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PubMed1985

The separate effects of coenzyme components may not be additive. Roles of pyridoxal and inorganic phosphate in aspartate aminotransferase apoenzymes.

Both cytosolic and mitochondrial aspartate transaminase can be resolved of pyridoxal phosphate. The resulting apoenzymes still bind individual structural components of the coenzyme. The separate contributions of coenzyme components to protein thermal stability have been independently assessed for phosphate ions (Pi) and for the pyridoxal or pyridoxamine components of the coenzyme. 31P NMR and differential scanning calorimetry reveal that the thermodynamic contributions of binding are not additive and are dissimilar for the two isozymes. High and low affinity sites for Pi binding are present in both apoenzymes with only the low affinity site being present in the holoenzyme forms. The contribution of both bound phosphates to increasing temperatures (Tm) and enthalpies (delta Hd) of denaturation differ between the isozymes and within sites. In either isozyme occupancy of the high affinity site by Pi produces only a 4- or 5- degree increase in the Tm value with respect to Pi-free apoenzyme. By contrast, in the mitochondrial apoenzyme, the presence of Pi at the second low affinity site increases the calorimetric parameters from Tm = 47 degrees C and delta Hd = 4.7 cal g-1 to Tm = 62 degrees C and delta Hd = 7 cal g-1. For cytosolic apoenzyme the respective changes are from 66 to 69.5 degrees C and 5.2 to 5.8 cal g-1. Addition of pyridoxal, but not pyridoxamine, displaces the high affinity Pi in both apoenzymes. This shows that the pyridine ring and Pi groups of pyridoxal-P bind exclusive of each other when they are not covalently linked as an ester, as in the coenzyme. The observation has been exploited as a method to prepare completely dephosphorylated mitochondrial apoenzyme. Electrostatic effects, structural differences in the phosphate binding pockets, and steric effects can be invoked to account for the Pi and pyridine binding behavior in the two proteins.

Animals↗
PubMed1999

Effect of NMDA antagonists on the activity of glutaminase and aspartate aminotransferase in the developing rat cerebellum.

Chronic treatment of rats from postnatal day 6 to 25 with drugs that interact with the N-methyl-D-aspartate (NMDA) receptor induced a differential effect on the activity of some enzymes involved in neurotransmitter synthesis. Two of these drugs ((5R,10S)-(+)-5-methyl-10,11 -dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate (MK-801) and 3-(2-carboxypiperazin-4-yl)propyl-1phosphonic acid (CPP)) caused a marked reduction (20-40%) of glutaminase and aspartate aminotransferase activity in the cerebellum. These changes were observed only at a very precise time of development (i.e. 10 to 19 postnatal day). The competitive antagonist, amino phosphonovaleric acid (APV), did not affect any of the enzymes studied at all tested ages. When animals were treated with NMDA only a slight, but significant, increase in the activity of glutaminase was observed at 9-11 postnatal day only. Any of the agonists or antagonists tested significantly affected the activity of lactate dehydrogenase as compared to control animals. Histologic observations of cerebella treated with the indicated drugs showed that only MK-801, and CPP to a lesser extent, induced a small reduction in the width of the internal granule layer. The body weight of animals treated with MK-801 was clearly reduced, but only in more mature rats (> 16 postnatal day), when animals did not show any alteration in the enzymes tested. These results support the suggestion that presynaptic influences, particularly from glutamatergic neurons, are critical to promote cerebellar granule neurons differentiation during critical periods of the cerebellar development.

2-Amino-5-phosphonovalerate↗
PubMed1975

[Old and new standardized color test for the determination of aspartate aminotransferase in the differential diagnosis of heart infarct and angina pectoris].

The usability of the new and old standardized colour test for the determination of the aspartate aminotransferase (GOT) (set of test instruments VEB Arzneimittelwerk Dresden) in the differential diagnosis between acute myocardial infarction and angina pectoris are compared. Concerning this problematics the new colour test does not evoke a better separation effect than the old one. Since with the change-over there were connected considerable uncertainties in the clinic it is recommended in case of a future standardisation to publish the regions of reference and first clinical experiences before the change-over.

Angina Pectoris↗
PubMed1995

The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments.

Here, a complete study is described of all the genes and isoenzymes for aspartate aminotransferase (AspAT) present in Arabidopsis thaliana. Four classes of cDNAs representing four distinct AspAT genes (ASP1-ASP4) have been cloned from Arabidopsis. Sequence analysis of the cDNAs suggests that the encoded proteins are targeted to different subcellular compartments. ASP1 encodes a mitochondrial form of AspAT, ASP3 encodes a chloroplastic/plastidic form of AspAT, whereas ASP2 and ASP4 each encode cytosolic forms of AspAT. Three distinct AspAT holoenzymes (AAT1-AAT3) were resolved by activity gel analysis. Organelle isolation reveals that AAT1 is mitochondrial-localized, AAT3 is plastid-localized, and AAT2 is cytosolic. Gene-specific Northern analysis reveals that each Asp mRNA accumulates differentially with respect to organ-type. However, the individual Asp mRNAs show no dramatic fluctuations in response to environmental stimuli such as light. Southern analysis reveals that four distinct nuclear genes probably represent the entire AspAT gene family in Arabidopsis. These molecular studies shed light on the subcellular synthesis of aspartate in Arabidopsis and suggest that some of the AspAT isoenzymes may play overlapping roles in plant nitrogen metabolism.

Amino Acid Sequence↗
PubMed1988

Crystallographic refinement by simulated annealing. Application to a 2.8 A resolution structure of aspartate aminotransferase.

Crystallographic refinement by simulated annealing with molecular dynamics has been applied to a 2.8 A (1 A = 0.1 nm) resolution X-ray structure of aspartate aminotransferase. Comparison of the refined structure and a structure obtained by combined restrained least-squares refinement and manual re-fitting shows a similar R factor, stereochemistry, and mean difference from the isomorphous replacement phase centroids. Crystallographic refinement by simulated annealing accomplished structural changes and improvements of the electron density maps that were not possible by using restrained least-squares refinement without manual re-fitting. Crystallographic refinement by simulated annealing can generate an ensemble of structures, each of which agrees with the diffraction information. Regions of large variations of the ensemble indicate either erroneously fitted or disordered segments of the macromolecule.

Aspartate Aminotransferases↗
PubMed1984

Multiple forms of aspartate aminotransferase as studied by liquid chromatography.

A convenient method, based on high-performance Liquid chromatography (HPLC) is described for the analysis of multiple forms of cytosolic aspartate aminotransferase. The method has a better resolution; in the same time it is drastically more rapid than the conventional chromatography on CM-Sephadex. The results suggest that transition forms arising from the heat denaturation of the enzyme can be detected by HPLC.

Animals↗
PubMed1981

Effect of glycerol on motility, viability, extracellular aspartate aminotransferase release and fertility of stallion semen before and after freezing.

The effect of different glycerol concentrations (0 to 5.3 per cent) on motility, viability and aspartate aminotransferase (AST) release of stallion spermatozoa was studied before and after deep-freezing. Addition of glycerol to a TRIS-fructose-egg yolk diluent used to extend stallion semen had no effect on motility and viability of spermatozoa and it did not increase AST release. Inclusion of glycerol in the extender only partially preserved the motility and viability of stallion semen during deep-freezing. A fertility trial revealed that concentrating stallion semen by centrifugation, followed by addition of the extender containing 7.5 per cent glycerol, had little effect on fertility and a 70 per cent conception rate was obtained in 10 mares after inseminations during one service period. Freezing and thawing horse spermatozoa, even in the presence of 7.5 per cent glycerol, significantly decreased the fertilisation rate.

Animals↗
PubMed2000

Glycation of aspartate aminotransferase and conformational flexibility.

Glycation of proteins alters biological function and changes cellular processes. Our study investigated the conformational changes that accompany glycation using the cardiac aspartate aminotransferase (cAAT). We examined the effects of brief and prolonged exposure of cAAT to glyceraldehyde (Glyc) and ribose 5-phosphate (R5P). When cAAT was briefly incubated (3.5 h) with Glyc (500 microM) or R5P (5 mM) at 37 degrees C, cAAT activity and 1-anilinonaphthalene 8-sulfonate (ANS) binding increased relative to control. After prolonged incubation (64 h) with Glyc (500 microM) or R5P (5 mM) at 37 degrees C, activity and ANS binding decreased relative to control. Furthermore, upon prolonged incubation of cAAT with 500 microM Glyc (14.5 h) or 2 mM R5P (64.25 h) at 37 degrees C, the denaturation curves shifted to the right relative to control. We conclude that upon brief incubation with Glyc and R5P, cAAT exhibited a more open and flexible structure and upon prolonged incubation, a more rigid structure.

Anilino Naphthalenesulfonates↗
PubMed1981

Preincubation of serum aspartate aminotransferase with pyridoxal 5'-phosphate in the SMAC: comparison with revised DuPont aca method and recommended IFCC method.

The method for continuous-flow assay of aspartate aminotransferase with the Technicon SMAC was modified to include preincubation of the serum enzyme with pyridoxal 5'-phosphate, to be consistent with the recommendations of IFCC and the Standards Committee of AACC. Preliminary estimates of the imprecision of the modified method on SMAC gave day-to-day standard deviations of 5.3 U/L at mean of 48 U/L (n = 66) and 6.2 U/L at 155 U/L (n = 61). Added bilirubin, sodium pyruvate, ascorbic acid, and endogenous lipids did not interfere. Comparison of results for 50 samples by this method with those by the manual IFCC method gave y = 1.1113x - 0.3 U/L, Sy/x = 4.4 U/L, and r = 0.997. Similar data are presented for the revised AST method for the DuPont aca discrete analyzer. Clinical data show that AST activities increase by as much as 200% when the serum is preincubated with pyridoxal 5'-phosphate.

Animals↗
PubMed1991

Association of gingival crevicular fluid aspartate aminotransferase levels with histopathology during ligature-induced periodontitis in the beagle dog.

Previous investigations have shown a clear association between the presence of the enzyme aspartate aminotransferase (AST) in gingival crevicular fluid (GCF) and clinical evidence of periodontal disease in humans, as well as in the beagle dog model. This paper describes a 26-week study that uses the beagle dog model of ligature-induced periodontitis in which GCF-AST (corrected for collection time) was correlated with microscopic evidence of tissue destruction in the periodontium at the sites of fluid collection. GCF and clinical data were collected at baseline, at optimal gingival health, during gingivitis, and after ligation. A cross-mouth design was implemented so that six premolar teeth in each dog were ligated for periods up to five weeks. Formalin-fixed tissues from the sites of GCF collection were prepared for light microscopy and evaluated for the presence of epithelial ulceration, bone resorption, and inflammatory cell infiltration. The relationship between GCF-AST levels and microscopic findings was analyzed by calculation of sensitivity and specificity and by plots of Receiver Operating Characteristics. These data revealed a correlation between elevated enzyme concentration and microscopic evidence of disease activity. Taken together with human studies, these results provide support for the use of AST as a marker of periodontal disease progression.

Alveolar Bone Loss↗
PubMed1988

Site-specific methylation of a strategic lysyl residue in aspartate aminotransferase.

Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination-like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected.

Amino Acid Sequence↗
PubMed1983

Properties of the active site lysyl residue of mitochondrial aspartate aminotransferase in solution.

Two vitamin B6 derivatives, N-bromoacetylpyridoxamine (BAPM) and its phosphate ester have been found to be affinity-labeling reagents for mitochondrial aspartate aminotransferase (EC 2.6.1.1). These derivatives were first shown to react with a critical sulfhydryl group in tryptophan synthase (Higgins, W., and Miles, E. W. (1978) J. Biol. Chem. 253, 4648-4652). In the apoaminotransferase, BAPM has now been found to inactivate by covalently modifying a critical lysyl residue, preventing reconstitution of the apoenzyme by pyridoxal 5'-phosphate. The dependence of the rate of inactivation upon the concentration of the reagent is consistent with a rapid equilibrium binary complex formation prior to the inactivation reaction. Both the dissociation constant for this complex and the rate of the reaction leading to inactivation are dependent on pH. BAPM binds best from pH 7.5 to 8.5. The rate of inactivation increases from pH 6 to 9. Succinate and phosphate competitively bind to the apoenzyme, protecting against BAPM inactivation. The C-5'-phosphorylated derivative is rapidly and tightly bound by the apotransaminase to form an inactive, noncovalent adduct. This bound reagent subsequently alkylates Lys-258. The rate of this covalent incorporation increases from pH 6 to 9 and is greater than the rate of BAPM modification at all pH values. The effect of pH on the reaction rates of both pyridoxal derivatives is interpreted to indicate protonation of Lys-258 at neutral pH values. These derivatives may also be analogs to a reaction intermediate different from those observed in other affinity-labeling studies. The ionization states of the Lys-258 epsilon-amino group apparently vary with the nature of the affinity label. These variations can be explained in terms of changing ionization states of Lys-258 in the steps of catalysis as well as in terms of the occupancy of charged sites on the protein by active site-directed substrates or inhibitory compounds.

Affinity Labels↗
PubMed1984

1H NMR studies of aspartate aminotransferase. Histidyl residues of cytosolic and mitochondrial isoenzymes.

200 MHz proton nuclear magnetic resonance spectra were compared between the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase (EC 2.6.1.1) from pig heart. The pattern of signal distribution in the whole spectral region differed considerably between the two isoenzymes, reflecting the difference in their amino acid sequences. A group of distinct signals were resolved at elevated temperatures (50 to 70 degrees C) in the low field region (9.0 to 7.5 ppm) of the spectra of both isoenzymes in the pyridoxal form. Most of these signals were also observable at 28 degrees C although some showed considerable line broadening. Among resonance lines in this spectral region, cAAT in the pyridoxal form showed four pH-titratable resonances with pKa of 9.54, 6.72, 5.69, and 4.87 at 28 degrees C. Variation in pK and line width of these signals indicated differences in the microenvironment of histidyl residues. On the other hand, mAAT showed six pH-titratable resonances with pKa of 6.73 (peak 2), 6.77 (peak 3), 6.07 (peak 4), 4.71 (peak 5), 4.54 (peak 6), and 4.33 (peak 7). Peaks 2, 3, and 4 were narrow and others were considerably broad. Thus, only part of the histidyl residues present in each isoenzyme (8 and 10 His/monomeric unit of cAAT and mAAT, respectively) appeared on the spectra as pH-titratable resonances. With both isoenzymes, chemical shift and pKa values of these signals obtained for the pyridoxal form were indistinguishable from those for the pyridoxamine form and the borohydride-reduced form. None of the observable signals were affected upon the interaction of cAAT with glutarate. By contrast, peaks 2 and 4 in mAAT showed subtle but distinct chemical shift changes upon complex formation with succinate, suggesting that these two resonances are due to histidyl residues located at the part of the enzyme molecule which undergoes a conformational change upon the interaction with the dicarboxylate.

Animals↗
PubMed1999

Relationship of the subgingival microbiota to a chairside test for aspartate aminotransferase in gingival crevicular fluid.

BACKGROUND: The aim of the present study was to evaluate the association between the occurrence of certain specific periodontal pathogens and aspartate aminotransferase (AST) levels in gingival crevicular fluid (GCF). METHODS: Thirty systemically healthy subjects with moderate to advanced periodontitis were selected. Within each subject, the AST contents of GCF from sites with probing depth between 5 mm and 7 mm were measured using a chairside colorimetric test. AST-positive site refers to one that had an AST level > or = 800 microIU. Subgingival plaque samples from one AST-positive and one negative site were collected for microbiological examination. One site with probing depth < or = 3 mm and no gingival inflammation was selected as a healthy control. Clinical parameters of the chosen sites, including the plaque index and gingival index scores, probing depth, and clinical attachment level were measured. Culture and immunofluorescence (IF) were used for detecting common periodontal pathogens, including Actinobacillus actinomycetemcomitans, Peptostreptococcus micros, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Capnocytophaga species, Prevotella intermedia, Prevotella melaninogenica, and Porphyromonas gingivalis. Logistic regression was used to analyze the correlation between the AST test and certain specific pathogens. RESULTS: The GCF scores and total cultivable bacterial counts were higher in AST-positive sites than either AST-negative or healthy sites. The prevalence and proportions of specific periodontal pathogens such as C rectus, E. corrodens, F. nucleatum, Capnocytophaga species, P. intermedia, and P. gingivalis were significantly higher in positive than in negative sites. In analyzing the correlation of the proportion of 6 pathogens with the AST test by logistic regression, only P. gingivalis showed a significant positive correlation. The odds ratio of having a high proportion of P. gingivalis in the presence of a positive AST test was 1.21. CONCLUSIONS: The present study showed that at AST-positive sites, there is a higher prevalence and higher proportion of certain periodontal pathogens. Although only the correlation of P. gingivalis and AST values was statistically significant, the results imply that certain periodontal pathogens may be associated with elevation of AST levels in GCF.

Adult↗
PubMed1997

Simultaneous assay for aspartate aminotransferase and guanase in human serum by high-performance liquid chromatography.

A simple high-performance liquid chromatography (HPLC) assay for the simultaneous determination of guanase and aspartate aminotransferase (AST) activities in a single serum sample is described. The method is based on direct detection of enzymatically formed products xanthine and glutamate, respectively. The procedure is sensitive, precise (C.V. below 2% for guanase and 3% for AST), suitable for routine purposes and requires only 100 microliters of sample. Kinetic measurements have shown the guanase activity to have an apparent Michaelis constant of 24.5 microM and the AST activity of 11.1 and 0.18 mM for aspartate and oxoglutarate, respectively, at 37 degrees C in Tris-HCl buffer (pH 7.5).

Aspartate Aminotransferases↗
PubMed1983

Interaction of a coenzyme analog with aspartate aminotransferase isoenzymes in the crystal.

The interaction between the coenzyme derivative 4'-N-(2,4-dinitro-5-fluorophenyl)-pyridoxamine 5'-phosphate with cytoplasmic and mitochondrial apo-aspartate aminotransferase in the crystalline state was investigated to establish whether the structural differences, known to exist between the active sites of the two isoenzymes in solution, are maintained in the crystal although they are not apparent from the available crystallographic data. In the crystal, as in solution, both apo-isoenzymes reversibly bind the coenzyme derivative and catalyze a slow cleavage reaction, by which pyridoxal 5'-phosphate is produced and bound to the active-site lysine. In the case of the cytoplasmic isoenzyme, however, in the crystal as in solution, the initial complex can follow an alternative reaction path that leads to the formation of a covalent bond between the active-site lysine and the C-5 of the 2,4-dinitrophenyl moiety of the reagent. Therefore, crystal-packing forces neither abolish the active site properties that are needed to cleave the specifically bound reagent and are common to the two isoenzymes nor mask the subtle differences that allow for the selective irreversible labeling of the cytoplasmic isoenzyme.

Animals↗
PubMed2000

Carnosine prevents the glycation-induced changes in electrophoretic mobility of aspartate aminotransferase.

Carbohydrate-derived aldehydes cause irreversible loss of protein function via glycation. We previously observed that glyceraldehyde 3-phosphate (Glyc3P) abolishes the enzyme activity of cardiac aspartate aminotransferase (cAAT). We also examined the protective effects of carnosine against Glyc3P-induced loss of enzyme activity. The present study looked at carnosine's prevention of Glyc3P-induced change in protein structure. Purified cAAT (2 mg protein/mL) was incubated with various concentrations of carnosine (1-20 mM) in the presence of Glyc3P (500 microM) for 4 days at 37 degrees C. Following incubation, samples were analyzed by SDS-polyacrylamide gel electrophoresis. Carnosine showed prevention of protein modification at carnosine-to-Glyc3P ratios of 10:1 or greater. There was a progressive loss of the unmodified cAAT protein band as Glyc3P concentration was increased. Additionally, the gel position of the Glyc3P-modified cAAT protein varied over time. The apparent molecular weight (MWapp) of the Glyc3P-modified cAAT protein that formed after 1 day at 37 degrees C (500 microM) was greater than its MWapp after 2 days, suggesting that a chemical rearrangement of the initial adduct occurs. These observations support the hypothesis that carnosine is an antiglycation agent and that its mechanism of action involves prevention of protein modification.

Aspartate Aminotransferases↗
PubMed1988

Apoenzyme of aspartate aminotransferase isoenzymes in maternal plasma during labor and following delivery.

In eight normal mothers the effects of pyridoxal 5'-phosphate (PLP) addition on the plasma activities of aspartate aminotransferase isoenzymes (AST-s and AST-m) during labor and following delivery were investigated. The AST-s activities with and without reactivation by PLP appeared to increase immediately after delivery and they were even higher on the 4th day postpartum. On the other hand, there were significant elevations in both the AST-m activities immediately and at 2 h after delivery, but not on the 4th day postpartum. Of AST isoenzymes measured in the nonstress test, only the relative activation rate of AST-m by PLP added was significantly higher than the control value. The present study may come to the following conclusions: 1) The relative activation rate of plasma AST-m activity by PLP may be a reliable index of vitamin B6 nutritional status during pregnancy. 2) The increases in AST-m activity with and without PLP added during labor suggest a minimal damage of mitochondria in skeletal, cardiac and uterine muscle cells. 3) The AST-s isoenzyme determinations with and without PLP may be especially useful as sensitive indication of erythrocyte and/or liver damage after delivery.

Adult↗