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PubMed1975

Surface-bound aspartate aminotransferase on collagen films. Compared properties with native enzyme.

Aspartate aminotransferase (L-aspartate : 2-oxoglutarate aminotransferase, EC 2.6.1.1) has been covalently bound to chemically activated collagen films. This enzyme had never previously been coupled to any other solid support. The coupling method, including acyl azide formation on the carrier, allowed coupling of many other enzymes. A systematic study of coupling conditions has been performed; influence of time of coupling and of concentration of coupling solution on the enzymatic activity retained on the film. Coupling solutions could be used for several successive couplings. To determine the yield of binding, N-[14C] ethylmaleimide-labelled enzyme was prepared fully active and bound to collagen films. After lyophilisation the film retained most of its activity when stored in buffer and the half-life of the enzymatic film was about ten months. pH Dependence and activation energy were about the same for soluble and coupled enzyme. Coupling protects against thermal denaturation and increases the stability of the enzyme; the enzymatic film could be used repeatedly. Kinetics were somewhat modified in the coupled enzyme as compared to the enzyme in solution. Glutamate appeared more available while oxaloacetate seemed to be limiting. These modifications might be due to the proteic support itself. The enzymatic films also revealed themselves as a good tool for industrial or clinical purposes as well as for studying the mechanism of enzyme action.

Aspartate Aminotransferases↗
PubMed1987

Expression of cDNAs encoding the precursor and the mature form of chicken mitochondrial aspartate aminotransferase in Escherichia coli.

Both the precursor and the mature form of chicken mitochondrial aspartate aminotransferase were synthesized in Escherichia coli. The precursor was found to sediment quantitatively together with insoluble cell material. In contrast, mature mitochondrial aspartate aminotransferase could be readily extracted from the cells and was indistinguishable from the enzyme isolated from chicken heart in all respects tested: specific activity 230 units mg-1; Mr 2 X 45,000; pI greater than 9; NH2-terminal sequence SSWWSHVEMG, the initiator methionine having been removed by the bacteria. Thus, the polypeptide chain representing mature mitochondrial aspartate aminotransferase is an autonomous folding unit which attains its functional spatial structure independently of the presence of the prepiece, trans-membrane passage, and proteolytic processing.

Amino Acid Sequence↗
PubMed1985

Cloning and sequence analysis of a cDNA encoding porcine mitochondrial aspartate aminotransferase precursor.

The primary structure of pig mitochondrial aspartate aminotransferase (mAspATase; L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) precursor was deduced from its cDNA sequence. A library of cDNA clones was constructed from pig liver poly(A)+ RNA by applying the vector/primer method of Okayama and Berg [Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170]. The library was screened for pig mAspATase sequences by using a mixture of eight oligodeoxyribonucleotides as a probe. The sequences of the probe were deduced from the known amino acid sequence of pig mAspATase residues 196-201. Two recombinant plasmids containing inserts of about 2500 and 2600 base pairs were selected for sequence analysis. The amino acid sequence predicted from the cDNA sequence shows that the pig mAspATase precursor consists of the mature enzyme of 401 amino acid residues and an amino-terminal segment of 29 amino acid residues called the "presequence" that contains four basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The sequence of this 29-amino acid mAspATase precursor segment was compared with the presequences of other mitochondrial enzymes.

Amino Acid Sequence↗
PubMed1997

Cloning and sequencing of aspartate aminotransferase from Thermus aquaticus YT1.

A 39-base oligonucleotide "guessmer" probe, based on partial N-terminal sequence analysis of the aspartate aminotransferase purified from Thermus aquaticus strain YT1, was used to screen a genomic library prepared from T. aquaticus DNA. A 1842 bp DNA fragment was isolated that proved to contain the coding sequence for the aspartate aminotransferase. The gene is 1152 bases long and codes for a protein of 383 amino acid residues. The amino acid sequence obtained showed 88.7%, 45.1% and 32.9% identity of sequence with those of thermostable aspartate aminotransferases from T. thermophilus, Bacillus YM2, and Sulfolobus solfataricus, respectively. It showed 39.1% identity with one of the gene products tentatively identified as aspartate aminotransferase from the methanogenic archaebacterium Methanococcus jannaschii. Neither the amino acid compositions nor the aligned amino acid sequences provides any obvious clue as to the origin of thermal stability in this group of enzymes.

Amino Acid Sequence↗
PubMed1991

The open/closed conformational equilibrium of aspartate aminotransferase. Studies in the crystalline state and with a fluorescent probe in solution.

Aspartate aminotransferase undergoes major shifts in the conformational equilibrium of the protein matrix during transamination. The present study defines the two conformational states of the enzyme by crystallographic analysis, examines the conditions under which the enzyme crystallizes in each of these conformations, and correlates these conditions with the conformational behaviour of the enzyme in solution, as monitored by a fluorescent reporter group. Cocrystallization of chicken mitochondrial aspartate aminotransferase with inhibitors and covalent coenzymesubstrate adducts yields three different crystal forms. Unliganded enzyme forms triclinic crystals of the open conformation, the structure of which has been solved (space group P1) [Ford, G. C., Eichele, G. & Jansonius, J. N. (1980) Proc. Natl Acad. Sci. USA 77, 2559-2563; Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H. & Christen, P. (1984) J. Mol. Biol. 174, 487-525]. Complexes of the enzyme with dicarboxylate ligands form monoclinic or orthorhombic crystals of the closed conformation. The results of structure determinations of the latter two crystal forms at 0.44 nm resolution are described here. In the closed conformation, the small domain has undergone a rigid-body rotation of 12-14 which closes the active-site pocket. Shifts in the conformational equilibrium of aspartate aminotransferase in solution, as induced by substrates, substrate analogues and specific dicarboxylic inhibitors, can be monitored by changes in the relative fluoresence yield of the enzyme labelled at Cys166 with monobromotrimethylammoniobimane. The pyridoxal and pyridoxamine forms of the labelled enzyme show the same fluorescence properties, whereas in the apoenzyme the fluorescence intensity is reduced by 30%. All active-site ligands, if added to the labelled pyridoxal enzyme at saturating concentrations, cause a decrease in the fluorescence intensity by 40-70% and a blue shift of maximally 5 nm. Comparison of the fluorescence properties of the enzyme in various functional states with the crystallographic data shows that both techniques probe the same conformational equilibrium. The conformational change that closes the active site seems to be ligand-induced in the reaction of the pyridoxal form of the enzyme and syncatalytic in the reverse reaction with the pyridoxamine enzyme.

Animals↗
PubMed1990

Drug-induced decrease of serum alanine and aspartate aminotransferase activity in the rat, as a result of treatment with oxodipine, a new calcium channel blocker.

Chronic oral administration of oxodipine, a new calcium channel blocker, resulted in a reduction in the blood enzyme activity of alanine and aspartate aminotransferase. The reductions were both time and dose related. The decline in enzyme activities was accompanied by microscopic hepatic changes, which in the opinion of the authors should have been associated with an increase in the enzyme activities of alanine aminotransferase and aspartate aminotransferase. The effect was only partially reversed one month after the cessation of oxodipine treatment.

Alanine Transaminase↗
PubMed1985

Distributions of aspartate aminotransferase and malate dehydrogenase activities in rat retinal layers.

Aspartate aminotransferase (AAT), an enzyme interconverting glutamate and aspartate, has been suggested to be a marker for glutamatergic and/or aspartatergic neurons. However, AAT, glutamate, and aspartate are also involved in cellular metabolism, e.g., the malate-aspartate shuttle. To investigate the extent to which AAT might be involved in these several functions in retina, the distribution of AAT activity in rat retinal layers was compared to that of malate dehydrogenase (MDH), an enzyme of aerobic metabolism proposed to be physically complexed with AAT in the malate-aspartate shuttle mechanism. The distribution of AAT activity in retinal layers closely paralleled that of MDH (correlation coefficient AAT versus MDH = 0.93). AAT activity was proportionately higher than MDH in the photoreceptor inner segments, containing a high density of mitochondria, and in the outer plexiform layer (OPL), containing photoreceptor terminals and bipolar and horizontal cell processes. The amount of total AAT activity in the inner segments related to the mitochondrial isoenzyme is almost twice that in the other layers tested, including the OPL. The correlation between AAT and MDH activities is consistent with AAT involvement in retinal energy metabolism, although other functions, such as neurotransmission, are possible.

Animals↗
PubMed1978

Optimization of methods for aspartate aminotransferase and alanine aminotransferase.

Conditions for accurate measurement of catalytic activity of aspartate aminotransferase and alanine aminotransferase in human serum have been reinvestigated. The basic variables (kind of buffer, buffer concentration, pH, ion effects, and the influence of pyridoxal-5-phosphate) can now be considered optimized. On this basis, the kinetic parameters of both aminotransferases were determined, i.e., Michaelis and inhibitor constants for substrates and reaction products. With a mathematical approach for two-substrate enzyme reactions the substrate concentrations were calculated from the viewpoints "most economical," "most convenient," and "lowest variability." Also the conditions for the indicator reactions have been newly defined with respect to a kinetic model. All calculated data were rechecked experimentally and it can be shown that both approaches fully agree. Furthermore, we show that the mathematical approach allows more precise recommendations for optimized methods. For technical reasons, the catalytic activity of aspartate aminotransferase in human serum can only be measured as a 0.96 fraction of its theoretical maximum velocity, the catalytic activity of alanine aminotransferase as a 0.91 fraction. The assay conditions for a Reference Method are finally described and recommendations are made for optimized routine methods for determination of the catalytic activity of these transferases in human serum.

Alanine Transaminase↗
PubMed1984

Pyridoxal-5'-phosphate-induced stimulation of aspartate aminotransferase and its isoenzymes in human myocardial biopsies and autopsies.

The extent to which pyridoxal-5'-phosphate stimulates the activities of aspartate aminotransferase and its cytoplasmic and mitochondrial isoenzymes was measured in six human left ventricular biopsies obtained freshly during open-heart surgery, and in 13 human left ventricular autopsies. A concentration of pyridoxal-5'-phosphate of 15 mumol/l for 1 h is sufficient to convert any apoenzyme to holo-enzyme. Pyridoxal-5'-phosphate-induced stimulation of aspartate aminotransferase is 26 +/- 6% (+/- SD) in myocardial biopsies (range 22-34%) and 112 +/- 54% in myocardial autopsies (range 35-200%). The extent of stimulation of the cytoplasmic and mitochondrial isoenzymes is 18 +/- 9% and 32 +/- 6%, respectively, in myocardial biopsies, and 150 +/- 57% and 100 +/- 62%, respectively, in myocardial autopsies. The greater extent and variation of the pyridoxal-5'-phosphate-induced stimulation of aspartate aminotransferase and its isoenzymes in myocardial autopsies compared to that in myocardial biopsies is caused by autolysis and its duration. Autolysis depresses myocardial aspartate aminotransferase activity measured in the absence of pyridoxal-5'-phosphate which effect is more prominent for the cytoplasmic than for the mitochondrial isoenzyme.

Adult↗
PubMed1987

Localization of glutamate, glutaminase, aspartate and aspartate aminotransferase in the rat midbrain periaqueductal gray.

Glutamate and aspartate are putative excitatory neurotransmitters in the central nervous system. The present study utilized novel monoclonal antibodies against fixative-modified glutamate and aspartate and polyclonal antisera against the amino acid synthesizing enzymes, glutaminase and aspartate aminotransferase, to analyze the distribution of these amino acids in the rodent midbrain periaqueductal gray. Glutamate-, aspartate-, glutaminase- and aspartate aminotransferase-like immunoreactive neurons, fibers and processes are present throughout the rostrocaudal length of the periaqueductal gray. Glutamate- and glutaminase-like immunoreactive neurons displayed a similar homogeneous pattern of distribution, being localized predominantly to the lateral and dorsal subdivisions of the periaqueductal gray. Co-localization experiments suggest that glutamate and glutaminase are in fact co-contained within the same PAG neurons. Aspartate aminotransferase-like immunoreactive neurons were distributed in a pattern similar to glutamate and glutaminase with the exception that fewer cells were stained in the dorsocaudal and the rostral third of the PAG. Aspartate-like immunoreactive neurons were less numerous than glutamate-like immunoreactive cells and were located in the lateral aspect of the PAG. These results demonstrate a specific and distinct distribution of glutamate and aspartate immunoreactive neurons and support recent data suggesting that glutamate and aspartate serve as excitatory neurotransmitters in the PAG.

Animals↗
PubMed1999

Selective determination of fish aspartate aminotransferase isoenzymes by their differential sensitivity to proteases.

Various proteases (proteinase K, subtilisin, trypsin and chymotrypsin) were used to study the selective inactivation of the aspartate aminotransferase (EC 2.6.1.1) isoenzymes of grey mullet (Mugil auratus Risso; Osteichthyes). The cytosolic isoenzyme was significantly inactivated by proteinase K, subtilisin and chymotrypsin, while the mitochondrial isoenzyme was sensitive only to proteinase K and to high doses of trypsin. Further identification of the aspartate aminotransferase isoenzymes was based on their discrete sensitivity toward chymotrypsin. Chymotrypsin (1 mg/ml) successfully inhibited purified cytosolic aspartate aminotransferase as well as cytosolic isoenzyme from plasma, whereas the mitochondrial form persisted unaffected. Similar results were obtained when examining liver and red muscle homogenates. This method revealed that the increased total activity of aspartate aminotransferase in fish plasma with induced acute liver injury, was partially a result of the mitochondrial isoenzyme leakage from damaged tissue.

Animals↗
PubMed1975

Partial purification and characterization of aspartate aminotransferases from seedling oat leaves.

As relatively little information is available on the properties of aspartate aminotransferase from photosynthetic tissue, isolation and characterization of the two major electrophoretically distinct forms of this enzyme from seedling oat leaf homogenates were undertaken. These two forms are designated I for the more anionic form and II for the less anionic form. Form I, 80 to 90% of the total activity, has been purified to a specific activity of 120 mumol/min/mg of protein (1100-fold) and is estimated to be 90 to 95% homogeneous, as judged by analytical polyacrylamide gel electrophoresis. Form II, 10 to 20% of the total activity, has been purified to a specific activity of approximately 6 mumol/min/mg of protein (300-fold). Both forms exhibit optimal activity at pH 7.5. Michaelis constants do not differ greatly between forms I and II and are similar to those reported for the pig heart cytosolic enzyme as well as aspartate aminotransferase from other plant sources. A molecular weight of 130,000 for the purified aspartate aminotransferase I was estimated by sedimentation equilibrium centrifugation; molecular weights of the two forms are similar as estimated by sucrose density gradient centrifugation. No activation by pyridoxal phosphate has been observed during purification.

Aspartate Aminotransferases↗
PubMed1994

Modulation of the activity of mitochondrial aspartate aminotransferase H352C by the redox state of the engineered interdomain disulfide bond.

Molecular modeling suggested that the large and small domain of mitochondrial aspartate aminotransferase might be linked by an engineered disulfide bond that could be expected to interfere with ligand-induced and syncatalytic changes in conformation and thus to assist in the elucidation of their significance for the catalytic mechanism. His-352, which is situated in the small domain close to Cys-166 of the large domain, was replaced with a cysteine residue by oligonucleotide-directed mutagenesis. Aspartate aminotransferase H352C, that had not been exposed to reducing conditions, in part contained a disulfide bond between Cys-166 and Cys-352. Exposure to a reducing agent cleaved the crosslink completely and produced an enzyme derivative with 8% of the activity of the wild type enzyme. Cu2+-mediated autoxidation resulted in complete formation of the disulfide bond and a decrease in enzymic activity to 2%. Independently of the redox state of the disulfide bond, the H352C substitution seems to shift the equilibrium from the open toward the closed conformation of the enzyme. This change in conformation was accompanied by an increase in the binding affinity for both the amino and oxo acid substrate by one order of magnitude. Apparently, 1-2 kcal/mol of the binding energy of the substrates are no longer diverted to shift the conformational equilibrium toward the closed conformation. The kcat/Km values were unchanged or even increased in the reduced form of the mutant enzyme and only slightly decreased in its oxidized form. Both the disulfide-independent decrease in enzymic activity, as observed in reduced aspartate aminotransferase H352C and also in two other mutant enzymes (C166H/H352C and H352Q), and the redox-dependent modulation of activity indicate that unhindered domain movements are essential for full catalytic competence of aspartate aminotransferase.

Animals↗
PubMed1979

Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver.

A method for the purification of mitochondrial isoenzyme of sheep liver aspartate aminotransferase (EC 2.6.1.1) is described. The final preparation is homogeneous by ultracentrifuge analyses and polyacrylamide-gel electrophoresis and has a high specific activity (182 units/mg). The molecular weight determined by sedimentation equilibrium is 87,100 +/- 680. The amino acid composition is presented; it is similar to that of other mitochondrial isoenzymes, but with a higher content of tyrosine and threonine. Subforms have been detected. On isoelectric focusing a broad band was obtained, with pI 9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. The Km for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 +/- 0.20 mM and 0.093 +/- 0.010 mM respectively; the corresponding values for the mitochondrial form were 0.40 +/- 0.12 mM and 0.98 +/- 0.14 mM. Cytoplasmic aspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25 mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH 7.4 inhibited cytoplasmic holoenzyme activity by up to about 60% and mitochondrial holoenzyme activity up to 40%. The apparent dissociation constants for pyridoxal 5'-phosphate were 0.23 micrometer (cytoplasmic) and 0.062 micrometer (mitochondrial) and for pyridoxamine 5'-phosphate they were 70 micrometer (cytoplasmic) and 40 micrometer (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37 degree C were 32 micrometer for the cytoplasmic isoenzyme and 19.5 micrometer for the mitochondrial form.

Amino Acids↗
PubMed1986

Experimental diabetes causes mitochondrial loss and cytoplasmic enrichment of pyridoxal phosphate and aspartate aminotransferase activity.

The streptozotocin diabetic rat was selected as a model to study how insulin deficiency alters vitamin B6 utilization by focusing on pyridoxal phosphate levels and aspartate aminotransferase activities in liver tissues. Diabetes of 15 weeks' duration lowered plasma pyridoxal phosphate levels by 84%. Normal plasma pyridoxal phosphate was 480 pmole/ml. Fractionation of liver into mitochondrial and extramitochondrial compartments demonstrated that diabetes caused a 43% diminution in mitochondrial pyridoxal phosphate per gram of liver. There was no cytoplasmic change in these diabetic rats. Mitochondrial aspartate aminotransferase activity was decreased 53% per gram of diabetic liver and cytoplasmic aspartate aminotransferase activity was elevated 3.4-fold. Damage to diabetic mitochondria during preparation procedures could not account for the rise in cytoplasmic aspartate aminotransferase activity. Electrophoresis showed that in the diabetic cytoplasm both cathodal and anodal forms of the enzyme were elevated. Speculations concerning mitochondrial loss and cytoplasmic gain of enzyme activity as well as those on the reduction of plasma pyridoxal phosphate in the diabetic rat are presented.

Animals↗
PubMedDate not supplied

Fluorescence polarization studies on the binding between glutamate dehydrogenase and cytoplasmic aspartate aminotransferase.

Polarization of fluorescence measurements on aspartate aminotransferase (from pig heart cytosol) labeled with fluorescein isothiocyanate have been used to detect the formation of a soluble complex of this protein wich glutamate dehydrogenase from bovine liver. The binding of the labeled transaminase to dehydrogenase is detectable at catalytic concentrations of the enzymes.

Animals↗
PubMed1991

[Crystallization of free aspartate aminotransferase from chicken heart cytosol].

Homogeneous aspartate aminotransferase (purity--99%, yield--70%) has been prepared from chicken heart cytosol. The purification procedure included fractionation with ammonium sulfate and ethanol and crystallization. Crystals (0.3 x 0.5 x 2 mm) of the free enzyme were prepared from ammonium sulfate solution and studied by X-ray analysis at 2.5 A resolution.

Ammonium Sulfate↗