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PubMed1997

A comparative study of the thermal inactivation of cytosolic and mitochondrial aspartate aminotransferases.

Rates of irreversible thermal inactivation of cytosolic and mitochondrial aspartate aminotransferases were measured over a large temperature range. Inactivation occurred by different kinetic pathways at high and low temperature with a transition point at about 60 degrees C. This suggests that the isoenzymes exist in different conformations above and below that temperature. Discontinuities in plots of ln(Vmax) against 1/T provided confirmatory evidence for this hypothesis. Activation parameters (deltaH and deltaS) for the thermal inactivation process were calculated in the high and low temperature ranges. At high temperature the greater rate of inactivation of the mitochondrial isoenzyme is determined largely by a high value of deltaS. This more than compensates for the fact that the deltaH is also greater for the mitochondrial isoenzyme indicative of greater intramolecular stabilising interactions compared with the cytosolic form. Thus the relative rates of inactivation are determined by the nature of the transition states rather than by intramolecular interactions in the folded proteins. At lower temperatures the kinetic stabilities of the isoenzymes reverse with the mitochondrial isoenzyme inactivating more slowly. This is largely because of a considerably smaller deltaS at low temperature which no longer compensates for the greater deltaH compared with the cytosolic isoenzyme.

Animals↗

PubMed1990

Properties of human liver cytosolic aspartate aminotransferase mRNAs generated by alternative polyadenylation site selection.

Human cytosolic aspartate aminotransferase (cAspAT) cDNA clones have been isolated from an adult human liver cDNA library. Among the clones, two cDNAs of 1550 and 1950 base pairs, respectively, have been characterized. These two cDNAs differ only in the lengths of their 3' noncoding regions and by the presence of one or two putative polyadenylation signals AATAAA. Northern blot analysis revealed two different mRNAs of 2.1 and 1.8 kbp in several human tissues, whereas Southern blot analysis suggested the existence of a single gene for the human cAspAT. The two mRNA species result from the alternative use of two polyadenylation signals. In the liver, the relative ratio of these mRNAs varies among different species and, in humans at least, during development. The properties of the two mRNAs were compared. The half-lives of the 2.1 and 1.8 kbp mRNAs, in the HepG2 cell line, are 8 and 12 h, respectively. The two mRNAs have similar and rather short poly(A) tracts of 20-50 nucleotides. Both mRNAs are capable of directing the in vitro synthesis of the cAspAT protein. We conclude that both the 2.1 and 1.8 kbp cAspAT mRNAs are functional and exhibit similar properties.

Amino Acid Sequence↗

PubMed1998

Ethanol up-regulates fatty acid uptake and plasma membrane expression and export of mitochondrial aspartate aminotransferase in HepG2 cells.

To explain the increased plasma mitochondrial aspartate aminotransferase (mAspAT) observed in alcoholics, we cultured HepG2 hepatoma cells in ethanol. Acute (24 hour) exposure to 0, 20, 40, or 80 mmol/L ethanol produced a dose-dependent (r = .98) increase in mAspAT messenger RNA (mRNA) of < or = thirteen-fold, with no significant change in the cellular content of mAspAT or of several other enzymes. The recovery of mAspAT in the medium over 24 hours of ethanol exposure correlated with both ethanol concentration and with mAspAT mRNA (r = .90), reaching 808% of cellular enzyme content/24 hours at 80 mmol/L. Recovery of all other enzymes studied was < or = 20% of cellular content and unaffected by ethanol. Plasma membrane mAspAT content also correlated with mAspAT mRNA (r = .96) and mitochondrial levels were unchanged. No mitochondrial morphologic abnormalities were observed at any ethanol concentration studied. In cells cultured chronically at 0 to 80 mmol/L ethanol, fatty acid uptake Vmax increased in parallel with plasma membrane expression of mAspAT (r = .98). Cellular triglyceride content was highly correlated with Vmax. Thus, the data suggest that: 1) the increased plasma mAspAT observed in alcoholics may reflect pharmacologic upregulation of mAspAT mRNA and of mAspAT synthesis by ethanol; and 2) increased mAspAT-mediated fatty acid uptake may contribute to alcoholic fatty liver.

Aspartate Aminotransferases↗

PubMed1987

Activity of serum aspartate aminotransferase isoenzymes in patients with acute myocardial infarction.

Activities of aspartate aminotransferase (AST) isoenzymes were determined in serial serum samples from 40 cases of acute myocardial infarction, and compared with activities of creatine kinase, CK-MB isoenzyme, lactate dehydrogenase, and alpha-hydroxybutyrate dehydrogenase for temporal changes. Cytosolic (soluble) AST (s-AST) and mitochondrial AST (m-AST) respectively increased 6.6 and 9.0 h after onset of chest pain. The median time at which serum m-AST activity peaked (15.8 U/L, range 6.4-53.5 U/L) was 47.8 h after the onset of infarction, 19.8 h later than the peak s-AST activity (171 U/L, range 53-517 U/L) and m-AST also disappeared from the serum more slowly than s-AST (p less than 0.001). Serum m-AST values were above normal for at least six days after the infarct. The ratio of m-AST to total AST in serum increased after myocardial infarction, being greatest (20%, range 11-32%) on the third day after onset. For individuals, peak activities of s-AST correlated well with total CK (r = 0.91) and CK-MB (r = 0.86) peak activities, indicating that s-AST also reflects the infarct size. However, m-AST correlated poorly with the enzymes commonly used in infarct diagnosis; it apparently provides different biological information.

Adult↗

PubMed1993

Influences of some low molecular compounds on enzymatic activity and isoelectric point of aspartate aminotransferase from rat liver.

The substances responsible for regulating cytosolic aspartate aminotransferase (AspATc) activity in the cytosolic fraction of rat liver were examined. AspATc was removed from the cytosolic fraction by passing the fraction through an affinity column to which anti-AspATc antiserum was conjugated. The unbound fraction from the column was found to decrease the activity of the purified AspATc. A fraction containing compounds of less than MW 1,000 was obtained by filtering the cytosolic fraction through a YM 2 membrane. This YM 2 filtrate decreased the activity of the purified enzyme; however, the enzymic activity was protected partially by the addition of 2-oxoglutarate or pyridoxal phosphate (PLP). The YM 2 filtrate also decreased the isoelectric points (pIs) of the purified enzyme. Influences of glucose and fructose on AspATc were examined, and fructose was found to decrease the enzymic activity and the pIs. Fructose was more effective on apoenzyme than holoenzyme, suggesting that fructose may bind to the Lys258 residue of AspATc which is the binding site of PLP. The effects of various amino acids including substrates on the enzymic activity were also examined. Some amino acids were found to decrease the enzymic activity to various extents, though the pIs were unaltered. These results suggest that under physiological conditions, AspATc activity is modified by various low molecular substances in various ways.

Amino Acids↗

PubMedDate not supplied

[Study of the reorientation of coenzyme in active sites of aspartate-aminotransferase isoenzymes using linear dichroism method].

Cytosolic and mitochondrial pig aspartate aminotransferases (cAAT and mAAT) and chicken cAAT were oriented in a compressed slab of polyacrylamide gel. Linear dichroism (LD) spectra of the pyridoxal and pyridoxamine forms of AATs and of complexes of the pyridoxal form with substrate analogues have been recorded. The tilt angles of the coenzyme at the intermediary steps of the transamination reaction have been calculated on the basis of reduced LD values (delta A/A), atomic coordinates of the coenzyme and directions of the transition dipole moments in the coenzyme ring. It was assumed that rotation of the coenzyme ring occurs around the C2-C5 axis in all cases except the enzyme complex with glutarate: in the latter case the direction N1-C4 was assumed to be a rotation axis. It has been found that formation of the enzyme complex with glutarate and protonation of the internal aldimine induce dissimilar reorientations of the coenzyme. As a result of protonation, the coenzyme tilts by 27 degrees in cAAT and 13 degrees in mAAT. Formation of the external aldimine with 2-methylaspartate is accompanied by tilting of the coenzyme ring by 44 degrees in cAAT and 39 degrees in mAAT. For the quinonoid complex with erythro-3-hydroxyaspartate, the tilt angles were found to be 63 degrees in cAAT and 53 degrees in mAAT. It was inferred that the basic features of the active site dynamics are similar in three AATs studied. The differences in the coenzyme tilt angles between cAAT and mAAT might be linked to catalytic peculiarities of the isoenzymes.

Animals↗

PubMed1983

Production and characterization of an antibody to cytosolic aspartate aminotransferase and immunolocalization of the enzyme in rat organs.

Cytosolic aspartate aminotransferase (c-AAT) was purified to homogeneity from porcine heart and immunized to rabbit for production of antiserum. The purity of this enzyme protein and the specificity of its antibody were judged by silver-stained-sodium dodecyl sulfate slab gel, Western blot transfer technique, and double immunodiffusion. The antibody against porcine heart c-AAT was found to cross-react with rat c-AAT but not with nine other different enzymes from the heart, liver, and muscle. Affinity purified antibody was used to localize this isoenzyme in the rat heart, liver, kidney, and cerebellum by indirect immunoperoxidase method. It was found that, in the rat heart muscle, c-AAT reaction product was present as a linear structure parallel to the muscle fiber and along the sarcolemma. Some cardiac muscle fibers contain more reaction products than the others. In the liver, reaction product was seen unevenly distributed in the hepatocytes. The Kupffer cells and endothelia were less stained. Most of the tubular epithelia of the loop of Henle in the kidney were intensely stained. But other tubular epithelia including convoluted and collecting tubules were sporadically and less stained. The basket and stellate cells and their neuronal processes and terminals in the cerebellum were markedly stained, but the Purkinje and granule cell bodies were weakly stained. For comparison of the staining intensity with enzyme activity in each organ, the c-AAT enzyme activity was simultaneously determined in those organs. This study indicates that the presence of c-AAT is specific in different organs and tissues.

Animals↗

PubMed2004

Genetic control of aspartate aminotransferase isoenzymes in Aegilops and Triticum species.

Zymograms of the aspartate aminotransferase (AAT, EC 2.6.1.1) activity in leaf extracts from Aegilops and Triticum species revealed three AAT zones, denoted according to the decreasing electrophoretic mobility towards the anode as AAT-1, AAT-2 and AAT-3. The AAT activity zymograms of subcellular fractions isolated from T. aestivum seedlings made it possible to establish that the AAT-1 zone is located in the mitochondria, AAT-2 in the chloroplasts and AAT-3 in the cytoplasm. Most of the total AAT activity from wheat leaves arises from the chloroplasts and cytoplasm. The AAT-3 zone exhibited the lowest electrophoretic mobility, but 3 isoenzymes occurring within were the most visibly separated. The occurrence of a single band in this zone at the AAT-3a position (closest to the anode) for the aneuploid CS3ASDt AABBDD line (the absence of long arms of the 3rd pair of homologous chromosomes in the A genome) and at the AAT-3c position for Ae. umbellulata (genome UU), as well as three bands in the whole zone for T. durum (AABB) and T. aestivum (AABBDD) each, made it possible to evaluate the subunit composition of isoenzymes in the AAT-3 zone. The band at the AAT-3a position in the zymogram is formed from bb dimers, AAT-3b from ab and AAT-3c from aa. By comparing the distribution of isoenzyme bands intensities (the result of enzymatic activity) with the mathematical models, the frequencies of the occurrence of the a and b subunits within AAT-3 zone were evaluated. In AAT-3 from T. durum, a and b occurred at the ratio of 0.54:0.46, and in that from T. aestivum - 0.62:0.38, respectively.

Aspartate Aminotransferases↗

PubMed1994

Post-translational modifications in aspartate aminotransferase from Sulfolobus solfataricus. Detection of N-epsilon-methyllysines by mass spectrometry.

Advanced mass spectrometric procedures have been extensively used to provide an accurate structural characterization of aspartate aminotransferase from Sulfolobus solfataricus. The amino acid sequence of this enzyme had previously been deduced from the DNA sequence. The accurate molecular mass of the protein, determined using electrospray mass spectrometry, demonstrated that the amino acid sequence deduced was correct and ruled out the possible presence of large covalent modifications which had been postulated to fit the much higher molecular mass obtained from previous SDS/PAGE experiments. The definition of the entire primary structure of aspartate aminotransferase from S. solfataricus was achieved by exploiting a new mass spectrometric mapping strategy. Initially, the molecular mass of relatively large protein fragments produced by CNBr hydrolysis was accurately determined using electrospray mass spectrometry. The protein regions where structural modifications had occurred were easily identified from their anomalous mass values. The corresponding CNBr fragments were then subdigested with suitable proteases and the resulting peptide mixtures were analysed by fast-atom-bombardment mass spectrometry. This mapping approach led to the detection of two partially modified lysine residues at positions 202 and 384, which had been converted to their N-epsilon-methyl derivatives to a substoichiometric extent.

Amino Acid Sequence↗

PubMed1986

Selective proteolysis of cytosolic aspartate aminotransferase by a new microbial protease.

A protease from Streptomyces violaceochromogenes (Murao, S., Nishino, Y., & Maeda, Y. (1984) Agric. Biol. Chem. 48, 2163-2166) is known to inactivate pig heart aspartate aminotransferase [EC 2.6.1.1]. Chemical analysis of the core proteins and peptide fragments produced upon proteolysis of the aminotransferase revealed that peptide bond cleavage occurred specifically at Leu 20 with concomitant inactivation. Neither inactivation nor peptide bond cleavage was observed with the mitochondrial isoenzyme. The proteolytically produced derivative 21-412 of the cytosolic isoenzyme retained approximately 0.1% enzymic activity for transamination with natural dicarboxylic substrates. The pyridoxal form of the derivative 21-412 was fully converted by cysteinesulfinate or alanine to the pyridoxamine form and conversely the pyridoxamine form of the derivative was also fully converted by 2-oxoglutarate or pyruvate into the pyridoxal form, indicating that the derivative was still catalytically competent. However, the rates of reaction with dicarboxylic substrates were much reduced whereas the rates with monocarboxylic substrates remained at an order of magnitude similar to that observed with the native enzyme. Thus the NH2-terminal segment appears to be an import structural component which determines the substrate specificity of aspartate aminotransferase for dicarboxylic keto and amino acids. A substantial alteration in the molecular structure accompanying the loss of the NH2-terminal 20 residues was also reflected by the decrease in heat stability and in the lowering of the pKa value for His 68, which is involved in the intersubunit interaction of this dimeric enzyme.

Animals↗

PubMed1978

An interlaboratory study of measurement of aspartate aminotransferase activity with use of purified enzyme materials.

The Center for Disease Control (CDC), the New York State Department of Health (NYSDH), the College of American Pathologists, and 23 manufacturers of diagnostic products participated in an interlaboratory study of aspartate aminotransferase (EC 2.6.1.1) methodologies. Six different lyophilized materials were prepared and characterized and then distributed to 293 laboratories for aspartate aminotransferase measurements. The specimens included one human serum; four catalytic concentrations of the cytoplasmic isoenzyme, two purified from human erythrocytes, and two from porcine heart; and one matrix bovine serum albumin (30 g/liter) blank. The purified isoenzymes were prepared in the matrix. We present data on Michaelis parameters (Km and Vmax), Arrhenius plots, activation with pyridoxal 5-phosphate, vial-to-vial variability, and stability on reconstitution. The 281 responses showed that most of the laboratories used NADH-detection methods (91.1%), monitored at 340 nm (79.4%), and reported results in U/liter (89.4%). The percentage of laboratories reporting use of reaction temperatures of 30 and 37 degrees C was evenly divided, i.e., 42.7 and 42%, respectively. Analytical values reported by participating laboratories were categorized by reporting temperature, instrument, and method. Results were most consistent for a selected group of laboratories that supplemented optimized reaction solutions with pyridoxal 5-phosphate.

Aspartate Aminotransferases↗

PubMed1982

Aspartate aminotransferase immunoreactivity in cochlea of guinea pig.

The distribution of aspartate aminotransferase-like immunoreactivity in the cochlea of the guinea pig was studied at the light microscopy level. Indirect immunofluorescence histochemistry using antisera against cytoplasmic aspartate aminotransferase prepared from pig heart was applied to surface preparations of the organ of Corti and cryostat sections of the cochlea. In the modiolus, immunofluorescence was localized to spiral ganglion cells and myelinated fibers of the auditory nerve and intraganglionic spiral bundles. In the organ of Corti, immunofluorescence was seen in upper tunnel crossing fibers and at the base of outer hair cells, following a distribution similar to that of the efferent innervation of the outer hair cells. Weak immunofluorescence was seen in the inner spiral bundle and tunnel spiral bundle, but was not present in all preparations. Immunofluorescence was not seen in inner hair cells, nor at the base of inner hair cells, and may have been absent from outer hair cells. It is concluded that spiral ganglion cells and myelinated auditory nerve axons contain aspartate aminotransferase-like immunoreactivity such immunoreactivity has previously been determined in auditory nerve endings inthe cochlear nucleus. Olivocochlear neurons that innervate outer hair cells also contain such immunoreactivity while other cochlear efferents contain little or none.

Animals↗

PubMed1980

Stereospecificity of sodium borohydride reduction of Schiff bases at the active site of aspartate aminotransferase.

Sodium boro[3H]hydride treatment of holoaspartate aminotransferase results in the reduction of the Schiff's base formed between pyridoxal phosphate and Lys 258. Treatment of the reduced enzyme with papain followed by acid hydrolysis liberates epsilon-N-[3H]pyridoxyl lysine which is degraded to [3H]pyridoxamine diHCl and stereochemically analyzed with apoaspartate aminotransferase. Sodium boro[3H]hydride treatment of active site carbamylated aspartate aminotransferase reconstituted with pyridoxyl phosphate and sodium aspartate results in the trapping of an enzyme x substrate complex through the reduction of the Schiff's base formed between pyridoxal phosphate and aspartate. Active site bound N-[3H]pyridoxyl aspartate is liberated by treatment with papain and degraded to [3H]pyridoxamine diHCl for stereochemical analysis. Borohydride reduction of the holoenzyme occurs from the re face of the pyridoxal phosphate Lys 258 Schiff's base. Similarly, reduction of active site carbamylated enzyme x substrate complex occurs from the re face of the pyridoxal phosphate-aspartate Schiff's base. These results indicate that when active site carbamylated enzyme binds substrate to pyridoxal phosphate it does so stereospecifically and without changing the face of the Schiff base that is available for reduction as compared to native enzyme.

Animals↗

PubMed1998

Chemical anatomy of excitatory endings in the dorsal cochlear nucleus of the rat: differential synaptic distribution of aspartate aminotransferase, glutamate, and vesicular zinc.

In order to identify cytochemical traits relevant to understanding excitatory neurotransmission in brainstem auditory nuclei, we have analyzed in the dorsal cochlear nucleus the synaptic distribution of aspartate aminotransferase, glutamate, and vesicular zinc, three molecules probably involved in different steps of excitatory glutamatergic signaling. High levels of glutamate immunolabeling were found in three classes of synaptic endings in the dorsal cochlear nucleus, as determined by quantitation of immunogold labeling. The first type included auditory nerve endings, the second were granule cell endings in the molecular layer, and the third very large endings, better described as "mossy." This finding points to a neurotransmitter role for glutamate in at least three synaptic populations in the dorsal cochlear nucleus. The same three types of endings enriched in glutamate immunoreactivity also contained histochemically detectable levels of aspartate aminotransferase activity, suggesting that this enzyme may be involved in the synaptic handling of glutamate in excitatory endings in the dorsal cochlear nucleus. There was also extrasynaptic localization of the enzyme. Zinc ions were localized exclusively in granule cell endings, as determined by a Danscher-selenite method, suggesting that this ion is involved in the operation of granule cell synapses in the dorsal cochlear nucleus.

Animals↗

PubMed2002

Structural characterization of the M* partly folded intermediate of wild type and P138A aspartate aminotransferase from Escherichia coli.

A combination of spectroscopic techniques, hydrogen/deuterium exchange, and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the topology of the monomeric M* partly folded intermediate of aspartate aminotransferase from Escherichia coli in wild type (WT) as well as in a mutant form in which the highly conserved cis-proline at position 138 was replaced by a trans-alanine (P138A). Fluorescence analysis indicates that, although M* is an off-pathway intermediate in the folding of WT aspartate aminotransferase from E. coli, it seems to coincide with an on-pathway folding intermediate for the P138A mutant. Spectroscopic data, hydrogen/deuterium exchange, and limited proteolysis experiments demonstrated the occurrence of conformational differences between the two M* intermediates, with P138A-M* being conceivably more compact than WT-M*. Limited proteolysis data suggested that these conformational differences might be related to a different relative orientation of the small and large domains of the protein induced by the presence of the cis-proline residue at position 138. These differences between the two M* species indicated that in WT-M* Pro138 is in the cis conformation at this stage of the folding process. Moreover, hydrogen/deuterium exchange results showed the occurrence of few differences in the native N(2) forms of WT and P138A, the spectroscopic features and crystallographic structures of which are almost superimposable.

Amino Acid Sequence↗

PubMed1993

The DNA sequence analysis of the HAP4-LAP4 region on chromosome XI of Saccharomyces cerevisiae suggests the presence of a second aspartate aminotransferase gene in yeast.

The nucleotide sequence of a 19,000 base pair region from the left arm of chromosome XI of Saccharomyces cerevisiae has been determined and analysed. It covers the HAP4-GFA1-LAP4 loci already described. As expected HAP4, GFA1 and LAP4 genes have been found and six new open reading frames (ORFs) with a coding capacity of more than 100 amino acid residues have been identified. One of them (YKL461) shows a high degree of identity with an aspartate aminotransferase gene. This raises the question of a second aspartate aminotransferase gene in yeast. A second ORF (YKL462) shows features compatible with a membranous localization. The other ORFs do not show a similarity with any known gene. A member of the highly repetitive 'CAT' DNA sequence is present.

Aspartate Aminotransferases↗

PubMed2004

Isolation, purification, and crystallization of aspartate aminotransferase from wheat grain.

A procedure for isolation and purification of aspartate aminotransferase from wheat grain includes chromatography on DEAE cellulose, acidification-alkalization, precipitation with protamine sulfate, fractionation with ammonium sulfate, and chromatography on hydroxyapatite. The yield of protein was 27% with 95% purity. Crystals of the enzyme (0.05 x 0.025 x 0.015 mm3) were obtained from ammonium sulfate solution.

Aspartate Aminotransferases↗

PubMed1990

Alanine and aspartate aminotransferase activities in muscles of diabetic rats.

Activities of alanine and aspartate aminotransferase in different muscle types and in the liver of streptozotocin diabetic rats were studied 1,2 and 3 days after administering of streptozotocin. It was shown that the activity of both enzymes was elevated in the "white" layer of the vastus lateralis, in the liver and in the heart, whereas it remained unchanged in the "red" layer of the same muscle, in the soleus and the diaphragm. It is concluded that the effect of acute insulin deficiency on the aminotransferase activity in skeletal muscles depends on the muscle fiber composition and does not appear in muscles with a high oxidative potential. These results indicate that muscle fiber composition should be taken into account when evaluating the role of insulin in amino acid metabolism in the muscle.

Alanine Transaminase↗