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Results for “ASPARTATE AMINOTRANSFERASE”

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PubMed1998

A model for the nucleotide-binding domains of ABC transporters based on the large domain of aspartate aminotransferase.

ABC transporters are a large superfamily of integral membrane proteins involved inATP-dependent transport across biological membranes. Members of this superfamily play roles in a number of phenomena of biomedical interest, including cystic fibrosis (CFTR) and multidrug resistance (P-glycoprotein, MRP). Most ABC transporters are predicted to consist of four domains, two membrane-spanning domains and two cytoplasmic domains. The latter contain conserved nucleotide-binding motifs. Attempts to determine the structure of ABC transporters and of their separate domains are in progress but have not yet been successful. To aid structure determination and possibly learn more about the domain boundaries, we set out to model nucleotide-binding domains (NBDs) of ABC transporters based on a known structure. Previous attempts to predict the 3D structure of NBDs were based solely on sequence similarity with known nucleotide-binding folds. We have analyzed the sequences of a number of nucleotide-binding domains with the algorithm THREADER, developed by D.T. Jones, and a possible fold was found in the structure of aspartate aminotransferase. We present a model for the N-terminal NBD of CFTR, based on the large domain of the A chain of aspartate aminotransferase. The model is refined using multiple sequence alignment, secondary structure prediction, and 3D-1D profiles. Our model seems to be in good agreement with known properties of nucleotide-binding domains and has some appealing characteristics compared with the previous models.

ATP-Binding Cassette Transporters↗

PubMed1987

Heterogeneity of aspartate aminotransferase (AAT) in bull semen.

Polymorphism of non-genetic character was discovered in aspartate aminotransferase (AAT) of bull semen. A relationship was found between the enzyme heterogeneity and susceptibility of plasmatic membranes of spermatozoa to cryogenic damage. The relation changed with the bull's age.

Animals↗

PubMed1992

Further studies on aspartate aminotransferase of thermophilic methanogens by analysis of general properties, bound cofactors, and subunit structures.

Aspartate aminotransferase (AspAT) [EC 2.6.1.1] of thermophilic methanogen was further characterized with the enzyme from Methanobacterium thermoautotrophicum strain FTF-INRA as well as M. thermoformicicum strain SF-4. AspAT of strain FTF-INRA was similar in the amino donor specificity to the enzyme of M. thermoformicicum strain SF-4, in that it was active on L-cysteine and L-cysteine sulfinate in addition to L-glutamate and L-aspartate. The enzymes gave similar absorption spectra having maxima at around 326 and 415 nm with no pH-dependent shift but were found to contain 1 mol of tightly bound pyridoxal 5'-phosphate (PLP) per subunit. Reconstitution of each apoenzyme with added PLP resulted in partial recovery of the original enzymatic activity, suggesting a significant conformational change of the active site region upon removal of the cofactor. Polyacrylamide gel electrophoresis (PAGE) and gel filtration analyses revealed a tetrameric structure (180 kDa) of identical subunits with a molecular mass of 43 kDa for each of these enzymes. Electric current was found to affect the interaction or affinity of each subunit, promoting dissociation of the native enzyme into the monomeric form. Alkaline treatment was effective only for dissociation of the enzyme from strain SF-4. They were distinguishable by the more rapid reassociation of the monomer to the native aggregated form in the enzyme of strain FTF-INRA.

Aspartate Aminotransferases↗

PubMed1998

Macroenzyme investigation and monitoring in children with persistent increase of aspartate aminotransferase of unexplained origin.

Ten children with asymptomatic persistent cryptogenic increased serum levels of aspartate aminotransferase (AST) were screened for detection and monitoring of AST macroenzyme (macroAST). MacroAST was found in 4 patients; their serum AST levels were significantly higher than in those without biochemical evidence of macroAST (mean +/- SD: 515 +/- 433 and 78 +/- 16 IU/L, respectively; P = .0095). MacroAST was a persistent, benign phenomenon and was probably not congenital.

Aspartate Aminotransferases↗

PubMed1981

On the pyridoxal-5'-phosphate stimulation of aspartate aminotransferase and alanine aminotransferase in serum and erythrocytes of patients undergoing chronic haemodialysis and with kidney transplants.

Aspartate aminotransferase (AST) determinations in erythrocytes of patients undergoing chronic haemodialysis and with kidney transplants showed that patients receiving vitamin B6 had a smaller relative stimulation rate of AST by pyridoxal-5'-phosphate (P-5-P). In contrast to these results in erythrocytes, the apoAST and apo-alanine aminotransferase (ALT) activities in serum were increased in patients as compared with those of healthy persons. The corresponding relative stimulation rates of AST and ALT by P-5-P addition to the reaction mixture were not changed in the haemodialysis patients, but in renal transplant recipients the relative stimulation rate of AST was significantly smaller and that of ALT was greater.

Alanine Transaminase↗

PubMed1992

Brønsted analysis of aspartate aminotransferase via exogenous catalysis of reactions of an inactive mutant.

Primary amines functionally replace lysine 258 by catalyzing both the 1,3-prototropic shift and external aldimine hydrolysis reactions with the inactive aspartate aminotransferase mutant K258A. This finding allows classical Brønsted analyses of proton transfer reactions to be applied to enzyme-catalyzed reactions. An earlier study of the reaction of K258A with cysteine sulfinate (Toney, M.D. & Kirsch, J.F., 1989, Science 243, 1485) provided a beta value of 0.4 for the 1,3-prototropic shift. The beta value reported here for the transamination of oxalacetate to aspartate is 0.6. The catalytic efficacy of primary amines is largely determined by basicity and molecular volume. The dependence of the rate constants for the reactions of K258A and K258M on amine molecular volume is nearly identical. This observation argues that the alkyl groups of the added amines do not occupy the position of the lysine 258 side chain in the wild type enzyme. Large primary C alpha and insignificant solvent deuterium kinetic isotope effects with amino acid substrates demonstrate that the amine nitrogen of the exogenous catalysts directly abstracts the labile proton in the rate-determining step.

Acetates↗

PubMed2006

Regulation of 2-oxoglutarate metabolism in rat liver by NADP-isocitrate dehydrogenase and aspartate aminotransferase.

Kinetic and regulatory properties of NADP-isocitrate dehydrogenase (NADP-IDH) and aspartate aminotransferase (AsAT) responsible for 2-oxoglutarate metabolism in the cytoplasm and mitochondria of rat liver were studied. Based on the subcellular location of these enzymes and their kinetic parameters (Km, Ksi) obtained with highly purified enzyme preparations, it is suggested that synthesis of 2-oxoglutarate should be mainly determined by cytoplasmic NADP-IDH (86% of the total activity in the cell), whereas its utilization should depend on cytoplasmic AsAT (78% of the total activity). AsAT from the rat liver was specified by substrate inhibition and also by changes in the enzyme affinity for the substrates under the influence of some intermediates of the tricarboxylic acid cycle: isocitrate, succinate, fumarate, and citrate. Key intermediates of nitrogen metabolism (glutamate, glutamine, and aspartate) are involved in the regulation of NADP-IDH and AsAT. These enzymes are regulated oppositely, and the catalytic activity of one enzyme can be stimulated concurrently with a decrease in the activity of the other. Obviously, carbon and nitrogen metabolism in the rat liver can be controlled through redistribution of 2-oxoglutarate between different metabolic processes via regulatory mechanisms influencing differently located forms of NADP-IDH and AsAT.

Animals↗

PubMed1986

[Catalytic properties of aspartate aminotransferase].

Analysis of Michaelis--Menten kinetics revealed that the enzyme in solution and the crystalline cytosolic aspartate aminotransferase (EC 2.6.1.1) possess a functional nonequivalence of active sites of the enzyme dimer for two substrates--aspartate and 2-oxoglutarate.

Animals↗

PubMed1987

Alanine and aspartate aminotransferase serum levels in burned patients: a long-term study.

Increased alanine and aspartate aminotransferase (ALT and AST) serum levels are usually considered expressions of cellular necrosis, especially in hepatocytes. They represent cellular damage due to burn which, according to many authors, becomes normal before discharge of patients. We studied 43 consecutive burned patients, both during and after recovery, from a minimum of 120 to a maximum of 640 days, and an average of 18.62 blood samples were taken from each patient. Hepatitis A and B markers were tested. Results showed a 67.44% increase in aminotransferases in patients during recovery and a 25.58% increase after discharge. No neopositivity was observed for hepatitis A and B markers. We therefore conclude that the increase of enzymes during recovery expresses a toxic-infective phase and this increase, contrary to what was believed, does not always drop to normal values at time of discharge. Instead, after discharge, higher values can be a manifestation of a Non-A Non-B hepatitis.

Alanine Transaminase↗

PubMed1993

Purification of aspartate aminotransferase from Thermus aquaticus.

A method is described for the purification of the enzyme aspartate aminotransferase from the thermophile Thermus aquaticus. The enzyme has been characterized with respect to its molecular weight on SDS PAGE and by amino acid analysis. Attempts to obtain N-terminal sequence data was unsuccessful, presumably due to a blocked N-terminus. The purified enzyme has been shown to be highly thermostable, having a half life of inactivation of about 6 hours at 100 degrees C, and to have a temperature optimum greater than 95 degrees C.

Amino Acids↗

PubMed1977

Kinetics of soluble and collagen-bound aspartate aminotransferase: diffusional effects with a two-substrate enzymatic reaction.

The kinetic properties of aspartate aminotransferase covalently bound to collagen are compared to those of the free enzyme. In the bound state, the enzyme exhibits a greater affinity for glutamate, but a lower affinity for oxalacetate. In order to assess precisely the contribution of diffusional limitations on the heterogeneous enzyme kinetics, a simple modeling of diffusional effects on a two-substrate enzymatic reaction is developed. According to this quantitative analysis, diffusional limitations for oxalacetate alone account for the increased and decreased enzyme affinities toward its two substrates. Consequently, coupling of the enzyme to collagen does not significantly affect its intrinsic kinetic properties.

Aspartate Aminotransferases↗

PubMed1990

Prolactin directly stimulates citrate production and mitochondrial aspartate aminotransferase of prostate epithelial cells.

Prolactin, in vitro, significantly increased citrate production, mAAT (mitochondrial aspartate aminotransferase) and pmAAT (precursor form of mAAT) activity of prostate epithelial cells derived from rat lateral prostate (LP) and pig prostate cultures. In contrast, prolactin had no effect on the cytosolic isozyme, cAAT. This prolactin effect appeared to be independent of testosterone. The phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate) induced the same effects as prolactin thereby indicating the involvement of protein kinase C. This report demonstrates that prolactin directly regulates citrate production of prostate epithelial cells and the availability of an in vitro model to elucidate the mechanism of action of prolactin.

Animals↗

PubMed1984

The complete amino acid sequence of aspartate aminotransferase from Escherichia coli: sequence comparison with pig isoenzymes.

The amino acid sequence of aspartate aminotransferase from E. coli B was determined by the alignment of seven cyanogen bromide peptides. The established sequence of the subunit was composed of 396 amino acid residues, and the molecular weight was calculated to be 43,573. The sequence was compared with those of the pig cytoplasmic and mitochondrial isoenzymes, showing that nearly 30% of all residues were invariant and that the E. coli enzyme exhibited the same degree of homology (about 40%) with either of them. Although majority of the residues were substituted, the functional residues constituting the active site structure were conserved.

Amino Acid Sequence↗

PubMed1975

Role of tryptophan, histidine and methionine residues in the catalytic activity of mitochondrial aspartate aminotransferase from beef kidney.

The role of tryptophan, methionine, and histidine residues in mitochondrial aspartate aminotransferase from beef kidney has been established by using N-bromosuccinimide, 2-hydroxy-5-nitrobenzylbromide, and tetraiodofluoresceine as specific chemical modifiers of the amino acid residues of the enzyme. Since N-bromosuccinimide promotes extensive inactivation of the enzyme and the chemical modification of 1.65 tryptophan and 3 methionine residues per enzymes protomer, 2-hydroxy-5-nitrobenzylbromide modifies once more 1.65 tryptophan residues per enzyme protomer but induces only 10% inactivation of the enzyme. Tetraiodofluoresceine exerts a 40% inactivation of the enzyme which is due to the chemical modification of 5.8 histidine res in

Animals↗

PubMed1966

Purification and general properties of aspartate aminotransferase of ox heart.

1. A five-step procedure for preparing highly purified aspartate aminotransferase from ox heart is described. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis in starch gel and in polyacrylamide gel. 3. The pure enzyme has an isoelectric point of about pH5, and E(1%) (1cm.) 14.40 at 278mmu. 4. The molecular weight of the pure enzyme was determined as 96000 by sedimentation equilibrium. 5. The pH optimum for the pure enzyme was about 8. It was determined by a new assay technique. 6. A difference in the electrophoretic migration rate between the enzyme from ox heart and brain and the enzyme from pig heart and brain suggests a species specificity rather than an organ specificity. 7. A new effect of deionization on the visible-absorption spectrum of the enzyme was observed.

Animals↗

PubMed1991

Rapid purification and thermostability of the cytoplasmic aspartate aminotransferase from carrot suspension cultures.

Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. In mammalian systems, there are two forms of AAT, a heat-stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography.

Journal Article↗

PubMed1987

Testosterone stimulation of mitochondrial aspartate aminotransferase levels and biosynthesis in rat ventral prostate.

The effects of testosterone on mitochondrial aspartate aminotransferase (mAAT) synthesis in rat ventral prostate was investigated. Procedures for the isolation, purification and characterization of AAT isozymes were developed and described. Purified mAAT preparations contained no demonstrable contaminating proteins. Prostatic mAAT was characterized as a cationic protein with an estimated mol. wt of 120,000. Cytoplasmic AAT (cAAT) isozyme was identified as an anionic protein with an estimated mol. wt of 132,000. A cytosolic cationic isozyme, similar to mAAT, was also identified as pre-mAAT. Testosterone administration to castrated rats resulted in significant increases in leucine incorporation into mAAT, in the level of mAAT, and in mAAT activity. These effects of testosterone were observed within 2 h of administration. Conversely, testosterone administration had none of these effects on cAAT or on non-AAT protein pool. Testosterone treatment did appear to increase leucine incorporation into pre-mAAT. Testosterone treatment in organ cultures and in prostate epithelial cell cultures resulted in the same stimulatory effects on mAAT as observed in the in vivo studies. The hormone was effective at the physiological concentration of 2 X 10(-9) M. These results indicated that testosterone has a rapid and specific effect on the biosynthesis of mAAT. This continues to support our proposal that testosterone regulates prostate citrate production via a stimulatory effect on mAAT which results in increased mitochondrial synthesis of citrate from aspartate.

Animals↗

PubMed1987

[31P-NMR spectra of aspartate aminotransferase from cytosol of the chicken heart].

31P NMR spectra of the cytosolic chicken aspartate aminotransferase have been recorded at 161.7 MHz in the pH range of 5.7 to 8.2. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; difference in the chemical shift at pH 5.7 and 8.2 is only 0.35 ppm. The monoanion-dianion transition of 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein--bound coenzyme is in dianionic form throughout the investigated pH range; the small pH-dependent change of chemical shift may be due to a protein conformational change that affects O-P-O bond angle. In the presence of the 0.1 M succinate, 31P chemical shift of the enzyme remains constant in the pH range of 5.0 to 8.3.

Animals↗