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PubMed2006

Analysis of aspartate aminotransferase and alkaline phosphatase in crevicular fluid from implants with and without peri-implantitis.

PURPOSE: The aim of this investigation was to determine the presence of aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in crevicular fluid collected from implants with and without clinical and radiographic signs of peri-implantitis. MATERIALS AND METHODS: There were 17 implants with symptoms of peri-implantitis in 12 subjects, including 4 females and 8 males, compared to 17 implants in 13 subjects, including 5 females and 8 males, with healthy peri-implant tissues. Filter paper strips were used to collect peri-implant crevicular fluid for 30 seconds in the base of the crevice/pocket. SPSS statistical software (SPSS, Inc., Chicago, IL) was used to determine AST and ALP activity. RESULTS: The results showed that there was a significant difference in the activity of AST and ALP between the 2 study groups (P < 0.0001). AST activity was significantly associated with the amount of bleeding on probing (P = 0.02), but no statistical correlation was found between ALP activity and increased amount of bleeding on probing (P = 0.05). CONCLUSIONS: Within the limits of this study, our results may suggest that peri-implant crevicular fluid analysis could be further investigated in longitudinal studies as a suitable diagnostic strategy in the evaluation of dental implants.

Alkaline Phosphatase↗
PubMed2006

Construction and evaluation of food-grade vectors for Lactococcus lactis using aspartate aminotransferase and alpha-galactosidase as selectable markers.

AIMS: We report development of two food-grade cloning vectors for Lactococcus lactis, which utilize either a lactococcal aspartate aminotransferase gene (aspC), or Bifidobacterium longumalpha-galactosidase gene (aglL) as selectable markers. METHODS AND RESULTS: The theta-replicon of lactococcal plasmid, pW563, was combined with aspC and a multiple cloning site. When electroporated into L. lactis JLS400 (AspC-), the resulting vector, pSUW611 (3.9 kbp), restores ability of the mutant to grow in milk thus allowing for selection of the transformants. The vector is stable during 100 generations of nonselective growth (0.2% loss per generation). The second vector, pSUW711 (5.1 kbp), was constructed by exchanging aspC with aglL under the control of usp45 promoter. Lactococcus lactis transformed with pSUW711 produced distinctive colonies within 48-72 h on melibiose-containing plates. Expression of two Lactobacillus helveticus peptidases was attempted using these new vehicles. Introduction of pepN on pSUW611 and pSUW711 into L. lactis led to a sixfold, or 27-fold increase in aminopeptidase activity, respectively. However, no changes in endopeptidase activity were recorded upon transformation with pSUW611 carrying pepO2 under control of three different promoters. Attempts were also made to construct high copy variants of pSUW711. CONCLUSIONS: The aspC and aglL can be employed as food-grade genetic markers for L. lactis. The vectors, pSUW611 and pSUW711, were successfully used to express Lact. helveticus PepN in L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: Two novel food-grade vectors were developed which provide simple and convenient selection and maintenance in L. lactis.

Aspartate Aminotransferases↗
PubMed2002

The role of the conserved Lys68*:Glu265 intersubunit salt bridge in aspartate aminotransferase kinetics: multiple forced covariant amino acid substitutions in natural variants.

The role of the Lys68*:Glu265 intersubunit salt bridge that is conserved (Csb) in all known aspartate aminotransferases (AATases), except those of animal cytosolic, Ac (His68*:Glu265), and plant mitochondrial, Pm (Met68*:Gln265), origins, was evaluated in the Escherichia coli AATase. Two double-mutant cycles, to K68M/E265Q and the charge reversed K68E/E265K, were characterized with the context dependence (C) and impact (I) formalism, previously defined for functional chimeric analysis. Mutations of Lys68* with Glu265 fixed are generally more deleterious than the converse mutations of Glu265 with Lys68* fixed, showing that buried negative charges have greater effects than buried positive charges in this context. Replacement of the charged Lys68*:Glu265 with the K68M/E265Q neutral pair introduces relatively small effects on the kinetic parameters. The differential sensitivity of k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) to salt bridge mutagenic replacements is shown by a linear-free energy relationship, in which the logarithms of the latter second order rate constants are generally decreased by a factor of two more than are those of the former. Thus, k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) are 133 and 442 mM(-1)s(-1) for the wild-type (WT) enzyme, respectively, but their relative order is reversed in the more severely compromised mutants (14.8 and 5.3 mM(-1)s(-1) for K68E). A Venn diagram illustrates apparent forced covariances of groups of amino acids that accompany the naturally occurring salt bridge replacements in the Pm and Ac classes. The more deeply rooted tree indicates that the Csb variant was the ancestral specie.

Amino Acid Sequence↗
PubMedDate not supplied

[Cytosol aspartate aminotransferase from the chicken heart: three-dimensional structure at 2.8 angstroms resolution and the characteristic conformation of various enzyme forms].

The spatial structure of cytosolic chicken aspartate aminotransferase (AAT) has been determined by X-ray crystallographic analysis at 2.8 A resolution. AAT consists of two chemically identical subunits. Each subunit can be subdivided into the large pyridoxal phosphate (PLP) binding domain and the small domain. The two active sites of AAT are situated in deep clefts at the subunit interface. The binding of PLP and 2-oxoglutarate is described. Conformations of the following enzyme forms have been compared by difference Fourier syntheses: the nonliganded PLP-form in phosphate and acetate buffers; the non-liganded pyridoxamine phosphate (PMP) form; complexes of the PLP-form with glutarate and 2-oxoglutarate. Lattice-induced dynamic asymmetry of the dimeric AAT molecules was revealed. In one subunit the small domain is mobile and shifted either toward the active site ("closed" conformation) or in the opposite direction ("open" conformation). The closed conformation is induced by the binding of dicarboxylate anions. In the second subunit the small domain is immobile and shifted toward the active site in all enzyme forms or complexes studied. In this subunit, there occurs a rotation of the PLP ring by approximately 20 degrees toward the substrate site. The rotation is observed when crystals are soaked in 0.6 saturated (NH4)2SO4 solution buffered with 0.3 M potassium phosphate, pH 7.5; it was explained by formation of an external aldimine between PLP and NH3. This aldimine is not formed in the presence of dicarboxylates or acetate. It was inferred that dicarboxylate or acetate anions stabilize the internal PLP-lysine aldimine and prevent its reaction with ammonia. Conversion of AAT from the PLP- to PMP-form is accompanied by rotation of the coenzyme ring by approximately 20 degrees; the rotation occurs in both subunits.

Animals↗
PubMed1984

Distribution of cytosolic and mitochondrial aspartate aminotransferase in normal, ischemic, and necrotic myocardium. An immunohistochemical study.

We utilized immunoperoxidase methods to study the distribution of both cytosolic or soluble(s) and mitochondrial (m) aspartate aminotransferase (AspAT) in normal, ischemic, and necrotic myocardium. Human myocardium was obtained from autopsy (n = 9) and surgery (n = 6). Cardiac tissue from 26 dogs with experimental myocardial infarction induced by closed-chest balloon occlusion and four dogs with myocardial ischemia without necrosis induced by a 50% reduction in left main coronary artery flow for 3 hours were studied. Duration of occlusion was 45 minutes (n = 1), 3 hours (n = 11), 5 to 6 hours (n = 10), or 15 to 24 hours (n = 4). Highly purified m- and s-AspAT and specific antibodies were prepared in our laboratory. In all cases, control experiments were performed. Microscopically normal human and dog myocardium uniformly stained for m- and s-AspAT. Necrotic myocardium from patients with infarcts showed markedly reduced immunostaining. In those dogs with myocardial necrosis, all dogs with coronary occlusion of 5 to 24 hours, and eight of 11 dogs with 3-hour occlusions, immunostaining was significantly reduced for both s- and m-AspAT in regions confirmed to be necrotic by triphenyl tetrazolium chloride and hematoxylin and eosin staining. Myocardial necrosis was confirmed in the 3-hour infarcts by electron microscopy. In the four dogs with a 50% reduction in left main flow for 3 hours, and one dog with a 45-minute coronary occlusion, ischemia was demonstrated by glycogen loss in period acid-Schiff-stained sections but there was no evidence of necrosis by electron microscopy or triphenyl tetrazolium chloride staining and there was no loss of immunostaining evident for s- or m-AspAT. Thus, s- and m-AspAT were visualized in normal and ischemic myocardium with decreased staining in necrotic tissue using immunoperoxidase techniques. Loss of both s- and m-AspAT can be demonstrated in human myocardium and in experimental canine myocardium as early as 3 hours after coronary occlusion and appears to be specific for irreversible myocardial injury. No depletion of isoenzyme can be detected by immunohistochemical techniques in tissue that is ischemic but not necrotic. Furthermore, by these immunoperoxidase techniques, loss of s- and m-AspAT from necrotic myocardium appears to be simultaneous.

Animals↗
PubMed1987

Purification and properties of the cytosolic and mitochondrial forms of aspartate aminotransferase and malate dehydrogenase from rat heart.

The present work describes the purification from rat heart of the mitochondrial and cytosolic forms of the enzymes of the malate--aspartate shuttle, aspartate aminotransferase (EC 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37), by a single procedure after the preparation of the original crude extract. In 10 purification steps, the four enzymes were obtained electrophoretically pure in yields ranging from 6 to 54% of their respective isoenzyme levels in the crude extract. Apoenzymes were formed from the aminotransferases by reacting them with cysteine sulfinate and dialyzing. Complete reconstitution was obtained after a brief incubation with pyridoxal phosphate. All four enzymes are dimers. The mitochondrial isoenzymes are of slightly lower molecular weight than their respective cytosolic forms. Michaelis constants and maximal velocities were derived by the use of primary and secondary plots. In general, the properties of the enzymes from rat heart are similar to the properties of the enzymes from other animal sources.

Animals↗
PubMed1986

Changes in mass and catalytic activity concentrations of aspartate aminotransferase isoenzymes in serum after a myocardial infarction.

We used an RIA and inhibition of enzyme activity to monitor the changes in mass and catalytic concentrations of the aspartate aminotransferase (EC 2.6.1.1;AST) isoenzymes in serum after myocardial infarction. Cytosolic (c-AST) and mitochondrial (m-AST) forms of AST were present in sera from all 38 of our patients. Although the immunological and catalytic concentrations of both isoenzymes correlated well with the size of the infarct, c-AST gave a better measure than did m-AST. About 20% of the total enzyme activity at peak activity was from the mitochondrial isoenzyme. Both isoenzyme activities peak at very nearly the same time, but m-AST has the longer half-life. Immunological evidence of the mitochondrial isoenzyme can be detected in serum for at least eight days after the infarct. The presence of left ventricular failure produces greater serum isoenzyme activities than in those without failure.

Adult↗
PubMed2003

Use of aspartate aminotransferase in diagnosing periodontal disease: a comparative study of clinical and microbiological parameters.

The objective of this study was to assess the association between the levels of enzyme aspartate aminotransferase (AST) in gingival crevicular fluid (GCF) with the BANA hydrolysis microbiological test (Perioscan) and clinical periodontal diagnostic measurements, such as bleeding on probing, plaque index, gingival index, probing depth, and attachment level in patients with chronic periodontitis using an enzymatic test (PocketWatch). One hundred and forty-seven sites were evaluated in 22 patients with a probing depth of > or = 5 mm at selected sites. AST and BANA enzymatic tests were carried out, and clinical parameters recorded. Pearson's chi-square and Fisher's exact tests were used for statistical analysis. There was no statistical correlation between AST levels and any of the analyzed parameters. The lack of any association between the factors studied does not indicate, however, that the latter cannot be used in diagnosing the actual periodontal condition of patients and/or sites. However, more research should be carried out to evaluate the true relationship between AST and periodontal disease.

Adult↗
PubMed2000

Analysis of aspartate aminotransferase in gingival crevicular fluid assessed by using PocketWatch: a longitudinal study with initial therapy.

AIM: The aim of this longitudinal study was to analyze the level of aspartate aminotransferase (AST) in gingival crevicular fluid (GCF) by using PocketWatch before and after initial therapy in patients with chronic adult periodontitis and to determine the relationship between AST and conventional measures of periodontal status, such as probing depth, clinical attachment level, bleeding on probing, and gingival index. METHOD: A total of 11 patients with chronic adult periodontitis were enrolled. Their periodontal status and AST levels in GCF were measured at baseline and post-initial therapy (the number of pockets=67), and statistically analyzed. RESULTS: There was a statistically significant difference in AST levels between diseased periodontal sites (1.2+/-0.7) and healthy sites (0.3+/-0.6, p<0.05), and between baseline and post-initial therapy (p<0.05). Improvements in clinical status were noted following periodontal therapy and there was a corresponding decrease in AST levels. CONCLUSIONS: In conclusion, it is suggested that AST levels may be a useful adjunct in the clinical assessment of periodontal disease sites, since AST level decreases when periodontal status improves.

Adult↗
PubMedDate not supplied

[Primary structure of cytoplasmic aspartate aminotransferase from pig heart muscle. Amino acid sequence of peptides from cyanogen bromide hydrolysate].

Amino acid sequences were determined for the six peptides from cyanogen bromide hydrolysis of cytoplasmic aspartate aminotransferase. These peptides accounted for 177 amino acid residues of the enzyme. Partial sequence of N-terminal peptide accounting for 212 amino acid residues of enzyme was also determined.

Amino Acid Sequence↗
PubMed1997

Prolactin regulation of mitochondrial aspartate aminotransferase and protein kinase C in human prostate cancer cells.

Citrate production is a major physiological function of the prostate that is regulated by testosterone and prolactin. Mitochondrial aspartate aminotransferase (mAAT) is a key enzyme in the metabolic pathway of prostate citrate production. In addition, prolactin stimulates expression of mAAT in the rat lateral prostate. In this report we establish the role of prolactin in the regulation of mAAT in two prostate cancer cell lines, LNCaP and PC-3. LNCaP cells respond to hormonal stimulation with increased secretion of prostate specific products. PC-3 cells, on the other hand, are testosterone independent and apparently do not respond to other growth factors either. Results showed that both LNCaP and PC-3 cells responded to prolactin with increased mAAT activity and an increased steady state level of mAAT mRNA. Prolactin also increased protein kinase C (PKC) activity in both these cell lines. Treatment of LNCaP and PC-3 cells with the phorbol ester 12-O-tetradecanoylphorbol (TPA) caused the same effect on mAAT activity and mRNA level as prolactin. The results suggest that the diacylglycerol-PKC signal transduction system mediates the prolactin effect on mAAT. In addition, these results also show that the prolactin effect on mAAT is independent of androgens since PC-3 cells reportedly lack androgen receptor expression. Thus, these results provide evidence that prolactin is a physiological regulator of prostate function in human as well as rat prostate. In addition, the results also show that though prostate cancer cells are androgen independent, they remain responsive to prolactin. This could have important implications for the treatment and management of prostate cancer.

Animals↗
PubMed1998

Macromolecular aspartate aminotransferase.

Macroenzymes are serum enzymes that have a greater molecular mass than the corresponding enzyme normally found in serum (Klonoff. West J Med 1980; 133: 392-407). Serum AST (aspartate aminotransferase) has rarely been reported to complex with immunoglobulins, resulting in an elevation in serum AST activity. Physicians should be aware of this condition so that patients are not subjected to invasive and costly procedures unnecessarily (Litin et al. Mayo Clin Proc 1987; 62: 681-687). We report on such a patient with a raised AST due to macroenzyme formation.

Adult↗
PubMed1989

cDNA cloning and expression of pig cytosolic aspartate aminotransferase in Escherichia coli: amino-terminal heterogeneity of expressed products and lack of its correlation with enzyme function.

A full-length cDNA encoding the pig cytosolic aspartate aminotransferase (EC 2.6.1.1) (cAspAT) was constructed from two overlapping cDNA clones. One clone (Lm pcAAT-8) isolated from a lambda gt10 pig heart cDNA library contained a 3' untranslated sequence, a poly(A) segment, and a part of the coding region for amino acid positions 127-412. Another clone (Lm pcAAT-107) isolated from a lambda gt10 primer extension library contained the coding region for amino acid positions 1-148 and a 5' untranslated sequence. Rejoining of the cDNA inserts of the two clones and recloning into pUC18 gave rise to a cDNA covering an entire coding sequence for pig cAspAT mRNA. Insertion into pKK223-3 yielded an expression plasmid, ppcAAT200. Escherichia coli JM105 cells transfected with ppcAAT200 overproduced pig cAspAT to an extent of about 3% of the total cellular soluble proteins. The expressed product was indistinguishable from the alpha subform of cAspAT isolated from pig heart in terms of specific activity, absorption spectra, molecular size, crystalline form, and immunological reactivity with anti pig cAspAT antibody. Compared with the amino-terminal sequence (Ala-Pro-Pro-) reported for pig heart cAspAT, the recombinant pig cAspAT showed heterogeneity in the amino-terminal sequence: Ala 1 (26%), Pro2 (54%), and Pro3 (19%). Construction of a mutant cAspAT with deletion of residues 1-3 and its comparison with the wild-type enzyme revealed that loss of the three amino-terminal residues does not affect the catalytic activity and structural integrity of the enzyme.

Amino Acid Sequence↗
PubMed2006

The level of serum ionised calcium, aspartate aminotransferase, insulin, glucose, betahydroxybutyrate concentrations and blood gas parameters in cows with left displacement of abomasum.

The purpose of this study was to determine concentrations of serum glucose, aspartate aminotransferase (AST), insulin, beta-hydroxybutyrate (BOH) and ionised calcium (Ica) in dairy cows with left displaced abomasum (LDA) and to compare these parameters before and after surgical correction of LDA. Eighteen Swiss-Holstein dairy cows with LDA were used in this study. Clinically healthy post parturient cows (n: 10) from a local dairy farm were used as control group. Blood samples were collected from the jugular vein from all the cows. Surgery was performed in cows with LDA. Blood samples from cows with LDA were collected 24 hours after surgery. The abomasum was repositioned followed by an omentopexy. Six of the 18 cows with LDA had clinical ketosis as detected with urine dipstick. The mean concentrations of insulin, BOH, glucose and AST in cows with LDA at admission time were increased compared with the healthy cows. But the mean concentration of ICa at admission time was slightly decreased compared with healthy cows. The mean BOH concentration was decreased 24 hours following surgery compared with values on admission time. However, the mean serum AST levels were increased both at admission time and 24 hours after surgery compared with healthy cows. The levels of blood gas parameters in cows with LDA were not significantly different in comparison with healthy cows, although hyperbasemia in six of 18 cows with LDA was determined. In conclusion, the results of this study indicated that serum insulin, glucose, AST and BOH levels were increased in dairy cows with LDA. Serum BOH and ICa levels were decreased 24 hours after surgery compared with values on admission time. All cows with LDA used in this study had subclinical/clinical ketosis. We could say that ketosis might be a risk factor for the displacement of the abomasum.

3-Hydroxybutyric Acid↗
PubMed2003

[Development of a method for the immunological measurement of aspartate aminotransferase with monoclonal antibodies].

BACKGROUND/AIMS: For laboratory diagnostics in liver diseases, many enzymes have been used for the assessment of hepatocellular function. Among them, two transaminases, alanine and aspartate aminotransferase, have been regarded as the most sensitive indicators of hepatocellular damage. However, the enhanced enzyme activities of the enzymes do not exactly indicate or represent the cause and progression of diseases in the patients with liver disease. To overcome such limitations, immunological methods have been suggested as one of the alternatives for the replacement or supplement of the conventional enzymatic analysis. METHODS: In the hope of developing a new assay system for measuring the AST concentration rather than its activity, we have developed a new assay using fluorescence labeled anti-AST monoclonal antibodies. Blood was obtained from a normal population of 234 patients and 43 liver disease patients. The linearity, limit of detection, and performance of the new assay system were tested and evaluated. The comparability of assay was examined with an ELISA and biochemical assays. RESULTS: The linearity fell in the range of 0-1 mg/L of AST (R=0.995), and the analytical detection limit was 12 microg/L of AST. The mean recovery of the control was 102.4 % in a working range. The precision of the intra- and inter-assay in a range of 50-800 microg/L was CVs < 7% and CVs < 6%, respectively. In the normal population, the mean AST concentration was 35.5 microg/L. The mean AST concentration in patients with liver disease was 266.5 microg/L. The new assay system correlated well with an ELISA and biochemical assay for quantification of AST concentration (R=0.92 and 0.88, respectively; N=43). CONCLUSIONS: We have developed a new immunological assay using generated monoclonal antibodies to human cytosolic AST and used them for the development of a fluorescent assay measuring the enzyme mass. Cytosolic AST mass in sera could be measured reproducibly by the immunological method. In conclusion, this study has provided us with a new type of tool for an accurate measurement of the enzyme amount in circulation.

Antibodies, Monoclonal↗
PubMed2006

Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism.

In this paper, we report the identification of genes from pine (PpAAT), Arabidopsis (AtAAT) and rice (OsAAT) encoding a novel class of aspartate aminotransferase (AAT, EC 2.6.1.1) in plants. The enzyme is unrelated to other eukaryotic AATs from plants and animals but similar to bacterial enzymes. Phylogenetic analysis indicates that this prokaryotic-type AAT is closely related to cyanobacterial enzymes, suggesting it might have an endosymbiotic origin. Interestingly, most of the essential residues involved in the interaction with the substrate and the attachment of pyridoxal phosphate cofactor in the active site of the enzyme were conserved in the deduced polypeptide. The polypeptide is processed in planta to a mature subunit of 45 kDa that is immunologically distinct from the cytosolic, mitochondrial and chloroplastic isoforms of AAT previously characterized in plants. Functional expression of PpAAT sequences in Escherichia coli showed that the processed precursor is assembled into a catalytically active homodimeric holoenzyme that is strictly specific for aspartate. These atypical genes are predominantly expressed in green tissues of pine, Arabidopsis and rice, suggesting a key role of this AAT in nitrogen metabolism associated with photosynthetic activity. Moreover, immunological analyses revealed that the plant prokaryotic-type AAT is a nuclear-encoded chloroplast protein. This implies that two plastidic AAT co-exist in plants: a eukaryotic type previously characterized and the prokaryotic type described here. The respective roles of these two enzymes in plant amino acid metabolism are discussed.

Amino Acid Sequence↗
PubMed1982

Circular-dichroism study of the interaction of aspartate-aminotransferase isoenzymes with a coenzyme analog.

The interaction between a coenzyme derivative, 4'-N-(2,4-dinitro-5-fluorophenyl)-pyridoxamine 5'-phosphate, and the apoenzyme of cytoplasmic and mitochondrial aspartate aminotransferase, was studied by circular dichroism. The specific complexes initially formed were characterized by their circular dichroic spectra. The spectra indicate that the complex is very probably the same for the two isoenzymes. In contrast the spectra recorded during further reaction, in agreement with previous results, monitor different reaction paths and characterize the irreversible labeling at the active site of the cytoplasmic enzyme and regeneration of pyridoxal 5'-phosphate in the mitochondrial enzyme. By following circular dichroic changes in the mitochondrial enzyme, initial kinetic characterization of the cleavage of 4'-N-(2,4-dinitro-5-fluorophenyl)-pyridoxamine 5'-phosphate to form pyridoxal 5'-phosphate at the active site, is provided.

Animals↗