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PubMed1994

Studies of the active-site lysyl residue of thermostable aspartate aminotransferase: combination of site-directed mutagenesis and chemical modification.

The gene technological substitution of the cysteinyl residue for the pyridoxal 5'-phosphate-binding lysyl residue (K239) of thermostable aspartate aminotransferase of Bacillus sp. YM-2 led to loss of the activity of the enzyme, which inherently contains no cysteinyl residues. The cysteinyl residue of the mutant enzyme was modified to lysine sulfur analog residues, S-(beta-aminoethyl)cysteine (SAEC), S-(beta-aminopropyl)cysteine (SAPC), and S-(beta-aminoethylthio)cysteine (SATC) with 2-bromoethylamine, 3-bromopropylamine, and 2-mercaptoethylamine, respectively. The modified mutant enzymes showed absorbance at 379 (K239SAEC), 400 (K239SAPC), and 365 nm (K239SATC), whereas the spectrum of the wild-type enzyme exhibited an absorption maximum at 360 nm derived from the internal Schiff base at pH 8.0. The absorption of the modified mutant enzymes at these wavelengths disappeared on reduction with NaCNBH3. This suggests that omega-amino groups of the introduced lysine sulfur analog residue form an internal Schiff base with pyridoxal 5'-phosphate. The modified mutant enzymes showed kcat values of 19.6-0.065% of that of the wild-type enzyme in the overall reaction, and were 10(6)-10(8) times more active than the K239C mutant enzyme. These results suggest that omega-amino groups of the introduced residues of the modified mutant enzyme serve as a catalytic base, and catalysis of the enzyme was affected by the length of the functional side-chain.

Aspartate Aminotransferases↗
PubMed1986

Ionization state of the coenzyme 5'-phosphate ester in cytosolic aspartate aminotransferase. A Fourier transform infrared spectroscopic study.

In order to determine the ionization state of the 5'-phosphate of bound pyridoxal phosphate, a Fourier transform infrared spectroscopic study of cytosolic aspartate aminotransferase has been carried out. Dianionic and monoanionic phosphate monoesters give rise to two bands each in the infrared spectrum [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498]. These bands can be identified in infrared spectra of the free coenzyme in solution. Due to interfering bands arising from the protein, only the band assigned to the symmetric stretching of the dianionic phosphate is observed in holoenzyme solutions. The integrated intensity of this band does not change with pH in the range 5.3-8.6, while for free pyridoxal phosphate, the integrated intensity of the same band changes with pH according to the pK value expected for the 5'-phosphate group in solution. Moreover, the value of the integrated intensity for the bound cofactor is close to the value given by free cofactor at pH 8-9. These results suggest that the 5'-phosphate of the bound cofactor remains mostly dianionic throughout the investigated pH range and disfavor other interpretations in terms of ionization of the phosphate group on the basis of the nuclear magnetic resonance 31P chemical shift-pH titration curve of holoenzyme [Schnackerz, K. D. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, E. A., Ed.) Part A, pp 195-208, Alan R. Liss, New York].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Cysteine-191 in aspartate aminotransferases appears to be conserved due to the lack of a neutral mutation pathway to the functional equivalent, alanine-191.

It was previously suggested that the conserved Cys-191 of aspartate aminotransferases (AATases) is conserved, not because it is essential, but because it is frozen in the sequence, with no neutral corridor to traverse to the similar phenotype of Ala-191 (Gloss et al., Biochemistry 31:32-39, 1992). This hypothesis has now been tested by additional mutations. All possible one-base mutations from Cys were made at position 191. All of these variants display kinetic parameters (kcat and kcat/KM values) that differ from the wild-type enzyme by 30% or more. The non-conserved cysteines that are predominantly Ala in other AATase sequences (Cys-82, Cys-192, and Cys-401) were mutated to Ser to test the corollary that a neutral Cys->Ala corridor does exist for these positions. These Cys->Ser mutations yielded enzymes with wild-type-like kinetic parameters. The pKa values of the internal aldimines of the mutants, Cys-191->Ser, Phe, Tyr, and Trp are higher than that of wild type by 0.6-0.8 pH units. The stabilities to urea denaturation of the Cys-191 mutants are similar to that of wild type, while those of the non-conserved cysteines show greater variation. Examination of the three-dimensional environment of the five cysteines showed that the van der Waals contacts of Cys-191 are more conserved than are those of the non-conserved cysteines. These data provide further support for the above hypothesis.

Alanine↗
PubMed1999

Microbiological features and crevicular fluid aspartate aminotransferase enzyme activity in early onset periodontitis patients.

Gingival crevicular fluid (GCF) reflects the immune and inflammatory reactions and the specific host-microbe interactions that lead to periodontal diseases. Aspartate aminotransferase enzyme (AST) is one of the components of GCF that is released as a result of cell death. In this study, periodontal sites (4 sites/patient) with a probing depth of > or =5 mm in early onset periodontitis (EOP) patients were first examined for the AST levels in GCF by the Periogard periodontal tissue monitor. To be eligible for the study, each of the patients had at least 1 AST positive site with clinical inflammatory changes (AST+, CIC+) and 1 AST negative site with no or minimum clinical inflammatory changes (AST-, CIC-). In 15 EOP patients who met the entry criteria, 30 AST+, CIC+ sites (1st group) and 19 AST-, CIC- sites (2nd group) were evaluated for microbiological variables. Certain microbial species, including Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Prevotella intermedia were detected more frequently (p<0.001, p<0.001 and p<0.05, respectively) in the 1st group, while gram-positive facultative organisms such as Actinomyces species were found more often (p<0.001) in the 2nd group. Parallel to the AST levels, the 2nd group had a lower number of total bacteria and proportion of obligate anaerobic and capnophilic micro-organisms than the first group (p<0.05 and p<0.05, respectively). Within the scope of this study, AST activity and microbiological data were found in agreement in the examined groups. These findings are encouraging and indicate the need for further studies to evaluate the ability of the AST test to differentiate the microbial flora of progressing sites and those that are inflamed, but not progressing.

Actinomyces↗
PubMed1988

The influence of long-term anticonvulsant therapy with diphenylhydantoin and carbamazepine on serum gamma-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase.

In 110 patients receiving long-term anti-convulsant monotherapy with diphenylhydantoin (DPH) and carbamazepine (CBZ) the serum activities of gamma-glutamyltransferase (gamma-GT), aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase (AP) were examined retrospectively. Elevated serum levels of gamma-GT and AP were seen in 91% and 39% of patients receiving DPH therapy compared to 64% and 14% of those receiving CBZ treatment. With all enzymes evaluated increases were more frequent and higher with DPH treatment than with CBZ. Frequency and extent of increased activity of gamma-GT were highly related to daily dosage in both preparations. The proportion of pathological enzyme levels was associated with age in DPH and CBZ therapies but not found to be significant. Sex differences in the frequency of increased enzyme activities could not be demonstrated. The results are discussed in the context of induction of the cytochrome P-450 system.

Adult↗
PubMed1998

Plasma aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH) and gamma-glutamyl transpeptidase (GGT) activities in water buffaloes with experimental subclinical fasciolosis.

The effect of chronic Fasciola hepatica infection on the activity of plasma aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH) and gamma-glutamyl transpeptidase (GGT) was investigated in water buffaloes dosed daily with 60 F. hepatica metacercariae over 20 days. Experimental fluke infection caused no clinical signs but provoked an increase in plasma level of IgG directed against F. hepatica from 4 weeks after infection. There was a significant increase in plasma AST from 6 weeks post-infection. Maximal values were reached at 14 weeks and remained significantly elevated by 23 weeks. Plasma GLDH was significantly elevated from 6 to 21 weeks post-infection. Significant increases in plasma GGT occurred from 8 to 26 weeks post-infection, reaching maximal values at 15 weeks. This study shows that plasma enzyme activities may be useful in studies of fluke-induced liver damage in water buffaloes.

Animals↗
PubMed1995

Severe preeclampsia with fulminant and extreme elevation of aspartate aminotransferase and lactate dehydrogenase levels: high risk for maternal death.

We report a subgroup of patients with fulminant hemolysis, elevated liver enzymes, low platelet count (HELLP) syndrome, manifesting extreme elevation of aspartate aminotransferase (AST; SGOT) and lactate dehydrogenase (LDH) levels and abnormal mental status. These gravidas are at high risk for mortality. Only four patients treated by the authors over a 10-year period have had AST more than 2000 IU/L and LDH more than 3000 IU/L in the HELLP syndrome. This report is based on retrospective chart review. All patients manifested disordered mental status, jaundice, intense hemolysis, and extreme hypertension. One patient had developed multiple organ system failure, was moribund at initial perinatal consultation, and died. The three others were treated with aggressive afterload reduction and plasma infusion or plasmapheresis; two survived. Fulminant HELLP syndrome occurs rarely, but marks a group of patients at high risk for mortality. Optimal therapy is unclear; early intervention, including afterload reduction, volume expansion, and consideration of plasma infusions or plasmapheresis, is recommended.

Adult↗
PubMed1991

Effects of pyridoxine supplementation on blood glucocorticoids level, aspartate aminotransferase activity and glucose tolerance pattern under acute hypoxic stress.

Studies were conducted on male adult rabbits to find out the changes in blood glucocorticoid levels along with the changes in aspartate aminotransferase activity in blood and the role of pyridoxine on the glucose tolerance pattern under hypoxic stress. Hypoxic stress was produced by exposing the animals to a simulated altitude of 7,000 m for 6 h. In the first set of experiments 10 rabbits were used. Blood haemoglobin level, plasma and erythrocyte glucocorticoid levels and erythrocyte GOT activity were measured just before and after the exposure to hypoxia. Erythrocyte GOT activity was measured both without and with 50 mg of pyridoxal phosphate addition to the incubation mixture. Glucocorticoid levels in plasma increased by 11% whereas in erythrocytes the increase was 55% after hypoxia. Percent stimulation of erythrocyte GOT activity with pyridoxal phosphate before exposure to hypoxia was 180% but increased to 321% after exposure. In the second set of experiments another 10 rabbits were used. First they were exposed to hypoxia without pyridoxine hydrochloride feeding and then after 7 days with 3 mg of pyridoxine hydrochloride feeding. For glucose tolerance tests the animals were fed with 1 g of glucose immediately after the hypoxic exposures. Plasma reduced glutathione (GSH), LDH and ICDH activities increased and GOT activity was depressed after hypoxic stress, but when the animals were fed pyridoxine hydrochloride prior to the exposure the enzyme activities remained unaltered after hypoxic stress. Pyridoxine hydrochloride did not alter the pattern of glucose tolerance after hypoxic stress.

Animals↗
PubMed1994

[Reestimation of aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio based on JSCC consensus method--changes of criteria for a differential diagnosis of hepatic disorders following the alteration from Karmen method to JSCC method].

We estimated clinical criteria of aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio that is measured by the consensus method of JSCC (Japan Society of Clinical Chemistry) for serum AST and ALT. The JSCC consensus method is closely correlated with both IFCC (International Federation of Clinical Chemistry) recommended method without pyridoxal phosphate and Karmen method recommended by the expert panel of liver function tests in Japanese Society of Gastroenterology. The slopes of the regression line is estimated to be 1.00 and 0.87 respectively. We propose that the decision making value of AST/ALT ratio to differentiate several types of liver diseases should be set to 0.87 measured by JSCC consensus method, instead of 1.0 measured by Karmen method.

Alanine Transaminase↗
PubMed1983

Vinblastine inhibits the maturation of the precursor of mitochondrial aspartate aminotransferase. Vincristine and six other cytoskeleton inhibitors do not show this effect.

Cytoskeleton inhibitors were tested in chicken embryo fibroblast cultures for possible effects on the import of the precursor of mitochondrial aspartate aminotransferase into mitochondria. Vinblastine (50 microM) increased the steady-state pool of the precursor 2.5-fold in pulse experiments with [35S]methionine. If the precursor was accumulated during a pulse in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and then chased under diluting CCCP, vinblastine (50 microM) prolonged the half-life of the precursor from 0.5 min in the control to 3 min. Other cytoskeleton inhibitors, i.e. vincristine (25 to 150 microM), colchicine (50 microM), nocodazole (50 microM), podophyllotoxin (50 microM), taxol (45 microM), cytochalasin D (20 microM) and phalloidin (25 microM) did not show this effect. The observed inhibition by vinblastine does not seem to relate to its action on microtubuli.

Alkaloids↗
PubMed1984

"In vivo" inhibition of murine liver aspartate aminotransferase by isoniazid.

Intraperitoneal administration of isoniazid (IN), an antituberculous agent, to mice or rats at a dose of 100 mg/kg body weight resulted in remarkable and rapid inhibition of liver cytosolic aspartate aminotransferase (AAT); inhibition was less marked with other antivitamin B6 compounds; AATs in other tissues and other aminotransferases in liver were less effectively inhibited by IN. The inhibition of liver cytosolic AAT was apparently irreversible in vivo; it was reversed only a little by gel filtration and dialysis to remove excess IN in vitro, although treatment with pyridoxal phosphate or 5'-deoxypyridoxal slowly restored the full activity. Attempts to inhibit the enzyme by in vitro treatments showed that IN itself was not an effective inhibitor, while the liver extracts from IN treated mice contained some strongly inhibitory substance. In addition, the extracts were shown to contain only trace amounts of IN. These results suggest that IN is metabolized to some other form which is markedly inhibitory to murine liver cytosolic AAT, and the metabolite binds to the coenzyme moiety of enzyme in a manner not readily dissociable.

Animals↗
PubMed1998

Plasma aspartate aminotransferase and creatine kinase activities in thoroughbred racehorses in relation to age, sex, exercise and training.

In order to investigate the effect of age, sex and month on the response of plasma aspartate aminotransferase (AST) and creatine kinase (CK) to exercise, blood samples were collected once a month between March and September from a group of 40 2- and 3-year-old (2yo and 3yo) thoroughbred racehorses (kept under the same managemental regimen) at rest before exercise (PRE) and at 2 (2H) and 24 h (24H) post-exercise. The absolute change in activities between the 2H and PRE samples (2H delta) and the 24H and PRE samples (24H delta) was also calculated. Age had a significant effect on all measured and calculated parameters for colts (C), apart from 24H delta CK but showed no effect in the fillies (F). Sex only had a significant effect in the 3yo in the 2H delta CK. In the 2yo, significant effects of sex were found for both CK and AST in the PRE, 2H and 24H samples. The effect of month varied according to the classification group with only the 2yoC not showing any significant effect on any parameter. Fillies were, in general, more likely than colts to show greater than a twofold increase in CK activity at 2H post-exercise and the number of animals showing such an increase decreased as the season progressed. Very little change in AST activities occurred with exercise.

Age Factors↗
PubMed1995

Aspartate aminotransferase isozymes in Panicum miliaceum L., an NAD-malic enzyme-type C4 plant: comparison of enzymatic properties primary structures, and expression patterns.

Panicum miliaceum has at least three isozymes of aspartate aminotransferase (AspAT); the cytosolic and mitochondrial isozymes (cAspAT and mAspAT) are major components and the third is a minor isozyme. Fractionation of leaf subcellular components showed that the minor isozyme was localized in plastids (pAspAT). We purified the three isozymes from green leaves of P. miliaceum. Both cAspAT and pAspAT consisted of triple subforms having the same molecular size but different isoelectric points. No substantial difference in enzymatic properties was observed among these isozymes besides the pH profiles. We isolated a full-length cDNA clone for pAspAT. This clone contains an open reading frame that encodes 457 amino acids. The amino-terminal region of the pAspAT precursor shares common features of plastid transit peptides. The amino acid sequence of P. miliaceum pAspAT shows higher similarity with other plant pAspATs than P. miliaceum cAspAT and mAspAT. The mRNA levels of the three isozymes were high in leaves compared with roots and mesocotyls. The three isozymes showed different expression patterns against environmental stimuli such as light and nitrate. The activities and protein levels of cAspAT and mAspAT increased during greening in accordance with those of phosphoenolpyruvate carboxylase and NAD-malic enzyme involved in the C4 pathway, primarily as a consequence of the increase in the levels of their mRNAs. By contrast, pAspAT was constitutively expressed during greening. The activity and protein levels of cAspAT and mAspAT selectively increased during recovery from an nitrogen deficit, primarily as a consequence of increase in the levels of their mRNAs while those of pAspAT remained unchanged.

Amino Acid Sequence↗
PubMed1984

[Relation between aspartate aminotransferase (AST) activity in the seminal fluid and indicators of boar ejaculate quality].

In a set of 154, and/or 260 ejaculates, collected from 72 boars, a relationship was studied between the activity of aspartate aminotransferase (AST) in seminal plasma and sperm concentration, and/or percent occurrence of morphologically abnormal spermatozoa. We failed to demonstrate any relation between the AST activity and sperm concentration (r = 0.023; P greater than 0.05). However, a statistically significant relation (P less than or equal to 0.05) was demonstrated between the AST activity and percent occurrence of morphologically abnormal spermatozoa (r = 0.343). Applying the above results and in agreement with literary data, determination of AST activity in the seminal plasma of boars can be considered as an important indicator of the cellular damage of sperms.

Animals↗
PubMed1984

Influence of substrate concentration on the activation of transketolase, aspartate aminotransferase, and glutathione reductase by coenzymes.

We investigated the mechanism by which the three most commonly measured enzymes in erythrocytes are activated by their respective coenzymes by determining the catalytic activity concentrations of transketolase (EC 2.2.1.1), aspartate aminotransferase (EC 2.6.1.1), and glutathione reductase (EC 1.6.4.2) in relation to various substrate concentrations. We conclude that the underlying mechanisms by which the enzymes are activated are not the same.

Aspartate Aminotransferases↗
PubMed1980

Chronic subclinical ovine fascioliasis: plasma glutamate dehydrogenase, gamma-glutamyl transpeptidase and aspartate aminotransferase activities and their significance as diagnostic aids.

The effect of low grade chronic Fasciola hepatica infection on the concentration of plasma glutamate dehydrogenase (GD), gamma-glutamyl transpeptidase (gamma-GT) and aspartate aminotransferase (AST) was investigated in sheep dosed daily with three (AL3), eight (AL8) or 14 (AL14) metacercariae over 22 weeks or given a single dose of 200 metacercariae. Significant increases in plasma GD activity first occurred after nine, 12 and 23 weeks and in gamma-GT activity after 12, 24 and 32 weeks for groups AL14, AL8 and AL3 respectively. Changes in AST activity were not as clearly related to dose level. In sheep with single infection, both GD and gamma-GT were capable of detecting liver damage resulting from the migration of 10 or more flukes. Plasma GD and gamma-GT activities are more sensitive indicators of liver cell damage in chronic subclinical fascioliasis than AST activity and gamma-GT may be more suitable as a diagnostic aid on account of its greater stability.

Animals↗
PubMedDate not supplied

Relationship of serum lactate dehydrogenase, creatine kinase, aspartate aminotransferase concentrations and severe intraventricular hemorrhage/leukomalacia in very low birth body weight preterm neonates.

Estimations of serum enzyme values are widely employed as valuable diagnostic aids in diseases. Most commonly employed enzymes include Aspartate aminotransferase (AST), Lactate dehydrogenase (LDH), and Creatine kinase (CK). The study was designed to determine the relationship of elevated postnatal serum LDH, CK, and AST concentrations within the first day of life and the risk of suffering severe intraventricular hemorrhage (IVH) and/or periventricular leukomalacia (PVL) in VLBW preterm newborns. 81 preterm neonates whose birth body weight < 1500 gm were enrolled. Serums were obtained for measurement within the first postnatal day. Cranial ultrasound scans were performed twice per week after birth until their body weight was above 2300 gm or postconceptional age above 40 weeks. Significant difference was noted in serum LDH and CK concentrations in severe IVH/PVL infants (p < 0.05). No difference was found in serum AST values. Compared with the cut-off values of 1933 IU/L of LDH concentration and 652 IU/L of CK, the predictive values revealed a sensitivity, specificity, negative predictive value and positive predictive value of 76.9%, 89.7%, 95.3% and 58.8%, respectively. In conclusion, higher serum LDH and/or CK concentrations within the first day of life were associated with risk for development of severe IVH/PVL.

Aspartate Aminotransferases↗
PubMed1996

The relation of microbiologic data to aspartate aminotransferase enzyme activity in gingival crevicular fluid.

Gingival crevicular fluid (GCF), reflects the immune and inflammatory reactions and is itself a location for specific host-microbe interactions that lead to periodontal diseases. Aspartate aminotransferase (AST) is one of the components of GCF that is released as a result of cell death. In this study, 40 periodontal sites in 10 early onset periodontitis patients before and after nonsurgical periodontal therapy, with and without local metronidazole administration, were first examined for the AST enzyme levels in GCF and then evaluated for microbiological and clinical variables. In each patient, 4 sites (one site/quadrant) with a probing depth of > or = 5 mm were selected and treated with separate treatment protocols. Certain microbial species including Prevotella intermedia, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans(A. a.) were found more often and/or in higher levels in AST active sites (36/40 first measurement--9/36 second measurement), while other species (Streptococcus and Actinomyces) were found more often and/or in higher levels in AST inactive sites (4/40 first measurement--8/36 second measurement). Eight post-treatment AST active sites revealed 1.5 mm of attachment loss, whereas 8 post-treatment AST inactive sites showed 1.37 mm of attachment gain. AST activity and microbiological-clinical data presenting such an agreement suggests that, AST level assessment would be beneficial as an adjunctive method alongside other clinical criteria, in guiding the clinician in periodontal treatment.

Adult↗