the polyadenylation site selection of a housekeeping gene can be tissue-specific.

Animals↗

PubMed1995

Androgen modulation of multiple transcription start sites of the mitochondrial aspartate aminotransferase gene in rat prostate.

Mitochondrial aspartate aminotransferase (mAAT) is one of two key enzymes in the pathway of citrate production in prostate. Expression of mAAT is modulated by testosterone and prolactin in prostate. We cloned the promoter and 5'-flanking region of the rat mAAT gene and sequenced 2.0 kilobases of the DNA. This fragment contains the 5'-regulatory promoter region that lacks a TATA and a CCAAT box but is G+C rich. The 5'-upstream flanking region contains sequences that have high homology with the consensus glucocorticoid response element/androgen response element (ARE) and a reported ARE sequence that is different from the consensus sequence. Functional transcription studies showed that a 481-base region containing the two ARE sequences was sufficient for androgen-regulated gene expression. There are multiple transcription start sites that are regulated by testosterone in prostate. In liver, on the other hand, castration did not affect transcription from any of the start sites. Therefore, these data provide evidence that transcriptional regulation of the rat pmAAT gene occurs through an ARE located in the 5'-region. In addition, not only is gene expression modulated by testosterone, but the effect of testosterone on transcription is cell specific.

Animals↗

PubMed1993

Protein folding in a cell-free translation system. The fate of the precursor to mitochondrial aspartate aminotransferase.

The precursor to rat mitochondrial aspartate aminotransferase (pmAspAT) can be expressed in and purified from Escherichia coli as a fully active enzyme with remarkable trypsin resistance. Only two sites within the presequence are readily hydrolyzed (Martinez-Carrion, M., Altieri, F., Iriarte, A., Mattingly, J. R., Youssef, J., and Wu, T. (1990) Ann. N.Y. Acad. Sci. 585, 346-356). In contrast, pmAspAT freshly synthesized in rabbit reticulocyte lysate is significantly less resistant to proteolysis and is completely digested by trypsin. Extended incubation of the pmAspAT translation product slowly converts it to a species with qualitatively the same trypsin resistance as the purified pmAspAT. In addition, this species binds pyridoxal 5'-phosphate, exhibits catalytic activity, and loses its ability to be imported into mitochondria. This process appears to reflect protein folding. The rate of folding is unaffected by the addition of cofactor or the depletion of endogenous cofactor and is not significantly affected by the concentration of translation product in the reaction. Agents that decrease the availability of ATP partially inhibit the folding, whereas the sulfhydryl alkylating reagent N-ethylmaleimide and the detergent Triton X-100 completely prevent the conversion. Although the folding of pmAspAT in reticulocyte lysate is slow, folding is rapid once the translation product is sequestered within the mitochondria as the mature form of the enzyme. These results are presented as a model for the in vivo folding of pyridoxal-dependent, oligomeric mitochondrial precursors in the presence of cytoplasmic components and for the fate of true mitochondrial precursor proteins when not imported.

Adenosine Triphosphate↗

PubMed1993

Structural features which control folding of homologous proteins in cell-free translation systems. The effect of a mitochondrial-targeting presequence on aspartate aminotransferase.

When the precursor to mitochondrial aspartate aminotransferase (pmAspAT) is synthesized in a rabbit reticulocyte lysate translation system (RRL), its properties are quite unlike those of the purified protein (Mattingly, J.R., Jr., Youssef, J., Iriarte, A., and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 3925-3937). These results suggest that molecular chaperones present in RRL modulate the folding of pmAspAT. To investigate the structural basis for this, we have used protease resistance to monitor the extent of folding for several related AspATs after synthesis in RRL and in wheat germ extract (WGE). In addition to pmAspAT, the following proteins were examined: the mature form of pmAspAT (delta 2-28 pmAspAT), its cytosolic counterpart (cAspAT), a chimeric protein consisting of the presequence of pmAspAT attached to the amino terminus of cAspAT (pcAspAT), and a pmAspAT variant in which the presequence and the amino-terminal domain of the mature enzyme are deleted (delta 2-57 pmAspAT). In RRL, delta 2-28 pmAspAT folds somewhat faster than intact pmAspAT, whereas the truncated delta 2-57 pmAspAT is unable to fold. In contrast, cAspAT and pcAspAT both fold with extreme rapidity. After synthesis in WGE, pmAspAT and delta 2-28 pmAspAT never acquire a protease-resistant conformation, whereas the folding of cAspAT and pcAspAT still occurs rapidly. We conclude that the presequence has only a minor role in determining the folding rate of the pmAspAT mitochondrial precursor protein in RRL or WGE and has no influence on the folding of the homologous cAspAT. Rather, the primary sequence of the mature part of the protein seems to dictate whether or how molecular chaperones regulate folding events.

Amino Acid Sequence↗

PubMed1982

Studies on the significance of serum mitochondrial aspartate aminotransferase activity following ischemic cardiac arrest.

Aspartate aminotransferase (EC 2.6.1.1:AST) is known to have two isoenzymes, one associated with the cytoplasm (c-AST) and the other with the mitochondria (m-AST). We studied the relationships of m-AST activity in the coronary sinus blood to left ventricular function, coronary blood flow, water content and high-energy phosphate stores of the left ventricle following hypothermic ischemic cardiac arrest. Under cardiopulmonary bypass with hypothermia of 20 degrees C of myocardial temperature, 120 min of aortic occlusion was employed in 15 mongrel dogs. Left ventricular function (peak left ventricular pressure, left ventricular end-diastolic pressure, max dp/dt, cardiac index, left ventricular stroke work index), coronary blood flow, myocardial oxygen consumption, myocardial enzyme activity (m-AST, CK-MB), myocardial water content and high-energy phosphate stores (adenosine triphosphate, creatine phosphate) of the subendocardium of the left ventricle were measured. Data was obtained in the control state, and after 0, 30 and 60 min of reperfusion. Significant negative correlations were obtained between m-AST activity and peak left ventricular pressure (r = -0.81, p less than 0.001), max dp/dt (r = -0.83, p less than 0.001), cardiac product (r = -0.73, p less than 0.01), coronary blood flow (r = -0.59, p less than 0.05), adenosine triphosphate level (r = 0.72, p less than 0.01) and creatine phosphate level (r = -0.72, p less than 0.02) after 60 min of reperfusion. Significant positive correlations were obtained between m-AST activity and left ventricular end-diastolic pressure (r=0.75, p less than 0.01) and water content (r = 0.78, p less than 0.01) after 60 min of reperfusion. These results led to the assumption that serum m-AST activity in the coronary venous blood is a useful index to evaluate the degree of myocardial injury.

Adenosine Triphosphate↗

PubMed1983

Clinical implications of differences between two recommended procedures for determination of aspartate aminotransferase.

We compared two officially recommended methods for determination of aspartate aminotransferase (EC 2.6.1.1): that of the International Federation of Clinical Chemistry (IFCC) and that of the Deutsche Gesellschaft für Klinische Chemie (DGKC). We used automated enzyme analyzers, initiating the reactions with 2-oxoglutarate. Normal values, 10-30 U/L (IFCC) and 7-18 U/L (DGKC), were apparently insensitive to intra-individual variations. Samples obtained from patients with heart disease showed a markedly different amount of activation with pyridoxal phosphate as compared with samples from other patient categories. Ratios for aspartate aminotransferase/alanine aminotransferase, as used in the differentiation of liver disease, can still be used with either method for determination of aspartate aminotransferase.

Alanine Transaminase↗

PubMed1976

Large-scale purification and some properties of the mitochondrial aspartate aminotransferase from pig heart.

A method has been developed which allows isolation of 0.3--0.5 g of mitochondrial aspartate aminotransferase in five days starting from 10 pig hearts; the method does not involve initial preparation of mitochondria. Mitochondrial malate dehydrogenase and the cytoplasmic aspartate aminotransferase may conveniently be recovered from side fractions. The product mitochondrial aspartate aminotransferase is homogeneous as judged by various electrophoretic techniques and by N-terminal analysis. Crystals of the enzyme have been obtained both from concentrated, essentially salt-free, solutions and from solutions of ammonium sulphate. The amino acid composition, N and C-terminal amino acid sequences and subunit molecular weight have been determined; these characteristic properties are compared with those of the cytoplasmic isozyme from the same source.

Amino Acid Sequence↗

PubMed1991

Heterogeneity of chicken liver cytosolic aspartate aminotransferase.

The isolated native molecular forms of chicken liver cytosolic aspartate aminotransferase give rise to two kinds of generation processes: (a) on storage, molecular forms are transformed into a series of variants with increasing anodic mobilities; and (b) addition of thiol reagents not only avoids the process, but causes the partial transformation of minor subforms into variants with higher isoelectric point values. In both cases the mobilities of each generated variant coincide with that of the corresponding native molecular form. The variants generated either by storage or in the presence of thiol reagents were separated by chromatofocusing. Several comparative studies have demonstrated the structural and functional identity between native molecular forms and 'in vitro' active generated variants of the enzyme. The results obtained suggest that native minor subforms arise from the major alpha form due to oxidation process and might represent intermediate species in the intracellular cytosolic aspartate aminotransferase turnover.

Animals↗