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Sample records for alanine aminotransferase aspartate

  1. Serum γ-Glutamyltransferase, Alanine Aminotransferase and Aspartate Aminotransferase Activity in Healthy Blood Donor of Different Ethnic Groups in Gorgan

    PubMed Central

    Mehrpouya, Masoumeh; Pourhashem, Zeinab

    2016-01-01

    Introduction Measure of liver enzymes may help to increase safety of blood donation for both blood donor and recipient. Determination of liver enzymes may prepare valuable clinical information. Aim To assess serum γ-Glutamyltransferase (GGT), Alanine Aminotransferase (ALT), and Aspartate Aminotransferase (AST) activities in healthy blood donors in different ethnic groups in Gorgan. Materials and Methods This study was performed in 450 healthy male blood donors, in three ethnic groups (Fars, Sistanee and Turkman) who attended Gorgan blood transfusion center. Liver enzymes (GGT, ALT and AST) were determined. Results Serum AST and ALT in three ethnic groups were significant except for serum GGT levels. There was significant correlation between family histories of liver disease and systolic blood pressure and AST in Fars, and GGT in Sistanee ethnic groups. Conclusion Several factors, such as age, family history of diabetes mellitus, family history of liver disease and smoking habit had no effect on some liver enzymes in different ethnic groups in this area. Variation of AST, ALT, and GGT enzyme activities in healthy subjects was associated with some subjects in our study groups. According to our study, it suggests that screening of AST and GGT enzymes in subjects with family history of liver disease is necessary in different ethnic groups. PMID:27630834

  2. Associations of White Blood Cell Count,Alanine Aminotransferase,and Aspartate Aminotransferase in the First Trimester withGestational Diabetes Mellitus.

    PubMed

    2016-06-10

    Objective To explore the associations of white blood cell (WBC) count,alanine aminotransferase (ALT),and aspartate aminotransferase(AST) in the first trimester of pregnancy with gestational diabetes mellitus (GDM). Methods Totally 725 GDM women and 935 women who remained euglycemic throughout pregnancy were enrolled in this study. Pre-pregnancy weight/height were recorded. WBC,ALT,and AST levels were detected between 8 and 12 weeks of pregnancy.At 24 to 28 weeks of pregnancy,the glucose and insulin levels were measured. The WBC,ALT,and AST levels were compared between two groups,and the associations of WBC,ALT,and AST levels with the blood glucose and insulin levels were retrospectively analyzed. Meanwhile,the potential associations of those factors with the occurrence of GDM were analzyed. Results WBC count [9.41(8.15,10.84)?10(9)/L vs. 9.04 (7.64,10.37)?10(9)/L,P=1.0?10(-5)] and ALT levels [18.00(12.00,30.00)U/L vs. 16.00 (11.00,26.00)U/L,P=0.004] in the first trimester of pregnancy were significantly increased in GDM subjects than in normal glucose tolerance(NGT)subjects;however,the AST level showed no significant difference between these two groups [41.00 (26.00,43.00)U/L vs. 41.00 (23.00,43.00)U/L,P=0.588]. Logistic regression analysis illustrated that elevated WBC count was an independent risk factor for GDM after adjustment for age,pre-pregnancy body mass index,blood pressure,and family history of diabetes(OR=1.119,P=0.001). The ROC curve revealed that threshold of WBC count was 7.965?10(9)/L(AUC=0.566,P=1?10(-5)),which had a sensitivity of 79.4% and a specificity of 31.3%. Multivariate linear regression analysis showed that homeostasis model assessment of insulin resistance was positively correlated with WBC count(B=0.051,P=0.022,R(2)=0.083);1-hour blood glucose after oral 50 grams of sugar (B=0.044,P=0.001,R(2)=0.044) and fasting plasma true insulin(B=0.214,P=0.032,R(2)=0.066) were positively correlated

  3. Elevated Preoperative Serum Alanine Aminotransferase/Aspartate Aminotransferase (ALT/AST) Ratio Is Associated with Better Prognosis in Patients Undergoing Curative Treatment for Gastric Adenocarcinoma

    PubMed Central

    Chen, Shu-Lin; Li, Jian-Pei; Li, Lin-Fang; Zeng, Tao; He, Xia

    2016-01-01

    The level of anine aminotransferase/aspartate aminotransferase (ALT/AST) ratio in the serum was often used to assess liver injury. Whether the ALT/AST ratio (LSR) was associated with prognosis for gastric adenocarcinoma (GA) has not been reported in the literature. Our aim was to investigate the prognostic value of the preoperative LSR in patients with GA. A retrospective study was performed in 231 patients with GA undergoing curative resection. The medical records collected include clinical information and laboratory results. We investigated the correlations between the preoperative LSR and overall survival (OS). Survival analysis was conducted with the Kaplan–Meier method, and Cox regression analysis was used to determine significant independent prognostic factors for predicting survival. A p value of <0.05 was considered to be statistically significant. A total of 231 patients were finally enrolled. The median overall survival was 47 months. Multivariate analysis indicated that preoperative LSR was an independent prognostic factor in GA. Patients with LSR ≤ 0.80 had a greater risk of death than those with LSR > 0.80. The LSR was independently associated with OS in patients with GA (hazard ratio: 0.610; 95% confidence interval: 0.388–0.958; p = 0.032), along with tumor stages (hazard ratio: 3.118; 95% confidence interval: 2.044–4.756; p < 0.001) and distant metastases (hazard ratio: 1.957; 95% confidence interval: 1.119–3.422; p = 0.019). Our study first established a connection between the preoperative LSR and patients undergoing curative resection for GA, suggesting that LSR was a simple, inexpensive, and easily measurable marker as a prognostic factor, and may help to identify high-risk patients for treatment decisions. PMID:27294917

  4. NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5'-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly-L-lysine.

    PubMed

    Chan-Huot, Monique; Dos, Alexandra; Zander, Reinhard; Sharif, Shasad; Tolstoy, Peter M; Compton, Shara; Fogle, Emily; Toney, Michael D; Shenderovich, Ilya; Denisov, Gleb S; Limbach, Hans-Heinrich

    2013-12-01

    Using (15)N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5'-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-L-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using (15)N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed (13)C and (15)N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-L-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism. PMID:24147985

  5. Studies on the influence of combustion exhaust gases and the products of their reaction with ammonia on the living organism. II. The influence on aspartate aminotransferase (AspAT) and alanine aminotransferase (AiAt) activities in the liver of guinea pig.

    PubMed

    Lewandowska-Tokarz, A; Stanosek, J; Ludyga, K; Kochanski, L

    1981-01-01

    The behaviour of aspartate aminotransferase (AspAT) an alanine aminotransferase (AIAT) in the whole homogenate and subcellular liver fractions of guinea pigs exposed to combustion exhaust gases and the neutralization products of these gases is presented in this paper. In the liver of animals exposed to the chronic action of combustion exhaust gases a decrease of both enzyme activities in the whole homogenate as well as in the subcellular fractions could be noted. Statistically significant changes are shown by AspAT. In the group of animals subjected to the action of neutralization products an increase of AIAT activity was observed. The activity of AspAT still shows a decrease, but less distinct in comparison with group I. An exception here is the mitochondrial fraction in which the AspAT activity is distinctly increased.

  6. Alanine aminotransferase controls seed dormancy in barley

    PubMed Central

    Sato, Kazuhiro; Yamane, Miki; Yamaji, Nami; Kanamori, Hiroyuki; Tagiri, Akemi; Schwerdt, Julian G.; Fincher, Geoffrey B.; Matsumoto, Takashi; Takeda, Kazuyoshi; Komatsuda, Takao

    2016-01-01

    Dormancy allows wild barley grains to survive dry summers in the Near East. After domestication, barley was selected for shorter dormancy periods. Here we isolate the major seed dormancy gene qsd1 from wild barley, which encodes an alanine aminotransferase (AlaAT). The seed dormancy gene is expressed specifically in the embryo. The AlaAT isoenzymes encoded by the long and short dormancy alleles differ in a single amino acid residue. The reduced dormancy allele Qsd1 evolved from barleys that were first domesticated in the southern Levant and had the long dormancy qsd1 allele that can be traced back to wild barleys. The reduced dormancy mutation likely contributed to the enhanced performance of barley in industrial applications such as beer and whisky production, which involve controlled germination. In contrast, the long dormancy allele might be used to control pre-harvest sprouting in higher rainfall areas to enhance global adaptation of barley. PMID:27188711

  7. Alanine aminotransferase controls seed dormancy in barley.

    PubMed

    Sato, Kazuhiro; Yamane, Miki; Yamaji, Nami; Kanamori, Hiroyuki; Tagiri, Akemi; Schwerdt, Julian G; Fincher, Geoffrey B; Matsumoto, Takashi; Takeda, Kazuyoshi; Komatsuda, Takao

    2016-01-01

    Dormancy allows wild barley grains to survive dry summers in the Near East. After domestication, barley was selected for shorter dormancy periods. Here we isolate the major seed dormancy gene qsd1 from wild barley, which encodes an alanine aminotransferase (AlaAT). The seed dormancy gene is expressed specifically in the embryo. The AlaAT isoenzymes encoded by the long and short dormancy alleles differ in a single amino acid residue. The reduced dormancy allele Qsd1 evolved from barleys that were first domesticated in the southern Levant and had the long dormancy qsd1 allele that can be traced back to wild barleys. The reduced dormancy mutation likely contributed to the enhanced performance of barley in industrial applications such as beer and whisky production, which involve controlled germination. In contrast, the long dormancy allele might be used to control pre-harvest sprouting in higher rainfall areas to enhance global adaptation of barley. PMID:27188711

  8. Aspartate Aminotransferase in Alfalfa Root Nodules 1

    PubMed Central

    Farnham, Mark W.; Griffith, Stephen M.; Miller, Susan S.; Vance, Carroll P.

    1990-01-01

    Aspartate aminotransferase (AAT) plays an important role in nitrogen metabolism in all plants and is particularly important in the assimilation of fixed N derived from the legume-Rhizoblum symbiosis. Two isozymes of AAT (AAT-1 and AAT-2) occur in alfalfa (Medicago sativa L.). Antibodies against alfalfa nodule AAT-2 do not recognize AAT-1, and these antibodies were used to study AAT-2 expression in different tissues and genotypes of alfalfa and also in other legume and nonlegume species. Rocket immunoelectrophoresis indicated that nodules of 38-day-old alfalfa plants contained about eight times more AAT-2 than did nodules of 7-day-old plants, confirming the nodule-enhanced nature of this isozyme. AAT-2 was estimated to make up 16, 15, 5, and 8 milligrams per gram of total soluble protein in mature nodules, roots, stems, and leaves, respectively, of effective N2-fixing alfalfa. The concentration of AAT-2 in nodules of ineffective non-N2-fixing alafalfa genotypes was about 70% less than that of effective nodules. Western blots of soluble protein from nodules of nine legume species indicated that a 40-kilodalton polypeptide that reacts strongly with AAT-2 antibodies is conserved in legumes. Nodule AAT-2 immunoprecipitation data suggested that amide- and ureide-type legumes may differ in expression and regulation of the enzyme. In addition, Western blotting and immunoprecipitations of AAT activity demonstrated that antibodies against alfalfa AAT-2 are highly cross-reactive with AAT enzyme protein in leaves of soybean (Glycine max L.), wheat (Triticum aestivum L.), and maize (Zea mays L.) and in roots of maize, but not with AAT in soybean and wheat roots. Results from this study indicate that AAT-2 is structurally conserved and localized in similar tissues among diverse species. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:16667896

  9. Radioimmunoassay of aspartate aminotransferase isoenzymes in human serum

    SciTech Connect

    Leung, F.Y.; Niblock, A.E.; Henderson, A.R.

    1984-08-01

    A description is given of the development of a sensitive, specific radioimmunoassay for the cytoplasmic and mitochondrial isoenzymes of human aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase; EC 2.6.1.1). Isoenzymes from human heart tissue were purified to homogeneity and used to raise high-titer antisera in rabbits. The antisera were partly purified by selective column chromatography. The Bolton-Hunter reagent was used to radioiodinate the isoenzymes. The assay requires 100 microL of serum, includes a solid-phase second-antibody separation, and can be completed in less than 3 h. There was no cross reactivity between the two isoenzymes. As little as 5 micrograms (50 pmol) of each aspartate aminotransferase can be measured per liter of serum.

  10. Eating a healthy lunch improves serum alanine aminotransferase activity

    PubMed Central

    2013-01-01

    Background Nutritional guidance and diet control play important roles in the treatment of obesity and non-alcoholic fatty liver. However, in Japan, nutritional guidance is difficult to provide in practice. Therefore, we evaluated the effects of providing the ‘once-a-day’ intervention of a healthy lunch on various metabolic parameters. Methods For a 1-month preparatory period, 10 subjects generally consumed the lunches that were provided by the worksite cafeteria. This was followed by a 1-week washout period, after which, the subjects consumed healthy, low-calorie, well-balanced lunches for a 1-month test period. After the preparatory and test periods, blood samples were obtained from all subjects. The serum levels of indices relevant to metabolic syndrome and fatty liver were measured. Results Serum alanine aminotransferase activity significantly decreased by 20.3% after the healthy intervention. However, the indices of metabolic syndrome did not significantly change. Analysis of the relationship between serum alanine aminotransferase activity and nutrient content indicated that the improvement of serum alanine aminotransferase status was due to the higher vegetable content and lower animal-source protein of the meals provided. Conclusions In summary, the ‘once-a-day’ intervention of providing a healthy lunch improved serum alanine aminotransferase status. A diet high in vegetables and low in animal-based protein is important in maintaining a healthy condition. PMID:24034595

  11. [Aspartate aminotransferase--key enzyme in the human systemic metabolism].

    PubMed

    Otto-Ślusarczyk, Dagmara; Graboń, Wojciech; Mielczarek-Puta, Magdalena

    2016-01-01

    Aspartate aminotransferase is an organ-nonspecific enzyme located in many tissues of the human body where it catalyzes reversible reaction of transamination. There are two aspartate aminotransferase isoforms--cytoplasmic (AST1) and mitochondrial (AST2), that usually occur together and interact with each other metabolically. Both isoforms are homodimers containing highly conservative regions responsible for catalytic properties of enzyme. The common feature of all aspartate aminotransfeses is Lys - 259 residue covalent binding with prosthetic group - pyridoxal phosphate. The differences in the primary structure of AST isoforms determine their physico-chemical, kinetic and immunological properties. Because of the low concentration of L-aspartate (L-Asp) in the blood, AST is the only enzyme, which supply of this amino acid as a substrate for many metabolic processes, such as urea cycle or purine and pyrimidine nucleotides in the liver, synthesis of L-arginine in the kidney and purine nucleotide cycle in the brain and the skeletal muscle. AST is also involved in D-aspartate production that regulates the metabolic activity at the auto-, para- and endocrine level. Aspartate aminotransferase is a part of the malate-aspartate shuttle in the myocardium, is involved in gluconeogenesis in the liver and kidney, glyceroneogenesis in the adipose tissue, and synthesis of neurotransmitters and neuro-glial pathway in the brain. Recently, the significant role of AST in glutaminolysis - normal metabolic pathway in tumor cells, was demonstrated. The article is devoted the role of AST, known primarily as a diagnostic liver enzyme, in metabolism of various human tissues and organs. PMID:27117097

  12. Crystal Structures of Aedes Aegypt Alanine Glyoxylate Aminotransferase

    SciTech Connect

    Han,Q.; Robinson, H.; Gao, Y.; Vogelaar, N.; Wilson, S.; Rizzi, M.; Li, J.

    2006-01-01

    Mosquitoes are unique in having evolved two alanine glyoxylate aminotransferases (AGTs). One is 3-hydroxykynurenine transaminase (HKT), which is primarily responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). Interestingly, XA is used by malaria parasites as a chemical trigger for their development within the mosquito. This 3-HK to XA conversion is considered the major mechanism mosquitoes use to detoxify the chemically reactive and potentially toxic 3-HK. The other AGT is a typical dipteran insect AGT and is specific for converting glyoxylic acid to glycine. Here we report the 1.75{angstrom} high-resolution three-dimensional crystal structure of AGT from the mosquito Aedes aegypti (AeAGT) and structures of its complexes with reactants glyoxylic acid and alanine at 1.75 and 2.1{angstrom} resolution, respectively. This is the first time that the three-dimensional crystal structures of an AGT with its amino acceptor, glyoxylic acid, and amino donor, alanine, have been determined. The protein is dimeric and adopts the type I-fold of pyridoxal 5-phosphate (PLP)-dependent aminotransferases. The PLP co-factor is covalently bound to the active site in the crystal structure, and its binding site is similar to those of other AGTs. The comparison of the AeAGT-glyoxylic acid structure with other AGT structures revealed that these glyoxylic acid binding residues are conserved in most AGTs. Comparison of the AeAGT-alanine structure with that of the Anopheles HKT-inhibitor complex suggests that a Ser-Asn-Phe motif in the latter may be responsible for the substrate specificity of HKT enzymes for 3-HK.

  13. Radiochemical microassay for aspartate aminotransferase activity in the nervous system

    SciTech Connect

    Garrison, D.; Beattie, J.; Namboodiri, M.A.

    1988-07-01

    A radiochemical procedure for measuring aspartate aminotransferase activity in the nervous system is described. The method is based on the exchange of tritium atoms at positions 2 and 3 of L-2,3-(/sup 3/H)aspartate with water when this amino acid is transaminated in the presence of alpha-ketoglutarate to form oxaloacetate. The tritiated water is separated from the radiolabeled aspartate by passing the reaction mixture over a cation exchange column. Confirmation that the radioactivity in the product is associated with water was obtained by separating it by anion exchange HPLC and by evaporation. The product formation is linear with time up to 120 min and with tissue in the 0.05- to 10-micrograms range. The apparent Km for aspartate in the rat brain homogenate is found to be 0.83 mM and that for alpha-ketoglutarate to be 0.12 mM. Methods that further improve the sensitivity of the assay are also discussed.

  14. Crystal structure of Saccharomyces cerevisiae cytosolic aspartate aminotransferase.

    PubMed Central

    Jeffery, C. J.; Barry, T.; Doonan, S.; Petsko, G. A.; Ringe, D.

    1998-01-01

    The crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 A resolution in the presence of the cofactor pyridoxal-5'-phosphate and the competitive inhibitor maleate. The structure was solved by the method of molecular replacement. The final value of the crystallographic R-factor after refinement was 23.1% with good geometry of the final model. The yeast cytoplasmic enzyme is a homodimer with two identical active sites containing residues from each subunit. It is found in the "closed" conformation with a bound maleate inhibitor in each active site. It shares the same three-dimensional fold and active site residues as the aspartate aminotransferases from Escherichia coli, chicken cytoplasm, and chicken mitochondria, although it shares less than 50% sequence identity with any of them. The availability of four similar enzyme structures from distant regions of the evolutionary tree provides a measure of tolerated changes that can arise during millions of years of evolution. PMID:9655342

  15. PPAR{alpha} regulates the hepatotoxic biomarker alanine aminotransferase (ALT1) gene expression in human hepatocytes

    SciTech Connect

    Thulin, Petra; Rafter, Ingalill; Stockling, Kenneth; Tomkiewicz, Celine; Norjavaara, Ensio; Aggerbeck, Martine; Hellmold, Heike; Ehrenborg, Ewa; Andersson, Ulf; Cotgreave, Ian; Glinghammar, Bjoern

    2008-08-15

    In this work, we investigated a potential mechanism behind the observation of increased aminotransferase levels in a phase I clinical trial using a lipid-lowering drug, the peroxisome proliferator-activated receptor (PPAR) {alpha} agonist, AZD4619. In healthy volunteers treated with AZD4619, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were elevated without an increase in other markers for liver injury. These increases in serum aminotransferases have previously been reported in some patients receiving another PPAR{alpha} agonist, fenofibrate. In subsequent in vitro studies, we observed increased expression of ALT1 protein and mRNA in human hepatocytes after treatment with fenofibric acid. The PPAR effect on ALT1 expression was shown to act through a direct transcriptional mechanism involving at least one PPAR response element (PPRE) in the proximal ALT1 promoter, while no effect of fenofibrate and AZD4619 was observed on the ALT2 promoter. Binding of PPARs to the PPRE located at - 574 bp from the transcriptional start site was confirmed on both synthetic oligonucleotides and DNA in hepatocytes. These data show that intracellular ALT expression is regulated by PPAR agonists and that this mechanism might contribute to increased ALT activity in serum.

  16. Alanine Aminotransferase-Old Biomarker and New Concept: A Review

    PubMed Central

    Liu, Zhengtao; Que, Shuping; Xu, Jing; Peng, Tao

    2014-01-01

    Measurement of serum alanine aminotransferase (ALT) is a common, readily available, and inexpensive laboratory assay in clinical practice. ALT activity is not only measured to detect liver disease, but also to monitor overall health. ALT activity is influenced by various factors, including viral hepatitis, alcohol consumption, and medication. Recently, the impact of metabolic abnormalities on ALT variation has raised concern due to the worldwide obesity epidemic. The normal ranges for ALT have been updated and validated considering the metabolic covariates in the various ethnic districts. The interaction between metabolic and demographic factors on ALT variation has also been discussed in previous studies. In addition, an extremely low ALT value might reflect the process of aging, and frailty in older adults has been raised as another clinically significant feature of this enzyme, to be followed with additional epidemiologic investigation. Timely updated, comprehensive, and systematic introduction of ALT activity is necessary to aid clinicians make better use of this enzyme. PMID:25013373

  17. Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants.

    PubMed

    de la Torre, Fernando; Cañas, Rafael A; Pascual, M Belén; Avila, Concepción; Cánovas, Francisco M

    2014-10-01

    In the chloroplasts and in non-green plastids of plants, aspartate is the precursor for the biosynthesis of different amino acids and derived metabolites that play distinct and important roles in plant growth, reproduction, development or defence. Aspartate biosynthesis is mediated by the enzyme aspartate aminotransferase (EC 2.6.1.1), which catalyses the reversible transamination between glutamate and oxaloacetate to generate aspartate and 2-oxoglutarate. Plastids contain two aspartate aminotransferases: a eukaryotic-type and a prokaryotic-type bifunctional enzyme displaying aspartate and prephenate aminotransferase activities. A general overview of the biochemistry, regulation, functional significance, and phylogenetic origin of both enzymes is presented. The roles of these plastidic aminotransferases in the biosynthesis of essential amino acids are discussed.

  18. Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV

    SciTech Connect

    Han, Q.; Robinson, H.; Cai, T.; Tagle, D. A.; Li, J.

    2011-10-01

    Mammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine aminotransferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14 of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of other KATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

  19. Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV

    PubMed Central

    Han, Qian; Robinson, Howard; Cai, Tao; Tagle, Danilo A.; Li, Jianyong

    2010-01-01

    Synopsis Mammalian mitochondrial aspartate aminotransferase (mAspAT) is recently reported to have kynurenine aminotransferase (KAT) activity and plays a role in the biosynthesis of kynurenic acid (KYNA) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen keto acids were tested for the co-substrate specificity of mouse mAspAT and fourteen of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor binding residues of mAspAT are similar to those of other KATs. The substrate binding residues of mAspAT are slightly different from those of other KATs. Our data provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain. PMID:20977429

  20. Alanine aminotransferase variants conferring diverse NUE phenotypes in Arabidopsis thaliana.

    PubMed

    McAllister, Chandra H; Good, Allen G

    2015-01-01

    Alanine aminotransferase (AlaAT, E.C. 2.6.1.2), is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes the reversible transfer of an amino group from alanine to 2-oxoglutarate to produce glutamate and pyruvate, or vice versa. It has been well documented in both greenhouse and field studies that tissue-specific over-expression of AlaAT from barley (Hordeum vulgare, HvAlaAT) results in a significant increase in plant NUE in both canola and rice. While the physical phenotypes associated with over-expression of HvAlaAT have been well characterized, the role this enzyme plays in vivo to create a more N efficient plant remains unknown. Furthermore, the importance of HvAlaAT, in contrast to other AlaAT enzyme homologues in creating this phenotype has not yet been explored. To address the role of AlaAT in NUE, AlaAT variants from diverse sources and different subcellular locations, were expressed in the wild-type Arabidopsis thaliana Col-0 background and alaat1;2 (alaat1-1;alaat2-1) knockout background in various N environments. The analysis and comparison of both the physical and physiological properties of AlaAT over-expressing transgenic plants demonstrated significant differences between plants expressing the different AlaAT enzymes under different external conditions. This analysis indicates that the over-expression of AlaAT variants other than HvAlaAT in crop plants could further increase the NUE phenotype(s) previously observed.

  1. Alanine-aminotransferase: an early marker for insulin resistance?

    PubMed

    Salazar, Martin R; Carbajal, Horacio A; Curciarello, Jose O; Aizpurua, Marcelo; Adrover, Raul E; Riondet, Beatriz

    2007-01-01

    In a population-based sample, after excluding alcohol consumption, hepatotoxic drugs and hepatitis B and C infected, we investigated if alanine-aminotransferase (ALT) was associated with metabolic syndrome and insulin resistance, and if this association was caused by non-alcoholic fatty liver disease (NAFLD). The sample (432 female and 119 male) was divided into two ALT thresholds corresponding to the 50th and 75th percentiles (P) (female > or = 15 and > or = 19 U/L; male > or = 17 and > or = 23 U/I, respectively). Blood pressure, body mass index, waist circumference, cholesterol, HDL cholesterol (HDLc), triglyceride (TG), TG/HDLc ratio, glycemia and homeostasis model assessment of insulin resistance (HOMA-IR) were compared between those above and below each ALT threshold. Female placed above the 50th P of ALT had higher levels of TG/HDLc ratio (p=0.029), glycemia (p=0.028), and homeostasis model assessment of insulin resistance, (p=0.045), and above the 75th P had higher SBP (p=0.036), DBP (p=0.018), TG (p=0.024), TG/HDLc ratio (p=0.028), glycemia (p=0.004) and HOMA-IR (p=0.0014). Male placed above the 50th P of ALT had higher BMI (p=0.017) and TG/HDLc ratio (p=0.048), and above the 75th P had lower values of HDLc (p=0.042). Only 16.5% of women and 14.5% of men, above the 75th P of ALT, showed an increase in liver brightness in the echography. This work shows in woman an early association of ALT with TG/HDLc ratio and HOMA-IR. Since the last two are independent predictors of cardiovascular risk, attention should be drawn to ALT values near the upper limit of the normal range even in the absence of NAFLD and obesity. PMID:17593595

  2. Irritable Bowel Syndrome May Be Associated with Elevated Alanine Aminotransferase and Metabolic Syndrome

    PubMed Central

    Lee, Seung-Hwa; Kim, Kwang-Min; Joo, Nam-Seok

    2016-01-01

    Purpose Recent studies have revealed close relationships between hepatic injury, metabolic pathways, and gut microbiota. The microorganisms in the intestine also cause irritable bowel syndrome (IBS). The aim of this study was to examine whether IBS was associated with elevated hepatic enzyme [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)], gamma-glutamyl transferase (γ-GT) levels, and metabolic syndrome (MS). Materials and Methods This was a retrospective, cross-sectional, case-control study. The case and control groups comprised subjects who visited our health promotion center for general check-ups from June 2010 to December 2010. Of the 1127 initially screened subjects, 83 had IBS according to the Rome III criteria. The control group consisted of 260 age- and sex-matched subjects without IBS who visited our health promotion center during the same period. Results Compared to control subjects, patients with IBS showed significantly higher values of anthropometric parameters (body mass index, waist circumference), liver enzymes, γ-GT, and lipid levels. The prevalences of elevated ALT (16.9% vs. 7.7%; p=0.015) and γ-GT (24.1% vs. 11.5%; p=0.037) levels were significantly higher in patients with IBS than in control subjects. A statistically significant difference was observed in the prevalence of MS between controls and IBS patients (12.7% vs. 32.5%; p<0.001). The relationships between elevated ALT levels, MS, and IBS remained statistically significant after controlling for potential confounding factors. Conclusion On the basis of our study results, IBS may be an important condition in certain patients with elevated ALT levels and MS. PMID:26632395

  3. Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro.

    PubMed Central

    Marra, E; Doonan, S; Saccone, C; Quagliariello, E

    1977-01-01

    1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo. PMID:883959

  4. Beta-alanine/alpha-ketoglutarate aminotransferase for 3-hydroxypropionic acid production

    DOEpatents

    Jessen, Holly Jean; Liao, Hans H.; Gort, Steven John; Selifonova, Olga V.

    2011-10-04

    The present disclosure provides novel beta-alanine/alpha ketoglutarate aminotransferase nucleic acid and protein sequences having increased biological activity. Also provided are cells containing such enzymes, as well as methods of their use, for example to produce malonyl semialdehyde and downstream products thereof, such as 3-hydroxypropionic acid and derivatives thereof.

  5. Beta-alanine/alpha-ketoglutarate aminotransferase for 3-hydroxypropionic acid production

    DOEpatents

    Jessen, Holly Jean; Liao, Hans H; Gort, Steven John; Selifonova, Olga V

    2014-11-18

    The present disclosure provides novel beta-alanine/alpha ketoglutarate aminotransferase nucleic acid and protein sequences having increased biological activity. Also provided are cells containing such enzymes, as well as methods of their use, for example to produce malonyl semialdehyde and downstream products thereof, such as 3-hydroxypropionic acid and derivatives thereof.

  6. Structural Insights into a Novel Class of Aspartate Aminotransferase from Corynebacterium glutamicum

    PubMed Central

    Son, Hyeoncheol Francis; Kim, Kyung-Jin

    2016-01-01

    Aspartate aminotransferase from Corynebacterium glutamicum (CgAspAT) is a PLP-dependent enzyme that catalyzes the production of L-aspartate and α-ketoglutarate from L-glutamate and oxaloacetate in L-lysine biosynthesis. In order to understand the molecular mechanism of CgAspAT and compare it with those of other aspartate aminotransferases (AspATs) from the aminotransferase class I, we determined the crystal structure of CgAspAT. CgAspAT functions as a dimer, and the CgAspAT monomer consists of two domains, the core domain and the auxiliary domain. The PLP cofactor is found to be bound to CgAspAT and stabilized through unique residues. In our current structure, a citrate molecule is bound at the active site of one molecule and mimics binding of the glutamate substrate. The residues involved in binding of the PLP cofactor and the glutamate substrate were confirmed by site-directed mutagenesis. Interestingly, compared with other AspATs from aminotransferase subgroup Ia and Ib, CgAspAT exhibited unique binding sites for both cofactor and substrate; moreover, it was found to have unusual structural features in the auxiliary domain. Based on these structural differences, we propose that CgAspAT does not belong to either subgroup Ia or Ib, and can be categorized into a subgroup Ic. The phylogenetic tree and RMSD analysis also indicates that CgAspAT is located in an independent AspAT subgroup. PMID:27355211

  7. A novel low molecular weight alanine aminotransferase from fasted rat liver.

    PubMed

    Vedavathi, M; Girish, K S; Kumar, M Karuna

    2006-01-01

    Alanine is the most effective precursor for gluconeogenesis among amino acids, and the initial reaction is catalyzed by alanine aminotransferase (AlaAT). Although the enzyme activity increases during fasting, this effect has not been studied extensively. The present study describes the purification and characterization of an isoform of AlaAT from rat liver under fasting. The molecular mass of the enzyme is 17.7 kD with an isoelectric point of 4.2; glutamine is the N-terminal residue. The enzyme showed narrow substrate specificity for L-alanine with Km values for alanine of 0.51 mM and for 2-oxoglutarate of 0.12 mM. The enzyme is a glycoprotein. Spectroscopic and inhibition studies showed that pyridoxal phosphate (PLP) and free -SH groups are involved in the enzymatic catalysis. PLP activated the enzyme with a Km of 0.057 mM. PMID:16487061

  8. Isolation and characterization of cytosolic alanine aminotransferase isoforms from starved rat liver.

    PubMed

    Vedavathi, M; Girish, K S; Kumar, M Karuna

    2004-12-01

    Alanine is the most effective precursor for gluconeogenesis among amino acids and the initial reaction is catalyzed by alanine aminotransferases (AlaATs). It is a less extensively studied enzyme under starvation and known to that the enzyme activity increases in liver under starvation. The present study describes the purification and characterization of two isoforms of alanine aminotransferases from starved male rat liver under starvation. The molecular mass of isoforms was found to be 17.7 and 112.2 kDa with isoelectric points of 4.2 and 5.3 respectively for AlaAT I and AlaAT II. Both the enzymes showed narrow substrate specificity for L-alanine with different Km for alanine and 2-oxoglutarate. Both the enzymes were glycoprotein in nature. Inhibition, modification and spectroscopic studies showed that both PLP and free-SH groups are directly involved in the enzymatic catalysis. PLP activated both the enzymes with a Km 0.057 mM and 0.2 mM for AlaAT I and II respectively. PMID:15663181

  9. Isolation and characterization of a gene coding for a novel aspartate aminotransferase from Rhizobium meliloti.

    PubMed Central

    Alfano, J R; Kahn, M L

    1993-01-01

    Aspartate aminotransferase (AAT) is an important enzyme in aspartate catabolism and biosynthesis and, by converting tricarboxylic acid cycle intermediates to amino acids, AAT is also significant in linking carbon metabolism with nitrogen metabolism. To examine the role of AAT in symbiotic nitrogen fixation further, plasmids encoding three different aminotransferases from Rhizobium meliloti 104A14 were isolated by complementation of an Escherichia coli auxotroph that lacks three aminotransferases. pJA10 contained a gene, aatB, that coded for a previously undescribed AAT, AatB. pJA30 encoded an aromatic aminotransferase, TatA, that had significant AAT activity, and pJA20 encoded a branched-chain aminotransferase designated BatA. Genes for the latter two enzymes, tatA and batA, were previously isolated from R. meliloti. aatB is distinct from but hybridizes to aatA, which codes for AatA, a protein required for symbiotic nitrogen fixation. The DNA sequence of aatB contained an open reading frame that could encode a protein 410 amino acids long and with a monomer molecular mass of 45,100 Da. The amino acid sequence of aatB is unusual, and AatB appears to be a member of a newly described class of AATs. AatB expressed in E. coli has a Km for aspartate of 5.3 mM and a Km for 2-oxoglutarate of 0.87 mM. Its pH optimum is between 8.0 and 8.5. Mutations were constructed in aatB and tatA and transferred to the genome of R. meliloti 104A14. Both mutants were prototrophs and were able to carry out symbiotic nitrogen fixation. Images PMID:8320232

  10. Aspartate aminotransferase and alanine aminotransferase activities in plasma: statistical distributions, individual variations, and reference values.

    PubMed

    Siest, G; Schiele, F; Galteau, M M; Panek, E; Steinmetz, J; Fagnani, F; Gueguen, R

    1975-07-01

    The determination of frequency value (percentile limits) and the classification of the different variation factors allow us to define more and more homogeneous subpopulations as we use these factors for sorting. Using as our study population those persons coming to the Centre for Preventive Medicine, we were able to: (a) Describe and measure the significance and importance of physiological variations or of variations attributed to age--the latter largely related only to excessive weight, which it seems to us is often the case. (b) Establish a classification for variation factors; the recapitulatory table should be useful to clinical chemists in helping physicians interpret a laboratory test result that falls within the zone of incertitude. (c) Suggest a preliminary group of reference values for healthy subjects, to be used in interpreting a laboratory test in this way.

  11. Uptake of aspartate aminotransferase into mitochondria in vitro depends on the transmembrane pH gradient.

    PubMed Central

    Passarella, S; Marra, E; Doonan, S; Languino, L R; Saccone, C; Quagliariello, E

    1982-01-01

    1. The effects of various inhibitors of electron transport and of oxidative phosphorylation and the effects of ionophores on the uptake of native aspartate aminotransferase into mitochondria were investigated. 2. Both antimycin and cyanide completely inhibited the uptake of the enzyme. On the other hand, uptake was stimulated to ATP and by oligomycin; however, the stimulation by ATP is inhibited by oligomycin. 3. The effects of ionophores of the valinomycin type in media containing K+ ions depended on the conditions used. Valinomycin alone stimulated the uptake of the enzyme, but in the presence of phosphate ions uptake was abolished. Nonactin was without effect at a low K+ concentration, but was stimulatory at 100 mM-KCl. Gramicidin also stimulated the uptake process. 4. Nigericin completely abolished uptake of aspartate aminotransferase into mitochondria. 5. The uptake of te enzyme was decreased by 18% in the absence of inhibitors or ionophores when the external pH was increased from 6.9 to 7.6. 6. These results indicate that ATP is not directly involved in the uptake of aspartate aminotransferase into mitochondria, neither is there a requirement for a cation gradient. Rather the uptake depends on the maintenance of a pH gradient across the mitochondrial inner membrane. PMID:7092821

  12. The anomalous kinetics of coupled aspartate aminotransferase and malate dehydrogenase. Evidence for compartmentation of oxaloacetate.

    PubMed Central

    Bryce, C F; Williams, D C; John, R A; Fasella, P

    1976-01-01

    Cytoplasmic aspartate aminotransferase and malate dehydrogenase were purified from pig heart. Kinetic parameters were determined for the separate reaction catalysed by each enzyme and used to predict the course of the coupled reaction: (see article). Although a lag phase should have been easily seen, none was detected. The same coupled reaction was also carried out by using radioactive aspartate in the presence of unlabelled oxaloacetate. The reaction was quenched with HClO4 after 70 ms and the specific radioactivity of the malate produced in this system was found to be essentially the same as that of the original aspartate. These results show that oxaloacetate produced by the aspartate aminotransferase is converted into malate by malate dehydrogenase before it equilibrates with the pool of unlabelled oxaloacetate and are consistent with a proposal that the enzymes are associated in a complex. However, no physical evidence of the existence of a complex could be found. An alternative means of compartmentation of the intermediate as an unstable isomer is considered. Images Fig. 2. Fig. 3. PMID:942372

  13. Synthesis of β-alanine from L-aspartate using L-aspartate-α-decarboxylase from Corynebacterium glutamicum.

    PubMed

    Shen, Yan; Zhao, Lianzhen; Li, Youran; Zhang, Liang; Shi, Guiyang

    2014-08-01

    β-Alanine is mainly produced by chemical methods in current industrial processes. Here, panD from Corynebacterium glutamicum encoding L-aspartate-α-decarboxylase (ADC) was cloned and expressed in Escherichia coli BL21(DE3). ADC C.g catalyzes the α-decarboxylation of L-aspartate to β-alanine. The purified ADC C.g was optimal at 55 °C and pH 6 with excellent stability at 16-37 °C and pH 4-7. A pH-stat directed, fed-batch feeding strategy was developed for enzymatic synthesis of β-alanine to keep the pH value within 6-7.2 and thus attenuate substrate inhibition. A maximum conversion of 97.2 % was obtained with an initial 5 g L-aspartate/l and another three feedings of 0.5 % (w/v) L-aspartate at 8 h intervals. The final β-alanine concentration was 12.85 g/l after 36 h. This is the first study concerning the enzymatic production of β-alanine by using ADC.

  14. Structures of aspartate aminotransferases from Trypanosoma brucei, Leishmania major and Giardia lamblia

    PubMed Central

    Abendroth, Jan; Choi, Ryan; Wall, Abigail; Clifton, Matthew C.; Lukacs, Christine M.; Staker, Bart L.; Van Voorhis, Wesley; Myler, Peter; Lorimer, Don D.; Edwards, Thomas E.

    2015-01-01

    The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm. PMID:25945710

  15. Structures of aspartate aminotransferases from Trypanosoma brucei, Leishmania major and Giardia lamblia.

    PubMed

    Abendroth, Jan; Choi, Ryan; Wall, Abigail; Clifton, Matthew C; Lukacs, Christine M; Staker, Bart L; Van Voorhis, Wesley; Myler, Peter; Lorimer, Don D; Edwards, Thomas E

    2015-05-01

    The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm. PMID:25945710

  16. Inhibition study of alanine aminotransferase enzyme using sequential online capillary electrophoresis analysis.

    PubMed

    Liu, Lina; Chen, Yuanfang; Yang, Li

    2014-12-15

    We report the study of several inhibitors on alanine aminotransferase (ALT) enzyme using sequential online capillary electrophoresis (CE) assay. Using metal ions (Na(+) and Mg(2+)) as example inhibitors, we show that evolution of the ALT inhibition reaction can be achieved by automatically and simultaneously monitoring the substrate consumption and product formation as a function of reaction time. The inhibition mechanism and kinetic constants of ALT inhibition with succinic acid and two traditional Chinese medicines were derived from the sequential online CE assay. Our study could provide valuable information about the inhibition reactions of ALT enzyme.

  17. Prodynorphine opioid peptides and aspartate aminotransferase studied in spinal cord and sensory neurons

    SciTech Connect

    Sweetnam, P.M.

    1985-01-01

    An objective of this research was to obtain evidence for the synthesis and release of newly discovered opioid peptides, such as dynorphin, in spinal cord and sensory neurons. Several specific antisera were used to visualize dynorphin and related peptides in spinal cord and dorsal root ganglion neurons in dissociated cell culture. Antisera specific for the midportion of the dynorphin molecule revealed a subpopulation of spinal cord neurons with dense immunoreactive dynorphin in cell perikarya, but none in their associated neurites. Antisera specific for either the amino or carboxy terminal sequences of the molecule produced intense immunoreactivity in both cell perikarya and neurites of spinal neurons. These data suggest the cleavage products of dynorphin and not the complete molecule are possible neurotransmitters in the spinal cord. Additional evidence in support of this hypothesis was derived from radioimmunoassays of these cells and their culture medium following depolarization induced by elevated extracellular potassium. Antisera against aspartate aminotransferase revealed no differentially elevated immunoreactive aspartate aminotransferase in tissue sections of spinal cord or dorsal root ganglia.

  18. Catalytic activity of non-cross-linked microcrystals of aspartate aminotransferase in poly(ethylene glycol).

    PubMed Central

    Kirsten, H; Christen, P

    1983-01-01

    The molar activity of crystalline mitochondrial aspartate aminotransferase is decreased to 10% of that of the enzyme in solution. The activity was measured in suspensions of non-cross-linked microcrystals (average dimensions 22 microns X 5 microns X 0.8 microns) in 30% (w/v) poly(ethylene glycol). Kinetic tests ruled out the possibility that diffusion of the substrate in the crystals is rate-limiting. The observed decrease in catalytic efficiency can be attributed exclusively to crystal-packing effects. A direct inhibition by poly(ethylene glycol) is excluded because poly(ethylene glycol), with average Mr 6000, cannot penetrate the liquid channels of the crystals, owing to its large Stokes radius. The crystals examined were triclinic and of the same habit as those used for high-resolution X-ray-crystallographic analysis [Ford, Eichele & Jansonius (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2559-2563]. The catalytic competence of crystalline aspartate aminotransferase confirms the relevance of the spatial model of this protein for the elucidation of its mechanism of action. Images Fig. 1. PMID:6870840

  19. The amino acid sequence of the aspartate aminotransferase from baker's yeast (Saccharomyces cerevisiae).

    PubMed Central

    Cronin, V B; Maras, B; Barra, D; Doonan, S

    1991-01-01

    1. The single (cytosolic) aspartate aminotransferase was purified in high yield from baker's yeast (Saccharomyces cerevisiae). 2. Amino-acid-sequence analysis was carried out by digestion of the protein with trypsin and with CNBr; some of the peptides produced were further subdigested with Staphylococcus aureus V8 proteinase or with pepsin. Peptides were sequenced by the dansyl-Edman method and/or by automated gas-phase methods. The amino acid sequence obtained was complete except for a probable gap of two residues as indicated by comparison with the structures of counterpart proteins in other species. 3. The N-terminus of the enzyme is blocked. Fast-atom-bombardment m.s. was used to identify the blocking group as an acetyl one. 4. Alignment of the sequence of the enzyme with those of vertebrate cytosolic and mitochondrial aspartate aminotransferases and with the enzyme from Escherichia coli showed that about 25% of residues are conserved between these distantly related forms. 5. Experimental details and confirmatory data for the results presented here are given in a Supplementary Publication (SUP 50164, 25 pages) that has been deposited at the British Library Document Supply Centre, Boston Spa. Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5. PMID:1859361

  20. Prevalence and Predictors of Elevated Aspartate Aminotransferase-to-Platelet Ratio Index in Latin American Perinatally HIV-infected Children

    PubMed Central

    Siberry, George K.; Cohen, Rachel A.; Harris, D. Robert; Cruz, Maria Leticia Santos; Oliveira, Ricardo; Peixoto, Mario F.; Cervi, Maria Celia; Hazra, Rohan; Pinto, Jorge A.

    2013-01-01

    Background Chronic liver disease has emerged as an important problem in adults with longstanding HIV infection, but data are lacking for children. We characterized elevated aspartate aminotransferase (AST)-to-platelet ratio index (APRI ), a marker of possible liver fibrosis, in perinatally HIV-infected children. Methods NISDI [NICHD (National Institute of Child Health and Human Development) International Site Development Initiative] enrolled HIV-infected children (ages 0.1-20.1 years) from five Latin American countries in an observational cohort from 2002–2009. Twice yearly visits included medical history, physical examination and laboratory evaluations. The prevalence (95% confidence interval [CI]) of APRI>1.5 was calculated and associations with demographic, HIV-related and liver-related variables were investigated in bivariate analyses. Results APRI was available for 1012 of 1032 children. APRI was >1.5 in 32 (3.2%, 95% CI: 2.2%-4.4%) including 2 of 4 participants with hepatitis B (HBV) infection. Factors significantly associated with APRI>1.5 (p<0.01 compared to APRI≤1.5) included country, younger age, past or current HBV, higher alanine aminotransferase, lower total cholesterol, higher log10 current viral load, lower current CD4 count, lower nadir CD4 count, use of hepatotoxic non-antiretroviral (ARV) medications, and no prior ARV use. Rates of APRI>1.5 varied significantly by current ARV regimen (p=0.0002), from 8.0% for no ARV to 3.2% for non-protease inhibitor (PI) regimens to 1.5% for PI-based regimens. Conclusions Elevated APRI occurred in approximately 3% of perinatally HIV-infected children. PI-based ARVs appeared protective while inadequate HIV control appeared to increase risk of elevated APRI. Additional investigations are needed to better assess potential subclinical, chronic liver disease in HIV-infected children. PMID:23799515

  1. Differential regulation of alanine aminotransferase homologues by abiotic stresses in wheat (Triticum aestivum L.) seedlings.

    PubMed

    Kendziorek, Maria; Paszkowski, Andrzej; Zagdańska, Barbara

    2012-06-01

    Wheat (Triticum aestivum L.) seedlings contain four alanine aminotransferase (AlaAT) homologues. Two of them encode AlaAT enzymes, whereas two homologues act as glumate:glyoxylate aminotransferase (GGAT). To address the function of the distinct AlaAT homologues a comparative examination of the changes in transcript level together with the enzyme activity and alanine and glutamate content in wheat seedlings subjected to low oxygen availability, nitrogen and light deficiency has been studied. Shoots of wheat seedlings were more tolerant to hypoxia than the roots as judging on the basis of enzyme activity and transcript level. Hypoxia induced AlaAT1 earlier in roots than in shoots, while AlaAT2 and GGAT were unaffected. The increase in AlaAT activity lagged behind the increase in alanine content. Nitrogen deficiency has little effect on the activity of GGAT. In contrast, lower activity of AlaAT and the level of mRNA for AlaAT1 and AlaAT2 in wheat seedlings growing on a nitrogen-free medium seems to indicate that AlaAT is regulated by the availability of nitrogen. Both AlaAT and GGAT activities were present in etiolated wheat seedlings but their activity was half of that observed in light-grown seedlings. Exposure of etiolated seedlings to light caused an increase in enzyme activities and up-regulated GGAT1. It is proposed that hypoxia-induced AlaAT1 and light-induced peroxisomal GGAT1 appears to be crucial for the regulation of energy availability in plants grown under unfavourable environmental conditions. Key message In young wheat seedlings, both AlaAT and GGAT are down-regulated by nitrogen deficiency, whereas AlaAT1 is upregulated by hypoxia and GGAT1 by light.

  2. Population differences of aspartate aminotransferase and peptidase in the bay mussel Mytilus edulis.

    PubMed

    Johnson, G; Utter, F M

    1975-01-01

    This investigation has demonstrated considerable heterogeneity among populations and some heterogeneity within populations in the distribution of alleles at two variant loci of Mytilus edulis. Although the causes of this variation remain obscure, some speculations have been made on the basis of available data. A cline for aspartate aminotransferase (AAT) alleles has been observed on the Pacific Coast. An immigration model has been proposed to explain the atypical ecological and genetic characteristics of large mussels found on Amchitka Island, Alaska. Marked differences were found in the distribution of peptidase alleles among collections from Southern California, the North Pacific Ocean, and New Jersey. Deviations from random distribution of phenotypes observed in comparisons made between large and small mussels from the New Jersey collection may reflect selection operating on these loci in this population.

  3. Macro-aspartate aminotransferase in a female with antibodies to hepatitis C virus.

    PubMed

    Collins, John; Ritter, Detlef; Bacon, Bruce R; Landt, Michael; Creer, Michael H

    2002-12-01

    Persistent elevation of aspartate aminotransferase (AST) activity in serum due to the presence of a macroenzyme form of AST (macro-AST) may lead to diagnostic confusion in many clinical conditions, particularly those associated with chronic liver disease. We describe a case of macro-AST arising in an adult female with a false-positive hepatitis C virus (HCV) RNA test result that was not accompanied by other biochemical or histologic evidence of liver disease. The presence of macro-AST in serum was confirmed utilizing size-exclusion, high performance liquid chromatography (HPLC) and Protein G-agarose beads to precipitate immune complexes of AST and immunoglobulin G followed by centrifugation and AST activity measurements in the supernatant. A brief review of the clinical enzymology of AST and methods used to quantify serum macro-AST activity is provided.

  4. The unfolding and refolding of cytoplasmic aspartate aminotransferase from pig heart.

    PubMed Central

    West, S M; Price, N C

    1989-01-01

    The unfolding of cytoplasmic aspartate aminotransferase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from protein fluorescence, c.d. and thiol-group reactivity indicated that the enzyme was unfolded in 6 M-GdnHCl. Spectroscopic studies showed that this unfolding was accompanied by dissociation of the pyridoxal 5'-phosphate cofactor. On dilution of the GdnHCl, re-activation of the enzyme occurred in reasonable yield, provided that dithiothreitol and pyridoxal 5'-phosphate were present. The regain of activity obeyed second-order kinetics. In the absence of added dithiothreitol and pyridoxal 5'-phosphate, substantial formation of high-Mr aggregates occurred. PMID:2775204

  5. Multiple adaptive losses of alanine-glyoxylate aminotransferase mitochondrial targeting in fruit-eating bats.

    PubMed

    Liu, Yang; Xu, Huihui; Yuan, Xinpu; Rossiter, Stephen J; Zhang, Shuyi

    2012-06-01

    The enzyme alanine-glyoxylate aminotransferase 1 (AGT) functions to detoxify glyoxylate before it is converted into harmful oxalate. In mammals, mitochondrial targeting of AGT in carnivorous species versus peroxisomal targeting in herbivores is controlled by two signal peptides that correspond to these respective organelles. Differential expression of the mitochondrial targeting sequence (MTS) is considered an adaptation to diet-specific subcellular localization of glyoxylate precursors. Bats are an excellent group in which to study adaptive changes in dietary enzymes; they show unparalleled mammalian dietary diversification as well as independent origins of carnivory, frugivory, and nectarivory. We studied the AGT gene in bats and other mammals with diverse diets and found that the MTS has been lost in unrelated lineages of frugivorous bats. Conversely, species exhibiting piscivory, carnivory, insectivory, and sanguinivory possessed intact MTSs. Detected positive selection in the AGT of ancestral fruit bats further supports adaptations related to evolutionary changes in diet.

  6. Multiple adaptive losses of alanine-glyoxylate aminotransferase mitochondrial targeting in fruit-eating bats.

    PubMed

    Liu, Yang; Xu, Huihui; Yuan, Xinpu; Rossiter, Stephen J; Zhang, Shuyi

    2012-06-01

    The enzyme alanine-glyoxylate aminotransferase 1 (AGT) functions to detoxify glyoxylate before it is converted into harmful oxalate. In mammals, mitochondrial targeting of AGT in carnivorous species versus peroxisomal targeting in herbivores is controlled by two signal peptides that correspond to these respective organelles. Differential expression of the mitochondrial targeting sequence (MTS) is considered an adaptation to diet-specific subcellular localization of glyoxylate precursors. Bats are an excellent group in which to study adaptive changes in dietary enzymes; they show unparalleled mammalian dietary diversification as well as independent origins of carnivory, frugivory, and nectarivory. We studied the AGT gene in bats and other mammals with diverse diets and found that the MTS has been lost in unrelated lineages of frugivorous bats. Conversely, species exhibiting piscivory, carnivory, insectivory, and sanguinivory possessed intact MTSs. Detected positive selection in the AGT of ancestral fruit bats further supports adaptations related to evolutionary changes in diet. PMID:22319153

  7. Effective disposal of nitrogen waste in blood-fed Aedes aegypti mosquitoes requires alanine aminotransferase.

    PubMed

    Mazzalupo, Stacy; Isoe, Jun; Belloni, Virginia; Scaraffia, Patricia Y

    2016-01-01

    To better understand the mechanisms responsible for the success of female mosquitoes in their disposal of excess nitrogen, we investigated the role of alanine aminotransferase (ALAT) in blood-fed Aedes aegypti. Transcript and protein levels from the 2 ALAT genes were analyzed in sucrose- and blood-fed A. aegypti tissues. ALAT1 and ALAT2 exhibit distinct expression patterns in tissues during the first gonotrophic cycle. Injection of female mosquitoes with either double-stranded RNA (dsRNA)-ALAT1 or dsRNA ALAT2 significantly decreased mRNA and protein levels of ALAT1 or ALAT2 in fat body, thorax, and Malpighian tubules compared with dsRNA firefly luciferase-injected control mosquitoes. The silencing of either A. aegypti ALAT1 or ALAT2 caused unexpected phenotypes such as a delay in blood digestion, a massive accumulation of uric acid in the midgut posterior region, and a significant decrease of nitrogen waste excretion during the first 48 h after blood feeding. Concurrently, the expression of genes encoding xanthine dehydrogenase and ammonia transporter (Rhesus 50 glycoprotein) were significantly increased in tissues of both ALAT1- and ALAT2-deficient females. Moreover, perturbation of ALAT1 and ALAT2 in the female mosquitoes delayed oviposition and reduced egg production. These novel findings underscore the efficient mechanisms that blood-fed mosquitoes use to avoid ammonia toxicity and free radical damage.-Mazzalupo, S., Isoe, J., Belloni, V., Scaraffia, P. Y. Effective disposal of nitrogen waste in blood-fed Aedes aegypti mosquitoes requires alanine aminotransferase.

  8. Expression and purification of a functional recombinant aspartate aminotransferase (AST) from Escherichia coli.

    PubMed

    Zou, Lihui; Zhao, Haijian; Wang, Daguang; Wang, Meng; Zhang, Chuanbao; Xiao, Fei

    2014-07-01

    Aspartate aminotransferase (AST; E.C. 2.6.1.1), a vitamin B6-dependent enzyme, preferentially promotes the mutual transformation of aspartate and α-ketoglutarate to oxaloacetate and glutamate. It plays a key role in amino acid metabolism and has been widely recommended as a biomarker of liver and heart damage. Our study aimed to evaluate the extensive preparation of AST and its application in quality control in clinical laboratories. We describe a scheme to express and purify the 6His-AST fusion protein. An optimized sequence coding AST was synthesized and transformed into Escherichia coli BL21 (DE3) strain for protein expression. Ideally, the fusion protein has a volumetric productivity achieving 900 mg/l cultures. After affinity chromatography, the enzyme activity of purified AST reached 150,000 U/L. Commutability assessment between the engineered AST and standard AST from Roche suggested that the engineered AST was the better candidate for the reference material. Moreover, the AST showed high stability during long-term storage at -20ºC. In conclusion, the highly soluble 6His-tagged AST can become a convenient tool for supplying a much better and cheaper standard or reference material for the clinical laboratory. PMID:24722375

  9. The sequences of the coenzyme-binding peptide in the cytoplasmic and the mitochondrial aspartate aminotransferases from sheep liver.

    PubMed Central

    Campos-Cavieres, M; Milstein, C P

    1975-01-01

    The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase. PMID:1180894

  10. Recombinant expression, purification and crystallographic studies of the mature form of human mitochondrial aspartate aminotransferase.

    PubMed

    Jiang, Xiuping; Wang, Jia; Chang, Haiyang; Zhou, Yong

    2016-02-01

    Mitochondrial aspartate aminotransferase (mAspAT) was recognized as a moonlighting enzyme because it has not only aminotransferase activity but also a high-affinity long-chain fatty acids (LCFA) binding site. This enzyme plays a key role in amino acid metabolism, biosynthesis of kynurenic acid and transport of the LCFA. Therefore, it is important to study the structure-function relationships of human mAspAT protein. In this work, the mature form of human mAspAT was expressed to a high level in Escherichia coli periplasmic space using pET-22b vector, purified by a combination of immobilized metal-affinity chromatography and cation exchange chromatography. Optimal activity of the enzyme occurred at a temperature of 47.5ºC and a pH of 8.5. Crystals of human mAspAT were grown using the hanging-drop vapour diffusion method at 277K with 0.1 M HEPES pH 6.8 and 25%(v/v) Jeffamine(®) ED-2001 pH 6.8. The crystals diffracted to 2.99 Å and belonged to the space group P1 with the unit-cell parameters a =56.7, b = 76.1, c = 94.2 Å, α =78.0, β =85.6, γ = 78.4º. Elucidation of mAspAT structure can provide a molecular basis towards understanding catalysis mechanism and substrate binding site of enzyme. PMID:26902786

  11. Comparison of Prothrombin Time and Aspartate Aminotransferase in Predicting Hepatotoxicity After Acetaminophen Overdose.

    PubMed

    Levine, Michael; O'Connor, Ayrn D; Padilla-Jones, Angela; Gerkin, Richard D

    2016-03-01

    Despite decades of experience with acetaminophen (APAP) overdoses, it remains unclear whether elevated hepatic transaminases or coagulopathy develop first. Furthermore, comparison of the predictive value of these two variables in determining hepatic toxicity following APAP overdoses has been poorly elucidated. The primary objective of this study is to determine the test characteristics of the aspartate aminotransferase (AST) and the prothrombin time (PT) in patients with APAP toxicity. A retrospective chart review of APAP overdoses treated with IV N-acetylcysteine at a tertiary care referral center was performed. Of the 304 subjects included in the study, 246 with an initial AST less than 1000 were analyzed to determine predictors of hepatic injury, defined as an AST exceeding 1000 IU/L. The initial AST >50 was 79.5 % sensitive and 82.6 % specific for predicting hepatic injury. The corresponding negative and positive predictive values were 95.5 and 46.3 %, respectively. In contrast, an initial abnormal PT had a sensitivity of 82.1 % and a specificity of 63.6 %. The negative and positive predictive values for initial PT were 94.9 and 30.2 %, respectively. Although the two tests performed similarly for predicting a composite endpoint of death or liver transplant, neither was a useful predictor. Initial AST performed better than the initial PT for predicting hepatic injury in this series of patients with APAP overdose. PMID:26341088

  12. Glycation of aspartate aminotransferase by methylglyoxal, effect of hydroxycitric and uric acid.

    PubMed

    Bousová, Iva; Bacílková, Eliska; Dobrijević, Sanja; Drsata, Jaroslav

    2009-11-01

    Glycation is a process closely related to the aging and pathogenesis of diabetic complications. Reactive alpha-dicarbonyl compounds (e.g., methylglyoxal) are formed during middle stage of glycation reaction. Compounds that would inhibit the glycation process have been seeked for years. The objective of this study was to investigate the inhibitory effect of hydroxycitric (0.25-2.5 mM) and uric acid (0.4-1.2 mM) on middle stage of protein glycation in vitro using the model containing aspartate aminotransferase (AST) and 0.5 mM methylglyoxal. Hydroxycitric acid, at all tested concentrations, reduced AST activity decrease and formation of fluorescent AGEs during incubation of the enzyme with methylglyoxal at 37 degrees C. This compound also prevented formation of high-molecular weight protein cross-links and changes in molecular charge of AST caused by glycation. Uric acid showed no positive anti-glycation activity. The results support the hypothesis that hydroxycitric acid has beneficial effects in controlling protein glycation. PMID:19449196

  13. Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase*

    PubMed Central

    Albert, Armando; Yunta, Cristina; Arranz, Rocío; Peña, Álvaro; Salido, Eduardo; Valpuesta, José María; Martín-Benito, Jaime

    2010-01-01

    Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism. PMID:20056599

  14. A Micro-Platinum Wire Biosensor for Fast and Selective Detection of Alanine Aminotransferase.

    PubMed

    Thuy, Tran Nguyen Thanh; Tseng, Tina T-C

    2016-01-01

    In this study, a miniaturized biosensor based on permselective polymer layers (overoxidized polypyrrole (Ppy) and Nafion(®)) modified and enzyme (glutamate oxidase (GlutOx)) immobilized micro-platinum wire electrode for the detection of alanine aminotransferase (ALT) was fabricated. The proposed ALT biosensor was measured electrochemically by constant potential amperometry at +0.7 V vs. Ag/AgCl. The ALT biosensor provides fast response time (~5 s) and superior selectivity towards ALT against both negatively and positively charged species (e.g., ascorbic acid (AA) and dopamine (DA), respectively). The detection range of the ALT biosensor is found to be 10-900 U/L which covers the range of normal ALT levels presented in the serum and the detection limit and sensitivity are found to be 8.48 U/L and 0.059 nA/(U/L·mm²) (N = 10), respectively. We also found that one-day storage of the ALT biosensor at -20 °C right after the sensor being fabricated can enhance the sensor sensitivity (1.74 times higher than that of the sensor stored at 4 °C). The ALT biosensor is stable after eight weeks of storage at -20 °C. The sensor was tested in spiked ALT samples (ALT activities: 20, 200, 400, and 900 U/L) and reasonable recoveries (70%~107%) were obtained. PMID:27240366

  15. Cellular and subcellular localization of hexokinase, glutamate dehydrogenase, and alanine aminotransferase in the honeybee drone retina.

    PubMed

    Veuthey, A L; Tsacopoulos, M; Millan de Ruiz, L; Perrottet, P

    1994-05-01

    Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina. PMID:8158142

  16. A Micro-Platinum Wire Biosensor for Fast and Selective Detection of Alanine Aminotransferase

    PubMed Central

    Thuy, Tran Nguyen Thanh; Tseng, Tina T.-C.

    2016-01-01

    In this study, a miniaturized biosensor based on permselective polymer layers (overoxidized polypyrrole (Ppy) and Nafion®) modified and enzyme (glutamate oxidase (GlutOx)) immobilized micro-platinum wire electrode for the detection of alanine aminotransferase (ALT) was fabricated. The proposed ALT biosensor was measured electrochemically by constant potential amperometry at +0.7 V vs. Ag/AgCl. The ALT biosensor provides fast response time (~5 s) and superior selectivity towards ALT against both negatively and positively charged species (e.g., ascorbic acid (AA) and dopamine (DA), respectively). The detection range of the ALT biosensor is found to be 10–900 U/L which covers the range of normal ALT levels presented in the serum and the detection limit and sensitivity are found to be 8.48 U/L and 0.059 nA/(U/L·mm2) (N = 10), respectively. We also found that one-day storage of the ALT biosensor at −20 °C right after the sensor being fabricated can enhance the sensor sensitivity (1.74 times higher than that of the sensor stored at 4 °C). The ALT biosensor is stable after eight weeks of storage at −20 °C. The sensor was tested in spiked ALT samples (ALT activities: 20, 200, 400, and 900 U/L) and reasonable recoveries (70%~107%) were obtained. PMID:27240366

  17. Clinical significance of serum alanine aminotransferase and lifestyle intervention in children with nonalcoholic fatty liver disease

    PubMed Central

    Kwon, Kyoung Ah; Chun, Peter

    2016-01-01

    Purpose This study aimed to investigate the clinical significance of serum alanine aminotransferase (ALT) levels in children with nonalcoholic fatty liver disease (NAFLD) and the effect of lifestyle intervention on NAFLD. Methods The clinical data of 86 children diagnosed with NAFLD were reviewed retrospectively. Forty-six patients belonged to the elevated ALT group and 40 to the normal ALT group. The clinical parameters of patients with NAFLD were also compared based on the status of ALT levels after lifestyle intervention. Results Patients with elevated ALT had significantly higher body mass index (BMI) scores than those with normal ALT (P<0.05). Of all the patients with elevated ALT, 89% exhibited moderate or severe degree of fatty change in the liver on ultrasonographic examination, whereas most patients with normal ALT exhibited mild or moderate degree changes. Liver biopsy was performed in 15 children with elevated ALT and all showed mild histological changes. Of all patients with elevated ALT, 49% achieved normal ALT levels after lifestyle intervention. Those with more severe histological changes tended to have continuously increasing ALT levels. There was no correlation between the normalization of posttreatment ALT level and BMI, as well as ultrasonographic findings at diagnosis. Conclusion ALT elevation in NAFLD is highly associated with higher BMI scores and more severe degree of fatty changes on ultrasonographic examination. Lifestyle intervention can significantly improve ALT in children with NAFLD. The degree of histologic changes appears to be a predictor of the treatment response to NAFLD. PMID:27721840

  18. Cellular and subcellular localization of hexokinase, glutamate dehydrogenase, and alanine aminotransferase in the honeybee drone retina.

    PubMed

    Veuthey, A L; Tsacopoulos, M; Millan de Ruiz, L; Perrottet, P

    1994-05-01

    Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina.

  19. Associations of functional alanine-glyoxylate aminotransferase 2 gene variants with atrial fibrillation and ischemic stroke.

    PubMed

    Seppälä, Ilkka; Kleber, Marcus E; Bevan, Steve; Lyytikäinen, Leo-Pekka; Oksala, Niku; Hernesniemi, Jussi A; Mäkelä, Kari-Matti; Rothwell, Peter M; Sudlow, Cathie; Dichgans, Martin; Mononen, Nina; Vlachopoulou, Efthymia; Sinisalo, Juha; Delgado, Graciela E; Laaksonen, Reijo; Koskinen, Tuomas; Scharnagl, Hubert; Kähönen, Mika; Markus, Hugh S; März, Winfried; Lehtimäki, Terho

    2016-01-01

    Asymmetric and symmetric dimethylarginines (ADMA and SDMA) impair nitric oxide bioavailability and have been implicated in the pathogenesis of atrial fibrillation (AF). Alanine-glyoxylate aminotransferase 2 (AGXT2) is the only enzyme capable of metabolizing both of the dimethylarginines. We hypothesized that two functional AGXT2 missense variants (rs37369, V140I; rs16899974, V498L) are associated with AF and its cardioembolic complications. Association analyses were conducted using 1,834 individulas with AF and 7,159 unaffected individuals from two coronary angiography cohorts and a cohort comprising patients undergoing clinical exercise testing. In coronary angiography patients without structural heart disease, the minor A allele of rs16899974 was associated with any AF (OR = 2.07, 95% CI 1.59-2.68), and with paroxysmal AF (OR = 1.98, 95% CI 1.44-2.74) and chronic AF (OR = 2.03, 95% CI 1.35-3.06) separately. We could not replicate the association with AF in the other two cohorts. However, the A allele of rs16899974 was nominally associated with ischemic stroke risk in the meta-analysis of WTCCC2 ischemic stroke cohorts (3,548 cases, 5,972 controls) and with earlier onset of first-ever ischemic stroke (360 cases) in the cohort of clinical exercise test patients. In conclusion, AGXT2 variations may be involved in the pathogenesis of AF and its age-related thromboembolic complications. PMID:26984639

  20. The enzymology of alanine aminotransferase (AlaAT) isoforms from Hordeum vulgare and other organisms, and the HvAlaAT crystal structure.

    PubMed

    Duff, Stephen M G; Rydel, Timothy J; McClerren, Amanda L; Zhang, Wenlan; Li, Jimmy Y; Sturman, Eric J; Halls, Coralie; Chen, Songyang; Zeng, Jiamin; Peng, Jiexin; Kretzler, Crystal N; Evdokimov, Artem

    2012-12-01

    In this paper we describe the expression, purification, kinetics and biophysical characterization of alanine aminotransferase (AlaAT) from the barley plant (Hordeum vulgare). This dimeric PLP-dependent enzyme is a pivotal element of several key metabolic pathways from nitrogen assimilation to carbon metabolism, and its introduction into transgenic plants results in increased yield. The enzyme exhibits a bi-bi ping-pong reaction mechanism with a K(m) for alanine, 2-oxoglutarate, glutamate and pyruvate of 3.8, 0.3, 0.8 and 0.2 mM, respectively. Barley AlaAT catalyzes the forward (alanine-forming) reaction with a k(cat) of 25.6 s(-1), the reverse (glutamate-forming) reaction with k(cat) of 12.1 s(-1) and an equilibrium constant of ~0.5. The enzyme is also able to utilize aspartate and oxaloacetate with ~10% efficiency as compared to the native substrates, which makes it much more specific than related bacterial/archaeal enzymes (that also have lower K(m) values). We have crystallized barley AlaAT in complex with PLP and l-cycloserine and solved the structure of this complex at 2.7 Å resolution. This is the first example of a plant AlaAT structure, and it reveals a canonical aminotransferase fold similar to structures of the Thermotoga maritima, Pyrococcus furiosus, and human enzymes. This structure bridges our structural understanding of AlaAT mechanism between three kingdoms of life and allows us to shed some light on the specifics of the catalysis performed by these proteins. PMID:22750542

  1. The enzymology of alanine aminotransferase (AlaAT) isoforms from Hordeum vulgare and other organisms, and the HvAlaAT crystal structure.

    PubMed

    Duff, Stephen M G; Rydel, Timothy J; McClerren, Amanda L; Zhang, Wenlan; Li, Jimmy Y; Sturman, Eric J; Halls, Coralie; Chen, Songyang; Zeng, Jiamin; Peng, Jiexin; Kretzler, Crystal N; Evdokimov, Artem

    2012-12-01

    In this paper we describe the expression, purification, kinetics and biophysical characterization of alanine aminotransferase (AlaAT) from the barley plant (Hordeum vulgare). This dimeric PLP-dependent enzyme is a pivotal element of several key metabolic pathways from nitrogen assimilation to carbon metabolism, and its introduction into transgenic plants results in increased yield. The enzyme exhibits a bi-bi ping-pong reaction mechanism with a K(m) for alanine, 2-oxoglutarate, glutamate and pyruvate of 3.8, 0.3, 0.8 and 0.2 mM, respectively. Barley AlaAT catalyzes the forward (alanine-forming) reaction with a k(cat) of 25.6 s(-1), the reverse (glutamate-forming) reaction with k(cat) of 12.1 s(-1) and an equilibrium constant of ~0.5. The enzyme is also able to utilize aspartate and oxaloacetate with ~10% efficiency as compared to the native substrates, which makes it much more specific than related bacterial/archaeal enzymes (that also have lower K(m) values). We have crystallized barley AlaAT in complex with PLP and l-cycloserine and solved the structure of this complex at 2.7 Å resolution. This is the first example of a plant AlaAT structure, and it reveals a canonical aminotransferase fold similar to structures of the Thermotoga maritima, Pyrococcus furiosus, and human enzymes. This structure bridges our structural understanding of AlaAT mechanism between three kingdoms of life and allows us to shed some light on the specifics of the catalysis performed by these proteins.

  2. Molecular Requirements for Peroxisomal Targeting of Alanine-Glyoxylate Aminotransferase as an Essential Determinant in Primary Hyperoxaluria Type 1

    PubMed Central

    Fodor, Krisztián; Wolf, Janina; Erdmann, Ralf; Schliebs, Wolfgang; Wilmanns, Matthias

    2012-01-01

    Alanine-glyoxylate aminotransferase is a peroxisomal enzyme, of which various missense mutations lead to irreversible kidney damage via primary hyperoxaluria type 1, in part caused by improper peroxisomal targeting. To unravel the molecular mechanism of its recognition by the peroxisomal receptor Pex5p, we have determined the crystal structure of the respective cargo–receptor complex. It shows an extensive protein/protein interface, with contributions from residues of the peroxisomal targeting signal 1 and additional loops of the C-terminal domain of the cargo. Sequence segments that are crucial for receptor recognition and hydrophobic core interactions within alanine-glyoxylate aminotransferase are overlapping, explaining why receptor recognition highly depends on a properly folded protein. We subsequently characterized several enzyme variants in vitro and in vivo and show that even minor protein fold perturbations are sufficient to impair Pex5p receptor recognition. We discuss how the knowledge of the molecular parameters for alanine-glyoxylate aminotransferase required for peroxisomal translocation could become useful for improved hyperoxaluria type 1 treatment. PMID:22529745

  3. Carnosine prevents glyceraldehyde 3-phosphate-mediated inhibition of aspartate aminotransferase.

    PubMed

    Swearengin, T A; Fitzgerald, C; Seidler, N W

    1999-08-01

    Post-mitotic tissues, such as the heart, exhibit high concentrations (20 mM) of carnosine (beta-alanyl-l-histidine). Carnosine may have aldehyde scavenging properties. We tested this hypothesis by examining its protective effects against inhibition of enzyme activity by glyceraldehyde 3-phosphate (Glyc3P). Glyc3P is a potentially toxic triose; Glyc3P inhibits the cardiac aspartate aminotransferase (cAAT) by non-enzymatic glycosylation (or glycation) of the protein. cAAT requires pyridoxal 5-phosphate (PyP) for catalysis. We observed that carnosine (20 mM) completely prevents the inhibition of cAAT activity by Glyc3P (5 mM) after brief incubation (30 min at 37 degrees C). After a prolonged incubation (3.25 h) of cAAT with Glyc3P (0.5 mM) at 37 degrees C, the protection by carnosine (20 mM) persisted but PyP availability was affected. In the absence of PyP from the assay medium, cAAT activities (plus Glyc3P) were 95 +/- 18.2 micromol/min per mg protein (mean +/- SD), minus carnosine and 100 +/- 2.4, plus carnosine; control activity was 172 +/- 3.9. When PyP (1.0 microM) was included in the assay medium, cAAT activities (plus Glyc3P) were 93 +/- 14.8, minus carnosine and 151 +/- 16.8, plus carnosine, P < 0. 001; control activity was 180 +/- 17.7. These data, which showed carnosine moderating the effects of both Glyc3P and PyP, suggest that carnosine may be an endogenous aldehyde scavenger.

  4. Postoperative aspartate aminotransferase to platelet ratio index change predicts prognosis for hepatocellular carcinoma.

    PubMed

    Peng, Wei; Li, Chuan; Wen, Tian-Fu; Yan, Lv-Nan; Li, Bo; Wang, Wen-Tao; Yang, Jia-Yin; Xu, Ming-Qing

    2016-07-01

    An elevated preoperative aspartate aminotransferase (AST) to platelet ratio index (APRI) is reported to be a prognostic factor for patients with hepatocellular carcinoma (HCC) after treatment. However, delta APRI (ΔAPRI), which represents the change from preoperative to postoperative APRI, has received little attention. The present study was designed to evaluate the prognostic value of ΔAPRI in patients with small HCC after liver resection.A retrospective cohort study analyzing 244 patients with small HCC who had undergone liver resection was conducted. Medical data were retrieved from our prospectively maintained database. Patients were divided into 2 groups according to ΔAPRI as follows: group A (ΔAPRI ≥0.02) and group B (ΔAPRI <0.02). The association of demographic and clinical data, overall survival (OS), and recurrence-free survival (RFS) were statistically compared in the 2 groups, and a multivariate analysis was used to identify prognostic factors.The 1, 3, and 5-year OS of patients in group A were 94.2%, 79.5%, and 62.3%, respectively, and 95.1%, 87.9%, and 84.6%, respectively, for patients in group B (P = 0.001). The corresponding 1, 3, and 5-year RFS was 69.0%, 44.7 %, and 28.1%, and 77.4%, 57.0%, and 54.2% for patients in the 2 groups, respectively (P = 0.009). The results of a multivariate analysis indicated that ΔAPRI was an independent prognostic factor for both OS (P = 0.001, hazard ratio 3.115, 95% confidence interval 1.642-5.912) and RFS (P = 0.006, hazard ratio 1.689, 95% confidence interval 1.163-2.452).A positive ΔAPRI after liver resection predicts decreased OS and RFS in patients with small HCC.

  5. Reconfiguration of N Metabolism upon Hypoxia Stress and Recovery: Roles of Alanine Aminotransferase (AlaAT) and Glutamate Dehydrogenase (GDH)

    PubMed Central

    Diab, Houssein; Limami, Anis M.

    2016-01-01

    In the context of climatic change, more heavy precipitation and more frequent flooding and waterlogging events threaten the productivity of arable farmland. Furthermore, crops were not selected to cope with flooding- and waterlogging-induced oxygen limitation. In general, low oxygen stress, unlike other abiotic stresses (e.g., cold, high temperature, drought and saline stress), received little interest from the scientific community and less financial support from stakeholders. Accordingly, breeding programs should be developed and agronomical practices should be adapted in order to save plants’ growth and yield—even under conditions of low oxygen availability (e.g., submergence and waterlogging). The prerequisite to the success of such breeding programs and changes in agronomical practices is a good knowledge of how plants adapt to low oxygen stress at the cellular and the whole plant level. In the present paper, we summarized the recent knowledge on metabolic adjustment in general under low oxygen stress and highlighted thereafter the major changes pertaining to the reconfiguration of amino acids syntheses. We propose a model showing (i) how pyruvate derived from active glycolysis upon hypoxia is competitively used by the alanine aminotransferase/glutamate synthase cycle, leading to alanine accumulation and NAD+ regeneration. Carbon is then saved in a nitrogen store instead of being lost through ethanol fermentative pathway. (ii) During the post-hypoxia recovery period, the alanine aminotransferase/glutamate dehydrogenase cycle mobilizes this carbon from alanine store. Pyruvate produced by the reverse reaction of alanine aminotransferase is funneled to the TCA cycle, while deaminating glutamate dehydrogenase regenerates, reducing equivalent (NADH) and 2-oxoglutarate to maintain the cycle function. PMID:27258319

  6. Reconfiguration of N Metabolism upon Hypoxia Stress and Recovery: Roles of Alanine Aminotransferase (AlaAT) and Glutamate Dehydrogenase (GDH).

    PubMed

    Diab, Houssein; Limami, Anis M

    2016-01-01

    In the context of climatic change, more heavy precipitation and more frequent flooding and waterlogging events threaten the productivity of arable farmland. Furthermore, crops were not selected to cope with flooding- and waterlogging-induced oxygen limitation. In general, low oxygen stress, unlike other abiotic stresses (e.g., cold, high temperature, drought and saline stress), received little interest from the scientific community and less financial support from stakeholders. Accordingly, breeding programs should be developed and agronomical practices should be adapted in order to save plants' growth and yield-even under conditions of low oxygen availability (e.g., submergence and waterlogging). The prerequisite to the success of such breeding programs and changes in agronomical practices is a good knowledge of how plants adapt to low oxygen stress at the cellular and the whole plant level. In the present paper, we summarized the recent knowledge on metabolic adjustment in general under low oxygen stress and highlighted thereafter the major changes pertaining to the reconfiguration of amino acids syntheses. We propose a model showing (i) how pyruvate derived from active glycolysis upon hypoxia is competitively used by the alanine aminotransferase/glutamate synthase cycle, leading to alanine accumulation and NAD⁺ regeneration. Carbon is then saved in a nitrogen store instead of being lost through ethanol fermentative pathway. (ii) During the post-hypoxia recovery period, the alanine aminotransferase/glutamate dehydrogenase cycle mobilizes this carbon from alanine store. Pyruvate produced by the reverse reaction of alanine aminotransferase is funneled to the TCA cycle, while deaminating glutamate dehydrogenase regenerates, reducing equivalent (NADH) and 2-oxoglutarate to maintain the cycle function. PMID:27258319

  7. Association between Elevated Alanine Aminotransferase and Urosepsis in Children with Acute Pyelonephritis

    PubMed Central

    Kim, Dongwan; Lee, Sung Hyun; Ryoo, Eell; Cho, Hye Kyung; Kim, Yun Mi

    2016-01-01

    Purpose The aim of this study is to investigate the association between elevated alanine aminotransferase (ALT) and urosepsis in children with acute pyelonephritis (APN). Methods We retrospectively identified all children who were managed in our hospital with APN during a decade period. In our study a diagnosis of APN was defined as having a positive urine culture and a positive (99m)Tc-dimercaptosuccinic acid scintigraphy. We compared those with elevated ALT and those with normal ALT according to the following variables: age, gender, duration of fever prior to admission, presence of hypotension, C-reactive protein (CRP), creatinine, presence of anemia, white blood cells count, platelet count, blood culture result, and grades of vesicoureteral reflux. In addition, the correlation between elevated ALT and positive blood culture was analyzed in detail. Results A total of 996 children were diagnosed with APN, of which 883 were included in the study. ALT was elevated in 81 children (9.2%). In the analysis of demographic characteristics, the number of children with elevated ALT was higher in children between 0 to 3 months, boys, and in those with positive blood culture (p=0.002, 0.036, and 0.010, respectively). In multivariate analysis of variables associated with positive blood culture, age younger than 3 months, elevated ALT, elevated CRP, and elevated creatinine showed statistical significance (p=0.004, 0.030, 0.043, and 0.044, respectively). Conclusion Our study demonstrates the association between elevated ALT and increased prevalence of urosepsis in addition to elevated CRP, elevated creatinine, and age younger than 3 months in children with APN. PMID:27066449

  8. Association between Serum Uric Acid and Elevated Alanine Aminotransferase in the General Population

    PubMed Central

    Chen, Shuang; Guo, Xiaofan; Yu, Shasha; Sun, Guozhe; Yang, Hongmei; Li, Zhao; Sun, Yingxian

    2016-01-01

    Background: Both the serum uric acid (SUA) level and elevated alanine aminotransferase (ALT) are related to metabolic syndrome. However, the association between SUA and elevated ALT has not been elucidated in the general population. The objective of this study was to investigate the association between SUA and elevated ALT in the general population of China; Methods: A total of 11,572 adults (≥35 years of age) participated in this survey. Elevated ALT was defined as >40 U/L. SUA ≥ 7.0 mg/dL in males or ≥6.0 mg/dL in females was defined as hyperuricemia. SUA within the reference range was divided into quartiles, and its associations with elevated ALT were evaluated by logistic regressions; Results: A total of 7.4% participants had elevated ALT. The prevalence of hyperuricemia was 14.9% in males and 7.3% in females. There was a significantly positive dose-response association between SUA levels and the prevalence of elevated ALT. After adjusting for potential confounders, a positive relationship for elevated ALT was observed in subjects with hyperuricemia (odds ratio [OR]: 2.032, 95% confidence interval [CI]: 1.443–2.861 for men; OR: 2.045, 95% CI: 1.221–3.425 for women, both p < 0.05). Within the reference range, the association between SUA and elevated ALT persisted in the fourth quartile (OR: 1.467, 95% CI: 1.063–2.025 for men; OR: 1.721, 95% CI: 1.146–2.585 for women, both p < 0.05); Conclusions: Our results indicated that an increased SUA level, even within the reference range, was independently associated with elevated ALT in Chinese adults. PMID:27563918

  9. Allele-specific characterization of alanine: glyoxylate aminotransferase variants associated with primary hyperoxaluria.

    PubMed

    Lage, Melissa D; Pittman, Adrianne M C; Roncador, Alessandro; Cellini, Barbara; Tucker, Chandra L

    2014-01-01

    Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive kidney stone disease caused by deficiency of the peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT), which is involved in glyoxylate detoxification. Over 75 different missense mutations in AGT have been found associated with PH1. While some of the mutations have been found to affect enzyme activity, stability, and/or localization, approximately half of these mutations are completely uncharacterized. In this study, we sought to systematically characterize AGT missense mutations associated with PH1. To facilitate analysis, we used two high-throughput yeast-based assays: one that assesses AGT specific activity, and one that assesses protein stability. Approximately 30% of PH1-associated missense mutations are found in conjunction with a minor allele polymorphic variant, which can interact to elicit complex effects on protein stability and trafficking. To better understand this allele interaction, we functionally characterized each of 34 mutants on both the major (wild-type) and minor allele backgrounds, identifying mutations that synergize with the minor allele. We classify these mutants into four distinct categories depending on activity/stability results in the different alleles. Twelve mutants were found to display reduced activity in combination with the minor allele, compared with the major allele background. When mapped on the AGT dimer structure, these mutants reveal localized regions of the protein that appear particularly sensitive to interactions with the minor allele variant. While the majority of the deleterious effects on activity in the minor allele can be attributed to synergistic interaction affecting protein stability, we identify one mutation, E274D, that appears to specifically affect activity when in combination with the minor allele.

  10. Vitamin E and changes in serum alanine aminotransferase levels in patients with non-alcoholic steatohepatitis

    PubMed Central

    Hoofnagle, J. H.; Van Natta, M. L.; Kleiner, D. E.; Clark, J. M.; Kowdley, K. V.; Loomba, R.; Neuschwander-Tetri, B. A.; Sanyal, A. J.; Tonascia, J.

    2013-01-01

    SUMMARY Background Non-alcoholic steatohepatitis (NASH) is a common cause of serum alanine aminotransferase (ALT) elevations and chronic liver disease, but it is unclear how well ALT elevations reflect the liver injury. Aim To assess how well changes in ALT elevations reflect improvements in liver histology in response to vitamin E therapy. Methods The vitamin E and placebo arms of the Pioglitazone vs. Vitamin E vs. Placebo in Non-alcoholic Steatohepatitis (PIVENS) trial were reassessed for associations among changes in ALT levels, body weight and liver histology. An ALT response was defined as a decrease to ≤40 U/L and by ≥30% of baseline. Liver biopsies taken before and after treatment were scored for non-alcoholic fatty liver disease activity (NAS) and fibrosis. Results ALT responses were more frequent among vitamin E (48%) than placebo (16%) recipients (P < 0.001). Among vitamin E recipients, ALT responses were associated with decreases in NAS (P < 0.001), but not fibrosis scores (P = 0.34), whereas among placebo recipients, ALT responses were associated with significant decreases in both (P < 0.05). Weight loss (≥2 kg) was also associated with ALT response (P < 0.001), improvements in NAS (P < 0.001) and fibrosis (P < 0.02), but vitamin E had an added effect both with and without weight loss. Weight gain (≥2 kg) was associated with lack of ALT response and worsening NAS and fibrosis scores in patients not on vitamin E. Conclusions Decrease of ALT levels to normal in patients with NASH is usually associated with improved histological activity. Management should stress the value of weight loss and strongly discourage weight gain. Vitamin E can improve both ALT levels and histology with and without weight loss. Clinical Trial Number: NCT00063622. PMID:23718573

  11. Diurnal Variation in Serum Alanine Aminotransferase Activity in the United States Population

    PubMed Central

    Everhart, James E.

    2012-01-01

    Goals & Background Serum alanine aminotransferase (ALT) activity has been reported to be greater in the afternoon than the early morning, but data are scarce. We examined diurnal variation of ALT in a national population-based sample. Study Participants in the 1999–2008 U.S. National Health and Nutrition Examination Survey were randomly assigned to morning (AM) (n=4,474 adolescents, 11,235 adults) or afternoon/evening (PM) (n=4,887 adolescents, 11,735 adults) examinations. We examined ALT distributions graphically and compared both geometric mean ALT and the prevalence of elevated ALT, defined as >31 IU/L for adolescent boys, >24 IU/L for adolescent girls, >43 IU/L for adult men and >30 IU/L for adult women, between AM and PM examination groups. Results The examination groups were similar with the exception in the AM group of a longer fasting time and slightly higher prevalence of diabetes among adolescents and viral hepatitis B among adult women. ALT distributions were similar between examination sessions among the four groups. Among adolescents and men, neither mean ALT nor prevalence of abnormal ALT differed by examination group. Among women, mean ALT was statistically significantly, but minimally higher in the PM (19.6 IU/L) than the AM group (19.1 IU/L; p=0.009). Among one subgroup, women with chronic viral hepatitis, there was a higher prevalence of abnormal ALT in the PM (p=0.018 in unadjusted analysis). Adjusting for liver injury risk factors had little effect on the difference in mean ALT. Conclusions In general, clinically significant diurnal variation in ALT activity was not found in the U.S. population. PMID:23164687

  12. Upper Limits of Normal for Alanine Aminotransferase Activity in the United States Population

    PubMed Central

    Ruhl, Constance E.; Everhart, James E.

    2011-01-01

    Background & Rationale Alanine aminotransferase (ALT) is an important test for liver disease, yet there is no generally accepted upper limit of normal (ULN) in the United States. Furthermore, the ability of ALT to differentiate persons with and without liver disease is uncertain. We examined cut-offs for ALT for their ability to discriminate between persons with positive hepatitis C virus (HCV) RNA and those at low risk for liver injury in the U.S. population. Methods Among adult participants in the 1999–2008 U.S. National Health and Nutrition Examination Survey, 259 were positive for serum HCV RNA and 3,747 were at low risk for liver injury (negative HCV RNA and hepatitis B surface antigen, low alcohol consumption, no evidence of diabetes, normal body mass index and waist circumference). Serum ALT activity was measured centrally. Results Maximum correct classification was achieved at ALT=29 IU/L for men (88% sensitivity, 83% specificity) and 22 (89% sensitivity, 82% specificity) for women. The cut-off for 95% sensitivity was an ALT=24 IU/L (70% specificity) for men and 18 (63% specificity) for women. The cut-off for 95% specificity was an ALT=44 IU/L (64% sensitivity) for men and 32 (59% sensitivity) for women. The area under the curve was 0.929 for men and 0.915 for women. If the cut-offs with the best correct classification were applied to the entire population, 36.4% of men and 28.3% of women would have had abnormal ALT. Conclusion ALT discriminates persons infected with HCV from those at low risk of liver disease, but would be considered elevated in a large proportion of the U.S. population. PMID:21987480

  13. Trunk Fat is Associated with Increased Serum Levels of Alanine Aminotransferase in the US

    PubMed Central

    Ruhl, Constance E.; Everhart, James E.

    2010-01-01

    Background & Aims Liver injury is associated with obesity and related measures such as body mass index (BMI) and waist circumference. The relationship between liver injury and body composition has not been evaluated in a population-based study. Methods Using data from a US population-based survey, we examined the contributions of body composition, measured by dual-energy x-ray absorptiometry (DXA), to increased serum alanine aminotransferase (ALT) activity among 11,821 adults without viral hepatitis. Trunk fat, extremity fat, trunk lean, and extremity lean mass were divided by height squared and used to categorize subjects into quintiles; logistic regression odds ratios (OR) were calculated for increased ALT. Results Increased ALT was associated with higher measures of fat and lean mass (p<0.001) after adjustment for alcohol consumption and other liver injury risk factors in separate models for each DXA measure. Trunk fat was associated with increased ALT (p≤0.001) in models also including any 1 of the other 3 measures. Extremity fat was independently inversely associated among women (p<0.001). Trunk and extremity lean mass were not independently related to increased ALT. In models that contained all 4 DXA measures, the OR (95% confidence interval) for increased ALT for the highest, relative to lowest, quintile of trunk fat/height squared was 13.8 (5.4-35.3) for men and 7.8 (3.9-15.8) for women. When BMI, waist circumference, and trunk fat were considered together, only trunk fat remained independently associated with increased ALT. Conclusions Trunk fat is a major body composition determinant of increased ALT, supporting the hypothesis that liver injury can be induced by metabolically active intra-abdominal fat. PMID:20060831

  14. Anthropometric Indices in Adults: Which Is the Best Indicator to Identify Alanine Aminotransferase Levels?

    PubMed Central

    Chen, Shuang; Guo, Xiaofan; Yu, Shasha; Zhou, Ying; Li, Zhao; Sun, Yingxian

    2016-01-01

    Background: To evaluate the correlations between serum alanine aminotransferase (ALT) levels and anthropometric indices including body mass index (BMI), waist circumference (WC), hip circumference (HC), waist-to-height ratio (WHtR), waist-to-hip ratio (WHR), and a new body index, the A Body Shape Index (ABSI) in Chinese adults. Methods: A multicenter, cross-sectional study was conducted in rural areas of China in 2012–2013, and 11,331 adults were included in our final analysis. Results: BMI, WC, HC, WHtR, WHR and ABSI were significantly positively correlated with ALT levels. Spearman rank test showed that WHtR (r = 0.346 for men, r = 0.282 for women, both p < 0.001) had the highest correlation coefficient for ALT level, whereas ABSI showed the lowest, and the correlation coefficient of each measure was higher in men than that in women. Comparing the lowest with the highest quintile of each anthropometric measure, the multivariate logistic model presented that WHtR had the superiority of identifying the presence of elevated ALT (OR 4.38; 95% CI 3.15–6.08 for men, OR 4.29; 95% CI 2.91–6.33 for women, both p < 0.001), and the ABSI was the poorest predictor in men (OR 2.51; 95% CI 1.93–3.27, p < 0.001). No association was observed for ABSI in women. Conclusions: Our results indicated that BMI, WC, HC, WHtR and WHR were able to determine elevated ALT presence, while ABSI was not capable. WHtR and to some extent BMI were the best body indices, for predicting the ALT levels in this population. Nevertheless, the predictive ability of ABSI as a novel body index was not superior compared to established anthropometric indices. PMID:26901214

  15. Follow-up of mild alanine aminotransferase elevation identifies hidden hepatitis C in primary care

    PubMed Central

    Helsper, Charles; van Essen, Gerrit; Frijling, Bernard D; de Wit, Niek J

    2012-01-01

    Background Hepatitis C (HCV) and hepatitis B (HBV) virus infection can lead to serious complications if left untreated, but often remain undetected in primary care. Mild alanine aminotransferase (ALT) elevations (30–100 IU/l) are commonly found and could be associated with viral hepatitis; unfortunately, these findings frequently remain without follow-up. Aim To determine if and how mild ALT elevation can be used to identify hidden HCV and HBV infection in primary care. Design and setting Primary care patients referred for liver enzyme testing were selected by a large primary care Diagnostic Centre (Saltro). Method First, 750 anonymous samples were collected in three categories of ALT elevation (30–50 IU/l, 50–70 IU/l, and 70–100 IU/l) and tested for HCV and HBV. Second, the national prevalence of each ALT elevation was estimated by analysing all annual ALT tests performed at Saltro. Results HCV prevalence was 1.6% and 1.2% in patients with an ALT of 50–70 IU/l and 70–100 IU/l respectively. In patients with an ALT of 30–50 IU/l, HCV prevalence was normal (≤0.1%). HBV prevalence was normal (≤0.4%) in all groups. The estimated number of ALT tests performed nationally each year in primary care was 1.1 million. An ALT of 30–50 IU/l was found in 21.1%, an ALT of 50–70 IU/l in 5.6%, and 2.6% had an ALT of 70–100 IU/l. Conclusion In primary care patients with an ALT level of 50–100 IU/l, HCV prevalence is tenfold the population prevalence, whereas HBV prevalence is not elevated. Therefore, diagnostic follow-up for HCV is indicated in these patients, even when other explanations for ALT elevation are present. PMID:22429439

  16. Upper Limits of Normal for Serum Alanine Aminotransferase Levels in Chinese Han Population

    PubMed Central

    Zheng, Ming-Hua; Shi, Ke-Qing; Fan, Yu-Chen; Liu, Wen-Yue; Lin, Xian-Feng; Li, Ling-Fei; Chen, Yong-Ping

    2012-01-01

    Background and Objectives Serum alanine aminotransferase (ALT) activity is the most common tool for the assessment of liver diseases. However, it is not clear whether the current normal ALT range really discriminate patients with or without liver diseases. The present study was to establish a new normal range of ALT and examine its ability to identify patients with hepatitis B or nonalcoholic fatty liver disease (NAFLD) in Chinese Han population. Methods 53037 adults were included in this study from January 1st 2008 to August 31st 2010. The 95th percentile of ALT in population with relative low risk factors for liver diseases was set as the new upper limits of normal ALT in gender-specific manner. Results The 95th percentile levels at low risk factors for liver diseases were achieved at 35 U/L for men and 23 U/L for women. The concordance statistics for detection were 0.873 (95%CI: 0.865–0.881) for HBV and 0.932 (95%CI: 0.927–0.937) for NAFLD in men while 0.857 (95%CI: 0.850–0.864) for HBV and 0.909 (95%CI: 0.903–0.915) for NAFLD in women. The median sensitivity of the current used ALT upper limit (40 U/L) was 6.6% for HBV and 29.7% for NAFLD and median specificity was 98.7% for men and 99.4% for women. Using our new-derived thresholds, the sensitivities ranged from 35.3% to 61.1% and the specificities were 94.8% for men and 94.6% for women. Conclusions Our results suggest that upper limits of ALT 35 U/L for men and 23 U/L for women in Chinese Han population. Re-consideration of normal limits of ALT should be recommended. Trial Registration ChiCTR.org ChiCTR-OCS-11001173 PMID:22962588

  17. The Effect of Artichoke Leaf Extract on Alanine Aminotransferase and Aspartate Aminotransferase in the Patients with Nonalcoholic Steatohepatitis.

    PubMed

    Rangboo, Vajiheh; Noroozi, Mostafa; Zavoshy, Roza; Rezadoost, Seyed Amirmansoor; Mohammadpoorasl, Asghar

    2016-01-01

    Background. Based on recent basic and clinical investigations, the extract of artichoke (Cynara scolymus) leaf has been revealed to be used for hepatoprotective and cholesterol reducing purposes. We aimed to assess the therapeutic effects of artichoke on biochemical and liver biomarkers in patients with nonalcoholic steatohepatitis (NASH). Methods. In a randomized double blind clinical trial, 60 consecutive patients suffering NASH were randomly assigned to receive Cynara scolymus extract (as 6 tablets per day consisting of 2700 mg extract of the herb) as the intervention group or placebo as the control group for two months. Results. Comparing changes in study markers following interventions showed improvement in liver enzymes. The levels of triglycerides and cholesterol were significantly reduced in the group treated with Cynara scolymus when compared to placebo group. To compare the role of Cynara scolymus use with placebo in changes in study parameters, multivariate linear regression models were employed indicating higher improvement in liver enzymes and also lipid profile particularly triglycerides and total cholesterol following administration of Cynara scolymus in comparison with placebo use. Conclusion. This study sheds light on the potential hepatoprotective activity and hypolipidemic effect of Cynara scolymus in management of NASH. This clinical trial is registered in the IRCT, Iranian Registry of Clinical Trials, by number IRCT2014070218321N1. PMID:27293900

  18. The Effect of Artichoke Leaf Extract on Alanine Aminotransferase and Aspartate Aminotransferase in the Patients with Nonalcoholic Steatohepatitis

    PubMed Central

    Rangboo, Vajiheh; Noroozi, Mostafa; Zavoshy, Roza; Rezadoost, Seyed Amirmansoor; Mohammadpoorasl, Asghar

    2016-01-01

    Background. Based on recent basic and clinical investigations, the extract of artichoke (Cynara scolymus) leaf has been revealed to be used for hepatoprotective and cholesterol reducing purposes. We aimed to assess the therapeutic effects of artichoke on biochemical and liver biomarkers in patients with nonalcoholic steatohepatitis (NASH). Methods. In a randomized double blind clinical trial, 60 consecutive patients suffering NASH were randomly assigned to receive Cynara scolymus extract (as 6 tablets per day consisting of 2700 mg extract of the herb) as the intervention group or placebo as the control group for two months. Results. Comparing changes in study markers following interventions showed improvement in liver enzymes. The levels of triglycerides and cholesterol were significantly reduced in the group treated with Cynara scolymus when compared to placebo group. To compare the role of Cynara scolymus use with placebo in changes in study parameters, multivariate linear regression models were employed indicating higher improvement in liver enzymes and also lipid profile particularly triglycerides and total cholesterol following administration of Cynara scolymus in comparison with placebo use. Conclusion. This study sheds light on the potential hepatoprotective activity and hypolipidemic effect of Cynara scolymus in management of NASH. This clinical trial is registered in the IRCT, Iranian Registry of Clinical Trials, by number IRCT2014070218321N1. PMID:27293900

  19. The Effect of Artichoke Leaf Extract on Alanine Aminotransferase and Aspartate Aminotransferase in the Patients with Nonalcoholic Steatohepatitis.

    PubMed

    Rangboo, Vajiheh; Noroozi, Mostafa; Zavoshy, Roza; Rezadoost, Seyed Amirmansoor; Mohammadpoorasl, Asghar

    2016-01-01

    Background. Based on recent basic and clinical investigations, the extract of artichoke (Cynara scolymus) leaf has been revealed to be used for hepatoprotective and cholesterol reducing purposes. We aimed to assess the therapeutic effects of artichoke on biochemical and liver biomarkers in patients with nonalcoholic steatohepatitis (NASH). Methods. In a randomized double blind clinical trial, 60 consecutive patients suffering NASH were randomly assigned to receive Cynara scolymus extract (as 6 tablets per day consisting of 2700 mg extract of the herb) as the intervention group or placebo as the control group for two months. Results. Comparing changes in study markers following interventions showed improvement in liver enzymes. The levels of triglycerides and cholesterol were significantly reduced in the group treated with Cynara scolymus when compared to placebo group. To compare the role of Cynara scolymus use with placebo in changes in study parameters, multivariate linear regression models were employed indicating higher improvement in liver enzymes and also lipid profile particularly triglycerides and total cholesterol following administration of Cynara scolymus in comparison with placebo use. Conclusion. This study sheds light on the potential hepatoprotective activity and hypolipidemic effect of Cynara scolymus in management of NASH. This clinical trial is registered in the IRCT, Iranian Registry of Clinical Trials, by number IRCT2014070218321N1.

  20. Correlation between liver cell necrosis and circulating alanine aminotransferase after ischaemia/reperfusion injuries in the rat liver.

    PubMed

    Knudsen, Anders R; Andersen, Kasper J; Hamilton-Dutoit, Stephen; Nyengaard, Jens R; Mortensen, Frank V

    2016-04-01

    Circulating liver enzymes such as alanine transaminase are often used as markers of hepatocellular damage. Ischaemia/reperfusion (I/R) injury is an inevitable consequence of prolonged liver ischaemia. The aim of this study was to examine the correlation between liver enzymes and volume of liver cell necrosis after ischaemia/reperfusion injuries, using design-unbiased stereological methods. Forty-seven male Wistar rats were subjected to 1 h of partial liver ischaemia, followed by either 4 or 24 h of reperfusion. Within each group, one-third of animals were subjected to ischaemic preconditioning and one-third to ischaemic postconditioning. At the end of reperfusion, blood and liver samples were collected for analysis. The volume of necrotic liver tissue was subsequently correlated to circulating markers of I/R injury. Correlation between histological findings and circulating markers was performed using Pearson's correlation coefficient. Alanine transferase peaked after 4 h of reperfusion; however, at this time-point, only mild necrosis was observed, with a Pearson's correlation coefficient of 0.663 (P = 0.001). After 24 h of reperfusion, alanine aminotransferase was found to be highly correlated to the degree of hepatocellular necrosis R = 0.836 (P = 0.000). Furthermore, alkaline phosphatase (R = 0.806) and α-2-macroglobulin (R = 0.655) levels were also correlated with the degree of necrosis. We show for the first time that there is a close correlation between the volume of hepatocellular necrosis and alanine aminotransferase levels in a model of I/R injury. This is especially apparent after 24 h of reperfusion. Similarly, increased levels of alkaline phosphatase and α-2-macroglobulin are correlated to the volume of liver necrosis. PMID:27292534

  1. Phylobiochemical characterization of class-Ib aspartate/prephenate aminotransferases reveals evolution of the plant arogenate phenylalanine pathway.

    PubMed

    Dornfeld, Camilla; Weisberg, Alexandra J; K C, Ritesh; Dudareva, Natalia; Jelesko, John G; Maeda, Hiroshi A

    2014-07-01

    The aromatic amino acid Phe is required for protein synthesis and serves as the precursor of abundant phenylpropanoid plant natural products. While Phe is synthesized from prephenate exclusively via a phenylpyruvate intermediate in model microbes, the alternative pathway via arogenate is predominant in plant Phe biosynthesis. However, the molecular and biochemical evolution of the plant arogenate pathway is currently unknown. Here, we conducted phylogenetically informed biochemical characterization of prephenate aminotransferases (PPA-ATs) that belong to class-Ib aspartate aminotransferases (AspAT Ibs) and catalyze the first committed step of the arogenate pathway in plants. Plant PPA-ATs and succeeding arogenate dehydratases (ADTs) were found to be most closely related to homologs from Chlorobi/Bacteroidetes bacteria. The Chlorobium tepidum PPA-AT and ADT homologs indeed efficiently converted prephenate and arogenate into arogenate and Phe, respectively. A subset of AspAT Ib enzymes exhibiting PPA-AT activity was further identified from both Plantae and prokaryotes and, together with site-directed mutagenesis, showed that Thr-84 and Lys-169 play key roles in specific recognition of dicarboxylic keto (prephenate) and amino (aspartate) acid substrates. The results suggest that, along with ADT, a gene encoding prephenate-specific PPA-AT was transferred from a Chlorobi/Bacteroidetes ancestor to a eukaryotic ancestor of Plantae, allowing efficient Phe and phenylpropanoid production via arogenate in plants today.

  2. Evaluation of Aspartate Aminotransferase-to-Platelet Ratio Index as a Non-Invasive Marker for Liver Cirrhosis

    PubMed Central

    Tripathi, B.K.; Gupta, B.; Bhandari, Bharti; Jalan, Divesh

    2015-01-01

    Introduction Liver biopsy is considered as a gold standard for the diagnosis of cirrhosis. Till date there is no non-invasive marker to replace it. Aim To investigate the effectiveness of Aspartate aminotransferase-to-platelet ratio index (APRI) as a non-invasive marker for liver cirrhosis. Materials and Methods Fifty-one patients with cirrhosis, identified on USG abdomen were included in study. Platelet count and Aspartate aminotransferase (AST) were done using haematology automatic analyser and automatic HITACHI-912 Auto Analyser respectively. APRI was calculated for every patient using the formula {(AST / ULN) x 100}/platelet count (109/L). Predictive accuracy was evaluated with a receiver-operating characteristics (ROC) curve. Results APRI correctly classified 49 (96.1%) patients of cirrhosis with area under the ROC curve of 0.973 (95% CI) at cut-off 0.65 with negative predictive value (NPV) and Positive predictive value (PPV) of 96% and 96.1% respectively. The sensitivity and specificity of the test was found to be 96% and 96.1% respectively. Conclusion APRI could identify cirrhosis with high degree of accuracy in the studied patients. PMID:26672800

  3. Deciphering the Role of Aspartate and Prephenate Aminotransferase Activities in Plastid Nitrogen Metabolism1[C][W][OPEN

    PubMed Central

    de la Torre, Fernando; El-Azaz, Jorge; Ávila, Concepción; Cánovas, Francisco M.

    2014-01-01

    Chloroplasts and plastids of nonphotosynthetic plant cells contain two aspartate (Asp) aminotransferases: a eukaryotic type (Asp5) and a prokaryotic-type bifunctional enzyme displaying Asp and prephenate aminotransferase activities (PAT). We have identified the entire Asp aminotransferase gene family in Nicotiana benthamiana and isolated and cloned the genes encoding the isoenzymes with plastidic localization: NbAsp5 and NbPAT. Using a virus-induced gene silencing approach, we obtained N. benthamiana plants silenced for NbAsp5 and/or NbPAT. Phenotypic and metabolic analyses were conducted in silenced plants to investigate the specific roles of these enzymes in the biosynthesis of essential amino acids within the plastid. The NbAsp5 silenced plants had no changes in phenotype, exhibiting similar levels of free Asp and glutamate as control plants, but contained diminished levels of asparagine and much higher levels of lysine. In contrast, the suppression of NbPAT led to a severe reduction in growth and strong chlorosis symptoms. NbPAT silenced plants exhibited extremely reduced levels of asparagine and were greatly affected in their phenylalanine metabolism and lignin deposition. Furthermore, NbPAT suppression triggered a transcriptional reprogramming in plastid nitrogen metabolism. Taken together, our results indicate that NbPAT has an overlapping role with NbAsp5 in the biosynthesis of Asp and a key role in the production of phenylalanine for the biosynthesis of phenylpropanoids. The analysis of NbAsp5/NbPAT cosilenced plants highlights the central role of both plastidic aminotransferases in nitrogen metabolism; however, only NbPAT is essential for plant growth and development. PMID:24296073

  4. Comparative study of dynamic structure of pig and chicken aspartate aminotransferases by measuring the rotational correlation time.

    PubMed

    Timofeev, V P; Dudich, I V; Volkenstein, M V

    1980-01-01

    The rotational correlation time of two homologous cytoplasmic aspartate aminotransferase molecules isolated from pig and chicken hearts was obtained by spin-labeling technique. The maleimide and iodoacetamide spin-labels modifying external SH-groups of a protein were used. In the interpretation of ESR spectra a rotational motion of nitroxide group relative to the protein molecule was taken into account. To determine the macromolecule rotational correlation time two methods of the immobilization of a protein molecule were used: 1) by means of increasing protein solution viscosity and 2) by fixation of the protein molecule on adsorbent. From comparison of experimental and theoretical values of rotational correlation time it was conclude that the both enzymes exhibits an intramolecular flexibility.

  5. An easy method for diagnosing macro-aspartate aminotransferase: a case series.

    PubMed

    Beşer, Omer Faruk; Laçinel, Sibel; Gülcü, Didem; Kutlu, Tufan; Cullu Çokuğraş, Fügen; Erkan, Tülay

    2014-10-01

    Macro-aspartate transaminase (macro-AST) must be considered when the aspartate transaminase (AST) level is chronically high without any liver, cardiac, or muscle disease. Many specialized laboratory techniques have been recommended for diagnosing macro-AST, including the polyethylene glycol immune precipitate technique, which is simple. This study presents a considerably easier method based on the studies of Davidson and Watson and Castiella et al. Our method is based on the decrease in the plasma AST level after storage of the macroenzyme at 2-8 °C for 5 days, and has the advantages of low cost, reliability, and practicality at any health center. In our eight cases of macro-AST, the AST activity at day 6 had decreased by more than 50% from day 1. This method is practical for primary healthcare facilities because of its easy application and accurate results, and obviated the need for unnecessary tests after diagnosis.

  6. The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes.

    PubMed Central

    Fotheringham, I G; Dacey, S A; Taylor, P P; Smith, T J; Hunter, M G; Finlay, M E; Primrose, S B; Parker, D M; Edwards, R M

    1986-01-01

    In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage. PMID:3521591

  7. Liver peroxisomal alanine:glyoxylate aminotransferase and the effects of mutations associated with Primary Hyperoxaluria Type I: An overview.

    PubMed

    Oppici, Elisa; Montioli, Riccardo; Cellini, Barbara

    2015-09-01

    Liver peroxisomal alanine:glyoxylate aminotransferase (AGT) (EC 2.6.1.44) catalyses the conversion of l-alanine and glyoxylate to pyruvate and glycine, a reaction that allows glyoxylate detoxification. Inherited mutations on the AGXT gene encoding AGT lead to Primary Hyperoxaluria Type I (PH1), a rare disorder characterized by the deposition of calcium oxalate crystals primarily in the urinary tract. Here we describe the results obtained on the biochemical features of AGT as well as on the molecular and cellular effects of polymorphic and pathogenic mutations. A complex scenario on the molecular pathogenesis of PH1 emerges in which the co-inheritance of polymorphic changes and the condition of homozygosis or compound heterozygosis are two important factors that determine the enzymatic phenotype of PH1 patients. All the reported data represent relevant steps toward the understanding of genotype/phenotype correlations, the prediction of the response of the patients to the available therapies, and the development of new therapeutic approaches. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

  8. Characterization of five putative aspartate aminotransferase genes in the N2-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120.

    PubMed

    Xu, Xinyi; Gu, Liping; He, Ping; Zhou, Ruanbao

    2015-06-01

    Aspartate and glutamate are two key amino acids used in biosynthesis of many amino acids that play vital role in cellular metabolism. Aspartate aminotransferases (AspATs) are required for channelling nitrogen (N(2)) between Glu and Asp in all life forms. Biochemical and genetic characterization of AspATs have been lacking in N(2)-fixing cyanobacteria. In this report, five putative AspAT genes (alr1039, all2340, alr2765, all4327 and alr4853) were identified in the N(2)-fixing heterocystous cyanobacterium Anabaena sp. PCC 7120. Five recombinant C-terminal hexahistidine-tagged AspATs (AspAT-H(6)) were overexpressed in Escherichia coli and purified to homogeneity. Biochemical analysis demonstrated that these five putative AspATs have authentic AspAT activity in vitro using aspartate as an amino donor. However, the enzymic activities of the five AspATs differed in vitro. Alr4853-H(6) showed the highest AspAT activity, while the enzymic activity for the other four AspATs ranged from 6.5 to 53.7 % activity compared to Alr4853 (100 %). Genetic characterization of the five AspAT genes was also performed by inactivating each individual gene. All of the five AspAT knockout mutants exhibited reduced diazotrophic growth, and alr4853 was further identified to be a Fox gene (requiring fixed N(2) for growth in the presence of oxygen). Four out of five P(aspAT)-gfp transcriptional fusions were constitutively expressed in both diazotrophic and nitrate-dependent growth conditions. Quantitative reverse transcriptase PCR showed that alr4853 expression was increased by 2.3-fold after 24 h of N(2) deprivation. Taken together, these findings add to our understanding of the role of AspATs in N(2)-fixing within heterocystous cyanobacteria.

  9. Predicting Three-Dimensional Conformations of Peptides Constructed of Only Glycine, Alanine, Aspartic Acid, and Valine

    NASA Astrophysics Data System (ADS)

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  10. Predicting three-dimensional conformations of peptides constructed of only glycine, alanine, aspartic acid, and valine.

    PubMed

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  11. BarR, an Lrp-type transcription factor in Sulfolobus acidocaldarius, regulates an aminotransferase gene in a β-alanine responsive manner.

    PubMed

    Liu, Han; Orell, Alvaro; Maes, Dominique; van Wolferen, Marleen; Lindås, Ann-Christin; Bernander, Rolf; Albers, Sonja-Verena; Charlier, Daniel; Peeters, Eveline

    2014-05-01

    In archaea, nothing is known about the β-alanine degradation pathway or its regulation. In this work, we identify and characterize BarR, a novel Lrp-like transcription factor and the first one that has a non-proteinogenic amino acid ligand. BarR is conserved in Sulfolobus acidocaldarius and Sulfolobus tokodaii and is located in a divergent operon with a gene predicted to encode β-alanine aminotransferase. Deletion of barR resulted in a reduced exponential growth rate in the presence of β-alanine. Furthermore, qRT-PCR and promoter activity assays demonstrated that BarR activates the expression of the adjacent aminotransferase gene, but only upon β-alanine supplementation. In contrast, auto-activation proved to be β-alanine independent. Heterologously produced BarR is an octamer in solution and forms a single complex by interacting with multiple sites in the 170 bp long intergenic region separating the divergently transcribed genes. In vitro, DNA binding is specifically responsive to β-alanine and site-mutant analyses indicated that β-alanine directly interacts with the ligand-binding pocket. Altogether, this work contributes to the growing body of evidence that in archaea, Lrp-like transcription factors have physiological roles that go beyond the regulation of α-amino acid metabolism.

  12. Associations of functional alanine-glyoxylate aminotransferase 2 gene variants with atrial fibrillation and ischemic stroke

    PubMed Central

    Seppälä, Ilkka; Kleber, Marcus E.; Bevan, Steve; Lyytikäinen, Leo-Pekka; Oksala, Niku; Hernesniemi, Jussi A.; Mäkelä, Kari-Matti; Rothwell, Peter M.; Sudlow, Cathie; Dichgans, Martin; Mononen, Nina; Vlachopoulou, Efthymia; Sinisalo, Juha; Delgado, Graciela E.; Laaksonen, Reijo; Koskinen, Tuomas; Scharnagl, Hubert; Kähönen, Mika; Markus, Hugh S.; März, Winfried; Lehtimäki, Terho

    2016-01-01

    Asymmetric and symmetric dimethylarginines (ADMA and SDMA) impair nitric oxide bioavailability and have been implicated in the pathogenesis of atrial fibrillation (AF). Alanine–glyoxylate aminotransferase 2 (AGXT2) is the only enzyme capable of metabolizing both of the dimethylarginines. We hypothesized that two functional AGXT2 missense variants (rs37369, V140I; rs16899974, V498L) are associated with AF and its cardioembolic complications. Association analyses were conducted using 1,834 individulas with AF and 7,159 unaffected individuals from two coronary angiography cohorts and a cohort comprising patients undergoing clinical exercise testing. In coronary angiography patients without structural heart disease, the minor A allele of rs16899974 was associated with any AF (OR = 2.07, 95% CI 1.59-2.68), and with paroxysmal AF (OR = 1.98, 95% CI 1.44–2.74) and chronic AF (OR = 2.03, 95% CI 1.35–3.06) separately. We could not replicate the association with AF in the other two cohorts. However, the A allele of rs16899974 was nominally associated with ischemic stroke risk in the meta-analysis of WTCCC2 ischemic stroke cohorts (3,548 cases, 5,972 controls) and with earlier onset of first-ever ischemic stroke (360 cases) in the cohort of clinical exercise test patients. In conclusion, AGXT2 variations may be involved in the pathogenesis of AF and its age-related thromboembolic complications. PMID:26984639

  13. Relationship of creatine kinase, aspartate aminotransferase, lactate dehydrogenase, and proteinuria to cardiomyopathy in the owl monkey (Aotus vociferans)

    SciTech Connect

    Gozalo, Alfonso S.; Chavera, Alfonso; Montoya, Enrique J.; Takano, Juan; Weller, Richard E.

    2008-02-01

    The purpose of this study was to determine serum reference values for crea- tine kinase (CK), aspartate aminotransferase (AST), and lactate dehydroge- nase (LDH) in captive-born and wild-caught owl monkeys to assess their usefulness for diagnosing myocardial disease. Urine samples were also collected and semi-quantitative tests performed. There was no statistically significant difference between CK, AST, and LDH when comparing both groups. However, when comparing monkeys with proteinuria to those without proteinuria, a statistically significant difference in CK value was observed (P = 0.021). In addition, the CK/AST ratio revealed that 29% of the animals included in this study had values suggesting cardiac infarction. Grossly, cardiac concentric hypertrophy of the left ventricle and small, pitted kidneys were the most common findings. Microscopically, myocardial fibrosis, contraction band necrosis, hypertrophy and hyperplasia of coronary arteries, medium-sized renal arteries, and afferent glomerular arteriolae were the most significant lesions, along with increased mesangial matrix and hypercellularity of glomeruli, Bowman’s capsule, and peritubular space fibroplasia. These findings suggest that CK, AST, and LDH along with urinalysis provide a reliable method for diagnosing cardiomyopathies in the owl monkey. In addition, CK/AST ratio, proteinuria, and the observed histological and ultrastructural changes suggest that Aotus vociferans suffer from arterial hypertension and chronic myocardial infarction.

  14. Elevation of retinol levels and suppression of alanine aminotransferase activity in the liver of taurine-deficient kittens.

    PubMed

    Lehmann, A; Knutsson, L; Bosaeus, I

    1990-10-01

    In taurine-deficient cats, the secretion of bile acids is impaired, and this impairment may reduce intestinal uptake of lipophilic vitamins. It was therefore hypothesized that retinol deficiency is involved in the generation of retinal lesions in taurine-deficient kittens. To this end, the concentration of retinol in plasma and liver was determined in taurine-deficient kittens. Further, the effects of taurine deficiency on amino acid concentrations of heart, liver and kidney were investigated. To see whether taurine deficiency adversely affects the liver, hepatic enzymes were measured in plasma and liver of kittens suffering from taurine deficiency. In addition, liver morphology, growth and food intake were studied. Taurine was the only amino acid whose concentration was consistently decreased in plasma of the experimental group. Unexpectedly, retinol level was increased in plasma and liver from taurine-depleted kittens. Several alterations were noted in amino acid concentrations in liver and kidney, but not in heart. Plasma alanine aminotransferase activity was diminished, probably reflecting decreased activity in the liver. Perivenular steatosis was found in both groups. Controls grew linearly, in contrast to deficient animals, which nevertheless consumed more food. The results demonstrate that retinol deficiency is not involved in taurine-deficiency retinopathy. Moreover, taurine is required for linear growth of juvenile cats and for the maintenance of hepatic and renal pools of certain amino acids. PMID:2213246

  15. A Novel Pathway for Metabolism of the Cardiovascular Risk Factor Homoarginine by alanine:glyoxylate aminotransferase 2

    PubMed Central

    Rodionov, Roman N.; Oppici, Elisa; Martens-Lobenhoffer, Jens; Jarzebska, Natalia; Brilloff, Silke; Burdin, Dmitrii; Demyanov, Anton; Kolouschek, Anne; Leiper, James; Maas, Renke; Cellini, Barbara; Weiss, Norbert; Bode-Böger, Stefanie M.

    2016-01-01

    Low plasma concentrations of L-homoarginine are associated with an increased risk of cardiovascular events, while homoarginine supplementation is protective in animal models of metabolic syndrome and stroke. Catabolism of homoarginine is still poorly understood. Based on the recent findings from a Genome Wide Association Study we hypothesized that homoarginine can be metabolized by alanine:glyoxylate aminotransferase 2 (AGXT2). We purified human AGXT2 from tissues of AGXT2 transgenic mice and demonstrated its ability to metabolize homoarginine to 6-guanidino-2-oxocaproic acid (GOCA). After incubation of HepG2 cells overexpressing AGXT2 with isotope-labeled homoarginine-d4 we were able to detect labeled GOCA in the medium. We injected wild type mice with labeled homoarginine and detected labeled GOCA in the plasma. We found that AGXT2 knockout (KO) mice have higher homoarginine and lower GOCA plasma levels as compared to wild type mice, while the reverse was true for AGXT2 transgenic (Tg) mice. In summary, we experimentally proved the presence of a new pathway of homoarginine catabolism – its transamination by AGXT2 with formation of GOCA and demonstrated that endogenous AGXT2 is required for maintenance of homoarginine levels in mice. Our findings may lead to development of novel therapeutic approaches for cardiovascular pathologies associated with homoarginine deficiency. PMID:27752063

  16. Genetic engineering of improved nitrogen use efficiency in rice by the tissue-specific expression of alanine aminotransferase.

    PubMed

    Shrawat, Ashok K; Carroll, Rebecka T; DePauw, Mary; Taylor, Gregory J; Good, Allen G

    2008-09-01

    Summary Nitrogen is quantitatively the most essential nutrient for plants and a major factor limiting crop productivity. One of the critical steps limiting the efficient use of nitrogen is the ability of plants to acquire it from applied fertilizer. Therefore, the development of crop plants that absorb and use nitrogen more efficiently has been a long-term goal of agricultural research. In an attempt to develop nitrogen-efficient plants, rice (Oryza sativa L.) was genetically engineered by introducing a barley AlaAT (alanine aminotransferase) cDNA driven by a rice tissue-specific promoter (OsAnt1). This modification increased the biomass and grain yield significantly in comparison with control plants when plants were well supplied with nitrogen. Compared with controls, transgenic rice plants also demonstrated significant changes in key metabolites and total nitrogen content, indicating increased nitrogen uptake efficiency. The development of crop plants that take up and assimilate nitrogen more efficiently would not only improve the use of nitrogen fertilizers, resulting in lower production costs, but would also have significant environmental benefits. These results are discussed in terms of their relevance to the development of strategies to engineer enhanced nitrogen use efficiency in crop plants. PMID:18510577

  17. Molecular beacon based bioassay for highly sensitive and selective detection of nicotinamide adenine dinucleotide and the activity of alanine aminotransferase.

    PubMed

    Tang, Zhiwen; Liu, Pei; Ma, Changbei; Yang, Xiaohai; Wang, Kemin; Tan, Weihong; Lv, Xiaoyuan

    2011-04-01

    We have developed a new approach to detect nicotinamide adenine dinucleotide (NAD(+)) with high specificity and sensitivity using molecular beacons (MBs) and employed it in the investigation of NAD(+) related biological processes, such as calorie restriction and alanine aminotransferase (ALT) activation. The E. coli DNA ligase would catalyze the ligation of two short oligonucleotides that complement with an MB only in the presence of NAD(+), resulting in the opening of the MB and the restoration of fluorescent signal. Thanks to the high sensitivity of the MB probe and the fidelity of E. coli DNA ligase toward its substrates, this approach can detect 0.3 nM NAD(+) with high selectivity against other NAD(+) analogs. This novel assay can also provide a convenient and robust way to analyze NAD(+) in biological samples such as cell lysate. As NAD(+) plays an essential role in many biochemical processes, this method can be used to investigate NAD(+) related life processes. For instance, the effect of calorie restriction on the intracellular NAD(+) level in MCF7 cells has been studied using this new assay. Moreover, this approach was also successfully used to analyze the activity of ALT. Therefore, this novel NAD(+) assay holds wide applicability as an analytical tool in biochemical and biomedical research.

  18. Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

    PubMed

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C; Abe, Keietsu

    2007-10-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

  19. Influence of Preoperative Serum Aspartate Aminotransferase (AST) Level on the Prognosis of Patients with Non-Small Cell Lung Cancer

    PubMed Central

    Chen, Shu-Lin; Xue, Ning; Wu, Mian-Tao; Chen, Hao; He, Xia; Li, Jian-Pei; Liu, Wan-Li; Dai, Shu-Qin

    2016-01-01

    The purpose of this work is to analyze preoperative serum aspartate aminotransferase (AST) levels and their effect on the prognosis of patients with non-small cell lung cancer (NSCLC) after surgical operation. These analyses were performed retrospectively in patients with NSCLC followed by surgery; participants were recruited between January 2004 and January 2008. All clinical information and laboratory results were collected from medical records. We explored the association between preoperative serum AST and recurrence-free survival (RFS), and the overall survival (OS) of NSCLC patients. Kaplan–Meier analysis and Cox multivariate analysis, stratified by the AST median value, were used to evaluate the prognostic effect. A chi-squared test was performed to compare clinical characteristics in different subgroups. A p-value of ≤0.05 was considered to be statistically significant. A total of 231 patients were enrolled. The median RFS and OS were 22 and 59 months, respectively. The AST levels were divided into two groups, using a cut-off value of 19 U/L: High AST (>19 U/L), n = 113 vs. low AST (≤19 U/L), n = 118. Multivariate analysis indicated that preoperative serum AST > 19 U/L (hazard ratio (HR) = 0.685, 95% confidence interval (CI): 0.493–0.994, p = 0.046 for RFS, HR = 0.646, 95% CI: 0.438–0.954, p = 0.028 for OS) was an independent prognostic factor for both RFS and OS. High preoperative serum AST levels may serve as a valuable marker to predict the prognosis of NSCLC after operation. PMID:27598151

  20. Diet and the frequency of the alanine:glyoxylate aminotransferase Pro11Leu polymorphism in different human populations.

    PubMed

    Caldwell, Elizabeth F; Mayor, Lianne R; Thomas, Mark G; Danpure, Christopher J

    2004-11-01

    The intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT) contains a Pro11Leu polymorphism that decreases its catalytic activity by a factor of three and causes a small proportion to be mistargeted from its normal intracellular location in the peroxisomes to the mitochondria. These changes are predicted to have significant effects on the synthesis and excretion of the metabolic end-product oxalate and the deposition of insoluble calcium oxalate in the kidney and urinary tract. Based on the evolution of AGT targeting in mammals, we have previously hypothesised that this polymorphism would be advantageous for individuals who have a meat-rich diet, but disadvantageous for those who do not. If true, the frequency distribution of Pro11Leu in different extant human populations should have been shaped by their dietary history so that it should be more common in populations with predominantly meat-eating ancestral diets than it is in populations in which the ancestral diets were predominantly vegetarian. In the present study, we have determined frequency of Pro11Leu in 11 different human populations with divergent ancestral dietary lifestyles. We show that the Pro11Leu allelic frequency varies widely from 27.9% in the Saami, a population with a very meat-rich ancestral diet, to 2.3% in Chinese, who are likely to have had a more mixed ancestral diet. FST analysis shows that the differences in Pro11Leu frequency between some populations (particularly Saami vs Chinese) was very high when compared with neutral loci, suggesting that its frequency might have been shaped by dietary selection pressure.

  1. Plasma Levels of Alanine Aminotransferase in the First Trimester Identify High Risk Chinese Women for Gestational Diabetes

    PubMed Central

    Leng, Junhong; Zhang, Cuiping; Wang, Peng; Li, Nan; Li, Weiqin; Liu, Huikun; Zhang, Shuang; Hu, Gang; Yu, Zhijie; Ma, Ronald CW; Chan, Juliana CN; Yang, Xilin

    2016-01-01

    Alanine aminotransferase (ALT) predicts type 2 diabetes but it is uncertain whether it also predicts gestational diabetes mellitus (GDM). We recruited 17359 Chinese women with ALT measured in their first trimester. At 24–28 weeks of gestation, all women underwent a 50-gram 1-hour glucose challenge test (GCT) followed by a 75-gram 2-hour oral glucose tolerance test if GCT result was ≥7.8 mmol/L. Restricted cubic spline analysis was used to examine full-range risk associations of ALT levels with GDM. Relative excess risk due to interaction, attributable proportion due to interaction and synergy index were used to estimate additive interaction between high ALT and overweight/obesity for GDM. Finally, 1332 (7.7%) women had GDM. ALT levels were positively associated with GDM risk without a clear threshold. Using ALT levels <22 U/L as the referent, the middle ALT levels (≥22 to <40 U/L) [odds ratio (OR) (95% confidence intervals): 1.41(1.21–1.65)] and high ALT levels (≥40 U/L) [1.62 (1.31–2.00)] were associated with increased GDM risk. Maternal overweight/obesity greatly enhanced the OR of ALT ≥22 U/L from 1.44 (1.23–1.69) to 3.46 (2.79–4.29) with significant additive interactions. In conclusion, elevated ALT levels in the first trimester even within normal range predicted GDM risk, further enhanced by overweight/obesity. PMID:27264612

  2. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase.

    PubMed

    Schumann, Gerhard; Bonora, Roberto; Ceriotti, Ferruccio; Férard, Georges; Ferrero, Carlo A; Franck, Paul F H; Gella, F Javier; Hoelzel, Wieland; Jørgensen, Poul Jørgen; Kanno, Takashi; Kessner, Art; Klauke, Rainer; Kristiansen, Nina; Lessinger, Jean-Marc; Linsinger, Thomas P J; Misaki, Hideo; Panteghini, Mauro; Pauwels, Jean; Schiele, Françoise; Schimmel, Heinz G; Weidemann, Gerhard; Siekmann, Lothar

    2002-07-01

    This paper is the fourth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of Gamma-Glutamyltransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of Gamma-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37 degrees C. A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30 degrees C IFCC reference method. Differences are tabulated and commented on in Appendix 2.

  3. Trihalomethane exposure and biomonitoring for the liver injury indicator, alanine aminotransferase, in the United States population (NHANES 1999–2006)

    PubMed Central

    Burch, James B.; Everson, Todd M.; Seth, Ratanesh K.; Wirth, Michael D.; Chatterjee, Saurabh

    2015-01-01

    Exposure to trihalomethanes (or THMs: chloroform, bromoform, bromodichloromethane, and dibromochloromethane [DBCM]) formed via drinking water disinfection has been associated with adverse reproductive outcomes and cancers of the digestive or genitourinary organs. However, few studies have examined potential associations between THMs and liver injury in humans, even though experimental studies suggest that these agents exert hepatotoxic effects, particularly among obese individuals. This study examined participants in the National Health and Nutrition Examination Survey (1999–2006, N = 2781) to test the hypothesis that THMs are associated with liver injury as assessed by alanine aminotransferase (ALT) activity in circulation. Effect modification by body mass index (BMI) or alcohol consumption also was examined. Associations between blood THM concentrations and ALT activity were assessed using unconditional multiple logistic regression to calculate prevalence odds ratios (ORs) with 95% confidence intervals (CIs) for exposure among cases with elevated ALT activity (men: >40 IU/L, women: >30 IU/L) relative to those with normal ALT, after adjustment for variables that may confound the relationship between ALT and THMs. Compared to controls, cases were 1.35 times more likely (95% CI: 1.02, 1.79) to have circulating DBCM concentrations exceeding median values in the population. There was little evidence for effect modification by BMI, although the association varied by alcohol consumption. Among non-drinkers, cases were more likely than controls to be exposed to DBCM (OR: 3.30, 95% CI: 1.37–7.90), bromoform (OR: 2.88, 95% CI: 1.21–6.81), or brominated THMs (OR: 4.00, 95% CI: 1.31–12.1), but no association was observed among participants with low, or moderate to heavy alcohol consumption. Total THM levels exceeding benchmark exposure limits continue to be reported both in the United States and globally. Results from this study suggest a need for further

  4. Trihalomethane exposure and biomonitoring for the liver injury indicator, alanine aminotransferase, in the United States population (NHANES 1999-2006).

    PubMed

    Burch, James B; Everson, Todd M; Seth, Ratanesh K; Wirth, Michael D; Chatterjee, Saurabh

    2015-07-15

    Exposure to trihalomethanes (or THMs: chloroform, bromoform, bromodichloromethane, and dibromochloromethane [DBCM]) formed via drinking water disinfection has been associated with adverse reproductive outcomes and cancers of the digestive or genitourinary organs. However, few studies have examined potential associations between THMs and liver injury in humans, even though experimental studies suggest that these agents exert hepatotoxic effects, particularly among obese individuals. This study examined participants in the National Health and Nutrition Examination Survey (1999-2006, N=2781) to test the hypothesis that THMs are associated with liver injury as assessed by alanine aminotransferase (ALT) activity in circulation. Effect modification by body mass index (BMI) or alcohol consumption also was examined. Associations between blood THM concentrations and ALT activity were assessed using unconditional multiple logistic regression to calculate prevalence odds ratios (ORs) with 95% confidence intervals (CIs) for exposure among cases with elevated ALT activity (men: >40IU/L, women: >30IU/L) relative to those with normal ALT, after adjustment for variables that may confound the relationship between ALT and THMs. Compared to controls, cases were 1.35 times more likely (95% CI: 1.02, 1.79) to have circulating DBCM concentrations exceeding median values in the study population. There was little evidence for effect modification by BMI, although the association varied by alcohol consumption. Among non-drinkers, cases were more likely than controls to be exposed to DBCM (OR: 3.30, 95% CI: 1.37, 7.90), bromoform (OR: 2.88, 95% CI: 1.21, 6.81), or brominated THMs (OR: 4.00, 95% CI: 1.31, 12.1), but no association was observed among participants with low, or moderate to heavy alcohol consumption. Total THM levels exceeding benchmark exposure limits continue to be reported both in the United States and globally. Results from this study suggest a need for further

  5. Analysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilisers during hypoxic stress.

    PubMed

    Rocha, Marcio; Sodek, Ladaslav; Licausi, Francesco; Hameed, Muhammad Waqar; Dornelas, Marcelo Carnier; van Dongen, Joost T

    2010-10-01

    Alanine aminotransferase (AlaAT) catalyses the reversible conversion of pyruvate and glutamate into alanine and oxoglutarate. In soybean, two subclasses were identified, each represented by two highly similar members. To investigate the role of AlaAT during hypoxic stress in soybean, changes in transcript level of both subclasses were analysed together with the enzyme activity and alanine content of the tissue. Moreover, the dependency of AlaAT activity and gene expression was investigated in relation to the source of nitrogen supplied to the plants. Using semi-quantitative PCR, GmAlaAT genes were determined to be highest expressed in roots and nodules. Under normal growth conditions, enzyme activity of AlaAT was detected in all organs tested, with lowest activity in the roots. Upon waterlogging-induced hypoxia, AlaAT activity increased strongly. Concomitantly, alanine accumulated. During re-oxygenation, AlaAT activity remained high, but the transcript level and the alanine content decreased. Our results show a role for AlaAT in the catabolism of alanine during the initial period of re-oxygenation following hypoxia. GmAlaAT also responded to nitrogen availability in the solution during waterlogging. Ammonium as nitrogen source induced both gene expression and enzyme activity of AlaAT more than when nitrate was supplied in the nutrient solution. The work presented here indicates that AlaAT might not only be important during hypoxia, but also during the recovery phase after waterlogging, when oxygen is available to the tissue again.

  6. Corticosterone, cortisol, triglycerides, aspartate aminotransferase and uric acid plasma concentrations during foie gras production in male mule ducks (Anas platyrhynchos × Cairina moschata).

    PubMed

    Flament, A; Delleur, V; Poulipoulis, A; Marlier, D

    2012-01-01

    1. Corticosterone, cortisol, triglycerides, aspartate aminotransferase (AST) and uric acid (UA) plasma concentration were measured at 8 (7 days after group housing), 12 (after 7 days of force feeding) and 13 weeks of age (at slaughter after 12 days of force feeding), and 45 min after an adrenocorticotrophic hormone (ACTH) stimulation test at 8 weeks of age in 12 male mule ducks in an on-farm experiment. 2. No significant increase of corticosterone was found during the force-feeding period compared with the concentration after housing. 3. Comparison of corticosterone and cortisol values indicates that cortisol can be considered as a reliable acute stress indicator in future routine examinations. 4. Plasma concentrations of triglycerides and aspartate aminotransferase increased progressively from pre-force feeding period to slaughtering. 5. Plasma concentrations of uric acid increased from the start at 8 weeks of age to the mid-force feeding period but no difference was noticed between the mid-force feeding period and slaughtering. 6. It is concluded that acute stress induced by force-feeding is similar at the beginning and end of the commercial production of foie gras.

  7. Alanine aminotransferase 1 (OsAlaAT1) plays an essential role in the regulation of starch storage in rice endosperm.

    PubMed

    Yang, Jungil; Kim, Sung-Ryul; Lee, Sang-Kyu; Choi, Heebak; Jeon, Jong-Seong; An, Gynheung

    2015-11-01

    Alteration of storage substances, in particular the major storage form starch, leads to floury endosperm. Because floury mutants have physical attributes for milling processes, identification and characterization of those mutants are valuable. In this study we identified a floury endosperm mutant caused by a T-DNA insertion in Oryza sativa alanine-aminotransferase1 (OsAlaAT1). OsAlaAT1 is localized in the cytosol and has aminotransferase enzyme activity. The osalaat1 mutant has less amylose and its amylopectin is structurally altered. OsAlaAT1 is predominantly expressed in developing seeds during active starch synthesis. AlaAT catalyzes the interconversion of pyruvate to alanine, and this pathway is activated under low-oxygen conditions. Consistently, OsAlaAT1 is induced by such conditions. Expression of the starch synthesis genes AGPases, OsSSI, OsSSIIa, and OsPPDKB is decreased in the mutant. Thus, our observations suggest that OsAlaAT1 plays an essential role in starch synthesis in developing seeds that are exposed to low concentrations of oxygen. PMID:26475189

  8. Characterization of amino acid aminotransferases of Methanococcus aeolicus.

    PubMed Central

    Xing, R Y; Whitman, W B

    1992-01-01

    Four aminotransferases were identified and characterized from Methanococcus aeolicus. Branched-chain aminotransferase (BcAT, EC 2.6.1.42), aspartate aminotransferase (AspAT, EC 2.6.1.1), and two aromatic aminotransferases (EC 2.6.1.57) were partially purified 175-, 84-, 600-, and 30-fold, respectively. The apparent molecular weight, substrate specificity, and kinetic properties of the BcAT were similar to those of other microbial BcATs. The AspAT had an apparent molecular weight of 162,000, which was unusually high. It had also a broad substrate specificity, which included activity towards alanine, a property which resembled the enzyme from Sulfolobus solfataricus. An additional alanine aminotransferase was not found in M. aeolicus, and this activity of AspAT could be physiologically significant. The apparent molecular weights of the aromatic aminotransferases (ArAT-I and ArAT-II) were 150,000 and 90,000, respectively. The methanococcal ArATs also had different pIs and kinetic constants. ArAT-I may be the major ArAT in methanococci. High concentrations of 2-ketoglutarate strongly inhibited valine, isoleucine, and alanine transaminations but were less inhibitory for leucine and aspartate transaminations. Aromatic amino acid transaminations were not inhibited by 2-ketoglutarate. 2-Ketoglutarate may play an important role in the regulation of amino acid biosynthesis in methanococci. PMID:1729242

  9. A novel C-S lyase from the latex-producing plant Taraxacum brevicorniculatum displays alanine aminotransferase and l-cystine lyase activity.

    PubMed

    Munt, Oliver; Prüfer, Dirk; Schulze Gronover, Christian

    2013-01-01

    We isolated a novel pyridoxal-5-phosphate-dependent l-cystine lyase from the dandelion Taraxacum brevicorniculatum. Real time qPCR analysis showed that C-S lyase from Taraxacum brevicorniculatum (TbCSL) mRNA is expressed in all plant tissues, although at relatively low levels in the latex and pedicel. The 1251 bp TbCSL cDNA encodes a protein with a calculated molecular mass of 46,127 kDa. It is homologous to tyrosine and alanine aminotransferases (AlaATs) as well as to an Arabidopsis thaliana carbon-sulfur lyase (C-S lyase) (SUR1), which has a role in glucosinolate metabolism. TbCSL displayed in vitrol-cystine lyase and AlaAT activities of 4 and 19nkatmg(-1) protein, respectively. However, we detected no in vitro tyrosine aminotransferase (TyrAT) activity and RNAi knockdown of the enzyme had no effect on phenotype, showing that TbCSL substrates might be channeled into redundant pathways. TbCSL is in vivo localized in the cytosol and functions as a C-S lyase or an aminotransferase in planta, but the purified enzyme converts at least two substrates specifically, and can thus be utilized for further in vitro applications.

  10. Estimation of free energy barriers in the cytoplasmic and mitochondrial aspartate aminotransferase reactions probed by hydrogen-exchange kinetics of C alpha-labeled amino acids with solvent

    SciTech Connect

    Julin, D.A.; Wiesinger, H.; Toney, M.D.; Kirsch, J.F. )

    1989-05-02

    The existence of the postulated quinonoid intermediate in the cytoplasmic aspartate amino-transferase catalyzed transamination of aspartate to oxaloacetate was probed by determining the extent of transfer of tritium from the C alpha position of tritiated L-aspartate to pyridoxamine 5'-phosphate in single turnover experiments in which washout from the back-reaction was obviated by product trapping. The maximum amount of transferred tritium observed was 0.7%, consistent either with a mechanism in which a fraction of the net transamination reaction proceeds through a quinonoid intermediate or with a mechanism in which this intermediate is formed off the main reaction pathway. It is shown that transfer of labeled hydrogen from the amino acid to cofactor cannot be used to differentiate a stepwise from a concerted transamination mechanism. The amount of tritium transferred is a function of the rate constant for torsional equilibration about the epsilon-amino group of Lys-258, the presumptive abstractor of the C alpha proton; the relative rate constants for hydrogen exchange with solvent versus cofactor protonation; and the tritium isotope effect on this ratio. The free energy barriers facing the covalent intermediate between aldimine and keto acid product (i.e., ketimine and possibly quinonoid) were evaluated relatively by comparing the rates of C alpha-hydrogen exchange in starting amino acid with the rates of keto acid formation. The value of theta (= kexge/kprod) was found to be 2.6 for the reaction of cytoplasmic isozyme with aspartate and ca. 0.5 for that of the mitochondrial form with glutamate.

  11. PsAAT3, an oomycete-specific aspartate aminotransferase, is required for full pathogenicity of the oomycete pathogen Phytophthora sojae.

    PubMed

    Wang, Rongbo; Zhang, Meixiang; Liu, Hong; Xu, Jing; Yu, Jia; He, Feng; Zhang, Xiong; Dong, Suomeng; Dou, Daolong

    2016-04-01

    Pathogen nutrient acquisition and metabolism are critical for successful infection and colonization. However, the nutrient requirements and metabolic pathways related to pathogenesis in oomycete pathogens are unknown. In this study, we bioinformatically identified Phytophthora sojae aspartate aminotransferases (AATs), which are key enzymes that coordinate carbon and nitrogen metabolism. We demonstrated that P. sojae encodes more AATs than the analysed fungi. Some of the AATs contained additional prephenate dehydratase and/or prephenate dehydrogenase domains in their N-termini, which are unique to oomycetes. Silencing of PsAAT3, an infection-inducible expression gene, reduced P. sojae pathogenicity on soybean plants and affected the growth under N-starving condition, suggesting that PsAAT3 is involved in pathogen pathogenicity and nitrogen utilisation during infection. Our results suggest that P. sojae and other oomycete pathogens may have distinct amino acid metabolism pathways and that PsAAT3 is important for its full pathogenicity. PMID:27020161

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

    PubMed

    Yang, Q; Mao, W H; Ferre, I; Bayón, J E; Mao, X Z; González-Gallego, J

    1998-07-31

    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.

  13. Elevation of alanine amino transferase and aspartate amino transferase produced by pyoverdin, a photolabile pigment of Pseudomonas fluorescens.

    PubMed

    Eraso, A J; Albesa, I

    1998-01-01

    The effect of three forms pyoverdin on mouse liver was studied. Significant increases of alanine amino transferase (ALT) and aspartate amino transferase (AST) were obtained in mice after ingestion of water with forms A and C. The effect on liver was more evident with A than with C. Pyoverdin was purified by means of salt saturation, solvent extractions and ion-exchange chromatography. Fluorescent peaks obtained in the presence of light were different from those eluted under dark conditions. The relative amounts of pyoverdin A, B and C varied when dark purification procedure was employed. Form A decreased while C increased in the absence of light. Optimum conditions for C were in the dark without iron. When C was exposed to light, it changed to form A. Fast Atom Bombardment (FAB) mass spectrometry of pyoverdin form C gave a form at M+ = 1324 m.u., which is 9 m.u. less than pyoverdin purified in the presence of light. The results suggest that light can influence pyoverdin stability and toxicity. PMID:9888631

  14. Interaction of aspartate aminotransferase with mercurochrome. Relationship of an exposed thiol group of the enzyme to the active centre.

    PubMed Central

    Kalogerakos, T G; Oikonomakos, N G; Dimitropoulos, C G; Karni-katsadima, I A; Evangelopoulos, A E

    1977-01-01

    Mercurochrome strongly inhibits aspartate transaminase and 2,3-dicarboxyethylated aspartate transaminase. The native enzyme exhibits a biphasic time-course of inactivation by mercurochrome with second-order rate constants 1.62 x 10(4) M-1 - min-1 and 2.15 x 10(3) M-1 - min-1, whereas the modified enzyme is inactivated more slowly (second-order rate constant 6.1 x 10(2) M-1 - min-1) under the same conditions. The inhibitor inactivates native and modified enzyme in the absence as well as in the presence of substrates. Mercurochrome-transaminase interaction is accompanied by a red shift in the absorption maximum of the fluorochrome of about 10 nm. Difference spectra of the mercurochrome-enzyme system versus mercurochrome, compared with analogous spectra of mercurochrome-ethanol, revealed that the spectral shifts recorded during mercurochrome-transaminase interaction are similar to those that occur when mercurochrome is dissolved in non-polar solvents. Studies of mercurochrome complexes with native or modified transaminase, isolated by chromatography on Sephadex G-25, revealed that native transaminase is able to conjugate with four mercurochrome molecules per molecule, but the modified enzyme is able to conjugate with only two mercurochrome molecules per molecule. PMID:73375

  15. The effects of the stress caused by experimental procedures on alanine aspartate, glutamate and glutamine in rat liver

    PubMed Central

    Heath, D. F.; George, D. R.; Rose, J. G.

    1971-01-01

    Rats were stressed by intravenous injection, tail-warming or moderate restraint for 30s, i.e. by stresses imposed by normal handling during experiment. Liver glutamate concentrations were greatly affected. The results were substantially the same in two varieties of rat (Wistar and Sprague–Dawley), in two laboratories, in experiments carried out by two sets of workers, and after all three stresses. The following detailed results refer to Wistar rats. 1. In starved rats at 20°C and 30°C and in post-absorptive rats at 20°C stress by injection raised liver glutamate concentrations from 1.54, 1.57 and 1.88μmol/g wet wt. 30s after injection to 3.4, 2.7 and 3.6μmol/g wet wt. respectively a few minutes later. In starved rats at 20°C the concentration then fell slowly to 2.3μmol/g wet wt., in starved rats at 30°C it remained steady, and in post-absorptive rats at 20°C it rose slowly to about 4.3μmol/g wet wt. The final values seemed fairly steady and corresponded to an `alert' state. 2. In starved rats at 20°C anaesthesia, with or without injection or cannulation during it, raised glutamate concentrations to the `alert' values, which were maintained for 2–3h. 3. Liver alanine concentration in post-absorptive rats initially fell from 1.5 to 0.8μmol/g, and then stayed fairly constant. 4. Aspartate and glutamine concentrations altered only in starved rats, and proportionately much less than those of glutamate. 5. The necessity for knowing the time-dependence of glutamate concentrations after experimental handling is emphasized. 6. There is no wholly satisfactory explanation of the observations. PMID:5145894

  16. γ-Glutamyl Transpeptidase in Men and Alanine Aminotransferase in Women are the Most Suitable Parameters Among Liver Function Tests for the Prediction of Metabolic Syndrome in Nonviral Hepatitis and Nonfatty Liver in the Elderly

    PubMed Central

    Pei, Dee; Hsia, Te-Lin; Chao, Ting-Ting; Lin, Jiunn-Diann; Hsu, Chun-Hsien; Wu, Chung-Ze; Hsieh, Chang-Hsun; Liang, Yao-Jen; Chen, Yen-Lin

    2015-01-01

    Background/Aims: Nonalchoholic fatty liver disease (NAFLD) has been reported as a hepatic manifestation of metabolic syndrome (MetS); it is common and accounts for 80% of the cases with abnormal liver function tests (LFTs). In addition, several studies have proved that there is a correlation between abnormal LFTs and MetS. Therefore, LFTs may represent the abnormal metabolic status of livers in the patients with MetS. To identify the early state of metabolic dysfunction, we investigate the value of LFTs for the future MetS development in the relatively healthy (non-NAFLD) elderly. Patients and Methods: A total of 16,912 subjects met the criteria for analysis. In the first stage of this study, subjects were enrolled in the cross-sectional study in order to find out the optimal cutoff value in different LFTs with higher chances to have MetS. In the second stage of the present study, subjects with MetS at baseline were excluded from the same study group, and a median 5.6-year longitudinal study was conducted on the rest of the group. Results: Among all LFTs, only aspartate aminotransferase in both genders and the α-fetal protein in women failed to show the significance in distinguishing subjects with MetS by the receiver operating characteristic curve. In the Kaplan–Meier plot, only γ-glutamyl transpeptidase (γ-GT) in men and the alanine aminotransferase (ALT) in women could be used to successfully separate subjects with higher risk of developing the MetS from those with lower risk. Finally, in the multivariant Cox regression model, similar results were identified. Still, the hazard ratio (HR) to have future MetS, γ-GT in men, and ALT in women showed significance (HR = 1.511 in men and 1.504 in women). Conclusion: Among all the different LFTs, γ-GT (>16 U/L) in male and ALT (>21 U/L) in female were the best predictors for the development of MetS in healthy elderly. These two liver markers could be an ancillary test in predicting future MetS development

  17. [Histochemical study of the activity of aspartate aminotransferase in the spinal cord, the medulla oblongata and the central cerebellar nuclei of several vertebrates].

    PubMed

    Garcia-Segura, L M; Martinez-Rodriguez, R; Toledano, A

    1976-01-01

    The aspartate aminotransferase activity (AAT) is reserched into the spinal cord, the medulla oblongata and the cerebellar nuclei of the rat, chicken, Lacerta lepida and Bufo calamitas. It's proved that the AAT activity shows in many locations, that are mainly: 1. In the nerve fibers 2. In the cytoplasmic membrane, and in the nuclear membrane of the neurons 3. In all neuronal cytoplasm, and 4. In the mitochondria of neurons and choroid plexus cells. The results base the idea that there's more than one pool of glutamic acid in relation to that AAT. It's suggested that the role that AAT plays is different in everyone of the described locations, and may be it's connected with transport phenomenons in the membrane, with energetic function on the mitochondria and with functions of the nerve impulse transmission in the synapsis. We remark, finally, the interest that the enzymatical works can have the time comming to establish homologies among similar structures of several animal's nervous system. PMID:1023552

  18. Prediction of the Risk of Hepatocellular Carcinoma in Chronic Hepatitis C Patients after Sustained Virological Response by Aspartate Aminotransferase to Platelet Ratio Index

    PubMed Central

    Lee, Keol; Sinn, Dong Hyun; Gwak, Geum-Youn; Cho, Hyun Chin; Jung, Sin-Ho; Paik, Yong-Han; Choi, Moon Seok; Lee, Joon Hyeok; Koh, Kwang Cheol; Paik, Seung Woon

    2016-01-01

    Background/Aims Following sustained virological response (SVR) for chronic hepatitis C (CHC) infection, patients with advanced fibrosis require regular monitoring for hepatocellular carcinoma (HCC). The aspartate aminotransferase to platelet ratio index (APRI) is a simple noninvasive surrogate marker known to reflect fibrosis. Methods We retrospectively analyzed 598 patients who achieved SVR with interferon-based therapy for CHC. Results Over a median of 5.1 years of follow-up, there were eight patients diagnosed with HCC and a 5-year cumulative incidence rate of 1.3%. The median pretreatment APRI was 0.83, which decreased to 0.29 after achieving SVR (p<0.001). Both the pre- and posttreatment indices were associated with HCC development. The 5-year cumulative HCC incidence rates were 0% and 2.8% for patients with pretreatment APRI <1.0 and ≥1.0, respectively (p=0.001) and 0.8% and 12.8% for patients with posttreatment APRI <1.0 and ≥1.0, respectively (p<0.001). Pretreatment APRI at a cutoff of 1.0 had a 100% negative predictive value until 10 years after SVR. Conclusions HCC development was observed among CHC patients who achieved SVR. The pre- and post-treatment APRI could stratify HCC risk, indicating that the APRI could be a useful marker to classify HCC risk in CHC patients who achieved SVR. However, given the small number of HCC patients, this finding warrants further validation. PMID:27114418

  19. Aspartate aminotransferase-lymphocyte ratio index and systemic immune-inflammation index predict overall survival in HBV-related hepatocellular carcinoma patients after transcatheter arterial chemoembolization.

    PubMed

    Yang, Zongguo; Zhang, Jianliang; Lu, Yunfei; Xu, Qingnian; Tang, Bozong; Wang, Qiang; Zhang, Wensi; Chen, Shishi; Lu, Lingqing; Chen, Xiaorong

    2015-12-15

    It has been suggested that lymphocytes play central roles in host antitumor immune responses and control cancer outcome. We reviewed the clinical parameters of 189 hepatocellular carcinoma (HCC) patients and investigated the prognostic significance of lymphocyte-related scores in HCC patients following transcatheter arterial chemoembolization (TACE). Survival analysis revealed that an elevated aspartate aminotransferase-lymphocyte ratio index (ALRI) > 57 and a systemic immune-inflammation index (SII) > 300 were negatively associated with overall survival in HBV-related HCC (HR = 2.181, P = 0.003 and HR = 2.453, P = 0.003; respectively). Spearman chi-square analysis showed that ALRI had a specificity of 82.4% and that SII index had a sensitivity of 71.9% for HCC overall survival. ALRI and SII had negative predictive values of 74.6% and 80%, respectively for HCC overall survival. Additionally, Barcelona Clinic Liver Cancer (BCLC) stage C patients had significantly higher ALRI and SII scores (both P < 0.0001) and poorer overall survival (HR = 3.618, P < 0.001). Additionally, HCC patients with portal vein tumor thrombosis (PVTT) had higher ALRI and SII scores (P < 0.0001 and P = 0.0059, respectively). In conclusion, as noninvasive, low cost, easily assessable and reproducible parameters, elevated ALRI and SII should be used as negative predictive factors for overall survival in HBV-related HCC in clinical practice.

  20. Diabetes-linked transcription factor HNF4α regulates metabolism of endogenous methylarginines and β-aminoisobutyric acid by controlling expression of alanine-glyoxylate aminotransferase 2

    PubMed Central

    Burdin, Dmitry V.; Kolobov, Alexey A.; Brocker, Chad; Soshnev, Alexey A.; Samusik, Nikolay; Demyanov, Anton V.; Brilloff, Silke; Jarzebska, Natalia; Martens-Lobenhoffer, Jens; Mieth, Maren; Maas, Renke; Bornstein, Stefan R.; Bode-Böger, Stefanie M.; Gonzalez, Frank; Weiss, Norbert; Rodionov, Roman N.

    2016-01-01

    Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of Agxt2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to Agxt2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in Agxt2 mRNA levels in Hepa 1–6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver Agxt2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of Agxt2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients. PMID:27752141

  1. Risk factors associated with hepatitis B or C markers or elevated alanine aminotransferase level among blood donors on a tropical island: the Guadeloupe experience.

    PubMed

    Fest, T; Viel, J F; Agis, F; Coffe, C; Dupond, J L; Hervé, P

    1992-10-01

    Donated blood is currently screened for hepatitis B surface antigen (HBsAg), antibody to hepatitis B core antigen (anti-HBc), antibody to hepatitis C virus (anti-HCV), and alanine aminotransferase (ALT) levels to prevent posttransfusion hepatitis. A prospective study of 2368 blood donors was carried out in Guadeloupe (French West Indies) with a view to determining the risk factors associated with serologic abnormalities. Blood donors included in the study had to complete a questionnaire. Statistical analysis was performed on the data thus obtained: 571 donations (24%) were positive for at least one of the four analyzed markers. The results were that 3.2 percent were positive for HBsAg, 22 percent for anti-HBc, and 0.8 percent for anti-HCV, and 1.4 percent had ALT > or = 45 IU per L. A good correlation was found between anti-HCV and elevated ALT. Transfusion history and two socioeconomic categories (working class, military personnel) were found to be risk factors. Other risk factors were lifelong residence in Guadeloupe (with risk increasing with the number of years), birthplace and current residence in the southern part of the island, and the existence of gastrointestinal discomfort unrelated to viral hepatitis (odds ratio = 2.98). The results of this study illustrate the difficulty of implementing a preventive policy against posttransfusion hepatitis in a tropical area. The unique epidemiologic situation of Guadeloupe as regards hepatitis B virus has led to more restrictive criteria for the acceptance of blood donors. PMID:1412685

  2. Comparison of measurements of canine plasma creatinine, glucose, proteins, urea, alanine aminotransferase, and alkaline phosphatase obtained with Spotchem SP 4430 and Vitros 250 analyzers.

    PubMed

    Trumel, C; Diquélou, A; Germain, C; Palanché, F; Braun, J P

    2005-12-01

    The suitability of the Spotchem 4430 benchtop biochemistry analyzer for canine blood samples was tested for creatinine, glucose, proteins, urea, alkaline phosphatases and alanine aminotransferase. Results obtained from whole blood and corresponding heparin plasma were identical except for proteins which were higher in plasma (n=10). Between series imprecision (n=10) was <5% for substrates and <10% for enzymes. Comparison of results from 100 Li-heparin samples with those measured with a Vitros 250 analyzer showed good correlation (r>0.93). The slopes of the Passing-Bablock's regression ranged from 0.90 to 1.20 and intercepts were low. The mean biases were low, except for creatinine for which the results obtained by Spotchem (Jaffe reaction) were about 20 micromol/L higher than with the Vitros (enzymatic reaction). The results of this study show that the Spotchem analyzer is suitable for use in canine whole blood or plasma when small numbers of tests are to be performed and large analyzers are not available. PMID:16054888

  3. Hepatic necroinflammation and severe liver fibrosis in patients with chronic hepatitis B with undetectable HBV DNA and persistently normal alanine aminotransferase.

    PubMed

    Alam, M M; Mahtab, M A; Akbar, S M F; Kamal, M; Rahman, S

    2014-12-01

    Both consensus and controversy remains regarding surrogacy of hepatitis B virus (HBV) deoxyribonucleic acid (DNA) and alanine aminotransferase (ALT), however, these markers are used to ascertain the extent of liver damages and to guide therapeutic options in patients with chronic hepatitis B. However, little is known about liver histology of patients with chronic hepatitis B with undetectable HBV DNA and persistently normal ALT. Thirty-five incidentally-detected patients with chronic HBV infection (assessed by expression of hepatitis B surface antigen for more than 6 months) with undetectable HBV DNA and normal serum ALT were enrolled in this study. Liver biopsy specimens were taken from all patients and the extent of hepatic necroinflammation and liver fibrosis were evaluated. Moderate degree of hepatic necroinflammation was detected in 2 of 35 patients and severe hepatic fibrosis was seen in 6 of 35 patients. Two patients with undetectable HBV DNA and sustained normal ALT had moderate hepatic necroinflammation and severe hepatic fibrosis. In spite of undetectable HBV DNA for prolonged period and persistently normal ALT, some patients with chronic hepatitis B express evidences of progressive liver diseases. Large scale studies in different races and geographical regions should be accomplished to develop insights about management of these patients. Studies about extent of liver diseases in these patients should be accomplished in Treatment recommendation and management strategies should be developed for these patients. PMID:26402972

  4. An archaeal glutamate decarboxylase homolog functions as an aspartate decarboxylase and is involved in β-alanine and coenzyme A biosynthesis.

    PubMed

    Tomita, Hiroya; Yokooji, Yuusuke; Ishibashi, Takuya; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-03-01

    β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5'-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4'-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the β-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for β-alanine biosynthesis in these organisms.

  5. Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis).

    PubMed

    Guha, Anirban; Gera, Sandeep; Sharma, Anshu

    2012-03-01

    Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis (SCM) with the aim of developing suitable diagnostic kit for SCM. Trace elements and enzyme activity in milk were estimated with Atomic absorption Spectrophotometer, GBC 932 plus and biochemical methods, respectively. Somatic cell count (SCC) was done microscopically. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. A statistically significant (p<0.01) increase in SCC, Fe, Zn, Co and LDH occurred in SCM milk containing gram positive bacterial agents only. ALP was found to be elevated in milk infected by both gram positive and negative bacteria. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×10(5) cells/ml of milk as the benchmark. Only ALP and Zn, the former being superior, were found to be suitable for diagnosis of SCM irrespective of etiological agents. LDH, Co and Fe can be introduced in the screening programs where Gram positive bacteria are omnipresent. It is recommended that both ALP and Zn be measured together in milk to diagnose buffalo SCM, irrespective of etiology. PMID:25049573

  6. Knockdown of a putative alanine aminotransferase gene affects amino acid content and flight capacity in the Colorado potato beetle Leptinotarsa decemlineata.

    PubMed

    Wan, Pin-Jun; Fu, Kai-Yun; Lü, Feng-Gong; Guo, Wen-Chao; Li, Guo-Qing

    2015-07-01

    Alanine aminotransferase (ALT) plays important physiological and biochemical roles in insect. In this study, a full-length Ldalt cDNA was cloned from Leptinotarsa decemlineata. It was ubiquitously expressed in the eggs, larvae, pupae and adults. In the adults, Ldalt mRNA was widely distributed in thorax muscles, fat body, midgut, foregut, hindgut, Malpighian tubules, ventral ganglion and epidermis, with the expression levels from the highest to the lowest. Two double-stranded RNAs (dsRNAs) (dsLdalt1 and dsLdalt2) targeting Ldalt were constructed and bacterially expressed. After adults fed on dsLdalt1- and dsLdalt2-immersed foliage for 3 day, Ldalt mRNA abundance was significantly decreased by 79.5 and 71.1 %, and ALT activities were significantly reduced by 64.5 and 67.6 %, respectively. Moreover, silencing Ldalt affected free amino acid contents. Lysine was decreased by 100.0 and 100.0 %, and arginine was reduced by 87.5 and 89.4 %, respectively, in the hemolymph from dsLdalt1- and dsLdalt2-ingested beetles, compared with control ones. In contrast, proline was increased by 88.7 and 96.4 %. Furthermore, ingestion of dsLdalt1 and dsLdalt2 significantly decreased flight speed, shortened flight duration time and flight distance. In addition, knocking down Ldalt significantly increased adult mortality. These data imply that LdALT plays important roles in amino acid metabolism and in flight in L. decemlineata.

  7. The Chaperoning Activity of Amino-oxyacetic Acid on Folding-Defective Variants of Human Alanine:Glyoxylate Aminotransferase Causing Primary Hyperoxaluria Type I.

    PubMed

    Oppici, Elisa; Montioli, Riccardo; Dindo, Mirco; Maccari, Laura; Porcari, Valentina; Lorenzetto, Antonio; Chellini, Sara; Voltattorni, Carla Borri; Cellini, Barbara

    2015-10-16

    The rare disease Primary Hyperoxaluria Type I (PH1) results from the deficit of liver peroxisomal alanine:glyoxylate aminotransferase (AGT), as a consequence of inherited mutations on the AGXT gene frequently leading to protein misfolding. Pharmacological chaperone (PC) therapy is a newly developed approach for misfolding diseases based on the use of small molecule ligands able to promote the correct folding of a mutant enzyme. In this report, we describe the interaction of amino-oxyacetic acid (AOA) with the recombinant purified form of two polymorphic species of AGT, AGT-Ma and AGT-Mi, and with three pathogenic variants bearing previously identified folding defects: G41R-Ma, G170R-Mi, and I244T-Mi. We found that for all these enzyme AOA (i) forms an oxime at the active site, (ii) behaves as a slow, tight-binding inhibitor with KI values in the nanomolar range, and (iii) increases the thermal stability. Furthermore, experiments performed in mammalian cells revealed that AOA acts as a PC by partly preventing the intracellular aggregation of G41R-Ma and by promoting the correct peroxisomal import of G170R-Mi and I244T-Mi. Based on these data, we carried out a small-scale screening campaign. We identified four AOA analogues acting as AGT inhibitors, even if only one was found to act as a PC. The possible relationship between the structure and the PC activity of these compounds is discussed. Altogether, these results provide the proof-of-principle for the feasibility of a therapy with PCs for PH1-causing variants bearing folding defects and provide the scaffold for the identification of more specific ligands. PMID:26161999

  8. Knockdown of a putative alanine aminotransferase gene affects amino acid content and flight capacity in the Colorado potato beetle Leptinotarsa decemlineata.

    PubMed

    Wan, Pin-Jun; Fu, Kai-Yun; Lü, Feng-Gong; Guo, Wen-Chao; Li, Guo-Qing

    2015-07-01

    Alanine aminotransferase (ALT) plays important physiological and biochemical roles in insect. In this study, a full-length Ldalt cDNA was cloned from Leptinotarsa decemlineata. It was ubiquitously expressed in the eggs, larvae, pupae and adults. In the adults, Ldalt mRNA was widely distributed in thorax muscles, fat body, midgut, foregut, hindgut, Malpighian tubules, ventral ganglion and epidermis, with the expression levels from the highest to the lowest. Two double-stranded RNAs (dsRNAs) (dsLdalt1 and dsLdalt2) targeting Ldalt were constructed and bacterially expressed. After adults fed on dsLdalt1- and dsLdalt2-immersed foliage for 3 day, Ldalt mRNA abundance was significantly decreased by 79.5 and 71.1 %, and ALT activities were significantly reduced by 64.5 and 67.6 %, respectively. Moreover, silencing Ldalt affected free amino acid contents. Lysine was decreased by 100.0 and 100.0 %, and arginine was reduced by 87.5 and 89.4 %, respectively, in the hemolymph from dsLdalt1- and dsLdalt2-ingested beetles, compared with control ones. In contrast, proline was increased by 88.7 and 96.4 %. Furthermore, ingestion of dsLdalt1 and dsLdalt2 significantly decreased flight speed, shortened flight duration time and flight distance. In addition, knocking down Ldalt significantly increased adult mortality. These data imply that LdALT plays important roles in amino acid metabolism and in flight in L. decemlineata. PMID:25868655

  9. Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine.

    PubMed

    Montioli, Riccardo; Oppici, Elisa; Dindo, Mirco; Roncador, Alessandro; Gotte, Giovanni; Cellini, Barbara; Borri Voltattorni, Carla

    2015-10-01

    Liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) enzyme, exists as two polymorphic forms, the major (AGT-Ma) and the minor (AGT-Mi) haplotype. Deficit of AGT causes Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive rare disease. Although ~one-third of the 79 disease-causing missense mutations segregates on AGT-Mi, only few of them are well characterized. Here for the first time the molecular and cellular defects of G47R-Mi are reported. When expressed in Escherichia coli, the recombinant purified G47R-Mi variant exhibits only a 2.5-fold reduction of its kcat, and its apo form displays a remarkably decreased PLP binding affinity, increased dimer-monomer equilibrium dissociation constant value, susceptibility to thermal denaturation and to N-terminal region proteolytic cleavage, and aggregation propensity. When stably expressed in a mammalian cell line, we found ~95% of the intact form of the variant in the insoluble fraction, and proteolyzed (within the N-terminal region) and aggregated forms both in the soluble and insoluble fractions. Moreover, the intact and nicked forms have a peroxisomal and a mitochondrial localization, respectively. Unlike what already seen for G41R-Mi, exposure of G47R-Mi expressing cells to pyridoxine (PN) remarkably increases the expression level and the specific activity in a dose-dependent manner, reroutes all the protein to peroxisomes, and rescues its functionality. Although the mechanism of the different effect of PN on the variants G47R-Mi and G41R-Mi remains elusive, the chaperoning activity of PN may be of value in the therapy of patients bearing the G47R mutation.

  10. Effect of Caffeine-Containing Beverage Consumption on Serum Alanine Aminotransferase Levels in Patients with Chronic Hepatitis C Virus Infection: A Hospital-Based Cohort Study

    PubMed Central

    Sasaki, Yachiyo; Ohfuji, Satoko; Fukushima, Wakaba; Tamori, Akihiro; Enomoto, Masaru; Habu, Daiki; Iwai, Shuji; Uchida-Kobayashi, Sawako; Fujii, Hideki; Shiomi, Susumu; Kawada, Norifumi; Hirota, Yoshio

    2013-01-01

    Introduction To date, there have been no prospective studies examining the effect of coffee consumption on serum alanine aminotransferase (ALT) level among individuals infected with the hepatitis C virus (HCV). We conducted a hospital-based cohort study among patients with chronic HCV infection to assess an association between baseline coffee consumption and subsequent ALT levels for 12 months. Materials and Methods From 1 August 2005 to 31 July 2006, total 376 HCV-RNA positive patients were recruited. A baseline questionnaire elicited information on the frequency of coffee consumption and other caffeine-containing beverages. ALT level as a study outcome was followed through the patients’ medical records during 12 months. The association between baseline beverage consumption and subsequent ALT levels was evaluated separately among patients with baseline ALT levels within normal range (≤45 IU/L) and among those with higher ALT levels (>45 IU/L). Results Among 229 patients with baseline ALT levels within normal range, 186 (81%) retained normal ALT levels at 12 months after recruitment. Daily drinkers of filtered coffee were three times more likely to preserve a normal ALT level than non-drinkers (OR=2.74; P=0.037). However, decaffeinated coffee drinkers had a somewhat inverse effect for sustained normal ALT levels, with marginal significance (OR=0.26; P=0.076). In addition, among 147 patients with higher baseline ALT levels, 39 patients (27%) had ALT reductions of ≥20 IU/L at 12 months after recruitment. Daily drinkers of filtered coffee had a significantly increased OR for ALT reduction (OR=3.79; P=0.034). However, in decaffeinated coffee drinkers, OR could not be calculated because no patients had ALT reduction. Conclusion Among patients with chronic HCV infection, daily consumption of filtered coffee may have a beneficial effect on the stabilization of ALT levels. PMID:24349501

  11. The association between non-alcoholic fatty liver disease and carotid atherosclerosis in subjects with within-reference range alanine aminotransferase levels.

    PubMed

    Kim, Kyung-Soo; Oh, Hyun-Ju; Kim, Dae-Jung; Kim, Soo-Kyung; Park, Seok Won; Cho, Yong-Wook; Huh, Kap-Bum

    2013-01-01

    Our aim was to investigate whether the evaluation of non-alcoholic fatty liver disease (NAFLD) by ultrasound provides additional benefit in assessing carotid atherosclerotic burden in subjects with alanine aminotransferase (ALT) concentrations within the reference range. This was a cross-sectional analysis of 769 healthy individuals (326 men and 443 women) with an ALT concentration ≤ 40 IU/L and alcohol consumption < 140 g/week. Mean carotid artery intima-media thickness (C-IMT) was measured using ultrasound. NAFLD was defined as a mild or greater degree of hepatic steatosis on ultrasound. Although all subjects had an ALT concentration within the reference range, the prevalence of NAFLD increased with increasing quartiles of ALT concentration (27.1%, 40.0%, 54.7%, 75.3% in men, P for trend < 0.001; 22.0%, 34.4%, 35.7%, 55.0% in women, P for trend < 0.001). In the 3rd and 4th quartiles of ALT concentration, women with NAFLD had a significantly higher C-IMT than those without NAFLD (0.671±0.019 mm vs. 0.742±0.025 mm, P=0.023 in Q3; 0.651±0.023 mm vs. 0.737±0.021 mm, P=0.005 in Q4). These differences remained significant even after adjusting for a broad spectrum of potential confounders. In contrast, although men with NAFLD tended to have a higher C-IMT than those without NAFLD in each quartile, these differences were not statistically significant. Women with an upper normal range ALT concentration showed increased C-IMT only when they had NAFLD. Therefore, in women with an elevated ALT level within the reference range, further evaluation for NAFLD, such as liver ultrasound, could potentially identify those patients at high risk for cardiovascular disease.

  12. High serum carotenoids are associated with lower risk for developing elevated serum alanine aminotransferase among Japanese subjects: the Mikkabi cohort study.

    PubMed

    Sugiura, Minoru; Nakamura, Mieko; Ogawa, Kazunori; Ikoma, Yoshinori; Yano, Masamichi

    2016-04-01

    Many recent studies have shown that antioxidant vitamins and/or carotenoids may reduce liver disease, but this association has not been well established with thorough longitudinal cohort studies. The objective of this study was to longitudinally investigate whether serum carotenoids at baseline are associated with the risk of developing elevated serum alanine aminotransferase (ALT) among Japanese subjects. We conducted a follow-up study of 1073 males and females aged between 30 and 79 years at baseline from the Mikkabi prospective cohort study. Those who participated in the baseline study and completed follow-up surveys were examined longitudinally. Exclusions included excessive alcohol consumption (≥60 g alcohol/d), hepatitis B and C and having a history of medication use for liver disease. A cohort of 213 males and 574 females free of elevated serum ALT (>30 IU/ml) at baseline was studied. Over a mean follow-up period of 7·4 (sd 3·1) years, thirty-one males and forty-nine females developed new elevated serum ALT. After adjustments for confounders, the hazard ratios for elevated serum ALT in the highest tertiles of basal serum β-carotene, β-cryptoxanthin and total provitamin A carotenoids against the lowest tertiles were 0·43 (95 % CI 0·22, 0·81), 0·51 (CI 0·27, 0·94) and 0·52 (CI 0·28, 0·97), respectively. For α-carotene and lycopene, borderline reduced risks were also observed; however, these were not significant. Our results further support the hypothesis that antioxidant carotenoids, especially provitamin A carotenoids, might help prevent earlier pathogenesis of non-alcoholic liver disease in Japanese subjects. PMID:26916997

  13. Enzymological and mutational analysis of a complex primary hyperoxaluria type I phenotype involving alanine: Glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting and intraperoxisomal aggregation

    SciTech Connect

    Danpure, C.J.; Purdue, P.E.; Allsop, J.; Lumb, M.J.; Jennings, P.R. ); Scheinman, J.I. ); Mauer, S.M. ); Davidson, N.O. )

    1993-08-01

    Primary hyperoxaluri type 1 (PH1) is a rare autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). Three unrelated PH1 patients, who possess a novel complex phenotype, are described. At the enzymological level, this phenotype is characterized by a complete, or nearly complete, absence of AGT catalytic activity and reduced AGT immunoreactivity. Unlike normal individuals in whom the AGT is confined to the peroxisomal matrix, the immunoreactive AGT in these three patients was distributed approximately equally between the peroxisomes and mitochondria. The peroxisomal AGT appeared to be aggregated into amorphous core-like structures in which no other peroxisomal enzymes could be identified. Mutational analysis of the AGT gene showed that two of the three patients were compound heterozygotes for two previously unrecognized point mutations which caused Gly41[yields]Arg and Phe152[yields]Iso amino acid substitutions. The third patient was shown to be a compound heterozygote for the Gly41[yields]Arg mutation and a previously recognized Gly170[yields]Arg mutation. All three patients were homozygous for the Pro11[yields]Leu polymorphism that had been found previously with a high allelic frequency in normal populations. It is suggested the the Phe152[yields]Iso and Gly170[yields]Arg substitutions, which are only eighteen residues apart and located in the same highly conserved internal region of 58 amino acids, might be involved in the inhibition of peroxisomal targeting and/or import of AGT and, in combination with the Pro11[yields]Leu polymorphism, be responsible for its aberrant mitochondrial compartmentalization. On the other hand, the Gly41[yields]Arg substitution, either in combination with the Pro11[yields]Leu polymorphism or by itself, is predicted to be responsible for the intraperoxisomal aggregation of the AGT protein. 50 refs., 8 figs., 4 tabs.

  14. Higher Ratio of Serum Alpha-Fetoprotein Could Predict Outcomes in Patients with Hepatitis B Virus-Associated Hepatocellular Carcinoma and Normal Alanine Aminotransferase

    PubMed Central

    Park, Joong-Won

    2016-01-01

    Background The role of serum alpha-fetoprotein (AFP) levels in the surveillance and diagnosis of hepatocellular carcinoma (HCC) is controversial. The aim of this study was to investigate the value of serially measured serum AFP levels in HCC progression or recurrence after initial treatment. Methods A total of 722 consecutive patients newly diagnosed with HCC and treated at the National Cancer Center, Korea, between January 2004 and December 2009 were enrolled. The AFP ratios between 4–8 weeks post-treatment and those at the time of HCC progression or recurrence were obtained. Multivariate logistic regression analysis was performed to correlate the post-treatment AFP ratios with the presence of HCC progression or recurrence. Results The etiology of HCC was related to chronic hepatitis B virus (HBV) infection in 562 patients (77.8%), chronic hepatitis C virus (HCV) infection in 74 (10.2%), and non-viral cause in 86 (11.9%). There was a significant decrease in serum AFP levels from the baseline to 4 to 8 weeks after treatment (median AFP, 319.6 ng/mL vs. 49.6 ng/mL; p< 0.001). Multivariate analysis showed that an AFP ratio > 1.0 was an independently associated with HCC progression or recurrence. Among the different causes of HCC analyzed, this association was significant only for HCC related to chronic hepatitis B (p< 0.001) and non-viral causes (p<0.05), and limited only to patients who had normal alanine aminotransferase (ALT) levels. Conclusion Serial measurements of serum AFP ratios could be helpful in detecting progression or recurrence in treated patients with HBV-HCC and normal ALT. PMID:27304617

  15. Inhibition of alanine:glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1

    PubMed Central

    1996-01-01

    Peroxisome-to-mitochondrion mistargeting of the homodimeric enzyme alanine:glyoxylate aminotransferase 1 (AGT) in the autosomal recessive disease primary hyperoxaluria type 1 (PH1) is associated with the combined presence of a normally occurring Pro(11)Leu polymorphism and a PH1-specific Gly170Arg mutation. The former leads to the formation of a novel NH2-terminal mitochondrial targeting sequence (MTS), which although sufficient to direct the import of in vitro-translated AGT into isolated mitochondria, requires the additional presence of the Gly170Arg mutation to function efficiently in whole cells. The role of this mutation in the mistargeting phenomenon has remained elusive. It does not interfere with the peroxisomal targeting or import of AGT. In the present study, we have investigated the role of the Gly170Arg mutation in AGT mistargeting. In addition, our studies have led us to examine the relationship between the oligomeric status of AGT and the peroxisomal and mitochondrial import processes. The results obtained show that in vitro-translated AGT rapidly forms dimers that do not readily exchange subunits. Although the presence of the Pro(11)Leu or Gly170Arg substitutions alone had no effect on dimerization, their combined presence abolished homodimerization in vitro. However, AGT containing both substitutions was still able to form heterodimers in vitro with either normal AGT or AGT containing either substitution alone. Expression of various combinations of normal and mutant, as well as epitope-tagged and untagged forms of AGT in whole cells showed that normal AGT rapidly dimerizes in the cytosol and is imported into peroxisomes as a dimer. This dimerization prevents mitochondrial import, even when the AGT possesses an MTS generated by the Pro(11)Leu substitution. The additional presence of the Gly170Arg substitution impairs dimerization sufficiently to allow mitochondrial import. Pharmacological inhibition of mitochondrial import allows AGT containing both

  16. Aspartate aminotransferase (AST) blood test

    MedlinePlus

    ... mono") Muscle disease or trauma Swollen and inflamed pancreas ( pancreatitis ) AST level may also increase after: Burns (deep) Heart procedures Seizure Surgery Pregnancy and exercise may also cause an increased AST level.

  17. Alanine-glyoxylate aminotransferase 2 (AGXT2) polymorphisms have considerable impact on methylarginine and β-aminoisobutyrate metabolism in healthy volunteers.

    PubMed

    Kittel, Anja; Müller, Fabian; König, Jörg; Mieth, Maren; Sticht, Heinrich; Zolk, Oliver; Kralj, Ana; Heinrich, Markus R; Fromm, Martin F; Maas, Renke

    2014-01-01

    Elevated plasma concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse clinical outcomes. Both methylarginines are substrates of alanine-glyoxylate aminotransferase 2 (AGXT2). It was the aim of the present study to simultaneously investigate the functional relevance and relative contributions of common AGXT2 single nucleotide polymorphisms (SNPs) to plasma and urinary concentrations of methylarginines as well as β-aminoisobutyrate (BAIB), a prototypic substrate of AGXT2. In a cohort of 400 healthy volunteers ADMA, SDMA and BAIB concentrations were determined in plasma and urine using HPLC-MS/MS and were related to the coding AGXT2 SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu). Volunteers heterozygous or homozygous for the AGXT2 SNP rs37369 had higher SDMA plasma concentrations by 5% and 20% (p = 0.002) as well as higher BAIB concentrations by 54% and 146%, respectively, in plasma and 237% and 1661%, respectively, in urine (both p<0.001). ADMA concentrations were not affected by both SNPs. A haplotype analysis revealed that the second investigated AGXT2 SNP rs16899974, which was not significantly linked to the other AGXT2 SNP, further aggravates the effect of rs37369 with respect to BAIB concentrations in plasma and urine. To investigate the impact of the amino acid exchange p.Val140Ile, we established human embryonic kidney cell lines stably overexpressing wild-type or mutant (p.Val140Ile) AGXT2 protein and assessed enzyme activity using BAIB and stable-isotope labeled [²H₆]-SDMA as substrate. In vitro, the amino acid exchange of the mutant protein resulted in a significantly lower enzyme activity compared to wild-type AGXT2 (p<0.05). In silico modeling of the SNPs indicated reduced enzyme stability and substrate binding. In conclusion, SNPs of AGXT2 affect plasma as well as urinary BAIB and SDMA concentrations linking methylarginine metabolism to the common genetic trait of hyper

  18. Performance of an Optimized Paper-Based Test for Rapid Visual Measurement of Alanine Aminotransferase (ALT) in Fingerstick and Venipuncture Samples

    PubMed Central

    Noubary, Farzad; Coonahan, Erin; Schoeplein, Ryan; Baden, Rachel; Curry, Michael; Afdhal, Nezam; Kumar, Shailendra; Pollock, Nira R.

    2015-01-01

    Background A paper-based, multiplexed, microfluidic assay has been developed to visually measure alanine aminotransferase (ALT) in a fingerstick sample, generating rapid, semi-quantitative results. Prior studies indicated a need for improved accuracy; the device was subsequently optimized using an FDA-approved automated platform (Abaxis Piccolo Xpress) as a comparator. Here, we evaluated the performance of the optimized paper test for measurement of ALT in fingerstick blood and serum, as compared to Abaxis and Roche/Hitachi platforms. To evaluate feasibility of remote results interpretation, we also compared reading cell phone camera images of completed tests to reading the device in real time. Methods 96 ambulatory patients with varied baseline ALT concentration underwent fingerstick testing using the paper device; cell phone images of completed devices were taken and texted to a blinded off-site reader. Venipuncture serum was obtained from 93/96 participants for routine clinical testing (Roche/Hitachi); subsequently, 88/93 serum samples were captured and applied to paper and Abaxis platforms. Paper test and reference standard results were compared by Bland-Altman analysis. Findings For serum, there was excellent agreement between paper test and Abaxis results, with negligible bias (+4.5 U/L). Abaxis results were systematically 8.6% lower than Roche/Hitachi results. ALT values in fingerstick samples tested on paper were systematically lower than values in paired serum tested on paper (bias -23.6 U/L) or Abaxis (bias -18.4 U/L); a correction factor was developed for the paper device to match fingerstick blood to serum. Visual reads of cell phone images closely matched reads made in real time (bias +5.5 U/L). Conclusions The paper ALT test is highly accurate for serum testing, matching the reference method against which it was optimized better than the reference methods matched each other. A systematic difference exists between ALT values in fingerstick and paired

  19. Expression, purification, crystallization, data collection and preliminary biochemical characterization of methicillin-resistant Staphylococcus aureus Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5′-phosphate-dependent aminotransferase

    SciTech Connect

    Seetharamappa, Jaldappagari; Oke, Muse; Liu, Huanting; McMahon, Stephen A.; Johnson, Kenneth A.; Carter, Lester; Dorward, Mark; Zawadzki, Michal; Overton, Ian M.; Niekirk, C. A. Johannes van; Graham, Shirley; Botting, Catherine H.; Taylor, Garry L.; White, Malcolm F.; Barton, Geoffrey J.; Coote, Peter J.; Naismith, James H.

    2007-05-01

    As part of work on S. aureus, the crystallization of Sar2028, a protein that is upregulated in MRSA, is reported. Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5′-phosphate-dependent aminotransferase with a molecular weight of 48 168 Da, was overexpressed in methicillin-resistant Staphylococcus aureus compared with a methicillin-sensitive strain. The protein was expressed in Escherichia coli, purified and crystallized. The protein crystallized in a primitive orthorhombic Laue group with unit-cell parameters a = 83.6, b = 91.3, c = 106.0 Å, α = β = γ = 90°. Analysis of the systematic absences along the three principal axes indicated the space group to be P2{sub 1}2{sub 1}2{sub 1}. A complete data set was collected to 2.5 Å resolution.

  20. Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

    PubMed Central

    Yoshikane, Yu; Yokochi, Nana; Ohnishi, Kouhei; Hayashi, Hideyuki; Yagi, Toshiharu

    2006-01-01

    Pyridoxamine–pyruvate aminotransferase is a PLP (pyridoxal 5′-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine–pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the α family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429–432]. The Kd value for pyridoxal determined by means of CD was 100-fold lower than the Km value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed. PMID:16545075

  1. Effects of pyridoxine on growth performance and plasma aminotransferases and homocysteine of white pekin ducks.

    PubMed

    Xie, Ming; Tang, Jing; Wen, Zhiguo; Huang, Wei; Hou, Shuisheng

    2014-12-01

    A dose-response experiment with seven supplemental pyridoxine levels (0, 0.66, 1.32, 1.98, 2.64, 3.30, and 3.96 mg/kg) was conducted to investigate the effects of pyridoxine on growth performance and plasma aminotransferases and homocysteine of White Pekin ducks and to estimate pyridoxine requirement for these birds. A total of 336 one-day-old male White Pekin ducks were divided to 7 experimental treatments and each treatment contained 8 replicate pens with 6 birds per pen. Ducks were reared in raised wire-floor pens from hatch to 28 d of age. At 28 d of age, the weight gain, feed intake, feed/gain, and the aspartate aminotransferase, alanine aminotransferase, and homocysteine in plasma of ducks from each pen were all measured. In our study, the pyridoxine deficiency of ducks was characterized by growth depression, decreasing plasma aspartate aminotransferase activity and increasing plasma homocysteine. The ducks fed vitamin B6-deficient basal diets had the worst weight gain and feed/gain among all birds and this growth depression was alleviated (p<0.05) when pyridoxine was supplemented to basal diets. On the other hand, plasma aspartate aminotransferase and homocysteine may be the sensitive indicators for vitamin B6 status of ducks. The ducks fed basal diets had much lower aspartate aminotransferase activity and higher homocysteine level in plasma compared with other birds fed pyridoxine-supplemented diets (p<0.05). According to quadratic regression, the supplemental pyridoxine requirements of Pekin ducks from hatch to 28 days of age was 2.44 mg/kg for feed/gain and 2.08 mg/kg for plasma aspartate aminotransferase and the corresponding total requirements of this vitamin for these two criteria were 4.37 and 4.01 mg/kg when the pyridoxine concentration of basal diets was included, respectively. All data suggested that pyridoxine deficiency could cause growth retardation in ducks and the deficiency of this vitamin could be indicated by decreasing plasma aspartate

  2. Hepatitis C virus-infected patients with a persistently normal alanine aminotransferase: do they exist and is this really a group with mild disease?

    PubMed

    Lawson, A

    2010-01-01

    Opinion varies on whether or not hepatitis C virus (HCV) infected patients with persistently normal aminotransferase (PNALT) levels represent a group with mild disease. To evaluate the risk of ALT flare and fibrosis progression in patients with PNALT followed up as part of the Trent HCV cohort. Treatment-naïve patients with an elevated ALT (n = 1140) or PNALT, the latter defined as either an ALT < or = 30 IU/L (n = 43) or an ALT < or = 40 IU/L (n = 87) on > or =2 occasions in the 6 months following diagnosis, and no ALT > 40 U/L were included. The likelihood of maintaining a PNALT < or = 30 IU/L was 42.2% and PNALT < or = 40 IU/L 41.7% at 3 years. The Ishak fibrosis score was > or =3 in 3.7%, 8.3% and 29.6% of patients with PNALT < or = 30 IU/L, PNALT < or = 40 IU/L and elevated ALT, respectively. Fibrosis progression between paired biopsies was similar for patients with PNALT < or = 30 IU/L (0.33 +/- 0.94 Ishak fibrosis points/year), PNALT < or = 40 IU/L (0.35 +/- 0.82) and elevated ALT (0.19 +/- 0.48). The majority of those defined as PNALT subsequently have an abnormal ALT. They have a similar risk of disease progression to other HCV infected patients and, therefore, warrant the same consideration with regard to treatment. PMID:19656289

  3. Evaluation of aminotransferase abnormality in dengue patients: A meta analysis.

    PubMed

    Wang, Xiao-Jun; Wei, Hai-Xia; Jiang, Shi-Chen; He, Cheng; Xu, Xiu-Juan; Peng, Hong-Juan

    2016-04-01

    Dengue virus is a type of flavivirus transmitted by Aedes mosquitoes. The symptoms of infection by this virus range from asymptomatic or mild symptomatic dengue fever (DF) to dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Significant abnormality in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) has been shown in a large number of dengue infection cases and to be indicator for liver injury provided that there are no other combined infections or liver injury. This study aims to assess the abnormal levels of liver aminotransferase in dengue patients. The related literature was searched in multiple databases, including PubMed, Embase, Google Scholar and Cochrane Library. The literature was selected through strict inclusion and exclusion criteria, and the quantitative synthesis of the liver aminotransferase abnormality was performed with R software. The fixed or random effects model was employed based on the results of the statistical test for homogeneity. In total, 15 studies were included. The proportion of AST abnormality with 95% confidence interval (95% CI) was 0.80 (95% CI: 0.56-0.92) in DHF patients and 0.75 (95% CI: 0.63-0.84) in DF patients; the proportion of ALT abnormality was 0.54 (95% CI: 0.34-0.73) in DHF patients and 0.52 (95% CI: 0.41-0.63) in DF patients. Serum ALT and AST levels may be indicators for evaluating liver injury in dengue infection and for diagnosis and treatment effect.

  4. Un-catalyzed peptide bond formation between two monomers of glycine, alanine, serine, threonine, and aspartic acid in gas phase: a density functional theory study

    NASA Astrophysics Data System (ADS)

    Bhunia, Snehasis; Singh, Ajeet; Ojha, Animesh K.

    2016-05-01

    In the present report, un-catalyzed peptide bond formation between two monomers of glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), and aspartic acid (Asp) has been investigated in gas phase via two steps reaction mechanism and concerted mechanism at B3LYP/6-31G(d,p) and M062X/6-31G(d,p) level of theories. The peptide bond is formed through a nucleophilic reaction via transition states, TS1 and TS2 in stepwise mechanism. The TS1 reveals formation of a new C-N bond while TS2 illustrate the formation of C=O bond. In case of concerted mechanism, C-N bond is formed by a single four-centre transition state (TS3). The energy barrier is used to explain the involvement of energy at each step of the reaction. The energy barrier (20-48 kcal/mol) is required for the transformation of reactant state R1 to TS1 state and intermediate state I1 to TS2 state. The large value of energy barrier is explained in terms of distortion and interaction energies for stepwise mechanism. The energy barrier of TS3 in concerted mechanism is very close to the energy barrier of the first transition state (TS1) of the stepwise mechanism for the formation of Gly-Gly and Ala-Ala di- peptide. However, in case of Ser-Ser, Thr-Thr and Asp-Asp di-peptide, the energy barrier of TS3 is relatively high than that of the energy barrier of TS1 calculated at B3LYP/6-31G(d,p) and M062X/6-31G(d,p) level of theories. In both the mechanisms, the value of energy barrier calculated at B3LYP/6-31G(d,p) level of theory is greater than that of the value calculated at M062X/6-31G(d,p) level of theory.

  5. Recurrent truncating mutations in alanine-glyoxylate aminotransferase gene in two South Indian families with primary hyperoxaluria type 1 causing later onset end-stage kidney disease

    PubMed Central

    Dutta, A. K.; Paulose, B. K.; Danda, S.; Alexander, S.; Tamilarasi, V.; Omprakash, S.

    2016-01-01

    Primary hyperoxaluria type 1 is an autosomal recessive inborn error of metabolism due to liver-specific peroxisomal enzyme alanine-glyoxylate transaminase deficiency. Here, we describe two unrelated patients who were diagnosed to have primary hyperoxaluria. Homozygous c.445_452delGTGCTGCT (p.L151Nfs*14) (Transcript ID: ENST00000307503; human genome assembly GRCh38.p2) (HGMD ID CD073567) mutation was detected in both the patients and the parents were found to be heterozygous carriers. Our patients developed end-stage renal disease at 23 years and 35 years of age. However, in the largest series published from OxalEurope cohort, the median age of end-stage renal disease for null mutations carriers was 9.9 years, which is much earlier than our cases. Our patients had slower progressions as compared to three unrelated patients from North India and Pakistan, who had homozygous c.302T>C (p.L101P) (HGMD ID CM093792) mutation in exon 2. Further, patients need to be studied to find out if c.445_452delGTGCTGCT mutation represents a founder mutation in Southern India. PMID:27512303

  6. Effects of test spills of chemically dispersed and nondispersed oil on the activity of aspartate aminotransferase and glucose-6-phosphate dehydrogenase in two intertidal bivalves, Mya arenaria and Mytilus edulis

    SciTech Connect

    Gilfillan, E.S.; Foster, J.; Gerber, R.; Hanson, S.A.; Page, D.S.; Vallas, D.

    1982-10-01

    In 1981, two test oil spills were made in Maine. One spill was 975 L (250 gal) of Murban crude oil; the other was 975 L of Murban crude oil premixed with 97 L (25 gal) of Corexit 9527. The uptake of the oil and its effects on enzymatic activity in two species of common intertidal bivalve mollusks, Mya arenaria and Mytilus edulis, were studied. Data were obtained on uptake and depuration of the oil for each species; data were also obtained on the activity of glucose-6-phosphate dehydrogenase and aspartate aminotransferase for each species. Data were collected both before and after each of the spills. Much less oil was taken up by the populations of animals exposed to chemically dispersed oil than by those exposed to nondispersed oil. Rates of depuration were the same for each species; they were also the same regardless of oil exposure. Significant long-term effects on enzyme activity were detected only in those animals exposed to nondispersed oil.

  7. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    PubMed

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  8. The peroxisome proliferator-activated receptor α agonist, AZD4619, induces alanine aminotransferase-1 gene and protein expression in human, but not in rat hepatocytes: Correlation with serum ALT levels.

    PubMed

    Thulin, Petra; Bamberg, Krister; Buler, Marcin; Dahl, Björn; Glinghammar, Björn

    2016-09-01

    Alanine aminotransferase (ALT) in serum is the standard biomarker for liver injury. We have previously described a clinical trial with a novel selective peroxisome proliferator-activated receptor α (PPARα) agonist (AZD4619), which unexpectedly caused increased serum levels of ALT in treated individuals without any other evidence of liver injury. We pinpointed a plausible mechanism through which AZD4619 could increase serum ALT levels; namely through the PPARα-specific activation of the human ALT1 gene at the transcriptional level. In the present study, we present data from the preceding rat toxicity study, demonstrating that AZD4619 had no effect on rat serum ALT activity levels, and further experiments were performed to elucidate the mechanisms responsible for this species-related difference. Our results revealed that AZD4619 increased ALT1 protein expression in a dose-dependent manner in human, but not in rat primary hepatocytes. Cloning of the human and rat ALT1 promoters into luciferase vectors confirmed that AZD4619 induced only the human, but not the rat ALT1 gene promoter in a dose-dependent manner. In PPARα-GAL4 reporter gene assays, AZD4619 was >100-fold more potent on the human vs. rat PPARα levels, explaining the differences in induction of the ALT1 gene between the species at the concentration range tested. These data demonstrate the usefulness of the human and rat ALT1 reporter gene assays for testing future drug candidates at the preclinical stage. In drug discovery projects, these assays elucidate whether elevations in ALT levels observed in vivo or in the clinic are due to metabolic effects rather than a toxic event in the liver. PMID:27430334

  9. Aspartate aminotransferase to platelet ratio index and sustained virologic response are associated with progression from hepatitis C associated liver cirrhosis to hepatocellular carcinoma after treatment with pegylated interferon plus ribavirin

    PubMed Central

    Ng, Khai-Jing; Tseng, Chih-Wei; Chang, Ting-Tsung; Tzeng, Shinn-Jia; Hsieh, Yu-Hsi; Hung, Tsung-Hsing; Huang, Hsiang-Ting; Wu, Shu-Fen; Tseng, Kuo-Chih

    2016-01-01

    Background The aim of this study was to evaluate the clinically significant predictors of hepatocellular carcinoma (HCC) development among hepatitis C virus (HCV) cirrhotic patients receiving combination therapy. Patients and methods One hundred and five compensated cirrhosis patients who received pegylated interferon plus ribavirin between January 2005 and December 2011 were enrolled. All the patients were examined with abdominal sonography and liver biochemistry at baseline, end of treatment, and every 3–6 months posttreatment. The occurrence of HCC was evaluated every 3–6 months posttreatment. Results A total of 105 patients were enrolled (mean age 58.3±10.4 years). The average follow-up time for each patient was 4.38 years (standard deviation 1.73 years; range 1.13–9.27 years). Fifteen (14.3%) patients developed HCC during follow-up period. Thirteen of them had high baseline aspartate aminotransferase to platelet ratio index (APRI) (ie, an APRI >2.0). Multivariate analysis showed that those without sustained virologic response (SVR) (hazard ratio [HR] 5.795; 95% confidence interval [CI] 1.370–24.5; P=0.017) and high APRI (HR 5.548; 95% CI 1.191–25.86; P=0.029) had a significantly higher risk of HCC occurrence. The cumulative incidence of HCC was significantly higher (P=0.009) in patients without SVR (3-year cumulative incidence 21.4%; 95% CI 7.4%–35.5%; 5-year cumulative incidence 31.1%; 95% CI 11.2%–51.1%) compared to those with SVR (3- and 5-year cumulative incidence 6.2%; 95% CI 0%–1.3%). Further, the cumulative incidence of HCC was significantly higher (P=0.006) in patients with high APRI (3-year cumulative incidence 21.8%; 95% CI 8.2%–35.3%; 5-year cumulative incidence 30.5%, 95% CI 11.8%–49.3%) compared to those with low APRI (3- and 5-year cumulative incidence 4.2%, 95% CI 0%–1.0%). Conclusion In HCV-infected cirrhotic patients who received combination therapy, APRI and SVR are the two major predictors of HCC development. PMID

  10. Simultaneous analysis of D-alanine, D-aspartic acid, and D-serine using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues.

    PubMed

    Karakawa, Sachise; Shimbo, Kazutaka; Yamada, Naoyuki; Mizukoshi, Toshimi; Miyano, Hiroshi; Mita, Masashi; Lindner, Wolfgang; Hamase, Kenji

    2015-11-10

    A highly sensitive and selective chiral LC-MS/MS method for D-alanine, D-aspartic acid and D-serine has been developed using the precolumn derivatization reagents, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag) or p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS). The thus N-tagged enantiomers of the derivatized amino acids were nicely separated within 20min using the cinchona alkaloid-based zwittterionic ion-exchange type enantioselective column, Chiralpak ZWIX(+). The selected reaction monitoring was applied for detecting the target d-amino acids in biological matrices. By using the present chiral LC-MS/MS method, the three d-amino acids and their l-forms could be simultaneously determined in the range of 0.1-500nmol/mL. Finally, the technique was successfully applied to rat plasma and tissue samples.

  11. Evolution of alanine:glyoxylate aminotransferase 1 peroxisomal and mitochondrial targeting. A survey of its subcellular distribution in the livers of various representatives of the classes Mammalia, Aves and Amphibia.

    PubMed

    Danpure, C J; Fryer, P; Jennings, P R; Allsop, J; Griffiths, S; Cunningham, A

    1994-08-01

    As part of a wider study on the molecular evolution of alanine:glyoxylate aminotransferase 1 (AGT1) intracellular compartmentalization, we have determined the subcellular distribution of immunoreactive AGT1, using postembedding protein A-gold immunoelectron microscopy, in the livers of various members of the classes Mammalia, Aves, and Amphibia. As far as organellar distribution is concerned, three categories could be distinguished. In members of the first category (type I), all, or nearly all, of the immunoreactive AGT1 was concentrated within the peroxisomes. In the second category (type II), AGT1 was found more evenly distributed in both peroxisomes and mitochondria. In the third category (type III), AGT1 was localized mainly within the mitochondria with much lower, but widely variable, amounts in the peroxisomes. Type I animals include the human, two great apes (gorilla, orangutan), two Old World monkeys (anubis baboon, Japanese macaque), a New World monkey (white-faced Saki monkey), a lago, morph (European rabbit), a bat (Seba's short-tailed fruit bat), two caviomorph rodents (guinea pig, orange-rumped agouti), and two Australian marsupials (koala, Bennett's wallaby). Type II animals include two New World monkeys (common marmoset, cotton-top tamarin), three prosimians (brown lemur, fat-tailed dwarf lemur, pygmy slow loris), five rodents (a hybrid crested porcupine, Colombian ground squirrel, laboratory rat, laboratory mouse, golden hamster), an American marsupial (grey short-tailed opossum), and a bird (raven). Type III animals include the large tree shrew, three insectivores (common Eurasian mole, European hedgehog, house shrew), four carnivores (domestic cat, ocelot, domestic dog, polecat ferret), and an amphibian (common frog). In addition to these categories, some animals (e.g. guinea pig, common frog) possessed significant amounts of cytosolic AGT1. Whereas the subcellular distribution of AGT1 in some orders (e.g. Insectivora and Carnivora) did not appear

  12. Clinicopathological features of choledocholithiasis patients with high aminotransferase levels without cholangitis

    PubMed Central

    Huh, Cheal Wung; Jang, Sung Ill; Lim, Beom Jin; Kim, Hee Wook; Kim, Jae Keun; Park, Jun Sung; Kim, Ja Kyung; Lee, Se Joon; Lee, Dong Ki

    2016-01-01

    Abstract Common bile duct (CBD) stones are generally associated with greater elevations of alkaline phosphatase and gamma-glutamyl transpeptidase levels than aspartate aminotransferase and alanine aminotransferase levels. However, some patients with CBD stones show markedly increased aminotransferase levels, sometimes leading to the misdiagnosis of liver disease. Therefore, the aim of this study was to investigate the clinicopathologic features of patients with CBD stones and high aminotransferase levels. This prospective cohort study included 882 patients diagnosed with CBD stones using endoscopic retrograde cholangiopancreatography (ERCP). Among these patients, 38 (4.3%) exhibited aminotransferase levels above 400 IU/L without cholangitis (gallstone hepatitis [GSH] group), and 116 (13.2%) exhibited normal aminotransferase levels (control group). We compared groups in terms of clinical features, laboratory test results, radiologic images, and ERCP findings such as CBD diameter, CBD stone diameter and number, and periampullary diverticulum. Liver biopsy was performed for patients in the GSH group. GSH patients were younger and more likely to have gallbladder stones than control patients, implying a higher incidence of gallbladder stone migration. Also, GSH patients experienced more severe, short-lasting abdominal pain. ERCP showed narrower CBDs in GSH patients than in control patients. Histological analysis of liver tissue from GSH patients showed no abnormalities except for mild inflammation. Compared with control patients, GSH patients were younger and showed more severe, short-lasting abdominal pain, which could be due to a sudden increase of CBD pressure resulting from the migration of gallstones through narrower CBDs. These clinical features could be helpful not only for the differential diagnosis of liver disease but also for investigating the underlying mechanisms of liver damage in obstructive jaundice. Moreover, we propose a new definition of

  13. In Vitro antioxidative activity of pumpkin seed (Cucurbita pepo) protein isolate and its In Vivo effect on alanine transaminase and aspartate transaminase in acetaminophen-induced liver injury in low protein fed rats.

    PubMed

    Nkosi, C Z; Opoku, A R; Terblanche, S E

    2006-09-01

    The antioxidative effects of pumpkin seed protein isolate (Cucurbita pepo) were investigated in vitro. The isolate exhibited about 80% radical scavenging activity, chelating activity of approximately 64% on Fe2+ ions and an inhibition of approximately 10% of xanthine oxidase. Subsequently the effects of the isolate on the plasma activity levels of alanine transaminase and aspartate transaminase against acetaminophen induced acute liver injury in low-protein fed male Sprague-Dawley rats were ascertained. The rats were maintained on a low-protein diet for 5 days and divided into three subgroups. Two subgroups were injected with acetaminophen and the other with an equivalent amount of polyethylene glycol 400. Two hours after intoxication one of the two subgroups was administered with the protein isolate. Rats from the different subgroups were killed at 24, 48 and 72 h after treatment. After 5 days on the low-protein diet the activity levels of the enzymes were significantly higher than their counterparts on a normal balanced diet. The administration of protein isolate after acetaminophen intoxication resulted in significantly reduced activity levels. It is concluded that the protein isolate has promising antioxidative properties. Furthermore, the isolate administration was effective in alleviating the detrimental effects associated with protein malnutrition and acetaminophen intoxication.

  14. The Crystal Structure of the Pseudomonas dacunhae Aspartate-[beta]-Decarboxylase Dodecamer Reveals an Unknown Oligomeric Assembly for a Pyridoxal-5′-Phosphate-Dependent Enzyme

    SciTech Connect

    Lima, Santiago; Sundararaju, Bakthavatsalam; Huang, Christina; Khristoforov, Roman; Momany, Cory; Phillips, Robert S.

    2010-09-01

    The Pseudomonas dacunhae L-aspartate-{beta}-decarboxylase (ABDC, aspartate 4-decarboxylase, aspartate 4-carboxylyase, E.C. 4.1.1.12) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the {beta}-decarboxylation of L-aspartate to produce L-alanine and CO{sub 2}. This catalytically versatile enzyme is known to form functional dodecamers at its optimal pH and is thought to work in conjunction with an L-Asp/L-Ala antiporter to establish a proton gradient across the membrane that can be used for ATP biosynthesis. We have solved the atomic structure of ABDC to 2.35 {angstrom} resolution using single-wavelength anomalous dispersion phasing. The structure reveals that ABDC oligomerizes as a homododecamer in an unknown mode among PLP-dependent enzymes and has highest structural homology with members of the PLP-dependent aspartate aminotransferase subfamily. The structure shows that the ABDC active site is very similar to that of aspartate aminotransferase. However, an additional arginine side chain (Arg37) was observed flanking the re-side of the PLP ring in the ABDC active site. The mutagenesis results show that although Arg37 is not required for activity, it appears to be involved in the ABDC catalytic cycle.

  15. Weaning Induced Hepatic Oxidative Stress, Apoptosis, and Aminotransferases through MAPK Signaling Pathways in Piglets

    PubMed Central

    Luo, Zhen; Zhu, Wei; Guo, Qi; Luo, Wenli; Zhang, Jing; Xu, Weina

    2016-01-01

    This study investigated the effects of weaning on the hepatic redox status, apoptosis, function, and the mitogen-activated protein kinase (MAPK) signaling pathways during the first week after weaning in piglets. A total of 12 litters of piglets were weaned at d 21 and divided into the weaning group (WG) and the control group (CG). Six piglets from each group were slaughtered at d 0 (d 20, referred to weaning), d 1, d 4, and d 7 after weaning. Results showed that weaning significantly increased the concentrations of hepatic free radicals H2O2 and NO, malondialdehyde (MDA), and 8-hydroxy-2′-deoxyguanosine (8-OHdG), while significantly decreasing the inhibitory hydroxyl ability (IHA) and glutathione peroxidase (GSH-Px), and altered the level of superoxide dismutase (SOD). The apoptosis results showed that weaning increased the concentrations of caspase-3, caspase-8, caspase-9 and the ratio of Bax/Bcl-2. In addition, aspartate aminotransferase transaminase (AST) and alanine aminotransferase (ALT) in liver homogenates increased after weaning. The phosphorylated JNK and ERK1/2 increased, while the activated p38 initially decreased and then increased. Our results suggested that weaning increased the hepatic oxidative stress and aminotransferases and initiated apoptosis, which may be related to the activated MAPK pathways in postweaning piglets. PMID:27807471

  16. Aspartic acid

    MedlinePlus

    ... Hormone production and release Normal nervous system function Plant sources of aspartic acid include: Legumes such as soybeans, garbanzo beans, and lentils Peanuts, almonds, walnuts, and flaxseeds Animal ...

  17. Antiretroviral Drugs and Risk of Chronic Alanine Aminotransferase Elevation in Human Immunodeficiency Virus (HIV)-Monoinfected Persons: The Data Collection on Adverse Events of Anti-HIV Drugs Study

    PubMed Central

    Kovari, Helen; Sabin, Caroline A.; Ledergerber, Bruno; Ryom, Lene; Reiss, Peter; Law, Matthew; Pradier, Christian; Dabis, Francois; d'Arminio Monforte, Antonella; Smith, Colette; de Wit, Stephane; Kirk, Ole; Lundgren, Jens D.; Weber, Rainer

    2016-01-01

    Background. Although human immunodeficiency virus (HIV)-positive persons on antiretroviral therapy (ART) frequently have chronic liver enzyme elevation (cLEE), the underlying cause is often unclear. Methods. Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) Study participants without chronic viral hepatitis were observed to the earliest of cLEE (elevated aminotransferase ≥6 months), death, last follow-up, or January 2, 2014. Antiretroviral treatment exposure was categorized as follows: no exposure and ongoing short- and long-term exposure (<2 or ≥2 years) after initiation. Association between development of cLEE and ART exposure was investigated using Poisson regression. Results. Among 21 485 participants observed for 105 413 person-years (PY), 6368 developed cLEE (incidence 6.04/100 PY; 95% confidence interval [CI], 5.89–6.19). Chronic liver enzyme elevation was associated with short-and long-term exposure to didanosine (<2 years rate ratio [RR] = 1.29, 95% CI, 1.11–1.49; >2 years RR = 1.26, 95% CI, 1.13–1.41); stavudine (<2 years RR = 1.51, 95% CI, 1.26–1.81; >2 years RR = 1.17, 95% CI, 1.03–1.32), and tenofovir disoproxil fumarate (<2 years RR = 1.55, 95% CI, 1.40–1.72; >2 years RR = 1.18, 95% CI, 1.05–1.32), but only short-term exposure to nevirapine (<2 years RR = 1.44, 95% CI, 1.29–1.61), efavirenz (<2 years RR = 1.14, 95% CI, 1.03–1.26), emtricitabine (<2 years RR = 1.18, 95% CI, 1.04–1.33), and atazanavir (<2 years RR = 1.20, 95% CI, 1.04–1.38). Chronic liver enzyme elevation was not associated with use of lamivudine, abacavir, and other protease inhibitors. Mortality did not differ between participants with and without cLEE. Conclusions. Although didanosine, stavudine, nevirapine, and efavirenz have been described to be hepatotoxic, we additionally observed a consistent association between tenofovir and cLEE emerging within the first 2 years after drug initiation. This novel tenofovir-cLEE signal should be

  18. β-alanine biosynthesis in Methanocaldococcus jannaschii.

    PubMed

    Wang, Yu; Xu, Huimin; White, Robert H

    2014-08-01

    One efficient approach to assigning function to unannotated genes is to establish the enzymes that are missing in known biosynthetic pathways. One group of such pathways is those involved in coenzyme biosynthesis. In the case of the methanogenic archaeon Methanocaldococcus jannaschii as well as most methanogens, none of the expected enzymes for the biosynthesis of the β-alanine and pantoic acid moieties required for coenzyme A are annotated. To identify the gene(s) for β-alanine biosynthesis, we have established the pathway for the formation of β-alanine in this organism after experimentally eliminating other known and proposed pathways to β-alanine from malonate semialdehyde, l-alanine, spermine, dihydrouracil, and acryloyl-coenzyme A (CoA). Our data showed that the decarboxylation of aspartate was the only source of β-alanine in cell extracts of M. jannaschii. Unlike other prokaryotes where the enzyme producing β-alanine from l-aspartate is a pyruvoyl-containing l-aspartate decarboxylase (PanD), the enzyme in M. jannaschii is a pyridoxal phosphate (PLP)-dependent l-aspartate decarboxylase encoded by MJ0050, the same enzyme that was found to decarboxylate tyrosine for methanofuran biosynthesis. A Km of ∼0.80 mM for l-aspartate with a specific activity of 0.09 μmol min(-1) mg(-1) at 70°C for the decarboxylation of l-aspartate was measured for the recombinant enzyme. The MJ0050 gene was also demonstrated to complement the Escherichia coli panD deletion mutant cells, in which panD encoding aspartate decarboxylase in E. coli had been knocked out, thus confirming the function of this gene in vivo.

  19. Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons.

    PubMed

    Dadsetan, Sherry; Bak, Lasse K; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Leke, Renata; Schousboe, Arne; Waagepetersen, Helle S

    2011-09-01

    It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5h with [U-(13)C]glucose to monitor de novo synthesis of alanine and glutamine in the absence and presence of 5.0 mM NH(4)Cl and 10 mM MSO. Ammonia exposure led to increased incorporation of label but not to a significant increase in the amount of these amino acids. However, in the presence of MSO, glutamine synthesis was blocked and synthesis of alanine increased leading to an elevated content intra- as well as extracellularly of this amino acid. Treatment with MSO led to a dramatic decrease in glutamine content and increased the intracellular contents of glutamate and aspartate. The large increase in alanine during exposure to MSO underlines the importance of the GDH and ALAT biosynthetic pathway for ammonia fixation, and it points to the use of a GS inhibitor to ameliorate the brain toxicity and edema induced by hyperammonemia, events likely related to glutamine synthesis.

  20. Glial cells transform glucose to alanine, which fuels the neurons in the honeybee retina.

    PubMed

    Tsacopoulos, M; Veuthey, A L; Saravelos, S G; Perrottet, P; Tsoupras, G

    1994-03-01

    The retina of honeybee drone is a nervous tissue with a crystal-like structure in which glial cells and photoreceptor neurons constitute two distinct metabolic compartments. The phosphorylation of glucose and its subsequent incorporation into glycogen occur in glia, whereas O2 consumption (QO2) occurs in the photoreceptors. Experimental evidence showed that glia phosphorylate glucose and supply the photoreceptors with metabolic substrates. We aimed to identify these transferred substrates. Using ion-exchange and reversed-phase HPLC and gas chromatography-mass spectrometry, we demonstrated that more than 50% of 14C(U)-glucose entering the glia is transformed to alanine by transamination of pyruvate with glutamate. In the absence of extracellular glucose, glycogen is used to make alanine; thus, its pool size in isolated retinas is maintained stable or even increased. Our model proposes that the formation of alanine occurs in the glia, thereby maintaining the redox potential of this cell and contributing to NH3 homeostasis. Alanine is released into the extracellular space and is then transported into photoreceptors using an Na(+)-dependent transport system. Purified suspensions of photoreceptors have similar alanine aminotransferase activity as glial cells and transform 14C-alanine to glutamate, aspartate, and CO2. Therefore, the alanine entering photoreceptors is transaminated to pyruvate, which in turn enters the Krebs cycle. Proline also supplies the Krebs cycle by making glutamate and, in turn, the intermediate alpha-ketoglutarate. Light stimulation caused a 200% increase of QO2 and a 50% decrease of proline and of glutamate. Also, the production of 14CO2 from 14C-proline was increased. The use of these amino acids would sustain about half of the light-induced delta QO2, the other half being sustained by glycogen via alanine formation. The use of proline meets a necessary anaplerotic function in the Krebs cycle, but implies high NH3 production. The results showed

  1. Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris).

    PubMed

    Harr, Kendal E; Allison, Kathryn; Bonde, Robert K; Murphy, David; Harvey, John W

    2008-06-01

    Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate aminotransferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 micromol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement

  2. Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris).

    PubMed

    Harr, Kendal E; Allison, Kathryn; Bonde, Robert K; Murphy, David; Harvey, John W

    2008-06-01

    Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate aminotransferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 micromol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement

  3. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci.

    PubMed

    Peralta, Guillermo Hugo; Bergamini, Carina Viviana; Hynes, Erica Rut

    2016-01-01

    Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor. PMID:27266631

  4. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci.

    PubMed

    Peralta, Guillermo Hugo; Bergamini, Carina Viviana; Hynes, Erica Rut

    2016-01-01

    Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor.

  5. Elevation of Alanine Aminotransferase Activity Occurs after Activation of the Cell-Death Signaling Initiated by Pattern-Recognition Receptors ‎but before Activation of Cytolytic Effectors in NK or CD8+ T Cells in the Liver During Acute HCV Infection

    PubMed Central

    Choi, Youkyung H.; Jin, Nancy; Kelly, Fiona; Sakthivel, SenthilKumar K.; Yu, Tianwei

    2016-01-01

    Pattern-recognition receptors (PRRs) promote host defenses against HCV infection by binding to their corresponding adapter molecules leading to the initiation of innate immune responses including cell death. We investigated the expression of PRR genes, biomarkers of liver cell-death, and T cell and NK cell activation/inhibition-related genes in liver and serum obtained from three experimentally infected chimpanzees with acute HCV infection, and analyzed the correlation between gene expression levels and clinical profiles. Our results showed that expression of hepatic RIG-I, TLR3, TLR7, 2OAS1, and CXCL10 mRNAs was upregulated as early as 7 days post-inoculation and peaked 12 to 83 days post-inoculation. All of the three HCV infected chimpanzees exhibited significant elevations of serum alanine aminotransferase (ALT) activity between 70 and 95 days after inoculation. Elevated levels of serum cytokeratin 18 (CK-18) and caspases 3 and 7 activity coincided closely with the rise of ALT activity, and were preceded by significant increases in levels of caspase 3 and caspase 7 mRNAs in the liver. Particularly we found that significant positive auto-correlations were observed between RIG-I, TLR3, CXCL10, 2OAS1, and PD-L1 mRNA and ALT activity at 3 to 12 days before the peak of ALT activity. However, we observed substantial negative auto-correlations between T cell and NK cell activation/inhibition-related genes and ALT activity at 5 to 32 days after the peak of ALT activity. Our results indicated cell death signaling is preceded by early induction of RIG-I, TLR3, 2OAS1, and CXCL10 mRNAs which leads to elevation of ALT activity and this signaling pathway occurs before the activation of NK and T cells during acute HCV infection. Our study suggests that PRRs and type I IFN response may play a critical role in development of liver cell injury related to viral clearance during acute HCV infection. PMID:27788241

  6. Correlation between HIV viral load and aminotransferases as liver damage markers in HIV infected naive patients: a concordance cross-sectional study

    PubMed Central

    Mata-Marín, José Antonio; Gaytán-Martínez, Jesús; Grados-Chavarría, Bernardo Horacio; Fuentes-Allen, José Luis; Arroyo-Anduiza, Carla Ileana; Alfaro-Mejía, Alfredo

    2009-01-01

    Abnormalities in liver function tests could be produced exclusively by direct inflammation in hepatocytes, caused by the human immunodeficiency virus (HIV). Mechanisms by which HIV causes hepatic damage are still unknown. Our aim was to determine the correlation between HIV viral load, and serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) as markers of hepatic damage in HIV naive infected patients. We performed a concordance cross-sectional study. Patients with antiviral treatment experience, hepatotoxic drugs use or co-infection were excluded. We used a Pearson's correlation coefficient to calculate the correlation between aminotransferases serum levels with HIV viral load. We enrolled 59 patients, 50 men and 9 women seen from 2006 to 2008. The mean (± SD) age of our subjects was 34.24 ± 9.5, AST 37.73 ± 29.94 IU/mL, ALT 43.34 ± 42.41 IU/mL, HIV viral load 199,243 ± 292,905 copies/mL, and CD4+ cells count 361 ± 289 cells/mm3. There was a moderately strong, positive correlation between AST serum levels and HIV viral load (r = 0.439, P < 0.001); and a weak correlation between ALT serum levels and HIV viral load (r = 0.276, P = 0.034); after adjusting the confounders in lineal regression model the correlation remained significant. Our results suggest that there is an association between HIV viral load and aminotransferases as markers of hepatic damage; we should improved recognition, diagnosis and potential therapy of hepatic damage in HIV infected patients. PMID:19878552

  7. [Evaluation of the analytic performance of blood collection tubes (BD Vacutainer SST) for the screening of anti-HIV, anti-HTLV, anti-HCV, anti-HBc, anti-CMV antibodies, and of HBs, P24 HIV antigens, and of alanine aminotransferase].

    PubMed

    Gobin, E; Desruelle, J M; Vigier, J P

    2001-02-01

    The Laboratory of Viral Diseases Immunology (Laboratoire d'Immunologie des Maladies Virales) of the Northern Region Blood Bank (Etablissement Français du Sang Nord de France) performs between 180.000 and 200.000 viral blood qualifications per year. The use of a serum gel separator evacuated tube should contribute to improve the quality of the pre-analytical phase. However, it must not impact negatively the analytical performances. We evaluated such tube within our specific environment and with the various reagents used in routine. The open study compared the BD Vacutainer plain tube (7 mL, non siliconised) with the BD Vacutainer SST tube (6 mL siliconised with serum gel separator) against the anti-HIV, anti-HTLV, anti-HCV, anti-HBc, anti-HBs, anti-CMV antibodies, the HBs, HIV P24 antigen and the alanine aminotransferase. The study objectives were to find potential gel interference; to verify the diagnostic sensitivity, reagents specificity, and reproducibility. The results analysis show: equivalent performances with the anti-HIV Ab (Anti HIV 1/2 recombinant--Biotest et Genscreen HIV 1/2--Sanofi), anti HIV WB Ab (New Lav Blot 1--Sanofi), anti-HBs Ab (Enzygnost anti-HBs micro--Behring), anti-HBc Ab (HBc Elisa Test System--Ortho), anti-CMV Ab (Enzygnost anti-CMV IgG + M--Behring) kits; lower performances with: The Vironostika HIV Uni Form II plus 0--Organon kit with a -3.5% signal decrease around the ratio R = 2.7 for positive anti-HIV Ab. The Elisa test System 3 Ag HBs-Ortho kit with an increase of the mean ratio of the negative Ag HBs samples; better performances with: the Vironostika HIV 1 Antigen--Organon kit with a +10% signal increase around the threshold ratio R = 1 for positive Ag HIV samples. This deserves further study to verify that the specificity is maintained. The HTLV Type 1 et 2 EIA--Ortho kit with +8% signal increase around the ratio R = 2 for positive anti-HTLV Ab samples without change of the specificity. The Ortho HCV 3.0 Elisa Test System and

  8. Meta-analysis of the influence of TM6SF2 E167K variant on Plasma Concentration of Aminotransferases across different Populations and Diverse Liver Phenotypes

    PubMed Central

    Sookoian, Silvia; Pirola, Carlos J.

    2016-01-01

    A nonsynonymous E167K (rs58542926 C/T) variant in TM6SF2 gene was recently associated with nonalcoholic fatty liver disease (NAFLD). We explored the association between E167K and plasma concentrations of alanine (ALT) and aspartate (AST) aminotransferases through a meta-analysis. We also estimated the strength of the effect across diverse liver phenotypes, including NAFLD and chronic viral hepatitis; fourteen studies were included. We found that ALT (p = 3.2 × 10−6, n = 94,414) and AST (p = 0007, n = 93,809) levels were significantly associated with rs58542926 in NAFLD. By contrast, rs58542926 was not associated with either ALT (p = 0.24, n = 4187) or AST (p = 0.17, n = 2678) levels in four studies on chronic hepatitis. In conclusion, the results of the pooled estimates in patients with NAFLD showed that carriers of the T allele (EK + KK), when compared with homozygous subjects for the C allele (EE genotype) have increased levels of aminotransferases; however, this increase represents –2.5 (9.8%) and 1.2 (5%) IU/L of ALT and AST respectively, which is fairly small compared with the large effect of PNPLA3- rs738409-G allele that is associated with a –28% increase in serum ALT. PMID:27278285

  9. Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris)

    USGS Publications Warehouse

    Harr, K.E.; Allison, K.; Bonde, R.K.; Murphy, D.; Harvey, J.W.

    2008-01-01

    Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate amino-transferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 ??mol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement

  10. An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes

    PubMed Central

    Richardson, Graham; Ding, Haizhen; Rocheleau, Tom; Mayhew, George; Reddy, Erin; Han, Qian; Christensen, Bruce M.; Li, Jianyong

    2010-01-01

    A major pathway of beta-alanine synthesis in insects is through the alpha-decarboxylation of aspartate, but the enzyme involved in the decarboxylation of aspartate has not been clearly defined in mosquitoes and characterized in any insect species. In this study, we expressed two putative mosquito glutamate decarboxylase-like enzymes of mosquitoes and critically analyzed their substrate specificity and biochemical properties. Our results provide clear biochemical evidence establishing that one of them is an aspartate decarboxylase and the other is a glutamate decarboxylase. The mosquito aspartate decarboxylase functions exclusively on the production of beta-alanine with no activity with glutamate. Likewise the mosquito glutamate decarboxylase is highly specific to glutamate with essentially no activity with aspartate. Although insect aspartate decarboxylase shares high sequence identity with glutamate decarboxylase, we are able to closely predict aspartate decarboxylase from glutamate decarboxylase based on the difference of their active site residues. PMID:19842059

  11. Non-enzymic beta-decarboxylation of aspartic acid.

    NASA Technical Reports Server (NTRS)

    Doctor, V. M.; Oro, J.

    1972-01-01

    Study of the mechanism of nonenzymic beta-decarboxylation of aspartic acid in the presence of metal ions and pyridoxal. The results suggest that aspartic acid is first converted to oxalacetic acid by transamination with pyridoxal which in turn is converted to pyridoxamine. This is followed by decarboxylation of oxalacetic acid to form pyruvic acid which transaminates with pyridoxamine to form alanine. The possible significance of these results to prebiotic molecular evolution is briefly discussed.

  12. Substrate Specificity and Structure of Human Aminoadipate Aminotransferase/kynurenine Aminotransferase II

    SciTech Connect

    Han,Q.; Cai, T.; Tagle, D.; Robinson, H.; Li, J.

    2008-01-01

    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to a-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested a-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with a-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  13. Substrate Specificity and Structure of Human aminoadipate aminotransferase/kynurenine aminotransferase II

    SciTech Connect

    Han, Q.; Cai, T; Tagle, D; Robinson, H; Li, J

    2009-01-01

    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to alpha-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested alpha-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with alpha-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  14. Alanine water complexes.

    PubMed

    Vaquero, Vanesa; Sanz, M Eugenia; Peña, Isabel; Mata, Santiago; Cabezas, Carlos; López, Juan C; Alonso, José L

    2014-04-10

    Two complexes of alanine with water, alanine-(H2O)n (n = 1,2), have been generated by laser ablation of the amino acid in a supersonic jet containing water vapor and characterized using Fourier transform microwave spectroscopy. In the observed complexes, water molecules bind to the carboxylic group of alanine acting as both proton donors and acceptors. In alanine-H2O, the water molecule establishes two intermolecular hydrogen bonds forming a six-membered cycle, while in alanine-(H2O)2 the two water molecules establish three hydrogen bonds forming an eight-membered ring. In both complexes, the amino acid moiety is in its neutral form and shows the conformation observed to be the most stable for the bare molecule. The microsolvation study of alanine-(H2O)n (n = 1,2) can be taken as a first step toward understanding bulk properties at a microscopic level.

  15. β-Alanine supplementation.

    PubMed

    Hoffman, Jay R; Emerson, Nadia S; Stout, Jeffrey R

    2012-01-01

    β-Alanine is rapidly developing as one of the most popular sport supplements used by strength/power athletes worldwide. The popularity of β-alanine stems from its unique ability to enhance intramuscular buffering capacity and thereby attenuating fatigue. This review will provide an overview of the physiology that underlies the mechanisms of action behind β-alanine, examine dosing schemes, and examine the studies that have been conducted on the efficacy of this supplement. In addition, the effect that β-alanine has on body mass changes or whether it can stimulate changes in aerobic capacity also will be discussed. The review also will begin to explore the potential health benefits that β-alanine may have on older adult populations. Discussion will examine the potential adverse effects associated with this supplement as well as the added benefits of combining β-alanine with creatine.

  16. The metabolism of histamine in the Drosophila optic lobe involves an ommatidial pathway: β-alanine recycles through the retina.

    PubMed

    Borycz, Janusz; Borycz, Jolanta A; Edwards, Tara N; Boulianne, Gabrielle L; Meinertzhagen, Ian A

    2012-04-15

    Flies recycle the photoreceptor neurotransmitter histamine by conjugating it to β-alanine to form β-alanyl-histamine (carcinine). The conjugation is regulated by Ebony, while Tan hydrolyses carcinine, releasing histamine and β-alanine. In Drosophila, β-alanine synthesis occurs either from uracil or from the decarboxylation of aspartate but detailed roles for the enzymes responsible remain unclear. Immunohistochemically detected β-alanine is present throughout the fly's entire brain, and is enhanced in the retina especially in the pseudocone, pigment and photoreceptor cells of the ommatidia. HPLC determinations reveal 10.7 ng of β-alanine in the wild-type head, roughly five times more than histamine. When wild-type flies drink uracil their head β-alanine increases more than after drinking l-aspartic acid, indicating the effectiveness of the uracil pathway. Mutants of black, which lack aspartate decarboxylase, cannot synthesize β-alanine from l-aspartate but can still synthesize it efficiently from uracil. Our findings demonstrate a novel function for pigment cells, which not only screen ommatidia from stray light but also store and transport β-alanine and carcinine. This role is consistent with a β-alanine-dependent histamine recycling pathway occurring not only in the photoreceptor terminals in the lamina neuropile, where carcinine occurs in marginal glia, but vertically via a long pathway that involves the retina. The lamina's marginal glia are also a hub involved in the storage and/or disposal of carcinine and β-alanine.

  17. Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzyme.

    PubMed

    Jomrit, Juntratip; Summpunn, Pijug; Meevootisom, Vithaya; Wiyakrutta, Suthep

    2011-02-25

    A sensitive non-radioactive method for determination of the stereospecificity of the C-4' hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in (2)H(2)O with a substrate amino acid resulted in PMP labeled with deuterium at C-4' in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The [4'-(2)H]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The (2)H at C-4' is retained with the PLP if the aminotransferase in question transfers C-4' hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the (2)H is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC-MS/MS for the presence or absence of (2)H. The method was highly sensitive that for the aminotransferase with ca. 50 kDa subunit molecular weight, only 2mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay.

  18. Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzyme

    SciTech Connect

    Jomrit, Juntratip; Summpunn, Pijug; Meevootisom, Vithaya; Wiyakrutta, Suthep

    2011-02-25

    Research highlights: {yields} Stereochemical mechanism of PLP enzymes is important but difficult to determine. {yields} This new method is significantly less complicated than the previous ones. {yields} This assay is as sensitive as the radioactive based method. {yields} LC-MS/MS positively identify the analyte coenzyme. {yields} The method can be used with enzyme whose apo form is unstable. -- Abstract: A sensitive non-radioactive method for determination of the stereospecificity of the C-4' hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in {sup 2}H{sub 2}O with a substrate amino acid resulted in PMP labeled with deuterium at C-4' in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The [4'-{sup 2}H]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The {sup 2}H at C-4' is retained with the PLP if the aminotransferase in question transfers C-4' hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the {sup 2}H is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC-MS/MS for the presence or absence of {sup 2}H. The method was highly sensitive that for the aminotransferase with ca. 50 kDa subunit molecular weight, only 2 mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay.

  19. Role of the Aspartate Transaminase and Platelet Ratio Index in Assessing Hepatic Fibrosis and Liver Inflammation in Adolescent Patients with HBeAg-Positive Chronic Hepatitis B.

    PubMed

    Zhijian, Yu; Hui, Li; Weiming, Yao; Zhanzhou, Lin; Zhong, Chen; Jinxin, Zheng; Hongyan, Wang; Xiangbin, Deng; Weizhi, Yang; Duoyun, Li; Xiaojun, Liu; Qiwen, Deng

    2015-01-01

    This study described an index of aspartate aminotransferase-to-platelet ratio index (APRI) to assess hepatic fibrosis with limited expense and widespread availability compared to the liver biopsy in adolescent patients with CHB. PMID:26236336

  20. An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis.

    PubMed

    Birsoy, Kıvanç; Wang, Tim; Chen, Walter W; Freinkman, Elizaveta; Abu-Remaileh, Monther; Sabatini, David M

    2015-07-30

    The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation. PMID:26232224

  1. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis

    PubMed Central

    Birsoy, Kıvanç; Wang, Tim; Chen, Walter; Freinkman, Elizaveta; Abu-Remaileh, Monther; Sabatini, David M.

    2015-01-01

    Summary The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation. PMID:26232224

  2. Critical aspartic acid residues in pseudouridine synthases.

    PubMed

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  3. [The effect of diet ratio of polyunsaturated fatty acids of omega-3 and omega-6 families on activity of aminotransferases and gamma-glutamyltransferase in rat blood serum].

    PubMed

    Ketsa, O V; Marchenko, M M

    2014-01-01

    The effect of diet fat compositions with various ratio of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) on alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) activities in blood serum of 45 white mongrel rats weighing 90-110 g (9 animals in group) has been investigated. Fat components in the semi-synthetic diet, compiled on the basis of AIN-93 diet, and sources of omega-6 and omega-3 PUFA were presented by sunflower oil, soybean oil and fish oil. It has been shown that four-week inclusion of linoleic acid (LA) and alpha-linolenic acid (alpha-LNA) in a ratio of 7:1 into the diet (soybean oil) as well as use of only omega-6 PUFA (sunflower oil) has lead to an increase in the activity of ALT and GGT in rat blood serum compared to control animals treated with the complex of linolenic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid through the mixture of sunflower oil and fish oil (9:1) with the ratio of omega-6 and omega-3 PUFA 7:1. Along with this, the AST:ALT ratio (de Ritis ratio) was lower (p < 0.05) as compared with the control group of rat, amounting respectively 0.92 +/- 0.08 and 0.79 +/- 0.12 vs 1.26 +/- 0.10. The use of high doses of omega-3 fatty acids (600 mg EPA and 400 mg DHA per kg of animal weight per day coming through fish oil) did not affect the activity of ALT and GGT, but increased AST serum activity (0.47 +/- 0.04 micromoles/min per mg protein) and the de Ritis ratio (2.53 +/- 0.23). The diet deprived with fat increased enzyme activity of ALT, AST and GGT in rat blood serum.

  4. Structure Expression and Function of kynurenine Aminotransferases in Human and Rodent Brains

    SciTech Connect

    Q Han; T Cai; D Tagle; J Li

    2011-12-31

    Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), an endogenous antagonist of N-methyl-D: -aspartate and alpha 7-nicotinic acetylcholine receptors. Abnormal KYNA levels in human brains are implicated in the pathophysiology of schizophrenia, Alzheimer's disease, and other neurological disorders. Four KATs have been reported in mammalian brains, KAT I/glutamine transaminase K/cysteine conjugate beta-lyase 1, KAT II/aminoadipate aminotransferase, KAT III/cysteine conjugate beta-lyase 2, and KAT IV/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase. KAT II has a striking tertiary structure in N-terminal part and forms a new subgroup in fold type I aminotransferases, which has been classified as subgroup Iepsilon. Knowledge regarding KATs is vast and complex; therefore, this review is focused on recent important progress of their gene characterization, physiological and biochemical function, and structural properties. The biochemical differences of four KATs, specific enzyme activity assays, and the structural insights into the mechanism of catalysis and inhibition of these enzymes are discussed.

  5. Effect of L-ornithine L-aspartate on Liver Injury Due to Acute Ethyl Alcohol Intoxication in Rats

    PubMed Central

    Durgun, HM; Ozhasenekler, A; Dursun, R; Basarali, MK; Turkcu, G; Orak, M; Ustundag, M; Guloglu, C

    2015-01-01

    ABSTRACT Objective: Ethyl alcohol is a substance that is widely used worldwide and known to exert toxic effects on liver. In this study, we aimed to examine the effect of L-ornithine L-aspartate (LOLA) on the toxicity of a single dose of ethyl alcohol in rats. Subjects and Method: We used 32 randomly selected male Sprague-Dawley rats weighing 200–250 g. The rats were grouped into four groups with each group containing eight rats: Group 1: the control group, Group 2: the ethyl alcohol group, Group 3: the LOLA group and Group 4: the ethyl alcohol+LOLA group. Ethyl alcohol was administered orally through a nasogastric tube at a dose of 6 g/kg after diluting with distilled water. One hour after ethyl alcohol administration, LOLA was administered to pre-specified groups orally through a nasogastric tube at a dose of 200 mg/kg after diluting with distilled water. Liver tissue and blood samples were obtained from all rats 24 hours later to study total antioxidant capacity (TAC), total oxidant status (TOS) and oxidative stress index (OSI) levels in liver samples, and aspartate aminotransferase (AST), alanine transferase (ALT), TAC, TOS and OSI levels in blood samples. Results: Serum TAC, TOS and OSI levels were higher in the groups that were administered ethyl alcohol. In addition, tissue TAC level was higher and TOS and OSI levels were lower in groups that were given ethyl alcohol. No significant changes were observed in serum and tissue TAC, TOS, OSI, ALT and AST levels in the LOLA administered groups. Conclusion: This study showed that LOLA was not biochemically effective and exerts no oxidative stress reducing activity in liver injury due to acute ethyl alcohol toxicity. PMID:26426168

  6. Aspartate carbamoyltransferase from rat liver

    PubMed Central

    Bresnick, E.; Mossé, Helena

    1966-01-01

    1. Aspartate-carbamoyltransferase activity was concentrated from rat-liver preparations. Only l-aspartate, β-benzyl-l-aspartate and β-erythro-hydroxy-dl-aspartate were carbamoylated enzymically. The Km for l-aspartate and carbamoyl phosphate have been determined by three methods: colorimetric procedure, radioactive assay with [14C]aspartate and an assay with [14C]carbamoyl phosphate. 2. The Km for aspartate has been determined as a function of the pH; the pK of the functional group at the active site of the enzyme, pKe, was at pH9·0. Enzymic activity was diminished in the presence of N-ethylmaleimide, p-hydroxymercuribenzoate and the heavy metals Ag+, Hg2+, or Zn2+. The inhibitions could be prevented by mercaptoethanol. These findings suggested the association of a thiol group with the enzymic activity. 3. Enzymic activity was also decreased by sodium lauryl sulphate, urea and dioxan. Competitive inhibition (with l-aspartate) was manifested by maleate, succinate, oxaloacetate, β-erythro-hydroxy-dl-aspartate and β-benzyl-l-aspartate. The Ki for most of these inhibitions has been determined. 4. The properties of the liver enzyme are compared with those of Escherichia coli aspartate carbamoyltransferase and the implications of the findings are discussed. PMID:5339547

  7. Structural and functional characterization of aspartate racemase from the acidothermophilic archaeon Picrophilus torridus.

    PubMed

    Aihara, Takayuki; Ito, Toshiya; Yamanaka, Yasuaki; Noguchi, Keiichi; Odaka, Masafumi; Sekine, Masae; Homma, Hiroshi; Yohda, Masafumi

    2016-07-01

    Functional and structural characterizations of pyridoxal 5'-phosphate-independent aspartate racemase of the acidothermophilic archaeon Picrophilus torridus were performed. Picrophilus aspartate racemase exhibited high substrate specificity to aspartic acid. The optimal reaction temperature was 60 °C, which is almost the same as the optimal growth temperature. Reflecting the low pH in the cytosol, the optimal reaction pH of Picrophilus aspartate racemase was approximately 5.5. However, the activity at the putative cytosolic pH of 4.6 was approximately 6 times lower than that at the optimal pH of 5.5. The crystal structure of Picrophilus aspartate racemase was almost the same as that of other pyridoxal 5'-phosphate -independent aspartate racemases. In two molecules of the dimer, one molecule contained a tartaric acid molecule in the catalytic site; the structure of the other molecule was relatively flexible. Finally, we examined the intracellular existence of D-amino acids. Unexpectedly, the proportion of D-aspartate to total aspartate was not very high. In contrast, both D-proline and D-alanine were observed. Because Picrophilus aspartate racemase is highly specific to aspartate, other amino acid racemases might exist in Picrophilus torridus. PMID:27094682

  8. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    SciTech Connect

    Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A.

    2013-04-01

    The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.

  9. Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode.

    PubMed Central

    Blankenfeldt, W.; Nowicki, C.; Montemartini-Kalisz, M.; Kalisz, H. M.; Hecht, H. J.

    1999-01-01

    The crystal structure of tyrosine aminotransferase (TAT) from the parasitic protozoan Trypanosoma cruzi, which belongs to the aminotransferase subfamily Igamma, has been determined at 2.5 A resolution with the R-value R = 15.1%. T. cruzi TAT shares less than 15% sequence identity with aminotransferases of subfamily Ialpha but shows only two larger topological differences to the aspartate aminotransferases (AspATs). First, TAT contains a loop protruding from the enzyme surface in the larger cofactor-binding domain, where the AspATs have a kinked alpha-helix. Second, in the smaller substrate-binding domain, TAT has a four-stranded antiparallel beta-sheet instead of the two-stranded beta-sheet in the AspATs. The position of the aromatic ring of the pyridoxal-5'-phosphate cofactor is very similar to the AspATs but the phosphate group, in contrast, is closer to the substrate-binding site with one of its oxygen atoms pointing toward the substrate. Differences in substrate specificities of T. cruzi TAT and subfamily Ialpha aminotransferases can be attributed by modeling of substrate complexes mainly to this different position of the cofactor-phosphate group. Absence of the arginine, which in the AspATs fixes the substrate side-chain carboxylate group by a salt bridge, contributes to the inability of T. cruzi TAT to transaminate acidic amino acids. The preference of TAT for tyrosine is probably related to the ability of Asn17 in TAT to form a hydrogen bond to the tyrosine side-chain hydroxyl group. PMID:10595543

  10. The narrow substrate specificity of human tyrosine aminotransferase--the enzyme deficient in tyrosinemia type II.

    PubMed

    Sivaraman, Sharada; Kirsch, Jack F

    2006-05-01

    Human tyrosine aminotransferase (hTATase) is the pyridoxal phosphate-dependent enzyme that catalyzes the reversible transamination of tyrosine to p-hydrophenylpyruvate, an important step in tyrosine metabolism. hTATase deficiency is implicated in the rare metabolic disorder, tyrosinemia type II. This enzyme is a member of the poorly characterized Igamma subfamily of the family I aminotransferases. The full length and truncated forms of recombinant hTATase were expressed in Escherichia coli, and purified to homogeneity. The pH-dependent titration of wild-type reveals a spectrum characteristic of family I aminotransferases with an aldimine pK(a) of 7.22. I249A mutant hTATase exhibits an unusual spectrum with a similar aldimine pK(a) (6.85). hTATase has very narrow substrate specificity with the highest enzymatic activity for the Tyr/alpha-ketoglutarate substrate pair, which gives a steady state k(cat) value of 83 s(-1). In contrast there is no detectable transamination of aspartate or other cosubstrates. The present findings show that hTATase is the only known aminotransferase that discriminates significantly between Tyr and Phe: the k(cat)/K(m) value for Tyr is about four orders of magnitude greater than that for Phe. A comparison of substrate specificities of representative Ialpha and Igamma aminotransferases is described along with the physiological significance of the discrimination between Tyr and Phe by hTATase as applied to the understanding of the molecular basis of phenylketonuria.

  11. Selected Cytokines Serve as Potential Biomarkers for Predicting Liver Inflammation and Fibrosis in Chronic Hepatitis B Patients With Normal to Mildly Elevated Aminotransferases

    PubMed Central

    Deng, Yong-Qiong; Zhao, Hong; Ma, An-Lin; Zhou, Ji-Yuan; Xie, Shi-Bin; Zhang, Xu-Qing; Zhang, Da-Zhi; Xie, Qing; Zhang, Guo; Shang, Jia; Cheng, Jun; Zhao, Wei-Feng; Zou, Zhi-Qiang; Zhang, Ming-Xiang; Wang, Gui-Qiang

    2015-01-01

    Abstract Previous studies of small cohorts have implicated several circulating cytokines with progression of chronic hepatitis B (CHB). However, to date there have been no reliable biomarkers for assessing histological liver damage in CHB patients with normal or mildly elevated alanine aminotransferase (ALT). The aim of the present study was to investigate the association between circulating cytokines and histological liver damage in a large cohort. Also, this study was designed to assess the utility of circulating cytokines in diagnosing liver inflammation and fibrosis in CHB patients with ALT less than 2 times the upper limit of normal range (ULN). A total of 227 CHB patients were prospectively enrolled. All patients underwent liver biopsy and staging by Ishak system. Patients with at least moderate inflammation showed significantly higher levels of CXCL-11, CXCL-10, and interleukin (IL)-2 receptor (R) than patients with less than moderate inflammation (P < 0.001). Patients with significant fibrosis had higher levels of IL-8 (P = 0.027), transforming growth factor alpha (TGF-α) (P = 0.011), IL-2R (P = 0.002), and CXCL-11 (P = 0.032) than the group without significant fibrosis. In addition, 31.8% and 29.1% of 151 patients with ALT < 2 × ULN had at least moderate inflammation and significant fibrosis, respectively. Multivariate analysis demonstrated that CXCL-11 was independently associated with at least moderate inflammation, and TGF-α and IL-2R independently correlated with significant fibrosis in patients with ALT < 2 × ULN. Based on certain cytokines and clinical parameters, an inflammation-index and fib-index were developed, which showed areas under the receiver operating characteristics curve (AUROC) of 0.75 (95% CI 0.66–0.84) for at least moderate inflammation and 0.82 (95% CI 0.75–0.90) for significant fibrosis, correspondingly. Compared to existing scores, fib-index was significantly superior to aspartate

  12. Selected Cytokines Serve as Potential Biomarkers for Predicting Liver Inflammation and Fibrosis in Chronic Hepatitis B Patients With Normal to Mildly Elevated Aminotransferases.

    PubMed

    Deng, Yong-Qiong; Zhao, Hong; Ma, An-Lin; Zhou, Ji-Yuan; Xie, Shi-Bin; Zhang, Xu-Qing; Zhang, Da-Zhi; Xie, Qing; Zhang, Guo; Shang, Jia; Cheng, Jun; Zhao, Wei-Feng; Zou, Zhi-Qiang; Zhang, Ming-Xiang; Wang, Gui-Qiang

    2015-11-01

    Previous studies of small cohorts have implicated several circulating cytokines with progression of chronic hepatitis B (CHB). However, to date there have been no reliable biomarkers for assessing histological liver damage in CHB patients with normal or mildly elevated alanine aminotransferase (ALT). The aim of the present study was to investigate the association between circulating cytokines and histological liver damage in a large cohort. Also, this study was designed to assess the utility of circulating cytokines in diagnosing liver inflammation and fibrosis in CHB patients with ALT less than 2 times the upper limit of normal range (ULN). A total of 227 CHB patients were prospectively enrolled. All patients underwent liver biopsy and staging by Ishak system. Patients with at least moderate inflammation showed significantly higher levels of CXCL-11, CXCL-10, and interleukin (IL)-2 receptor (R) than patients with less than moderate inflammation (P < 0.001). Patients with significant fibrosis had higher levels of IL-8 (P = 0.027), transforming growth factor alpha (TGF-α) (P = 0.011), IL-2R (P = 0.002), and CXCL-11 (P = 0.032) than the group without significant fibrosis. In addition, 31.8% and 29.1% of 151 patients with ALT < 2 × ULN had at least moderate inflammation and significant fibrosis, respectively. Multivariate analysis demonstrated that CXCL-11 was independently associated with at least moderate inflammation, and TGF-α and IL-2R independently correlated with significant fibrosis in patients with ALT < 2 × ULN. Based on certain cytokines and clinical parameters, an inflammation-index and fib-index were developed, which showed areas under the receiver operating characteristics curve (AUROC) of 0.75 (95% CI 0.66-0.84) for at least moderate inflammation and 0.82 (95% CI 0.75-0.90) for significant fibrosis, correspondingly. Compared to existing scores, fib-index was significantly superior to aspartate aminotransferase

  13. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    PubMed Central

    Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A.

    2013-01-01

    The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-­aminotransferases. PMID:23519665

  14. Value of two noninvasive methods to detect progression of fibrosis among HCV carriers with normal aminotransferases.

    PubMed

    Colletta, Cosimo; Smirne, Carlo; Fabris, Carlo; Toniutto, Pierluigi; Rapetti, Rachele; Minisini, Rosalba; Pirisi, Mario

    2005-10-01

    The course of hepatitis C virus (HCV) infection carriers with normal/near-normal aminotransferases (NALT) is usually mild; however, in a few, fibrosis progression occurs. We aimed to verify whether monitoring by liver biopsy might be replaced by noninvasive methods and to identify factors associated with fibrosis progression in patients with persistently normal alanine aminotransferases. We studied 40 untreated HCV-RNA-positive subjects (22 male; median age, 44 years), who underwent two liver biopsies, with a median interval of 78.5 months, during which alanine aminotransferase concentrations (median number of determinations: 12) never exceeded 1.2 times the upper normal limit. Within 9 months from the second biopsy, they were tested by the shear elasticity probe (Fibroscan) and the artificial intelligence algorithm FibroTest. METAVIR fibrosis scores were analyzed in relationship to demographic, clinical, and viral parameters. Weighted kappa analysis was used to verify whether the results of noninvasive methods agreed with histology. Significant fibrosis (> or = F2), present at the first biopsy in only one patient (2.5%), was observed at the second biopsy in 14 patients (35%). At multivariate analysis, excess alcohol consumption in the past (>20 g/d; P = .017) and viral load (>8.0 x 10(6) copies/mL; P = .021) were independent predictors of progression. In identifying patients with significant fibrosis, inter-rater agreement was excellent for Fibroscan (weighted kappa = 1.0), and poor for FibroTest (weighted kappa = -0.041). In conclusion, among HCV carriers with NALT, Fibroscan is superior to the FibroTest in the noninvasive identification of fibrosis, for which excess alcohol consumption in the past and high viral load represent risk factors.

  15. Uptake and metabolism of (14C)-aspartate by developing kernels of maize (Zea mays L. )

    SciTech Connect

    Muhitch, M.J. )

    1990-05-01

    Pulse-chase experiments were performed to determine the metabolic fate of (14C)-aspartate in the pedicel region and subsequent uptake into the endosperm. Kernels were removed from the cob, leaving the pedicel attached but removing glumes, palea, and lemma. The basal tips were incubated in (14C)-aspartate for 0.5 h, followed by a 2 h chase period with unlabeled aspartate. In contrast to a previous study in which 70% of the 14C from aspartate was recovered in the organic acid fraction (Lyznik, et al., Phytochemistry 24: 425, 1985), only 20 to 25% of the radioactivity found in the 2 h chase period. While a small amount of the 14C transiently appeared in alanine at the beginning of the chase period, the most heavily labeled non-fed amino acid was glutamine, which accounted for 21% of the radioactivity within the pedicel amino acid fraction by 0.5 h into the chase period. There was no evidence for asparagine synthesis within the pedicel region of the kernel. 14C recovered from the endosperm in the form of amino acids were aspartate (60%), glutamine (20%), glutamate (15%), and alanine (5%). These results suggest that some of the maternally supplied amino acids undergo metabolic conversion to other amino acids before being taken up by the endosperm.

  16. Bilirubin Test

    MedlinePlus

    ... test in conjunction with other laboratory tests ( alkaline phosphatase , aspartate aminotransferase , alanine aminotransferase ) when someone shows signs ... Gilbert syndrome, due to low levels of the enzyme that produces conjugated bilirubin If conjugated (direct) bilirubin ...

  17. Structural characterization of AtmS13, a putative sugar aminotransferase involved in indolocarbazole AT2433 aminopentose biosynthesis

    PubMed Central

    Singh, Shanteri; Kim, Youngchang; Wang, Fengbin; Bigelow, Lance; Endres, Michael; Kharel, Madan K.; Babnigg, Gyorgy; Bingman, Craig A.; Joachimiak, Andrzej; Thorson, Jon S.; Phillips, George N.

    2015-01-01

    AT2433 from Actinomadura melliaura is an indolocarbazole antitumor antibiotic structurally distinguished by its unique aminodideoxypentose-containing disaccharide moiety. The corresponding sugar nucleotide-based biosynthetic pathway for this unusual sugar derives from comparative genomics where AtmS13 has been suggested as the contributing sugar aminotransferase (SAT). Determination of the AtmS13 X-ray structure at 1.50 Å resolution reveals it as a member of the aspartate aminotransferase fold type I (AAT-I). Structural comparisons of AtmS13 with homologous SATs that act upon similar substrates implicate potential active site residues that contribute to distinctions in sugar C5 (hexose versus pentose) and/or sugar C2 (deoxy versus hydroxyl) substrate specificity. PMID:26061967

  18. Phosphoserine aminotransferase in soybean root nodules : demonstration and localization.

    PubMed

    Reynolds, P H; Blevins, D G

    1986-05-01

    Phosphoserine aminotransferase activity was detected in the plant and bacteroid fractions from soybean (Glycine max) root nodules. Both total and specific activities increased in the plant fraction during nodule development. Serine-pyruvate aminotransferase activity was not detectable in the plant or bacteroid fractions of these nodules. Sucrose density gradient fractionation indicated a proplastid localization for phosphoserine aminotransferase. The data presented support a role for this enzyme in carbon supply to purine biosynthesis in the pathway of ureide biogenesis in soybean nodules.

  19. Ornithine aminotransferase vs. GABA aminotransferase. Implications for the design of new anticancer drugs

    PubMed Central

    Lee, Hyunbeom; Juncosa, Jose I.; Silverman, Richard B.

    2015-01-01

    Ornithine aminotransferase (OAT) and γ-aminobutyric acid aminotransferase (GABA-AT) are classified under the same evolutionary subgroup and share a large portion of structural, functional, and mechanistic features. Therefore, it is not surprising that many molecules that bind to GABA-AT also bind well to OAT. Unlike GABA-AT, OAT had not been viewed as a potential therapeutic target until recently; consequently, the number of therapeutically viable molecules that target OAT is very limited. In this review the two enzymes are compared with respect to their active site structures, catalytic and inactivation mechanisms, and selective inhibitors. Insight is offered that could aid in the design and development of new selective inhibitors of OAT for the treatment of cancer. PMID:25145640

  20. Aspartate protects Lactobacillus casei against acid stress.

    PubMed

    Wu, Chongde; Zhang, Juan; Du, Guocheng; Chen, Jian

    2013-05-01

    The aim of this study was to investigate the effect of aspartate on the acid tolerance of L. casei. Acid stress induced the accumulation of intracellular aspartate in L. casei, and the acid-resistant mutant exhibited 32.5 % higher amount of aspartate than that of the parental strain at pH 4.3. Exogenous aspartate improved the growth performance and acid tolerance of Lactobacillus casei during acid stress. When cultivated in the presence of 50 mM aspartate, the biomass of cells increased 65.8 % compared with the control (without aspartate addition). In addition, cells grown at pH 4.3 with aspartate addition were challenged at pH 3.3 for 3 h, and the survival rate increased 42.26-fold. Analysis of the physiological data showed that the aspartate-supplemented cells exhibited higher intracellular pH (pHi), intracellular NH4 (+) content, H(+)-ATPase activity, and intracellular ATP pool. In addition, higher contents of intermediates involved in glycolysis and tricarboxylic acid cycle were observed in cells in the presence of aspartate. The increased contents of many amino acids including aspartate, arginine, leucine, isoleucine, and valine in aspartate-added cells may contribute to the regulation of pHi. Transcriptional analysis showed that the expression of argG and argH increased during acid stress, and the addition of aspartate induced 1.46- and 3.06-fold higher expressions of argG and argH, respectively, compared with the control. Results presented in this manuscript suggested that aspartate may protect L. casei against acid stress, and it may be used as a potential protectant during the production of probiotics. PMID:23292549

  1. Molecular cloning and enzymological characterization of pyridoxal 5'-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473.

    PubMed

    Washio, Tsubasa; Kato, Shiro; Oikawa, Tadao

    2016-09-01

    We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5'-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on L-cysteic acid and L-cysteine sulfinic acid in addition to D- and L-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. D-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells. PMID:27438592

  2. Effects of Glutamate and Aspartate on Serum Antioxidative Enzyme, Sex Hormones, and Genital Inflammation in Boars Challenged with Hydrogen Peroxide

    PubMed Central

    Ni, Hengjia; Lu, Lu; Deng, Jinpin; Fan, Wenjun

    2016-01-01

    Background. Oxidative stress is associated with infertility. This study was conducted to determine the effects of glutamate and aspartate on serum antioxidative enzymes, sex hormones, and genital inflammation in boars suffering from oxidative stress. Methods. Boars were randomly divided into 4 groups: the nonchallenged control (CON) and H2O2-challenged control (BD) groups were fed a basal diet supplemented with 2% alanine; the other two groups were fed the basal diet supplemented with 2% glutamate (GLU) or 2% aspartate (ASP). The BD, GLU, and ASP groups were injected with hydrogen peroxide (H2O2) on day 15. The CON group was injected with 0.9% sodium chloride solution on the same day. Results. Dietary aspartate decreased the malondialdehyde (MDA) level in serum (P < 0.05) compared with the BD group. Additionally, aspartate maintained serum luteinizing hormone (LH) at a relatively stable level. Moreover, glutamate and aspartate increased transforming growth factor-β1 (TGF-β1) and interleukin-10 (IL-10) levels in the epididymis and testis (P < 0.05) compared with the BD group. Conclusion. Both glutamate and aspartate promoted genital mRNA expressions of anti-inflammatory factors after oxidative stress. Aspartate more effectively decreased serum MDA and prevented fluctuations in serum sex hormones after H2O2 challenge than did glutamate. PMID:27777497

  3. Kynurenine Aminotransferase Isozyme Inhibitors: A Review

    PubMed Central

    Nematollahi, Alireza; Sun, Guanchen; Jayawickrama, Gayan S.; Church, W. Bret

    2016-01-01

    Kynurenine aminotransferase isozymes (KATs 1–4) are members of the pyridoxal-5’-phosphate (PLP)-dependent enzyme family, which catalyse the permanent conversion of l-kynurenine (l-KYN) to kynurenic acid (KYNA), a known neuroactive agent. As KATs are found in the mammalian brain and have key roles in the kynurenine pathway, involved in different categories of central nervous system (CNS) diseases, the KATs are prominent targets in the quest to treat neurodegenerative and cognitive impairment disorders. Recent studies suggest that inhibiting these enzymes would produce effects beneficial to patients with these conditions, as abnormally high levels of KYNA are observed. KAT-1 and KAT-3 share the highest sequence similarity of the isozymes in this family, and their active site pockets are also similar. Importantly, KAT-2 has the major role of kynurenic acid production (70%) in the human brain, and it is considered therefore that suitable inhibition of this isozyme would be most effective in managing major aspects of CNS diseases. Human KAT-2 inhibitors have been developed, but the most potent of them, chosen for further investigations, did not proceed in clinical studies due to the cross toxicity caused by their irreversible interaction with PLP, the required cofactor of the KAT isozymes, and any other PLP-dependent enzymes. As a consequence of the possibility of extensive undesirable adverse effects, it is also important to pursue KAT inhibitors that reversibly inhibit KATs and to include a strategy that seeks compounds likely to achieve substantial interaction with regions of the active site other than the PLP. The main purpose of this treatise is to review the recent developments with the inhibitors of KAT isozymes. This treatise also includes analyses of their crystallographic structures in complex with this enzyme family, which provides further insight for researchers in this and related studies. PMID:27314340

  4. Aspartate-90 and arginine-269 of hamster aspartate transcarbamylase affect the oligomeric state of a chimaeric protein with an Escherichia coli maltose-binding domain.

    PubMed Central

    Qiu, Y; Davidson, J N

    1998-01-01

    Residues Asp-90 and Arg-269 of Escherichia coli aspartate transcarbamylase seem to interact at the interface of adjacent catalytic subunits. Alanine substitutions at the analogous positions in the hamster aspartate transcarbamylase of a chimaeric protein carrying an E. coli maltose-binding domain lead to changes in both the kinetics of the enzyme and the quaternary structure of the protein. The Vmax for the Asp-90-->Ala and Arg-269-->Ala substitutions is decreased to 1/21 and 1/50 respectively, the [S]0.5 for aspartate is increased 540-fold and 826-fold respectively, and the [S]0.5 for carbamoyl phosphate is increased 60-fold for both. These substitutions decrease the oligomeric size of the protein. Whereas the native chimaeric protein behaves as a pentamer, the Asp-90 variant is a trimer and the Arg-269 variant is a dimer. The altered enzymes also exhibit marked decreases in thermal stability and are inactivated at much lower concentrations of urea than is the unaltered enzyme. Taken together, these results are consistent with the hypothesis that both Asp-90 and Arg-269 have a role in the enzymic function and structural integrity of hamster aspartate transcarbamylase. PMID:9425105

  5. Vibrational dynamics of crystalline L-alanine

    SciTech Connect

    Bordallo, H.N.; Eckert, J.; Barthes, M.

    1997-11-01

    The authors report a new, complete vibrational analysis of L-alanine and L-alanine-d{sub 4} which utilizes IINS intensities in addition to frequency information. The use of both isotopomers resulted in a self-consistent force field for and assignment of the molecular vibrations in L-alanine. Some details of the calculation as well as a comparison of calculated and observed IINS spectra are presented. The study clarifies a number of important issues on the vibrational dynamics of this molecule and presents a self-consistent force field for the molecular vibrations in crystalline L-alanine.

  6. Catalytic Stereoinversion of L-Alanine to Deuterated D-Alanine.

    PubMed

    Moozeh, Kimia; So, Soon Mog; Chin, Jik

    2015-08-01

    A combination of an achiral pyridoxal analogue and a chiral base has been developed for catalytic deuteration of L-alanine with inversion of stereochemistry to give deuterated D-alanine under mild conditions (neutral pD and 25 °C) without the use of any protecting groups. This system can also be used for catalytic deuteration of D-alanine with retention of stereochemistry to give deuterated D-alanine. Thus a racemic mixture of alanine can be catalytically deuterated to give an enantiomeric excess of deuterated D-alanine. While catalytic deracemization of alanine is forbidden by the second law of thermodynamics, this system can be used for catalytic deracemization of alanine with deuteration. Such green and biomimetic approach to catalytic stereocontrol provides insights into efficient amino acid transformations.

  7. Activity of the lactate-alanine shuttle is independent of glutamate-glutamine cycle activity in cerebellar neuronal-astrocytic cultures.

    PubMed

    Bak, Lasse K; Sickmann, Helle M; Schousboe, Arne; Waagepetersen, Helle S

    The glutamate-glutamine cycle describes the neuronal release of glutamate into the synaptic cleft, astrocytic uptake, and conversion into glutamine, followed by release for use as a neuronal glutamate precursor. This only explains the fate of the carbon atoms, however, and not that of the ammonia. Recently, a role for alanine has been proposed in transfer of ammonia between glutamatergic neurons and astrocytes, denoted the lactate-alanine shuttle (Waagepetersen et al. [ 2000] J. Neurochem. 75:471-479). The role of alanine in this context has been studied further using cerebellar neuronal cultures and corresponding neuronal-astrocytic cocultures. A superfusion paradigm was used to induce repetitively vesicular glutamate release by N-methyl-D-aspartate (NMDA) in the neurons, allowing the relative activity dependency of the lactate-alanine shuttle to be assessed. [(15)N]Alanine (0.2 mM), [2-(15)N]/[5-(15)N]glutamine (0.25 mM), and [(15)N]ammonia (0.3 mM) were used as precursors and cell extracts were analyzed by mass spectrometry. Labeling from [(15)N]alanine in glutamine, aspartate, and glutamate in cerebellar cocultures was independent of depolarization of the neurons. Employing glutamine with the amino group labeled ([2-(15)N]glutamine) as the precursor, an activity-dependent increase in the labeling of both glutamate and aspartate (but not alanine) was observed in the cerebellar neurons. When the amide group of glutamine was labeled ([5-(15)N]glutamine), no labeling could be detected in the analyzed metabolites. Altogether, the results of this study support the existence of the lactate-alanine shuttle and the associated glutamate-glutamine cycle. No direct coupling of the two shuttles was observed, however, and only the glutamate-glutamine cycle seemed activity dependent.

  8. Soluble Serum CD81 Is Elevated in Patients with Chronic Hepatitis C and Correlates with Alanine Aminotransferase Serum Activity

    PubMed Central

    Welker, Martin-Walter; Reichert, David; Susser, Simone; Sarrazin, Christoph; Martinez, Yolanda; Herrmann, Eva; Zeuzem, Stefan; Piiper, Albrecht; Kronenberger, Bernd

    2012-01-01

    Aim Cellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81 in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the exosomal serum fraction is associated with HCV replication and inflammatory activity. Patients and Methods Four cohorts were investigated, patients with chronic hepatitis C (n = 37), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with long term sustained virologic response (SVR, n = 7), and healthy volunteers (n = 23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry. Results Soluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p = 0.03) and cured patients (p = 0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR had similar soluble CD81 levels as healthy controls (p>0.2). Overall, soluble CD81 levels were associated with ALT levels (r = 0.334, p = 0.016) and severe liver fibrosis (p = 0.027). Conclusion CD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be associated with inflammatory activity and severity of fibrosis. PMID:22355327

  9. Functional Characterization of Alanine Racemase from Schizosaccharomyces pombe: a Eucaryotic Counterpart to Bacterial Alanine Racemase

    PubMed Central

    Uo, Takuma; Yoshimura, Tohru; Tanaka, Naotaka; Takegawa, Kaoru; Esaki, Nobuyoshi

    2001-01-01

    Schizosaccharomyces pombe has an open reading frame, which we named alr1+, encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1+ gene in Escherichia coli and purified the gene product (Alr1p), with an Mr of 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent Km and Vmax values as follows: for l-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for d-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but l-serine and l-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of l-alanine, respectively. S. pombe uses d-alanine as a sole nitrogen source, but deletion of the alr1+ gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for l-alanine coupled with racemization plays a major role in the catabolism of d-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses l-alanine but not d-alanine as a sole nitrogen source. Moreover, d-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1+ gene enabled S. cerevisiae to grow efficiently on d-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of d-alanine. PMID:11244061

  10. Contribution of cysteine aminotransferase and mercaptopyruvate sulfurtransferase to hydrogen sulfide production in peripheral neurons.

    PubMed

    Miyamoto, Ryo; Otsuguro, Ken-Ichi; Yamaguchi, Soichiro; Ito, Shigeo

    2014-07-01

    Hydrogen sulfide (H2 S) is a gaseous neuromodulator produced from L-cysteine. H2 S is generated by three distinct enzymatic pathways mediated by cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT). This study investigated the relative contributions of these three pathways to H2 S production in PC12 cells (rat pheochromocytoma-derived cells) and the rat dorsal root ganglion. CBS, CAT, and MPST, but not CSE, were expressed in the cells and tissues, and appreciable amounts of H2 S were produced from L-cysteine in the presence of α-ketoglutarate, together with dithiothreitol. The production of H2 S was inhibited by a CAT inhibitor (aminooxyacetic acid), competitive CAT substrates (L-aspartate and oxaloacetate), and RNA interference (RNAi) against MPST. Immunocytochemistry revealed a mitochondrial localization of MPST in PC12 cells and dorsal root ganglion neurons, and the amount of H2 S produced by CAT/MPST at pH 8.0, a physiological mitochondrial matrix pH, was comparable to that produced by CSE and CBS in the liver and the brain, respectively. Furthermore, H2 S production was markedly increased by alkalization. These results indicate that CAT and MPST are primarily responsible for H2 S production in peripheral neurons, and that the regulation of mitochondrial metabolism may influence neuronal H2 S generation. In the peripheral nervous system, hydrogen sulfide (H2 S) has been implicated in neurogenic pain or hyperalgesia. This study provides evidence that H2 S is synthesized in peripheral neurons through two mitochondrial enzymes, cysteine aminotransferase (CAT) and mercaptopyruvate sulfurtransferase (MPST). We propose that mitochondrial metabolism plays key roles in the physiology and pathophysiology of the peripheral nervous system via regulation of neuronal H2 S production. PMID:24611772

  11. Ingesting a preworkout supplement containing caffeine, creatine, β-alanine, amino acids, and B vitamins for 28 days is both safe and efficacious in recreationally active men.

    PubMed

    Kendall, Kristina L; Moon, Jordan R; Fairman, Ciaran M; Spradley, Brandon D; Tai, Chih-Yin; Falcone, Paul H; Carson, Laura R; Mosman, Matt M; Joy, Jordan M; Kim, Michael P; Serrano, Eric R; Esposito, Enrico N

    2014-05-01

    The purpose of this study was to determine the safety and efficacy of consuming a preworkout supplement (SUP) containing caffeine, creatine, β-alanine, amino acids, and B vitamins for 28 days. We hypothesized that little to no changes in kidney and liver clinical blood markers or resting heart rate and blood pressure (BP) would be observed. In addition, we hypothesized that body composition and performance would improve in recreationally active males after 28 days of supplementation. In a double-blind, placebo-controlled study, participants were randomly assigned to ingest one scoop of either the SUP or placebo every day for 28 days, either 20 minutes before exercise or ad libitum on nonexercise days. Resting heart rate and BP, body composition, and fasting blood samples were collected before and after supplementation. Aerobic capacity as well as muscular strength and endurance were also measured. Significant (P < .05) main effects for time were observed for resting heart rate (presupplementation, 67.59 ± 7.90 beats per minute; postsupplementation, 66.18 ± 7.63 beats per minute), systolic BP (presupplementation, 122.41 ± 11.25 mm Hg; postsupplementation, 118.35 ± 11.58 mm Hg), blood urea nitrogen (presupplementation, 13.12 ± 2.55 mg/dL; postsupplementation, 15.24 ± 4.47 mg/dL), aspartate aminotransferase (presupplementation, 34.29 ± 16.48 IU/L; postsupplementation, 24.76 ± 4.71 IU/L), and alanine aminotransferase (presupplementation, 32.76 ± 19.72 IU/L; postsupplementation, 24.88 ± 9.68 IU/L). Significant main effects for time were observed for body fat percentage (presupplementation, 15.55% ± 5.79%; postsupplementation, 14.21% ± 5.38%; P = .004) and fat-free mass (presupplementation, 70.80 ± 9.21 kg; postsupplementation, 71.98 ± 9.27 kg; P = .006). A significant decrease in maximal oxygen consumption (presupplementation, 47.28 ± 2.69 mL/kg per minute; postsupplementation, 45.60 ± 2.81 mL/kg per minute) and a significant increase in percentage of

  12. Ingesting a preworkout supplement containing caffeine, creatine, β-alanine, amino acids, and B vitamins for 28 days is both safe and efficacious in recreationally active men.

    PubMed

    Kendall, Kristina L; Moon, Jordan R; Fairman, Ciaran M; Spradley, Brandon D; Tai, Chih-Yin; Falcone, Paul H; Carson, Laura R; Mosman, Matt M; Joy, Jordan M; Kim, Michael P; Serrano, Eric R; Esposito, Enrico N

    2014-05-01

    The purpose of this study was to determine the safety and efficacy of consuming a preworkout supplement (SUP) containing caffeine, creatine, β-alanine, amino acids, and B vitamins for 28 days. We hypothesized that little to no changes in kidney and liver clinical blood markers or resting heart rate and blood pressure (BP) would be observed. In addition, we hypothesized that body composition and performance would improve in recreationally active males after 28 days of supplementation. In a double-blind, placebo-controlled study, participants were randomly assigned to ingest one scoop of either the SUP or placebo every day for 28 days, either 20 minutes before exercise or ad libitum on nonexercise days. Resting heart rate and BP, body composition, and fasting blood samples were collected before and after supplementation. Aerobic capacity as well as muscular strength and endurance were also measured. Significant (P < .05) main effects for time were observed for resting heart rate (presupplementation, 67.59 ± 7.90 beats per minute; postsupplementation, 66.18 ± 7.63 beats per minute), systolic BP (presupplementation, 122.41 ± 11.25 mm Hg; postsupplementation, 118.35 ± 11.58 mm Hg), blood urea nitrogen (presupplementation, 13.12 ± 2.55 mg/dL; postsupplementation, 15.24 ± 4.47 mg/dL), aspartate aminotransferase (presupplementation, 34.29 ± 16.48 IU/L; postsupplementation, 24.76 ± 4.71 IU/L), and alanine aminotransferase (presupplementation, 32.76 ± 19.72 IU/L; postsupplementation, 24.88 ± 9.68 IU/L). Significant main effects for time were observed for body fat percentage (presupplementation, 15.55% ± 5.79%; postsupplementation, 14.21% ± 5.38%; P = .004) and fat-free mass (presupplementation, 70.80 ± 9.21 kg; postsupplementation, 71.98 ± 9.27 kg; P = .006). A significant decrease in maximal oxygen consumption (presupplementation, 47.28 ± 2.69 mL/kg per minute; postsupplementation, 45.60 ± 2.81 mL/kg per minute) and a significant increase in percentage of

  13. Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

    PubMed

    Pinto, John T; Krasnikov, Boris F; Alcutt, Steven; Jones, Melanie E; Dorai, Thambi; Villar, Maria T; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J L

    2014-11-01

    Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites. PMID:25231977

  14. Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine*

    PubMed Central

    Pinto, John T.; Krasnikov, Boris F.; Alcutt, Steven; Jones, Melanie E.; Dorai, Thambi; Villar, Maria T.; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J. L.

    2014-01-01

    Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites. PMID:25231977

  15. Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

    PubMed

    Pinto, John T; Krasnikov, Boris F; Alcutt, Steven; Jones, Melanie E; Dorai, Thambi; Villar, Maria T; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J L

    2014-11-01

    Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites.

  16. Tyrosine aminotransferase activity in the benzene intoxicated rat

    SciTech Connect

    Bong, M.; Michalska, A.; Laskowska-Klita, T.; Szymczyk, T.

    1985-01-01

    The toxic effect of hydrocarbon solvents on hepatic metabolism manifests itself by changes in the enzymatic pattern of blood serum. Changes in the activity of phosphatases as well as leucine aminopeptidase, glutamine aminotransferase, sorbitol dehydrogenase and ..gamma..-glutamyltransferase were observed in rats intoxicated with different fractions of benzene. Therefore it seemed reasonable to investigate the effect of benzene fraction of petroleum on cellular metabolism. The results of the present work concern the activity of tyrosine aminotransferase, the enzyme involved in catabolism of aromatic amino acid which is known to be under both hormonal and stress dependent control. Changes in tyrosine aminotransferase activity effect the level of tyrosine oxidation as well as the metabolic conversion of this amino acid into tyramine, tyroxin, adrenaline and noradrenaline.

  17. Alanine increases blood pressure during hypotension

    NASA Technical Reports Server (NTRS)

    Conlay, L. A.; Maher, T. J.; Wurtman, R. J.

    1990-01-01

    The effect of L-alanine administration on blood pressure (BP) during haemorrhagic shock was investigated using anesthetized rats whose left carotid arteries were cannulated for BP measurement, blood removal, and drug administration. It was found that L-alanine, in doses of 10, 25, 50, 100, and 200 mg/kg, increased the systolic BP of hypotensive rats by 38 to 80 percent (while 100 mg/kg pyruvate increased BP by only 9.4 mmhg, not significantly different from saline). The results suggest that L-alanine might influence cardiovascular function.

  18. Structural analysis and mutant growth properties reveal distinctive enzymatic and cellular roles for the three major L-alanine transaminases of Escherichia coli.

    PubMed

    Peña-Soler, Esther; Fernandez, Francisco J; López-Estepa, Miguel; Garces, Fernando; Richardson, Andrew J; Quintana, Juan F; Rudd, Kenneth E; Coll, Miquel; Vega, M Cristina

    2014-01-01

    In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5'-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.

  19. Oral administration of D-alanine in monkeys robustly increases plasma and cerebrospinal fluid levels but experimental D-amino acid oxidase inhibitors had minimal effect.

    PubMed

    Rojas, Camilo; Alt, Jesse; Ator, Nancy A; Wilmoth, Heather; Rais, Rana; Hin, Niyada; DeVivo, Michael; Popiolek, Michael; Tsukamoto, Takashi; Slusher, Barbara S

    2016-09-01

    Hypofunction of the N-methyl-d-aspartate (NMDA) receptor is thought to exacerbate psychosis in patients diagnosed with schizophrenia. Consistent with this hypothesis, D-alanine, a co-agonist at the glycine site of the NMDA receptor, was shown to improve positive and cognitive symptoms when used as add-on therapy for schizophrenia treatment. However, D-alanine had to be administered at high doses (~7 g) to observe clinical effects. One possible reason for the high dose is that D-alanine could be undergoing oxidation by D-amino acid oxidase (DAAO) before it reaches the brain. If this is the case, the dose could be reduced by co-administration of D-alanine with a DAAO inhibitor (DAAOi). Early studies with rodents showed that co-administration of D-alanine with 5-chloro-benzo[d]isoxazol-3-ol (CBIO), a prototype DAAOi, significantly enhanced the levels of extracellular D-alanine in the frontal cortex compared with D-alanine alone. Further, the use of CBIO reduced the dose of D-alanine needed to attenuate prepulse inhibition deficits induced by dizocilpine. The objective of the work reported herein was to confirm the hypothesis that DAAO inhibition can enhance D-alanine exposure in a species closer to humans: non-human primates. We report that while oral D-alanine administration to baboons (10 mg/kg) enhanced D-alanine plasma and CSF levels over 20-fold versus endogenous levels, addition of experimental DAAOi to the regimen exhibited a 2.2-fold enhancement in plasma and no measurable effect on CSF levels. The results provide caution regarding the utility of DAAO inhibition to increase D-amino acid levels as treatment for patients with schizophrenia. PMID:27287825

  20. 1.2 Å resolution crystal structure of the periplasmic aminotransferase PvdN from Pseudomonas aeruginosa.

    PubMed

    Drake, Eric J; Gulick, Andrew M

    2016-05-01

    The Gram-negative pathogen Pseudomonas aeruginosa uses a nonribosomal peptide synthetase (NRPS) biosynthetic cluster for the production of a peptide siderophore. In addition to four multimodular NRPS proteins, the biosynthetic pathway also requires several additional enzymes involved in the production of nonproteinogenic amino acids and maturation of the peptide product. Among the proteins that are required for the final steps in pyoverdine synthesis is PvdN, a pyridoxal phosphate-dependent enzyme that catalyzes an uncharacterized step in pyoverdine production. This study reports the high-resolution structure of PvdN bound to a PLP cofactor solved by multi-wavelength anomalous dispersion (MAD). The PvdN model shows high structural homology to type I aspartate aminotransferases and also contains positive density that suggests an uncharacterized external aldimine. PMID:27139833

  1. Solved? The reductive radiation chemistry of alanine.

    PubMed

    Pauwels, Ewald; De Cooman, Hendrik; Waroquier, Michel; Hole, Eli O; Sagstuen, Einar

    2014-02-14

    The structural changes throughout the entire reductive radiation-induced pathway of l-α-alanine are solved on an atomistic level with the aid of periodic DFT and nudged elastic band (NEB) simulations. This yields unprecedented information on the conformational changes taking place, including the protonation state of the carboxyl group in the "unstable" and "stable" alanine radicals and the internal transformation converting these two radical variants at temperatures above 220 K. The structures of all stable radicals were verified by calculating EPR properties and comparing those with experimental data. The variation of the energy throughout the full radiochemical process provides crucial insight into the reason why these structural changes and rearrangements occur. Starting from electron capture, the excess electron quickly localizes on the carbon of a carboxyl group, which pyramidalizes and receives a proton from the amino group of a neighboring alanine molecule, forming a first stable radical species (up to 150 K). In the temperature interval 150-220 K, this radical deaminates and deprotonates at the carboxyl group, the detached amino group undergoes inversion and its methyl group sustains an internal rotation. This yields the so-called "unstable alanine radical". Above 220 K, triggered by the attachment of an additional proton on the detached amino group, the radical then undergoes an internal rotation in the reverse direction, giving rise to the "stable alanine radical", which is the final stage in the reductive radiation-induced decay of alanine.

  2. β-Alanine supplementation and military performance.

    PubMed

    Hoffman, Jay R; Stout, Jeffrey R; Harris, Roger C; Moran, Daniel S

    2015-12-01

    During sustained high-intensity military training or simulated combat exercises, significant decreases in physical performance measures are often seen. The use of dietary supplements is becoming increasingly popular among military personnel, with more than half of the US soldiers deployed or garrisoned reported to using dietary supplements. β-Alanine is a popular supplement used primarily by strength and power athletes to enhance performance, as well as training aimed at improving muscle growth, strength and power. However, there is limited research examining the efficacy of β-alanine in soldiers conducting operationally relevant tasks. The gains brought about by β-alanine use by selected competitive athletes appears to be relevant also for certain physiological demands common to military personnel during part of their training program. Medical and health personnel within the military are expected to extrapolate and implement relevant knowledge and doctrine from research performed on other population groups. The evidence supporting the use of β-alanine in competitive and recreational athletic populations suggests that similar benefits would also be observed among tactical athletes. However, recent studies in military personnel have provided direct evidence supporting the use of β-alanine supplementation for enhancing combat-specific performance. This appears to be most relevant for high-intensity activities lasting 60-300 s. Further, limited evidence has recently been presented suggesting that β-alanine supplementation may enhance cognitive function and promote resiliency during highly stressful situations.

  3. 21 CFR 172.540 - DL-Alanine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false DL-Alanine. 172.540 Section 172.540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD ADDITIVES....540 DL-Alanine. DL-Alanine (a racemic mixture of D- and L-alanine; CAS Reg. No. 302-72-7) may...

  4. Secreted fungal aspartic proteases: A review.

    PubMed

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application. PMID:27137097

  5. Secreted fungal aspartic proteases: A review.

    PubMed

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application.

  6. Dataset of cocoa aspartic protease cleavage sites.

    PubMed

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-09-01

    The data provide information in support of the research article, "The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors" (Janek et al., 2016) [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. PMID:27508221

  7. A multinuclear NMR relaxation study of the interaction of divalent metal ions with L-aspartic acid.

    PubMed

    Khazaeli, S; Viola, R E

    1984-09-01

    Carbon-13 spin-lattice relaxation times, T1, have been measured for aqueous solutions of L-aspartic acid, L-alanine, O-phospho-L-serine, and 2-mercapto-L-succinic acid in the presence of the paramagnetic metal ions, Cu2+ and Mn2+, and Mg2+ as a diamagnetic control, at ambient temperature and neutral pH. Nitrogen-15, oxygen-17 and proton relaxation times were also obtained for L-aspartic acid and phosphorus-31 relaxation times for O-phospho-L-serine under similar conditions. The structures of these complexes in solution were determined from the various metal ion-nuclei distances calculated from the paramagnetically-induced relaxation. These results indicate that the Cu2+ interaction with L-aspartic acid is through alpha-amino and beta-carboxyl groups while Mn2+ coordinates most strongly through alpha- and beta-carboxyl groups, with the possibility of a weak interaction through the amino group. An examination of the coordination of these divalent metal ions to an analog of L-aspartic acid in which the beta-carboxyl group is replaced by a phosphate group (O-phospho-L-serine) indicated that Cu2+ coordination is now probably through the alpha-amino and phosphate groups, while this analog is a monodentate ligand for Mn2+ coordinating through the phosphate group. Removal of the beta-carboxyl group (L-alanine) also results in Cu2+ coordination through the alpha-carboxyl and alpha-amino groups, and the same ligand interactions are observed with Mn2+. Replacement of the alpha-amino group of L-aspartic acid with an -SH group (2-mercapto-L-succinate) is sufficient to eliminate any specific coordination with either Cu2+ or Mn2+. PMID:6491655

  8. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases.

    PubMed

    Liu, Pingyang; Torrens-Spence, Michael P; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2013-02-01

    Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development. PMID:22718265

  9. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases.

    PubMed

    Liu, Pingyang; Torrens-Spence, Michael P; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2013-02-01

    Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development.

  10. Alanine racemase from the acidophile Acetobacter aceti.

    PubMed

    Francois, Julie A; Kappock, T Joseph

    2007-01-01

    Acetobacter aceti converts ethanol to acetic acid, and survives acetic acid exposure by tolerating cytoplasmic acidification. Alanine racemase (Alr) is a pyridoxal 5' phosphate (PLP) -dependent enzyme that catalyzes the interconversion of the d- and l-isomers of alanine and has a basic pH optimum. Since d-alanine is essential for peptidoglycan biosynthesis, Alr must somehow function in the acidic cytoplasm of A. aceti. We report the partial purification of native A. aceti Alr (AaAlr) and evidence that it is a rather stable enzyme. The C-terminus of AaAlr has a strong resemblance to the ssrA-encoded protein degradation signal, which thwarted initial protein expression experiments. High-activity AaAlr forms lacking a protease recognition sequence were expressed in Escherichia coli and purified. Biophysical and enzymological experiments confirm that AaAlr is intrinsically acid-resistant, yet has the catalytic properties of an ordinary Alr.

  11. Molecular cloning of human ornithine aminotransferase mRNA

    SciTech Connect

    Inana, G.; Totsuka, S.; Redmond, M.; Dougherty, T.; Nagle, J.; Shiono, T.; Ohura, T. Kominami, E.; Katunuma, N.

    1986-03-01

    The isolation and characterization of a cDNA clone for the mRNA of human ornithine aminotransferase (OATase; ornithine-oxo-acid aminotransferase; L-ornithine:2-oxo-acid aminotransferase, EC 2.6.1.13), a nonabundant mitochondrial matrix enzyme that is severely deficient in a hereditary chorioretinal degenerative disease (gyrate atrophy), is described. Human liver, retina, and retinoblastoma (Y79) mRNAs were prepared and tested for the OATase mRNA content by in vitro translation, immunoprecipitation, and NaDodSO/sub 4//PAGE. The retinoblastoma cells were found to be expressing this enzyme at a relatively high level. The primary translation product of the OATase mRNA is larger than the pure OATase protein on NaDodSO/sub 4//PAGE. lambdagt11 cDNA libraries were prepared from the human mRNAs, and the recombinant clones were immunoscreened as plaques with two different preparations of rabbit anti-human OATase antibodies. The amino acid sequences of seven tryptic peptides (115 amino acid residues) of the pure human OATase were obtained by microsequencing. When the tryptic peptide and cDNA-derived amino acid sequences were compared, homologies in 111 of 115 residues, including a match of 20 consecutive residues, were observed. An RNA blot hybridization of /sup 32/P-labeled OATase cDNA to normal human retina and retinoblastoma mRNAs demonstrated an OATase mRNA species of approx. = 2.2 kilobases.

  12. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  13. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  14. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  15. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  16. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  17. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  18. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  19. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  20. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  1. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  2. Infrared Spectroscopy of Alanine in Solid Parahydrogen

    NASA Astrophysics Data System (ADS)

    Toh, Shin Yi; Wong, Ying-Tung Angel; Djuricanin, Pavle; Momose, Takamasa

    2014-06-01

    Amino acids are the building blocks of biological molecules, and thus the investigation of their physical and chemical properties would allow for further understanding of their functions in biological systems. In addition, the existence of amino acids in interstellar space has been discussed for many years, but it is still under intense debate. The effect of UV radiation on amino acids is one of the keys for their search in interstellar space, where strong UV radiation exists. In this experiment, conformational compositions of alpha and beta alanine and their UV photolysis were investigated via matrix-isolation FTIR spectroscopy and quantum chemical calculations. Solid parahydrogen was used as the matrix, which provides higher resolution spectra than other noble gas matrices. We have identified several stable conformers for both alpha and beta alanine in solid parahydrogen. A clear correlation between conformational ratio and sublimation temperature was found for beta alanine. Furthermore, it was found that UV photolysis of alanine yields not only its conformational changes, but also photodissociation into a CO2 molecule and fragments. Observed spectra and their analysis will be discussed in relation to interstellar chemistry.

  3. Crystal structures of the PLP- and PMP-bound forms of BtrR, a dual functional aminotransferase involved in butirosin biosynthesis.

    PubMed

    Popovic, Bojana; Tang, Xiao; Chirgadze, Dimitri Y; Huang, Fanglu; Blundell, Tom L; Spencer, Jonathan B

    2006-10-01

    The aminotransferase (BtrR), which is involved in the biosynthesis of butirosin, a 2-deoxystreptamine (2-DOS)-containing aminoglycoside antibiotic produced by Bacillus circulans, catalyses the pyridoxal phosphate (PLP)-dependent transamination reaction both of 2-deoxy-scyllo-inosose to 2-deoxy-scyllo-inosamine and of amino-dideoxy-scyllo-inosose to 2-DOS. The high-resolution crystal structures of the PLP- and PMP-bound forms of BtrR aminotransferase from B. circulans were solved at resolutions of 2.1 A and 1.7 A with R(factor)/R(free) values of 17.4/20.6 and 19.9/21.9, respectively. BtrR has a fold characteristic of the aspartate aminotransferase family, and sequence and structure analysis categorises it as a member of SMAT (secondary metabolite aminotransferases) subfamily. It exists as a homodimer with two active sites per dimer. The active site of the BtrR protomer is located in a cleft between an alpha helical N-terminus, a central alphabetaalpha sandwich domain and an alphabeta C-terminal domain. The structures of the PLP- and PMP-bound enzymes are very similar; however BtrR-PMP lacks the covalent bond to Lys192. Furthermore, the two forms differ in the side-chain conformations of Trp92, Asp163, and Tyr342 that are likely to be important in substrate selectivity and substrate binding. This is the first three-dimensional structure of an enzyme from the butirosin biosynthesis gene cluster.

  4. Experimental and computational thermochemical study of α-alanine (DL) and β-alanine.

    PubMed

    da Silva, Manuel A V Ribeiro; da Silva, Maria das Dores M C Ribeiro; Santos, Ana Filipa L O M; Roux, Maria Victoria; Foces-Foces, Concepción; Notario, Rafael; Guzmán-Mejía, Ramón; Juaristi, Eusebio

    2010-12-16

    This paper reports an experimental and theoretical study of the gas phase standard (p° = 0.1 MPa) molar enthalpies of formation, at T = 298.15 K, of α-alanine (DL) and β-alanine. The standard (p° = 0.1 MPa) molar enthalpies of formation of crystalline α-alanine (DL) and β-alanine were calculated from the standard molar energies of combustion, in oxygen, to yield CO2(g), N2(g), and H2O(l), measured by static-bomb combustion calorimetry at T = 298.15 K. The vapor pressures of both amino acids were measured as function of temperature by the Knudsen effusion mass-loss technique. The standard molar enthalpies of sublimation at T = 298.15 K was derived from the Clausius−Clapeyron equation. The experimental values were used to calculate the standard (p° = 0.1 MPa) enthalpy of formation of α-alanine (DL) and β-alanine in the gaseous phase, Δ(f)H(m)°(g), as −426.3 ± 2.9 and −421.2 ± 1.9 kJ·mol(−1), respectively. Standard ab initio molecular orbital calculations at the G3 level were performed. Enthalpies of formation, using atomization reactions, were calculated and compared with experimental data. Detailed inspections of the molecular and electronic structures of the compounds studied were carried out.

  5. Brain alanine formation as an ammonia-scavenging pathway during hyperammonemia: effects of glutamine synthetase inhibition in rats and astrocyte-neuron co-cultures.

    PubMed

    Dadsetan, Sherry; Kukolj, Eva; Bak, Lasse K; Sørensen, Michael; Ott, Peter; Vilstrup, Hendrik; Schousboe, Arne; Keiding, Susanne; Waagepetersen, Helle S

    2013-08-01

    Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of (15)NH4(+) in alanine during acute hyperammonemia. We observed a fourfold increased amount of (15)NH4 incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to (15)NH4Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of (15)NH4 into alanine together with increased (15)N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy.

  6. Secretion of d-alanine by Escherichia coli.

    PubMed

    Katsube, Satoshi; Sato, Kazuki; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

    2016-07-01

    Escherichia coli has an l-alanine export system that protects the cells from toxic accumulation of intracellular l-alanine in the presence of l-alanyl-l-alanine (l-Ala-l-Ala). When a DadA-deficient strain was incubated with 6.0 mM l-Ala-l-Ala, we detected l-alanine and d-alanine using high-performance liquid chromatography (HPLC) analysis at a level of 7.0 mM and 3.0 mM, respectively, after 48 h incubation. Treatment of the culture supernatant with d-amino acid oxidase resulted in the disappearance of a signal corresponding to d-alanine. Additionally, the culture supernatant enabled a d-alanine auxotroph to grow without d-alanine supplementation, confirming that the signal detected by HPLC was authentic d-alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, alr or dadX, the extracellular level of d-alanine increased to 11.5 mM and 8.5 mM, respectively, under similar conditions, suggesting that increased metabolic flow from l-alanine to d-alanine enhanced d-alanine secretion. When high-density DadA-deficient cells preloaded with l-Ala-l-Ala were treated with 20 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), secretion of both l-alanine and d-alanine was enhanced ~twofold compared with that in cells without CCCP treatment. In contrast, the ATPase inhibitor dicyclohexylcarbodiimide did not exert such an effect on the l-alanine and d-alanine secretion. Furthermore, inverted membrane vesicles prepared from DadA-deficient cells lacking the l-alanine exporter AlaE accumulated [3H]D-alanine in an energy-dependent manner. This energy-dependent accumulation of [3H]D-alanine was strongly inhibited by CCCP. These results indicate that E. coli has a transport system(s) that exports d-alanine and that this function is most likely modulated by proton electrochemical potential. PMID:27166225

  7. [Ulysses retrotransposon aspartate proteinase (Drosophila virilis)].

    PubMed

    Volkov, D A; Savvateeva, L V; Dergousova, N I; Rumsh, L D

    2002-01-01

    Retrotransposones are mobile genetic elements occurring in genomes of bacteria, plants or animals. Retrotransposones were found to contain nucleotide sequences encoding proteins which are homological to retroviral aspartic proteinases. Our research has been focused on Ulysses which is mobile genetic element found in Drosophila virilis. We suggested a primary structure of Ulysses proteinase using comparative analysis of amino acid sequences of retroviral proteinases and proteinases from retrotransposones. The appropriate cDNA fragment has been cloned and expressed in E. coli. The purification of recombinant protein (12 kD) has been carried out by affinity chromatography using pepstatine-agarose. The obtained protein has proteolytic activity at optimum pH 5.5 like the majority of aspartic proteinases.

  8. L-alanine uptake in membrane vesicles from Mytilus edulis gills

    SciTech Connect

    Pajor, A.M.; Wright, S.H.

    1986-03-05

    Previous studies have shown that gills from M. edulis can accumulate L-alanine from seawater by a saturable process specific for ..cap alpha..-neutral amino acids. This uptake occurs against chemical gradients in excess of 10/sup 6/ to 1. To further characterize this uptake, membrane vesicles were prepared from M. edulis gill tissue by differential centrifugation. Enrichments of putative enzyme markers (relative to that in combined initial fractions) were as follows: ..gamma..-Glutamyltranspeptidase, 25-30x; Alkaline Phosphatase, 5-6x; K/sup +/-dependent para-Nitrophenyl Phosphatase, 3-5x; Succinate Dehydrogenase 0.1-0.2x. These results suggest that the preparation is enriched in plasma membranes, although histochemical studies will be needed to verify this. The time course of /sup 14/C-L-alanine uptake in the presence of inwardly-directed Na/sup +/ gradient showed a transient overshoot (3-5 fold) at 10 minutes which decreased to equilibrium after six hours. The size of the overshoot and early uptake rates depended on the size of the inwardly-directed Na/sup +/ gradient. No overshoot was seen in the presence of inwardly-directed gradients of LiCl or choline-Cl, or with equilibrium concentrations NaCl or mannitol. A reduced overshoot was seen with a gradient of NaSCN. A small overshoot was seen with an inwardly-directed gradient of KCl. Transport of L-alanine included saturable and diffusive components. Uptake of 6 ..mu..M L-alanine was inhibited more than 80% by 100 ..mu..M ..cap alpha..-zwitterionic amino acids (alanine, leucine, glycine); by 30 to 75% by proline, aspartate and lysine; and less than 20% by a ..beta..-amino acid, taurine. The results of these experiments agree with those from intact gill studies and support the hypothesis that L-alanine is transported into gill epithelial cells by a secondary active transport process involving Na/sup +/.

  9. 21 CFR 172.540 - DL-Alanine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true DL-Alanine. 172.540 Section 172.540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Agents and Related Substances § 172.540 DL-Alanine. DL-Alanine (a racemic mixture of D- and...

  10. 21 CFR 172.540 - DL-Alanine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false DL-Alanine. 172.540 Section 172.540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Agents and Related Substances § 172.540 DL-Alanine. DL-Alanine (a racemic mixture of D- and...

  11. On the existence of 'L-alanine cadmium bromide'.

    PubMed

    Srinivasan, Bikshandarkoil R

    2013-12-01

    It is argued that the recently reported nonlinear optical crystal L-alanine cadmium bromide, grown by slow solvent evaporation method at room temperature [P. Ilayabarathi, J. Chandrasekaran, Spectrochim. Acta 96A (2012) 684-689] is the well-known L-alanine crystal. The isolation of L-alanine crystal is explained due to fractional crystallization.

  12. 21 CFR 172.540 - DL-Alanine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false DL-Alanine. 172.540 Section 172.540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Agents and Related Substances § 172.540 DL-Alanine. DL-Alanine (a racemic mixture of D- and...

  13. 21 CFR 172.540 - DL-Alanine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false DL-Alanine. 172.540 Section 172.540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Agents and Related Substances § 172.540 DL-Alanine. DL-Alanine (a racemic mixture of D- and...

  14. Earthworms accumulate alanine in response to drought.

    PubMed

    Holmstrup, Martin; Slotsbo, Stine; Henriksen, Per G; Bayley, Mark

    2016-09-01

    Earthworms have ecologically significant functions in tropical and temperate ecosystems and it is therefore important to understand how these animals survive during drought. In order to explore the physiological responses to dry conditions, we simulated a natural drought incident in a laboratory trial exposing worms in slowly drying soil for about one month, and then analyzed the whole-body contents of free amino acids (FAAs). We investigated three species forming estivation chambers when soils dry out (Aporrectodea tuberculata, Aporrectodea icterica and Aporrectodea longa) and one species that does not estivate during drought (Lumbricus rubellus). Worms subjected to drought conditions (< -2MPa) substantially increased the concentration of FAAs and in particular alanine that was significantly upregulated in all tested species. Alanine was the most important FAA reaching 250-650μmolg(-1) dry weight in dehydrated Aporrectodea species and 300μmolg(-1) dry weight in L. rubellus. Proline was only weakly upregulated in some species as were a few other FAAs. Species forming estivation chambers (Aporrectodea spp.) did not show a better ability to conserve body water than the non-estivating species (L. rubellus) at the same drought level. These results suggest that the accumulation of alanine is an important adaptive trait in drought tolerance of earthworms in general. PMID:27107492

  15. Biocatalytic potential of vanillin aminotransferase from Capsicum chinense

    PubMed Central

    2014-01-01

    Background The conversion of vanillin to vanillylamine is a key step in the biosynthetic route towards capsaicinoids in pungent cultivars of Capsicum sp. The reaction has previously been annotated to be catalysed by PAMT (putative aminotransferase; [GenBank: AAC78480.1, Swiss-Prot: O82521]), however, the enzyme has previously not been biochemically characterised in vitro. Results The biochemical activity of the transaminase was confirmed by direct measurement of the reaction with purified recombinant enzyme. The enzyme accepted pyruvate, and oxaloacetate but not 2-oxoglutarate as co-substrate, which is in accordance with other characterised transaminases from the plant kingdom. The enzyme was also able to convert (S)-1-phenylethylamine into acetophenone with high stereo-selectivity. Additionally, it was shown to be active at a broad pH range. Conclusions We suggest PAMT to be renamed to VAMT (vanillin aminotransferase, abbreviation used in this study) as formation of vanillin from vanillylamine could be demonstrated. Furthermore, due to high stereoselectivity and activity at physiological pH, VAMT is a suitable candidate for biocatalytic transamination in a recombinant whole-cell system. PMID:24712445

  16. Functional characterization of aromatic amino acid aminotransferase involved in 2-phenylethanol biosynthesis in isolated rose petal protoplasts.

    PubMed

    Hirata, Hiroshi; Ohnishi, Toshiyuki; Ishida, Haruka; Tomida, Kensuke; Sakai, Miwa; Hara, Masakazu; Watanabe, Naoharu

    2012-03-15

    In rose flowers, 2-phenylethanol (2PE) is biosynthesized from l-phenylalanine (l-Phe) via phenylacetaldehyde (PAld) by the actions of two enzymes, pyridoxal-5'-phosphate (PLP)-dependent aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). We here report that Rosa 'Yves Piaget' aromatic amino acid aminotransferase produced phenylpyruvic acid (PPA) from l-Phe in isolated petal protoplasts. We have cloned three full length cDNAs (RyAAAT1-3) of aromatic amino acid aminotransferase families based on rose EST database and homology regions. The RyAAATs enzymes were heterogeneously expressed in Escherichia coli and characterized biochemically. The recombinant RyAAAT3 showed the highest activity toward l-Phe in comparison with l-tryptophan, l-tyrosine, d-Phe, glycine, and l-alanine, and showed 9.7-fold higher activity with l-Phe rather than PPA as a substrate. RyAAAT3 had an optimal activity at pH 9 and at 45-55°C with α-ketoglutaric acid, and was found to be a PLP dependent enzyme based on the inhibition test using Carbidopa, an inhibitor of PLP-dependent enzymes. The transcript of RyAAAT3 was expressed in flowers as well as other organs of R. 'Yves Piaget'. RNAi suppression of RyAAAT3 decreased 2PE production, revealing the involvement of RyAAAT3 in 2PE biosynthesis in rose protoplasts and indicating that rose protoplasts have potentially two different 2PE biosynthetic pathways, the AADC route and the new route via PPA from l-Phe. PMID:22236980

  17. Functional characterization of aromatic amino acid aminotransferase involved in 2-phenylethanol biosynthesis in isolated rose petal protoplasts.

    PubMed

    Hirata, Hiroshi; Ohnishi, Toshiyuki; Ishida, Haruka; Tomida, Kensuke; Sakai, Miwa; Hara, Masakazu; Watanabe, Naoharu

    2012-03-15

    In rose flowers, 2-phenylethanol (2PE) is biosynthesized from l-phenylalanine (l-Phe) via phenylacetaldehyde (PAld) by the actions of two enzymes, pyridoxal-5'-phosphate (PLP)-dependent aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). We here report that Rosa 'Yves Piaget' aromatic amino acid aminotransferase produced phenylpyruvic acid (PPA) from l-Phe in isolated petal protoplasts. We have cloned three full length cDNAs (RyAAAT1-3) of aromatic amino acid aminotransferase families based on rose EST database and homology regions. The RyAAATs enzymes were heterogeneously expressed in Escherichia coli and characterized biochemically. The recombinant RyAAAT3 showed the highest activity toward l-Phe in comparison with l-tryptophan, l-tyrosine, d-Phe, glycine, and l-alanine, and showed 9.7-fold higher activity with l-Phe rather than PPA as a substrate. RyAAAT3 had an optimal activity at pH 9 and at 45-55°C with α-ketoglutaric acid, and was found to be a PLP dependent enzyme based on the inhibition test using Carbidopa, an inhibitor of PLP-dependent enzymes. The transcript of RyAAAT3 was expressed in flowers as well as other organs of R. 'Yves Piaget'. RNAi suppression of RyAAAT3 decreased 2PE production, revealing the involvement of RyAAAT3 in 2PE biosynthesis in rose protoplasts and indicating that rose protoplasts have potentially two different 2PE biosynthetic pathways, the AADC route and the new route via PPA from l-Phe.

  18. Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine.

    PubMed

    Borodina, Irina; Kildegaard, Kanchana R; Jensen, Niels B; Blicher, Thomas H; Maury, Jérôme; Sherstyk, Svetlana; Schneider, Konstantin; Lamosa, Pedro; Herrgård, Markus J; Rosenstand, Inger; Öberg, Fredrik; Forster, Jochen; Nielsen, Jens

    2015-01-01

    Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the β-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of β-alanine and its subsequent conversion into 3HP using a novel β-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3gL(-1) with a 0.14±0.0C-molC-mol(-1) yield on glucose in 80h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.

  19. Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine.

    PubMed

    Borodina, Irina; Kildegaard, Kanchana R; Jensen, Niels B; Blicher, Thomas H; Maury, Jérôme; Sherstyk, Svetlana; Schneider, Konstantin; Lamosa, Pedro; Herrgård, Markus J; Rosenstand, Inger; Öberg, Fredrik; Forster, Jochen; Nielsen, Jens

    2015-01-01

    Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the β-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of β-alanine and its subsequent conversion into 3HP using a novel β-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3gL(-1) with a 0.14±0.0C-molC-mol(-1) yield on glucose in 80h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production. PMID:25447643

  20. Glutamate and aspartate are decreased in the skin in amyotrophic lateral sclerosis

    NASA Technical Reports Server (NTRS)

    Ono, S.; Yamauchi, M.

    1992-01-01

    We measured the levels of amino acids in biopsied skin from eight patients with amyotrophic lateral sclerosis (ALS) and seven controls. The most conspicuous changes in ALS patients were as follows. First, the contents of the acidic amino acids glutamate and aspartate were significantly decreased in ALS, and were negatively and significantly associated with the duration of illness. Second, the levels of the collagen-associated amino acids hydroxyproline, proline, glycine, alanine, and hydroxylysine were significantly decreased in ALS, and correlated inversely with the duration of illness. These results suggest that there are abnormalities of acidic amino acids and collagen-associated amino acids in the skin of patients with ALS. These changes may underlie the pathogenesis of ALS.

  1. Diurnal variations in response of rat liver tyrosine aminotransferase activity to food intake.

    PubMed

    Kato, H; Saito, M

    1980-01-01

    Effects of fasting and refeeding on the hepatic tyrosine aminotransferase activity were examined in rats that had been fed during the night. The tyrosine aminotransferase activity showed clear diurnal variations, with a maximal activity after the feeding time. The tyrosine aminotransferase rhythm persisted even under starvation, though the amplitude decreased remarkably. When the starved rats were refed at night, the tyrosine aminotransferase activity increased rapidly to a high level, but it increased slowly to a rather lower level when they were refed in daytime.

  2. D-alanine incorporation into macromolecules and effects of D-alanine deprivation on active transport in Bacillus subtilis.

    PubMed

    Clark, V L; Young, F E

    1978-03-01

    An auxotroph of Bacillus subtilis 168 unable to synthesize D-alanine loses the ability to support endogenously energized transport when deprived of D-alanine. Revertants of the mutant retain transport activity. The loss of transport is specific for substrates taken up by active transport; substrates taken up by group translocation are transported at normal rates. The loss of transport can be retarded by pretreatment of the cells with inhibitors of protein synthesis. Since the loss of transport could be due to an alteration in a D-alanine-containing polymer, we investigated the incorporation of D-[14C]alanine into macromolecules. The major D-alanine-containing polymers in B. subtilis are peptidoglycan and teichoic acid, with 4 to 6% of the D-[14C]alanine label found in trypsin-soluble material. Whereas the peptidoglycan and teichoic acid undergo turnover, the trypsin-soluble material does not. Treatment of the trypsin-soluble material with Pronase releases free D-alanine. Analysis of acid-hydrolyzed trypsin-soluble material indicated that approximately 75% of the radioactivity is present as D-alanine, with the remainder present as L-alanine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified D-[14C]alanine-labeled membranes indicated the presence of two peaks of radioactivity (molecular weights, 230,000 and 80,000) that could be digested by trypsin. The results suggest that D-alanine may be covalently bound to cellular proteins.

  3. Aspartate Decarboxylase is Required for a Normal Pupa Pigmentation Pattern in the Silkworm, Bombyx mori

    PubMed Central

    Dai, Fangyin; Qiao, Liang; Cao, Cun; Liu, Xiaofan; Tong, Xiaoling; He, Songzhen; Hu, Hai; Zhang, Li; Wu, Songyuan; Tan, Duan; Xiang, Zhonghuai; Lu, Cheng

    2015-01-01

    The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of β-alanine and N-β-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of β-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera. PMID:26077025

  4. Aspartate Decarboxylase is Required for a Normal Pupa Pigmentation Pattern in the Silkworm, Bombyx mori.

    PubMed

    Dai, Fangyin; Qiao, Liang; Cao, Cun; Liu, Xiaofan; Tong, Xiaoling; He, Songzhen; Hu, Hai; Zhang, Li; Wu, Songyuan; Tan, Duan; Xiang, Zhonghuai; Lu, Cheng

    2015-06-16

    The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of β-alanine and N-β-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of β-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera.

  5. Crystallization and preliminary crystallographic analysis of d-alanine-d-alanine ligase from Streptococcus mutans

    SciTech Connect

    Lu, Yong-Zhi; Sheng, Yu; Li, Lan-Fen; Tang, De-Wei; Liu, Xiang-Yu; Zhao, Xiaojun; Liang, Yu-He Su, Xiao-Dong

    2007-09-01

    A potential target for antibiotic drug design, d-alanine-d-alanine ligase from S. mutans, was expressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. d-Alanine-d-alanine ligase is encoded by the gene ddl (SMU-599) in Streptococcus mutans. This ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. To study the structure and function of this ligase, the gene ddl was amplified from S. mutans genomic DNA and cloned into the expression vector pET28a. The protein was expressed in soluble form in Escherichia coli strain BL21 (DE3). Homogeneous protein was obtained using a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Purified protein was crystallized and the cube-shaped crystal diffracted to 2.4 Å. The crystal belongs to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 79.50, c = 108.97 Å. There is one molecule per asymmetric unit.

  6. Alteration of substrate specificity of alanine dehydrogenase

    PubMed Central

    Fernandes, Puja; Aldeborgh, Hannah; Carlucci, Lauren; Walsh, Lauren; Wasserman, Jordan; Zhou, Edward; Lefurgy, Scott T.; Mundorff, Emily C.

    2015-01-01

    The l-alanine dehydrogenase (AlaDH) has a natural history that suggests it would not be a promising candidate for expansion of substrate specificity by protein engineering: it is the only amino acid dehydrogenase in its fold family, it has no sequence or structural similarity to any known amino acid dehydrogenase, and it has a strong preference for l-alanine over all other substrates. By contrast, engineering of the amino acid dehydrogenase superfamily members has produced catalysts with expanded substrate specificity; yet, this enzyme family already contains members that accept a broad range of substrates. To test whether the natural history of an enzyme is a predictor of its innate evolvability, directed evolution was carried out on AlaDH. A single mutation identified through molecular modeling, F94S, introduced into the AlaDH from Mycobacterium tuberculosis (MtAlaDH) completely alters its substrate specificity pattern, enabling activity toward a range of larger amino acids. Saturation mutagenesis libraries in this mutant background additionally identified a double mutant (F94S/Y117L) showing improved activity toward hydrophobic amino acids. The catalytic efficiencies achieved in AlaDH are comparable with those that resulted from similar efforts in the amino acid dehydrogenase superfamily and demonstrate the evolvability of MtAlaDH specificity toward other amino acid substrates. PMID:25538307

  7. Structural Analysis of QdtB, an Aminotransferase Required for the Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-[alpha]-D-glucose

    SciTech Connect

    Thoden, James B.; Schaffer, Christina; Messner, Paul; Holden, Hazel M.

    2009-05-21

    3-Acetamido-3,6-dideoxy-{alpha}-D-glucose or Quip3NAc is an unusual deoxyamino sugar found in the O-antigens of some Gram-negative bacteria and in the S-layers of Gram-positive bacteria. It is synthesized in these organisms as a dTDP-linked sugar via the action of five enzymes. The focus of this investigation is on QdtB from Thermoanaerobacterium thermosaccharolyticum E207-71, a PLP-dependent aminotransferase that catalyzes the penultimate step in the production of dTDP-Quip3NAc. For this analysis, the enzyme was crystallized in the presence of its product, dTDP-Quip3N, and the structure was solved and refined to 2.15 {angstrom} resolution. QdtB is a dimer, and its overall fold places it into the well-characterized aspartate aminotransferase superfamily. Electron density corresponding to the bound product reveals the presence of a Schiff base between C-4' of the PLP cofactor and the amino nitrogen of the sugar. Those amino acid side chains involved in binding the dTDP-sugar into the active site include Tyr 183, His 309, and Tyr 310 from subunit 1 and Lys 219 from subunit 2. Notably there is a decided lack of interactions between the pyranosyl C-4' hydroxyl of the dTDP-sugar and the protein. In keeping with this observation, we show that QdtB can also turn over dTDP-3-acetamido-3,6-dideoxy-{alpha}-D-galactose. This investigation represents the first structural analysis of a sugar-modifying aminotransferase with a bound product in its active site that functions at the C-3' rather than the C-4' position of the hexose.

  8. L-aspartic acid transport by cat erythrocytes

    SciTech Connect

    Chen, C.W.; Preston, R.L.

    1986-03-01

    Cat and dog red cells are unusual in that they have no Na/K ATPase and contain low K and high Na intracellularly. They also show significant Na dependent L-aspartate (L-asp) transport. The authors have characterized this system in cat RBCs. The influx of /sup 3/H-L-asp (typically 2..mu..M) was measured in washed RBCs incubated for 60 s at 37/sup 0/C in medium containing 140 mM NaCl, 5 mM Kcl, 2 mM CaCl/sub 2/, 15 mM MOPS pH 7.4, 5 mM glucose, and /sup 14/C-PEG as a space marker. The cells were washed 3 times in the medium immediately before incubation which was terminated by centrifuging the RBCs through a layer of dibutylphthalate. Over an L-asp concentration range of 0.5-1000..mu..M, influx obeyed Michaelis-Menten kinetics with a small added linear diffusion component. The Kt and Jmax of the saturable component were 5.40 +/- 0.34 ..mu..M and 148.8 +/- 7.2 ..mu..mol 1. cell/sup -1/h/sup -1/ respectively. Replacement of Na with Li, K, Rb, Cs or choline reduce influx to diffusion. With the addition of asp analogues (4/sup +/M L-asp, 40/sup +/M inhibitor), the following sequence of inhibition was observed (range 80% to 40% inhib.): L-glutamate > L-cysteine sulfonate > D-asp > L-cysteic acid > D-glutamate. Other amino acids such as L-alanine, L-proline, L-lysine, L-cysteine, and taurine showed no inhibition (<5%). These data suggest that cat red cells contain a high-affinity Na dependent transport system for L-asp, glutamate, and closely related analogues which resembles that found in the RBCs of other carnivores and in neural tissues.

  9. International society of sports nutrition position stand: Beta-Alanine.

    PubMed

    Trexler, Eric T; Smith-Ryan, Abbie E; Stout, Jeffrey R; Hoffman, Jay R; Wilborn, Colin D; Sale, Craig; Kreider, Richard B; Jäger, Ralf; Earnest, Conrad P; Bannock, Laurent; Campbell, Bill; Kalman, Douglas; Ziegenfuss, Tim N; Antonio, Jose

    2015-01-01

    The International Society of Sports Nutrition (ISSN) provides an objective and critical review of the mechanisms and use of beta-alanine supplementation. Based on the current available literature, the conclusions of the ISSN are as follows: 1) Four weeks of beta-alanine supplementation (4-6 g daily) significantly augments muscle carnosine concentrations, thereby acting as an intracellular pH buffer; 2) Beta-alanine supplementation currently appears to be safe in healthy populations at recommended doses; 3) The only reported side effect is paraesthesia (tingling), but studies indicate this can be attenuated by using divided lower doses (1.6 g) or using a sustained-release formula; 4) Daily supplementation with 4 to 6 g of beta-alanine for at least 2 to 4 weeks has been shown to improve exercise performance, with more pronounced effects in open end-point tasks/time trials lasting 1 to 4 min in duration; 5) Beta-alanine attenuates neuromuscular fatigue, particularly in older subjects, and preliminary evidence indicates that beta-alanine may improve tactical performance; 6) Combining beta-alanine with other single or multi-ingredient supplements may be advantageous when supplementation of beta-alanine is high enough (4-6 g daily) and long enough (minimum 4 weeks); 7) More research is needed to determine the effects of beta-alanine on strength, endurance performance beyond 25 min in duration, and other health-related benefits associated with carnosine. PMID:26175657

  10. International society of sports nutrition position stand: Beta-Alanine.

    PubMed

    Trexler, Eric T; Smith-Ryan, Abbie E; Stout, Jeffrey R; Hoffman, Jay R; Wilborn, Colin D; Sale, Craig; Kreider, Richard B; Jäger, Ralf; Earnest, Conrad P; Bannock, Laurent; Campbell, Bill; Kalman, Douglas; Ziegenfuss, Tim N; Antonio, Jose

    2015-01-01

    The International Society of Sports Nutrition (ISSN) provides an objective and critical review of the mechanisms and use of beta-alanine supplementation. Based on the current available literature, the conclusions of the ISSN are as follows: 1) Four weeks of beta-alanine supplementation (4-6 g daily) significantly augments muscle carnosine concentrations, thereby acting as an intracellular pH buffer; 2) Beta-alanine supplementation currently appears to be safe in healthy populations at recommended doses; 3) The only reported side effect is paraesthesia (tingling), but studies indicate this can be attenuated by using divided lower doses (1.6 g) or using a sustained-release formula; 4) Daily supplementation with 4 to 6 g of beta-alanine for at least 2 to 4 weeks has been shown to improve exercise performance, with more pronounced effects in open end-point tasks/time trials lasting 1 to 4 min in duration; 5) Beta-alanine attenuates neuromuscular fatigue, particularly in older subjects, and preliminary evidence indicates that beta-alanine may improve tactical performance; 6) Combining beta-alanine with other single or multi-ingredient supplements may be advantageous when supplementation of beta-alanine is high enough (4-6 g daily) and long enough (minimum 4 weeks); 7) More research is needed to determine the effects of beta-alanine on strength, endurance performance beyond 25 min in duration, and other health-related benefits associated with carnosine.

  11. Vesicular GABA transporter (VGAT) transports β-alanine.

    PubMed

    Juge, Narinobu; Omote, Hiroshi; Moriyama, Yoshinori

    2013-11-01

    Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In this study, we show that VGAT recognizes β-alanine as a substrate. Proteoliposomes containing purified VGAT transport β-alanine using Δψ but not ΔpH as a driving force. The Δψ-driven β-alanine uptake requires Cl(-). VGAT also facilitates Cl(-) uptake in the presence of β-alanine. A previously described VGAT mutant (Glu213Ala) that disrupts GABA and glycine transport similarly abrogates β-alanine uptake. These findings indicated that VGAT transports β-alanine through a mechanism similar to those for GABA and glycine, and functions as a vesicular β-alanine transporter. Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In the present study, we showed that proteoliposomes containing purified VGAT transport β-alanine using Δψ as a driving force. VGAT also facilitates Cl(-) uptake. Our findings indicated that VGAT functions as a vesicular β-alanine transporter.

  12. Crystal Structure of Ll-Diaminopimelate Aminotransferase From 'Arabidopsis Thaliana': a Recently-Discovered Enzyme in the Biosynthesis of L-Lysine By Plants And 'Chlamydia'

    SciTech Connect

    Watanabe, N.; Cherney, M.M.; van Belkum, M.J.; Marcus, S.L.; Flegel, M.D.; Clay, M.D.; Deyholos, M.K.; Vederas, J.C.; James, M.N.G.

    2007-07-13

    The essential biosynthetic pathway to l-Lysine in bacteria and plants is an attractive target for the development of new antibiotics or herbicides because it is absent in humans, who must acquire this amino acid in their diet. Plants use a shortcut of a bacterial pathway to l-Lysine in which the pyridoxal-5-phosphate (PLP)-dependent enzyme ll-diaminopimelate aminotransferase (LL-DAP-AT) transforms l-tetrahydrodipicolinic acid (L-THDP) directly to LL-DAP. In addition, LL-DAP-AT was recently found in Chlamydia sp., suggesting that inhibitors of this enzyme may also be effective against such organisms. In order to understand the mechanism of this enzyme and to assist in the design of inhibitors, the three-dimensional crystal structure of LL-DAP-AT was determined at 1.95 Angstroms resolution. The cDNA sequence of LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT) was optimized for expression in bacteria and cloned in Escherichia coli without its leader sequence but with a C-terminal hexahistidine affinity tag to aid protein purification. The structure of AtDAP-AT was determined using the multiple-wavelength anomalous dispersion (MAD) method with a seleno-methionine derivative. AtDAP-AT is active as a homodimer with each subunit having PLP in the active site. It belongs to the family of type I fold PLP-dependent enzymes. Comparison of the active site residues of AtDAP-AT and aspartate aminotransferases revealed that the PLP binding residues in AtDAP-AT are well conserved in both enzymes. However, Glu97* and Asn309* in the active site of AtDAP-AT are not found at similar positions in aspartate aminotransferases, suggesting that specific substrate recognition may require these residues from the other monomer. A malate-bound structure of AtDAP-AT allowed LL-DAP and L-glutamate to be modeled into the active site. These initial three-dimensional structures of LL-DAP-AT provide insight into its substrate specificity and catalytic mechanism.

  13. Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate.

    PubMed

    Moreno, Miguel Angel; Alonso, Ana; Alcolea, Pedro Jose; Abramov, Ariel; de Lacoba, Mario García; Abendroth, Jan; Zhang, Sunny; Edwards, Thomas; Lorimer, Don; Myler, Peter John; Larraga, Vicente

    2014-12-01

    Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT) has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs. PMID:25516846

  14. Ornithine-δ-Aminotransferase Inhibits Neurogenesis During Xenopus Embryonic Development

    PubMed Central

    Peng, Ying; Cooper, Sandra K.; Li, Yi; Mei, Jay M.; Qiu, Shuwei; Borchert, Gregory L.; Donald, Steven P.; Kung, Hsiang-fu; Phang, James M.

    2015-01-01

    Purpose. In humans, deficiency of ornithine-δ-aminotransferase (OAT) results in progressive degeneration of the neural retina (gyrate atrophy) with blindness in the fourth decade. In this study, we used the Xenopus embryonic developmental model to study functions of the OAT gene on embryonic development. Methods. We cloned and sequenced full-length OAT cDNA from Xenopus oocytes (X-OAT) and determined X-OAT expression in various developmental stages of Xenopus embryos and in a variety of adult tissues. The phenotype, gene expression of neural developmental markers, and enzymatic activity were detected by gain-of-function and loss-of-function manipulations. Results. We showed that X-OAT is essential for Xenopus embryonic development, and overexpression of X-OAT produces a ventralized phenotype characterized by a small head, lack of axial structure, and defective expression of neural developmental markers. Using X-OAT mutants based on mutations identified in humans, we found that substitution of both Arg 180 and Leu 402 abrogated both X-OAT enzymatic activity and ability to modulate the developmental phenotype. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development. Conclusions. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development, but it is essential for Xenopus embryonic development. The Arg 180 and Leu 402 are crucial for these effects of the OAT molecule in development. PMID:25783604

  15. Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate

    PubMed Central

    Moreno, Miguel Angel; Alonso, Ana; Alcolea, Pedro Jose; Abramov, Ariel; de Lacoba, Mario García; Abendroth, Jan; Zhang, Sunny; Edwards, Thomas; Lorimer, Don; Myler, Peter John; Larraga, Vicente

    2014-01-01

    Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT) has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs. PMID:25516846

  16. Medial temporal N-acetyl aspartate in pediatric major depression

    PubMed Central

    MacMaster, Frank P.; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S. Preeya; Buhagiar, Christian; Rosenberg, David R.

    2008-01-01

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD-case control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  17. Medial temporal N-acetyl-aspartate in pediatric major depression.

    PubMed

    MacMaster, Frank P; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S Preeya; Buhagiar, Christian; Rosenberg, David R

    2008-10-30

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD case-control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in the left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  18. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  19. Aspartic acid racemization in tooth enamel from living humans.

    PubMed Central

    Helfman, P M; Bada, J L

    1975-01-01

    The aspartic acid in human tooth enamel shows increasing racemization with age. This increase is not seen in the metabolically active protein hemoglobin. The rate constant for the racemization reaction of aspartic acid in human tooth enamel was found to be 8.29 X 10(-4) yr-1. This rate constant suggests that in any protein with a long in vivo lifetime, D-aspartic acid will accumulate with age (about 8% of total aspartic acid in enamel will be the D-enantiomer after 60 years). Thus, racemization may play some role in the aging process affecting metabolically stable tissues in long-lived homeotherms. Aspartic acid racemization in toogh enamel also provides a biochronological tool for assessing the age of living mammals. PMID:1059082

  20. Alanine repeats influence protein localization in splicing speckles and paraspeckles.

    PubMed

    Chang, Shuo-Hsiu; Chang, Wei-Lun; Lu, Chia-Chen; Tarn, Woan-Yuh

    2014-12-16

    Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ.

  1. Beta-alanine as a small molecule neurotransmitter.

    PubMed

    Tiedje, K E; Stevens, K; Barnes, S; Weaver, D F

    2010-10-01

    This review discusses the role of beta-alanine as a neurotransmitter. Beta-alanine is structurally intermediate between alpha-amino acid (glycine, glutamate) and gamma-amino acid (GABA) neurotransmitters. In general, beta-alanine satisfies a number of the prerequisite classical criteria for being a neurotransmitter: beta-alanine occurs naturally in the CNS, is released by electrical stimulation through a Ca(2+) dependent process, has binding sites, and inhibits neuronal excitability. beta-Alanine has 5 recognized receptor sites: glycine co-agonist site on the NMDA complex (strychnine-insensitive); glycine receptor site (strychnine sensitive); GABA-A receptor; GABA-C receptor; and blockade of GAT protein-mediated glial GABA uptake. Although beta-alanine binding has been identified throughout the hippocampus, limbic structures, and neocortex, unique beta-alaninergic neurons with no GABAergic properties remain unidentified, and it is impossible to discriminate between beta-alaninergic and GABAergic properties in the CNS. Nevertheless, a variety of data suggest that beta-alanine should be considered as a small molecule neurotransmitter and should join the ranks of the other amino acid neurotransmitters. These realizations open the door for a more comprehensive evaluation of beta-alanine's neurochemistry and for its exploitation as a platform for drug design.

  2. Mechanisms of itch evoked by β-alanine.

    PubMed

    Liu, Qin; Sikand, Parul; Ma, Chao; Tang, Zongxiang; Han, Liang; Li, Zhe; Sun, Shuohao; LaMotte, Robert H; Dong, Xinzhong

    2012-10-17

    β-Alanine, a popular supplement for muscle building, induces itch and tingling after consumption, but the underlying molecular and neural mechanisms are obscure. Here we show that, in mice, β-alanine elicited itch-associated behavior that requires MrgprD, a G-protein-coupled receptor expressed by a subpopulation of primary sensory neurons. These neurons exclusively innervate the skin, respond to β-alanine, heat, and mechanical noxious stimuli but do not respond to histamine. In humans, intradermally injected β-alanine induced itch but neither wheal nor flare, suggesting that the itch was not mediated by histamine. Thus, the primary sensory neurons responsive to β-alanine are likely part of a histamine-independent itch neural circuit and a target for treating clinical itch that is unrelieved by anti-histamines.

  3. Use of β-alanine as an ergogenic aid.

    PubMed

    Derave, Wim

    2013-01-01

    Despite the large variety of so-called ergogenic supplements used by the sporting community, only few of them are effectively supported by scientific proof. One of the recent evidence-based supplements that entered the market is β-alanine. β-Alanine is the rate-limiting precursor for the synthesis of the dipeptide carnosine (β-alanyl-L-histidine) in human muscle. The chronic daily ingestion of β-alanine can markedly elevate muscle carnosine content, which results in improved exercise capacity, especially in sports that include high-intensity exercise episodes. The use of β-alanine is exponentially growing in recent years. This chapter aims to (1) discuss the scientific basis and physiological background of β-alanine and its synthesis product carnosine, and (2) translate these scientific findings to practical applications in sports.

  4. Metabolic fingerprint of ischaemic cardioprotection: importance of the malate-aspartate shuttle.

    PubMed

    Nielsen, Torsten Toftegaard; Støttrup, Nicolaj Brejnholt; Løfgren, Bo; Bøtker, Hans Erik

    2011-08-01

    The convergence of cardioprotective intracellular signalling pathways to modulate mitochondrial function as an end-target of cytoprotective stimuli is well described. However, our understanding of whether the complementary changes in mitochondrial energy metabolism are secondary responses or inherent mechanisms of ischaemic cardioprotection remains incomplete. In the heart, the malate-aspartate shuttle (MAS) constitutes the primary metabolic pathway for transfer of reducing equivalents from the cytosol into the mitochondria for oxidation. The flux of MAS is tightly linked to the flux of the tricarboxylic acid cycle and the electron transport chain, partly by the amino acid l-glutamate. In addition, emerging evidence suggests the MAS is an important regulator of cytosolic and mitochondrial calcium homeostasis. In the isolated rat heart, inhibition of MAS during ischaemia and early reperfusion by the aminotransferase inhibitor aminooxyacetate induces infarct limitation, improves haemodynamic responses, and modulates glucose metabolism, analogous to effects observed in classical ischaemic preconditioning. On the basis of these findings, the mechanisms through which MAS preserves mitochondrial function and cell survival are reviewed. We conclude that the available evidence is supportive of a down-regulation of mitochondrial respiration during lethal ischaemia with a gradual 'wake-up' during reperfusion as a pivotal feature of ischaemic cardioprotection. Finally, comments on modulating myocardial energy metabolism by the cardioprotective amino acids glutamate and glutamine are given.

  5. Tyrosine aminotransferase contributes to benzylisoquinoline alkaloid biosynthesis in opium poppy.

    PubMed

    Lee, Eun-Jeong; Facchini, Peter J

    2011-11-01

    Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5'-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and α-ketoglutarate, with apparent K(m) values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.

  6. Tyrosine Aminotransferase: Biochemical and Structural Properties and Molecular Dynamics Simulations

    SciTech Connect

    P Mehere; Q Han; J Lemkul; C Vavricka; H Robinson; D Bevan; J Li

    2011-12-31

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  7. Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations

    SciTech Connect

    Mehere, P.; Robinson, H.; Han, Q.; Lemkul, J. A.; Vavricka, C. J.; Bevan, D. R.; Li, J.

    2010-11-01

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  8. Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

    SciTech Connect

    Han, Q.; Robinson, H; Cai, T; Tagle, D; Li, J

    2009-01-01

    Kynurenine aminotransferase III (KAT III) has been considered to be involved in the production of mammalian brain kynurenic acid (KYNA), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. The enzyme was identified based on its high sequence identity with mammalian KAT I, but its activity toward kynurenine and its structural characteristics have not been established. In this study, the biochemical and structural properties of mouse KAT III (mKAT III) were determined. Specifically, mKAT III cDNA was amplified from a mouse brain cDNA library, and its recombinant protein was expressed in an insect cell protein expression system. We established that mKAT III is able to efficiently catalyze the transamination of kynurenine to KYNA and has optimum activity at relatively basic conditions of around pH 9.0 and at relatively high temperatures of 50 to 60C. In addition, mKAT III is active toward a number of other amino acids. Its activity toward kynurenine is significantly decreased in the presence of methionine, histidine, glutamine, leucine, cysteine, and 3-hydroxykynurenine. Through macromolecular crystallography, we determined the mKAT III crystal structure and its structures in complex with kynurenine and glutamine. Structural analysis revealed the overall architecture of mKAT III and its cofactor binding site and active center residues. This is the first report concerning the biochemical characteristics and crystal structures of KAT III enzymes and provides a basis toward understanding the overall physiological role of mammalian KAT III in vivo and insight into regulating the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

  9. Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations.

    PubMed

    Mehere, Prajwalini; Han, Qian; Lemkul, Justin A; Vavricka, Christopher J; Robinson, Howard; Bevan, David R; Li, Jianyong

    2010-11-01

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using α-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 Å resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  10. Comparison of EPR response of alanine and Gd₂O₃-alanine dosimeters exposed to TRIGA Mainz reactor.

    PubMed

    Marrale, M; Schmitz, T; Gallo, S; Hampel, G; Longo, A; Panzeca, S; Tranchina, L

    2015-12-01

    In this work we report some preliminary results regarding the analysis of electron paramagnetic resonance (EPR) response of alanine pellets and alanine pellets added with gadolinium used for dosimetry at the TRIGA research reactor in Mainz, Germany. Two set-ups were evaluated: irradiation inside PMMA phantom and irradiation inside boric acid phantom. We observed that the presence of Gd2O3 inside alanine pellets increases the EPR signal by a factor of 3.45 and 1.24 in case of PMMA and boric acid phantoms, respectively. We can conclude that in the case of neutron beam with a predominant thermal neutron component the addition of gadolinium oxide can significantly improve neutron sensitivity of alanine pellets. Monte Carlo (MC) simulations of both response of alanine and Gd-added alanine pellets with FLUKA code were performed and a good agreement was achieved for pure alanine dosimeters. For Gd2O3-alanine deviations between MC simulations and experimental data were observed and discussed. PMID:26315099

  11. Comparison of EPR response of alanine and Gd₂O₃-alanine dosimeters exposed to TRIGA Mainz reactor.

    PubMed

    Marrale, M; Schmitz, T; Gallo, S; Hampel, G; Longo, A; Panzeca, S; Tranchina, L

    2015-12-01

    In this work we report some preliminary results regarding the analysis of electron paramagnetic resonance (EPR) response of alanine pellets and alanine pellets added with gadolinium used for dosimetry at the TRIGA research reactor in Mainz, Germany. Two set-ups were evaluated: irradiation inside PMMA phantom and irradiation inside boric acid phantom. We observed that the presence of Gd2O3 inside alanine pellets increases the EPR signal by a factor of 3.45 and 1.24 in case of PMMA and boric acid phantoms, respectively. We can conclude that in the case of neutron beam with a predominant thermal neutron component the addition of gadolinium oxide can significantly improve neutron sensitivity of alanine pellets. Monte Carlo (MC) simulations of both response of alanine and Gd-added alanine pellets with FLUKA code were performed and a good agreement was achieved for pure alanine dosimeters. For Gd2O3-alanine deviations between MC simulations and experimental data were observed and discussed.

  12. Aspartate inhibits Staphylococcus aureus biofilm formation.

    PubMed

    Yang, Hang; Wang, Mengyue; Yu, Junping; Wei, Hongping

    2015-04-01

    Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp.

  13. Aspartate inhibits Staphylococcus aureus biofilm formation.

    PubMed

    Yang, Hang; Wang, Mengyue; Yu, Junping; Wei, Hongping

    2015-04-01

    Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp. PMID:25687923

  14. Toxicological evaluation of the hydro-alcohol extract of the dry leaves of Peumus boldus and boldine in rats.

    PubMed

    Almeida, E R; Melo, A M; Xavier, H

    2000-03-01

    The hydro-alcohol extract of the dry leaves of Peumus boldus and boldine, showed abortive and teratogenic action and changes in the blood levels of bilirubin, cholesterol, glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and urea in rats. The long term administration of the extract and boldine did not cause histological modification during a period of 90 days. PMID:10685105

  15. [Markers of viral hepatitis B and D and levels of alanine aminotransferase in military blood donors: a profile of 30,000 blood donations in 1989].

    PubMed

    Boulesteix, G; Bourin, P; Fabre, G; Blanchard de Vaucouleurs, A; Molinié, C; Denee, J M; Buisson, Y; Schill, H; Joussemet, M

    1990-01-01

    Serologic data for B and D viral hepatitis are studied on 30,000 military blood donors. Because of legal norms of blood products for transfusion 761 donations (2.53% have been destroyed). Exclusion criteria for viral B hepatitis and ALT are independent. In this study the prevalency of HBV infections is significantly lower than for other blood centers: probably in account of the young age of military blood donors.

  16. β-Alanine supplementation for athletic performance: an update.

    PubMed

    Bellinger, Phillip M

    2014-06-01

    β-alanine supplementation has become a common practice among competitive athletes participating in a range of different sports. Although the mechanism by which chronic β-alanine supplementation could have an ergogenic effect is widely debated, the popular view is that β-alanine supplementation augments intramuscular carnosine content, leading to an increase in muscle buffer capacity, a delay in the onset of muscular fatigue, and a facilitated recovery during repeated bouts of high-intensity exercise. β-alanine supplementation appears to be most effective for exercise tasks that rely heavily on ATP synthesis from anaerobic glycolysis. However, research investigating its efficacy as an ergogenic aid remains equivocal, making it difficult to draw conclusions as to its effectiveness for training and competition. The aim of this review was to update, summarize, and critically evaluate the findings associated with β-alanine supplementation and exercise performance with the most recent research available to allow the development of practical recommendations for coaches and athletes. A critical review of the literature reveals that when significant ergogenic effects have been found, they have been generally shown in untrained individuals performing exercise bouts under laboratory conditions. The body of scientific data available concerning highly trained athletes performing single competition-like exercise tasks indicates that this type of population receives modest but potentially worthwhile performance benefits from β-alanine supplementation. Recent data indicate that athletes may not only be using β-alanine supplementation to enhance sports performance but also as a training aid to augment bouts of high-intensity training. β-alanine supplementation has also been shown to increase resistance training performance and training volume in team-sport athletes, which may allow for greater overload and superior adaptations compared with training alone. The ergogenic

  17. Preparation and Characterisation of Pva Doped with Beta Alanine

    NASA Astrophysics Data System (ADS)

    Bhuvaneshwari, R.; Karthikeyan, S.; Rajeswari, N.; Selvasekarapandian, S.; Sanjeeviraja, C.

    2013-07-01

    Pure PVA has been doped with different amount of β - alanine. Film has been prepared by Solution Casting Technique using water as a solvent. The Complex formation between the PVA and β - alanine has been confirmed by FTIR. The Pure PVA conductivity is in the order 10-10 Scm-1 at ambient temperature. The conductivity has been found to increase to the order 10-6 when doped with 10% β - alanine. In this paper characterization of a PVA doped with β-ala has been studied using XRD, FTIR, AC impedance analysis and the results are reported.

  18. Aspartic acid 413 is important for the normal allosteric functioning of ADP-glucose pyrophosphorylase

    SciTech Connect

    Greene, T.W.; Woodbury, R.L.; Okita, T.W.

    1996-11-01

    As part of a structure-function analysis of the higher-plant ADP-glucose pyrophosphorylase (AGP), we used a random mutagenesis approach in combination with a novel bacterial complementation system to isolate over 100 mutants that were defective in glycogen production. One mutant of the large subunit M27 was identified by its capacity to only partially complement a mutation in the structural gene for the bacterial AGP (glg C), as determined by its light-staining phenotype when cells were exposed to I{sub 2} vapors. Enzyme-linked immunosorbent assay and enzymatic pyrophosphorylysis assays of M27 cell extracts showed that the level of expression and AGP activity was comparable to those of cells that expressed the wildtype recombinant enzyme. Kinetic analysis indicated that the M27 AGP displays normal Michaelis constant values for the substrates glucose-1-phosphate and ATP but requires 6- to 10-fold greater levels of 3-phosphoglycerate (3-PGA) than the wild-type recombinant enzyme for maximum activation. DNA sequence analysis showed that M27 contains a single point mutation that resulted in the replacement of aspartic acid 413 to alanine. Substitution of a lysine residue at this site almost completely abolished activation by 3-PGA. Aspartic acid 413 is adjacent to a lysine residue that was previously identified by chemical modification studies to be important in the binding of 3-PGA. The kinetic properties of M27 corroborate the importance of this region in the allosteric regulation of a higher-plant AGP. 28 refs., 3 figs., 1 tab.

  19. REVERSAL OF d-CYCLOSERINE INHIBITION OF BACTERIAL GROWTH BY ALANINE

    PubMed Central

    Zygmunt, Walter A.

    1962-01-01

    Zygmunt, Walter A. (Mead Johnson & Co., Evansville, Ind.). Reversal of d-cycloserine inhibition of bacterial growth by alanine. J. Bacteriol. 84:154–156. 1962.—Reversal of the antibacterial activity of d-4-amino-3-isoxazolidone by alanine in bacterial cultures actively growing on chemically defined media was compared in cultures requiring exogenous alanine and those capable of its synthesis. dl-Alanine was the most effective reversal agent in Pediococcus cerevisiae, an alanine-requiring organism, and d-alanine was effective in Escherichia coli and Staphylococcus aureus, organisms synthesizing alanine. With all three cultures, l-alanine was the least effective reversal agent. PMID:16561951

  20. Relationships between serum aminotransferase levels, liver histologies and virological status in patients with chronic hepatitis C in Taiwan.

    PubMed

    Luo, J C; Hwang, S J; Lai, C R; Lu, C L; Li, C P; Tsay, S H; Wu, J C; Chang, F Y; Lee, S D

    1998-07-01

    In patients with chronic hepatitis C, the relationships between serum alanine aminotransferase (ALT) levels, histological liver injury and serum hepatitis C virus (HCV) RNA titres remain controversial. To evaluate these relationships, 93 Chinese patients with histological diagnosis of chronic hepatitis C were enrolled for this study. Serum ALT levels, HCV-RNA titres and HCV genotypes were examined. The histology was evaluated according to a modified histological activity score based on the degree of periportal necro-inflammation, intralobular necro-inflammation, portal inflammation, total necro-inflammation and fibrosis. The mean serum ALT level was significantly higher in patients with severe intralobular necro-inflammation activity than in patients with mild or no activity (P = 0.013). However, scores of intralobular activity were only weakly correlated with serum ALT levels (r = 0.27) and could not be used to adequately predict ALT values. Serum ALT levels showed no significant correlation with the scores of portal inflammation, periportal necro-inflammation, total necro-inflammation and fibrosis. Also, there was no significant difference in the mean serum ALT level among different serum HCV-RNA levels and HCV genotypes. Serum HCV-RNA titres and genotypes showed no significant correlation with liver histology and serum HCV-RNA titres were only weakly correlated with the total necro-inflammatory score (r = 0.27). In conclusion, although serum ALT levels were higher in patients with more severe intralobular necro-inflammatory activity, the correlation was not strong enough to adequately predict ALT values. Serum HCV-RNA titres and genotypes also showed no significant correlation with serum ALT levels and liver histologies.

  1. Dose response of alanine detectors irradiated with carbon ion beams

    SciTech Connect

    Herrmann, Rochus; Jaekel, Oliver; Palmans, Hugo; Sharpe, Peter; Bassler, Niels

    2011-04-15

    Purpose: The dose response of the alanine detector shows a dependence on particle energy and type when irradiated with ion beams. The purpose of this study is to investigate the response behavior of the alanine detector in clinical carbon ion beams and compare the results to model predictions. Methods: Alanine detectors have been irradiated with carbon ions with an energy range of 89-400 MeV/u. The relative effectiveness of alanine has been measured in this regime. Pristine and spread out Bragg peak depth-dose curves have been measured with alanine dosimeters. The track structure based alanine response model developed by Hansen and Olsen has been implemented in the Monte Carlo code FLUKA and calculations were compared to experimental results. Results: Calculations of the relative effectiveness deviate less than 5% from the measured values for monoenergetic beams. Measured depth-dose curves deviate from predictions in the peak region, most pronounced at the distal edge of the peak. Conclusions: The used model and its implementation show a good overall agreement for quasimonoenergetic measurements. Deviations in depth-dose measurements are mainly attributed to uncertainties of the detector geometry implemented in the Monte Carlo simulations.

  2. Relationship Between Hepatic Steatosis and the Elevation of Aminotransferases in HBV-Infected Patients With HBe-Antigen Negativity and a Low Viral Load

    PubMed Central

    Enomoto, Hirayuki; Aizawa, Nobuhiro; Nishikawa, Hiroki; Ikeda, Naoto; Sakai, Yoshiyuki; Takata, Ryo; Hasegawa, Kunihiro; Nakano, Chikage; Nishimura, Takashi; Yoh, Kazunori; Ishii, Akio; Takashima, Tomoyuki; Iwata, Yoshinori; Iijima, Hiroko; Nishiguchi, Shuhei

    2016-01-01

    Abstract Nonalcoholic fatty liver disease has been suggested to be associated with alanine aminotransferase (ALT) elevation in hepatitis B virus (HBV)-infected patients with HBe antigen (HBeAg)-negativity and a low HBV-DNA level. However, few studies have evaluated the association according to histological findings of the liver. Among a total of 198 HBV-infected patients who received a percutaneous liver biopsy, we studied the histological and laboratory findings of HBeAg-negative patients without receiving nucleoside/nucleotide analogues treatment (N = 70) in order to evaluate whether hepatic steatosis and its related metabolic disorders were associated with an elevation in ALT levels in HBeAg-negative patients. In HBeAg-negative patients with a high serum HBV-DNA level (≥2000 IU/mL), the level of HBV-DNA was the only significant factor related to ALT elevation. However, in HBeAg-negative patients with a low HBV-DNA level, the serum ferritin level, and histologically observed hepatic steatosis were significantly associated factors with ALT elevation. When we evaluated 2 metabolic variables (serum ferritin and fasting insulin) that are suggested to be relevant to the presence of progressive disease in Japanese patients, we found that the rate of metabolic disorders was significantly higher among patients with a high ALT level and a low HBV-DNA level than it was among those with other conditions. The triglyceride level and the frequency of moderate or severe hepatic steatosis were significantly higher in patients with a low HBV-DNA level than in those with a high HBV-DNA level. Histologically proven hepatic steatosis and its related metabolic disorders are suggested to be involved in the elevation of aminotransferases of HBeAg-negative patients, particularly those with low HBV-DNA levels. PMID:27124068

  3. Noncovalent and covalent functionalization of a (5, 0) single-walled carbon nanotube with alanine and alanine radicals.

    PubMed

    Rajarajeswari, Muthusivarajan; Iyakutti, Kombiah; Kawazoe, Yoshiyuki

    2012-02-01

    We have systematically investigated the noncovalent and covalent adsorption of alanine and alanine radicals, respectively, onto a (5, 0) single-walled carbon nanotube using first-principles calculation. It was found that XH···π (X = N, O, C) interactions play a crucial role in the non-ovalent adsorption and that the functional group close to the carbon nanotube exhibits a significant influence on the binding strength. Noncovalent functionalization of the carbon nanotube with alanine enhances the conductivity of the metallic (5, 0) nanotube. In the covalent adsorption of each alanine radical onto a carbon nanotube, the binding energy depends on the adsorption site on CNT and the electronegative atom that binds with the CNT. The strongest complex is formed when the alanine radical interacts with a (5, 0) carbon nanotube through the amine group. In some cases, the covalent interaction of the alanine radical introduces a half-filled band at the Fermi level due to the local sp (3) hybridization, which modifies the conductivity of the tube.

  4. Structure of D-alanine-D-alanine ligase from Yersinia pestis: nucleotide phosphate recognition by the serine loop.

    PubMed

    Tran, Huyen Thi; Hong, Myoung Ki; Ngo, Ho Phuong Thuy; Huynh, Kim Hung; Ahn, Yeh Jin; Wang, Zhong; Kang, Lin Woo

    2016-01-01

    D-Alanyl-D-alanine is an essential precursor of bacterial peptidoglycan and is synthesized by D-alanine-D-alanine ligase (DDL) with hydrolysis of ATP; this reaction makes DDL an important drug target for the development of antibacterial agents. Five crystal structures of DDL from Yersinia pestis (YpDDL) were determined at 1.7-2.5 Å resolution: apo, AMP-bound, ADP-bound, adenosine 5'-(β,γ-imido)triphosphate-bound, and D-alanyl-D-alanine- and ADP-bound structures. YpDDL consists of three domains, in which four loops, loop 1, loop 2 (the serine loop), loop 3 (the ω-loop) and loop 4, constitute the binding sites for two D-alanine molecules and one ATP molecule. Some of them, especially the serine loop and the ω-loop, show flexible conformations, and the serine loop is mainly responsible for the conformational change in substrate nucleotide phosphates. Enzyme-kinetics assays were carried out for both the D-alanine and ATP substrates and a substrate-binding mechanism was proposed for YpDDL involving conformational changes of the loops.

  5. Structure of D-alanine-D-alanine ligase from Yersinia pestis: nucleotide phosphate recognition by the serine loop.

    PubMed

    Tran, Huyen Thi; Hong, Myoung Ki; Ngo, Ho Phuong Thuy; Huynh, Kim Hung; Ahn, Yeh Jin; Wang, Zhong; Kang, Lin Woo

    2016-01-01

    D-Alanyl-D-alanine is an essential precursor of bacterial peptidoglycan and is synthesized by D-alanine-D-alanine ligase (DDL) with hydrolysis of ATP; this reaction makes DDL an important drug target for the development of antibacterial agents. Five crystal structures of DDL from Yersinia pestis (YpDDL) were determined at 1.7-2.5 Å resolution: apo, AMP-bound, ADP-bound, adenosine 5'-(β,γ-imido)triphosphate-bound, and D-alanyl-D-alanine- and ADP-bound structures. YpDDL consists of three domains, in which four loops, loop 1, loop 2 (the serine loop), loop 3 (the ω-loop) and loop 4, constitute the binding sites for two D-alanine molecules and one ATP molecule. Some of them, especially the serine loop and the ω-loop, show flexible conformations, and the serine loop is mainly responsible for the conformational change in substrate nucleotide phosphates. Enzyme-kinetics assays were carried out for both the D-alanine and ATP substrates and a substrate-binding mechanism was proposed for YpDDL involving conformational changes of the loops. PMID:26894530

  6. EPR/alanine dosimetry for two therapeutic proton beams

    NASA Astrophysics Data System (ADS)

    Marrale, Maurizio; Carlino, Antonio; Gallo, Salvatore; Longo, Anna; Panzeca, Salvatore; Bolsi, Alessandra; Hrbacek, Jan; Lomax, Tony

    2016-02-01

    In this work the analysis of the electron paramagnetic resonance (EPR) response of alanine pellets exposed to two different clinical proton beams employed for radiotherapy is performed. One beam is characterized by a passive delivery technique and is dedicated to the eyes treatment (OPTIS2 beam line). Alanine pellets were irradiated with a 70 MeV proton beam corresponding to 35 mm range in eye tissue. We investigated how collimators with different sizes and shape used to conform the dose to the planned target volume influence the delivered dose. For this purpose we performed measurements with varying the collimator size (Output Factor) and the results were compared with those obtained with other dosimetric techniques (such as Markus chamber and diode detector). This analysis showed that the dosimeter response is independent of collimator diameter if this is larger than or equal to 10 mm. The other beam is characterized by an active spot-scanning technique, the Gantry1 beam line (maximum energy 230 MeV), and is used to treat deep-seated tumors. The dose linearity of alanine response in the clinical dose range was tested and the alanine dose response at selected locations in depth was measured and compared with the TPS planned dose in a quasi-clinical scenario. The alanine response was found to be linear in the dose in the clinical explored range (from 10 to 70 Gy). Furthermore, a depth dose profile in a quasi-clinical scenario was measured and compared to the dose computed by the Treatment Planning System PSIPLAN. The comparison of calibrated proton alanine measurements and TPS dose shows a difference under 1% in the SOBP and a "quenching" effect up to 4% in the distal part of SOBP. The positive dosimetric characteristics of the alanine pellets confirm the feasibility to use these detectors for "in vivo" dosimetry in clinical proton beams.

  7. Evidence for the generation of transaminase inhibitor(s) during ethanol metabolism by rat liver homogenates: a potential mechanism for alcohol toxicity.

    PubMed

    Solomon, L R

    1987-08-01

    Since ethanol consumption decreases hepatic aminotransferase activities in vivo, mechanisms of ethanol-mediated transaminase inhibition were explored in vitro using mitochondria-depleted rat liver homogenates. When homogenates were incubated at 37 degrees with 50 mM ethanol for 1 hr, alanine aminotransferase decreased by 20%, while aspartate aminotransferase was unchanged. After 2 hr, aspartate aminotransferase decreased by 20% and by 3 hr, alanine and aspartate aminotransferases were decreased by 31 and 23%, respectively. Levels of acetaldehyde generated during ethanol oxidation were 525 +/- 47 microM at 1 hr, 855 +/- 14 microM at 2 hr, and 1293 +/- 140 microM at 3 hr. Although inhibition of alcohol oxidation with methylpyrazole or cyanide markedly decreased ethanol-mediated transaminase inhibition, neither incubation with acetate nor generation of reducing equivalents by oxidation of lactate, malate, xylitol, or sorbitol altered the activity of either enzyme. However, semicarbazide, an aldehyde scavenger, prevented inhibition of both aminotransferases by ethanol. Moreover, incubation with 5 mM acetaldehyde for 1 hr inhibited alanine and aspartate aminotransferases by 36 and 26%, respectively. Cyanamide, an aldehyde dehydrogenase inhibitor, had little effect on ethanol-mediated transaminase inhibition. Thus, metabolism of ethanol by rat liver homogenates produces transaminase inhibition similar to that described in vivo and this effect requires acetaldehyde generation but not acetaldehyde oxidation. Since addition of pyridoxal 5'-phosphate to assay mixes did not reverse ethanol effects, aminotransferase inhibition does not result from displacement of vitamin B6 coenzymes.

  8. Evidence for the generation of transaminase inhibitor(s) during ethanol metabolism by rat liver homogenates: a potential mechanism for alcohol toxicity.

    PubMed

    Solomon, L R

    1987-08-01

    Since ethanol consumption decreases hepatic aminotransferase activities in vivo, mechanisms of ethanol-mediated transaminase inhibition were explored in vitro using mitochondria-depleted rat liver homogenates. When homogenates were incubated at 37 degrees with 50 mM ethanol for 1 hr, alanine aminotransferase decreased by 20%, while aspartate aminotransferase was unchanged. After 2 hr, aspartate aminotransferase decreased by 20% and by 3 hr, alanine and aspartate aminotransferases were decreased by 31 and 23%, respectively. Levels of acetaldehyde generated during ethanol oxidation were 525 +/- 47 microM at 1 hr, 855 +/- 14 microM at 2 hr, and 1293 +/- 140 microM at 3 hr. Although inhibition of alcohol oxidation with methylpyrazole or cyanide markedly decreased ethanol-mediated transaminase inhibition, neither incubation with acetate nor generation of reducing equivalents by oxidation of lactate, malate, xylitol, or sorbitol altered the activity of either enzyme. However, semicarbazide, an aldehyde scavenger, prevented inhibition of both aminotransferases by ethanol. Moreover, incubation with 5 mM acetaldehyde for 1 hr inhibited alanine and aspartate aminotransferases by 36 and 26%, respectively. Cyanamide, an aldehyde dehydrogenase inhibitor, had little effect on ethanol-mediated transaminase inhibition. Thus, metabolism of ethanol by rat liver homogenates produces transaminase inhibition similar to that described in vivo and this effect requires acetaldehyde generation but not acetaldehyde oxidation. Since addition of pyridoxal 5'-phosphate to assay mixes did not reverse ethanol effects, aminotransferase inhibition does not result from displacement of vitamin B6 coenzymes. PMID:3663401

  9. Rescue of Na+ affinity in aspartate 928 mutants of Na+,K+-ATPase by secondary mutation of glutamate 314.

    PubMed

    Holm, Rikke; Einholm, Anja P; Andersen, Jens P; Vilsen, Bente

    2015-04-10

    The Na(+),K(+)-ATPase binds Na(+) at three transport sites denoted I, II, and III, of which site III is Na(+)-specific and suggested to be the first occupied in the cooperative binding process activating phosphorylation from ATP. Here we demonstrate that the asparagine substitution of the aspartate associated with site III found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood causes a dramatic reduction of Na(+) affinity in the α1-, α2-, and α3-isoforms of Na(+),K(+)-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na(+)-coordinating residues in site III. Remarkably, the Na(+) affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na(+) binding and does not affect the E1-E2 conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na(+) affinity is likely intrinsic to the Na(+) binding pocket, and the underlying mechanism could be a tightening of Na(+) binding at Na(+) site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na(+),K(+) pump function in intact cells. Rescue of Na(+) affinity and Na(+) and K(+) transport by second-site mutation is unique in the history of Na(+),K(+)-ATPase and points to new possibilities for treatment of neurological patients carrying Na(+),K(+)-ATPase mutations.

  10. Biochemical properties and crystal structure of a β-phenylalanine aminotransferase from Variovorax paradoxus.

    PubMed

    Crismaru, Ciprian G; Wybenga, Gjalt G; Szymanski, Wiktor; Wijma, Hein J; Wu, Bian; Bartsch, Sebastian; de Wildeman, Stefaan; Poelarends, Gerrit J; Feringa, Ben L; Dijkstra, Bauke W; Janssen, Dick B

    2013-01-01

    By selective enrichment, we isolated a bacterium that can use β-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg(-1) for (S)-β-phenylalanine at 30°C and 33 U mg(-1) at the optimum temperature of 55°C. The β-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted β-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-β-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic β-amino acids to yield (R)-β-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the β-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic β-amino acid-converting aminotransferases may be identified.

  11. Biochemical and Structural Characterization of a Ureidoglycine Aminotransferase in the Klebsiella pneumoniae Uric Acid Catabolic Pathway

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2010-09-03

    Many plants, fungi, and bacteria catabolize allantoin as a mechanism for nitrogen assimilation. Recent reports have shown that in plants and some bacteria the product of hydrolysis of allantoin by allantoinase is the unstable intermediate ureidoglycine. While this molecule can spontaneously decay, genetic analysis of some bacterial genomes indicates that an aminotransferase may be present in the pathway. Here we present evidence that Klebsiella pneumoniae HpxJ is an aminotransferase that preferentially converts ureidoglycine and an {alpha}-keto acid into oxalurate and the corresponding amino acid. We determined the crystal structure of HpxJ, allowing us to present an explanation for substrate specificity.

  12. Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites.

    PubMed

    Polonais, Valerie; Shea, Michael; Soldati-Favre, Dominique

    2011-08-01

    Aspartic proteases are important virulence factors for pathogens and are recognized as attractive drug targets. Seven aspartic proteases (ASPs) have been identified in Toxoplasma gondii genome. Bioinformatics and phylogenetic analyses regroup them into five monophyletic groups. Among them, TgASP1, a coccidian specific aspartic protease related to the food vacuole plasmepsins, is associated with the secretory pathway in non-dividing cells and relocalizes in close proximity to the nascent inner membrane complex (IMC) of daughter cells during replication. Despite a potential role for TgASP1 in IMC formation, the generation of a conventional knockout of the TgASP1 gene revealed that this protease is not required for T. gondii tachyzoite survival or for proper IMC biogenesis.

  13. On the existence of "L-threonine formate", "L-alanine lithium chloride" and "bis L-alanine lithium chloride" crystals.

    PubMed

    Petrosyan, A M; Ghazaryan, V V; Fleck, M

    2013-03-15

    We argue that the recently reported crystals "L-threonine formate" as well as "L-alanine lithium chloride" and "bis L-alanine lithium chloride" actually are the well-known crystals L-threonine and L-alanine, respectively.

  14. Post-Irradiation Study of the Alanine Dosimeter

    PubMed Central

    Desrosiers, Marc F.

    2014-01-01

    Post-irradiation stability of high-dose dosimeters has traditionally been an important measurement influence quantity. Though the exceptional stability of the alanine dosimeter response with time has rendered this factor a non-issue for routine work, the archival quality of the alanine dosimeter has not been characterized. Here the alanine pellet dosimeter response is measured up to seven years post-irradiation for a range of absorbed doses. This long-term study is accompanied by an examination of the environmental influence quantities (e.g., ambient light) on the relatively short-term (3–4 month) stability of both pellet and film commercial dosimeters. Both dosimeter types demonstrated exceptional stability in the short term and proved to be relatively insensitive to common influence quantities. The long-term data revealed a complex dose-dependent response trend. PMID:26601033

  15. Morphosynthesis of alanine mesocrystals by pH control.

    PubMed

    Ma, Yurong; Cölfen, Helmut; Antonietti, Markus

    2006-06-01

    Crystallization of DL-alanine is studied as a single polymorph model case to analyze the different modes of crystallization of polar organic molecules in absence of any structure directing additives. Depending on supersaturation, which is controlled either by temperature or by pH, and in the absence of additives, crystallization by mesoscale assembly of nanoparticles is found over a wide range of conditions, leading to so-called mesocrystals. This supplements the classical molecule-based crystallization mechanism, which is identified at lower supersaturations and at pH values away from the isoelectric point (IEP). The resulting alanine crystals are characterized by SEM, XRD, and single-crystal analysis. Time-resolved conductivity measurements and dynamic light scattering of the reaction solutions reveal information about precursor structures and reaction kinetics. A formation mechanism is proposed for the alanine mesocrystals. PMID:16771332

  16. First-principles studies of pure and fluorine substituted alanines

    NASA Astrophysics Data System (ADS)

    Ahmad, Sardar; Vaizie, Hamide; Rahnamaye Aliabad, H. A.; Ahmad, Rashid; Khan, Imad; Ali, Zahid; Jalali-Asadabadi, S.; Ahmad, Iftikhar; Khan, Amir Abdullah

    2016-05-01

    This paper communicates the structural, electronic and optical properties of L-alanine, monofluoro and difluoro substituted alanines using density functional calculations. These compounds exist in orthorhombic crystal structure and the calculated structural parameters such as lattice constants, bond angles and bond lengths are in agreement with the experimental results. L-alanine is an indirect band gap insulator, while its fluorine substituted compounds (monofluoroalanine and difluoroalanine) are direct band gap insulators. The substitution causes reduction in the band gap and hence these optically tailored direct wide band gap materials have enhanced optical properties in the ultraviolet (UV) region of electromagnetic spectrum. Therefore, optical properties like dielectric function, refractive index, reflectivity and energy loss function are also investigated. These compounds have almost isotropic nature in the lower frequency range while at higher energies, they have a significant anisotropic nature.

  17. Tolerance of Arc repressor to multiple-alanine substitutions.

    PubMed

    Brown, B M; Sauer, R T

    1999-03-01

    Arc repressor mutants containing from three to 15 multiple-alanine substitutions have spectral properties expected for native Arc proteins, form heterodimers with wild-type Arc, denature cooperatively with Tms equal to or greater than wild type, and, in some cases, fold as much as 30-fold faster and unfold as much as 50-fold slower than wild type. Two of the mutants, containing a total of 14 different substitutions, also footprint operator DNA in vitro. The stability of some of the proteins with multiple-alanine mutations is significantly greater than that predicted from the sum of the single substitutions, suggesting that a subset of the wild-type residues in Arc may interact in an unfavorable fashion. Overall, these results show that almost half of the residues in Arc can be replaced by alanine en masse without compromising the ability of this small, homodimeric protein to fold into a stable, native-like structure. PMID:10051581

  18. [Effects of ß-alanine supplementation on athletic performance].

    PubMed

    Domínguez, Raúl; Hernández Lougedo, Juan; Maté-Muñoz, José Luis; Garnacho-Castaño, Manuel Vicente

    2014-10-06

    Carnosine, dipeptide formed by amino acids ß-alanine and L-histidine, has important physiological functions among which its antioxidant and related memory and learning. However, in connection with the exercise, the most important functions would be associated with muscle contractility, improving calcium sensitivity in muscle fibers, and the regulatory function of pH. Thus, it is proposed that carnosine is the major intracellular buffer, but could contribute to 7-10% in buffer or buffer capacity. Since carnosine synthesis seems to be limited by the availability of ß-alanine supplementation with this compound has been gaining increasing popularity among the athlete population. Therefore, the objective of this study literature review was to examine all those research works have shown the effect of ß-alanine supplementation on athletic performance. Moreover, it also has attempted to establish a specific dosage that maximizing the potential benefits, minimize paresthesia, the main side effect presented in response to supplementation.

  19. Administration of thimerosal to infant rats increases overflow of glutamate and aspartate in the prefrontal cortex: protective role of dehydroepiandrosterone sulfate.

    PubMed

    Duszczyk-Budhathoki, Michalina; Olczak, Mieszko; Lehner, Malgorzata; Majewska, Maria Dorota

    2012-02-01

    Thimerosal, a mercury-containing vaccine preservative, is a suspected factor in the etiology of neurodevelopmental disorders. We previously showed that its administration to infant rats causes behavioral, neurochemical and neuropathological abnormalities similar to those present in autism. Here we examined, using microdialysis, the effect of thimerosal on extracellular levels of neuroactive amino acids in the rat prefrontal cortex (PFC). Thimerosal administration (4 injections, i.m., 240 μg Hg/kg on postnatal days 7, 9, 11, 15) induced lasting changes in amino acid overflow: an increase of glutamate and aspartate accompanied by a decrease of glycine and alanine; measured 10-14 weeks after the injections. Four injections of thimerosal at a dose of 12.5 μg Hg/kg did not alter glutamate and aspartate concentrations at microdialysis time (but based on thimerosal pharmacokinetics, could have been effective soon after its injection). Application of thimerosal to the PFC in perfusion fluid evoked a rapid increase of glutamate overflow. Coadministration of the neurosteroid, dehydroepiandrosterone sulfate (DHEAS; 80 mg/kg; i.p.) prevented the thimerosal effect on glutamate and aspartate; the steroid alone had no influence on these amino acids. Coapplication of DHEAS with thimerosal in perfusion fluid also blocked the acute action of thimerosal on glutamate. In contrast, DHEAS alone reduced overflow of glycine and alanine, somewhat potentiating the thimerosal effect on these amino acids. Since excessive accumulation of extracellular glutamate is linked with excitotoxicity, our data imply that neonatal exposure to thimerosal-containing vaccines might induce excitotoxic brain injuries, leading to neurodevelopmental disorders. DHEAS may partially protect against mercurials-induced neurotoxicity. PMID:22015977

  20. Synthesis and In Vitro Evaluation of Aspartate Transcarbamoylase Inhibitors

    PubMed Central

    Coudray, Laëtitia; Pennebaker, Anne F.; Montchamp, Jean-Luc

    2009-01-01

    The design, synthesis, and evaluation of a series of novel inhibitors of aspartate transcarbamoylase (ATCase) are reported. Several submicromolar phosphorus-containing inhibitors are described, but all-carboxylate compounds are inactive. Compounds were synthesized to probe the postulated cyclic transition-state of the enzyme-catalyzed reaction. In addition, the associated role of the protonation state at the phosphorus acid moiety was evaluated using phosphinic and carboxylic acids. Although none of the synthesized inhibitors is more potent than N-phosphonacetyl-L-aspartate (PALA), the compounds provide useful mechanistic information, as well as the basis for the design of future inhibitors and/or prodrugs. PMID:19828320

  1. Atomic Layer Deposition of L-Alanine Polypeptide

    SciTech Connect

    Fu, Yaqin; Li, Binsong; Jiang, Ying-Bing; Dunphy, Darren R.; Tsai, Andy; Tam, Siu-Yue; Fan, Hongyou Y.; Zhang, Hongxia; Rogers, David; Rempe, Susan; Atanassov, Plamen; Cecchi, Joseph L.; Brinker, C. Jeffrey

    2014-10-30

    L-Alanine polypeptide thin films were synthesized via atomic layer deposition (ALD). Rather, instead of using an amino acid monomer as the precursor, an L-alanine amino acid derivatized with a protecting group was used to prevent self-polymerization, increase the vapor pressure, and allow linear cycle-by-cycle growth emblematic of ALD. Moreover, the successful deposition of a conformal polypeptide film has been confirmed by FTIR, TEM, and Mass Spectrometry, and the ALD process has been extended to polyvaline.

  2. Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides of DL-alanine and N-(tert-butoxycarbonyl)-DL-alanine

    NASA Technical Reports Server (NTRS)

    Profy, A. T.; Usher, D. A.

    1984-01-01

    The aminoacylation of diinosine monophosphate was studied experimentally. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-DL-alanine, a 40 percent enantiomeric excess of the isomer was incorporated at the 2' site and the positions of equilibrium for the reversible 2'-3' migration reaction differed for the D and L enantiomers. The reactivity of the nucleoside hydroxyl groups was found to decrease on the order 2'(3') less than internal 2' and less than 5', and the extent of the reaction was affected by the concentration of the imidazole buffer. Reaction of IpI with imidazolide of unprotected DL-alanine, by contrast, led to an excess of the D isomer at the internal 2' site. Finally, reaction with the N-carboxy anhydride of DL-alanine occurred without stereoselection. These results are found to be relevant to the study of the evolution of optical chemical activity and the origin of genetically directed protein synthesis.

  3. Combined acoustic radiation force impulse, aminotransferase to platelet ratio index and Forns index assessment for hepatic fibrosis grading in hepatitis B

    PubMed Central

    Dong, Chang-Feng; Xiao, Jia; Shan, Ling-Bo; Li, Han-Ying; Xiong, Yong-Jia; Yang, Gui-Lin; Liu, Jing; Yao, Si-Min; Li, Sha-Xi; Le, Xiao-Hua; Yuan, Jing; Zhou, Bo-Ping; Tipoe, George L; Liu, Ying-Xia

    2016-01-01

    AIM: To investigate the combined diagnostic accuracy of acoustic radiation force impulse (ARFI), aspartate aminotransferase to platelet ratio index (APRI) and Forns index for a non-invasive assessment of liver fibrosis in patients with chronic hepatitis B (CHB). METHODS: In this prospective study, 206 patients had CHB with liver fibrosis stages F0-F4 classified by METAVIR and 40 were healthy volunteers were measured by ARFI, APRI and Forns index separately or combined as indicated. RESULTS: ARFI, APRI or Forns index demonstrated a significant correlation with the histological stage (all P < 0.001). According to the AUROC of ARFI and APRI for evaluating fibrotic stages more than F2, ARFI showed an enhanced diagnostic accuracy than APRI (P < 0.05). The combined measurement of ARFI and APRI exhibited better accuracy than ARFI alone when evaluating ≥ F2 fibrotic stage (Z = 2.77, P = 0.006). Combination of ARFI, APRI and Forns index did not obviously improve the diagnostic accuracy compared to the combination of ARFI and APRI (Z = 0.958, P = 0.338). CONCLUSION: ARFI + APRI showed enhanced diagnostic accuracy than ARFI or APRI alone for significant liver fibrosis and ARFI + APRI + Forns index shows the same effect with ARFI + APRI. PMID:27190578

  4. [Regulation of key enzymes of L-alanine biosynthesis by Brevibacterium flavum producer strains].

    PubMed

    Melkonian, L O; Avetisova, G E; Ambartsumian, A A; Chakhalian, A Kh; Sagian, A S

    2013-01-01

    The mechanisms of L-alanine overproduction by Brevibacterium flavum producer strains were studied. It was shown that beta-CI-L-alanine is an inhibitor of some key enzymes involved in the synthesis of L-alanine, including alanine transaminase and valine-pyruvate transaminase. Two highly active B. flavum GL1 and GL1 8 producer strains, which are resistant to the inhibitory effect of beta-Cl-L-alanine, were obtained using a parental B. flavum AA5 producer strain, characterized by a reduced activity of alanine racemase (>or=98%). It was demonstrated that the increased L-alanine synthesis efficiency observed in the producer strains developed in this work is associated with the absence of inhibition of alanine transaminase by the end product of the biosynthesis reaction, as well as with the effect of derepression of both alanine transaminase and valine-pyruvate transaminase synthesis by the studied compound.

  5. A stereo-inverting D-phenylglycine aminotransferase from Pseudomonas stutzeri ST-201: purification, characterization and application for D-phenylglycine synthesis.

    PubMed

    Wiyakrutta, S; Meevootisom, V

    1997-07-01

    D-phenylglycine aminotransferase (D-PhgAT) from a newly isolated soil bacterium, Pseudomonas stutzeri ST-201, was purified to electrophoretic homogeneity and characterized. The molecular weight (M(r)) of the native enzyme was estimated to be 92,000. It is composed of two subunits identical in molecular weight (M(r)) = 47,500). The isoelectric point (pI) of the native enzyme was 5.0. The enzyme catalyzed reversible transamination specific for D-phenylglycine or D-4-hydroxyphenylglycine in which 2-oxoglutarate was an exclusive amino group acceptor and was converted into L-glutamic acid. Neither the D- nor L-isomer of phenylalanine, tyrosine, alanine, valine, leucine, isoleucine or serine could serve as a substrate. The enzyme was most active at alkaline pH with maximum activity at pH 9-10. The temperature for maximum activity was 35-45 degrees C. The apparent K(m) values for D-phenylglycine and for 2-oxoglutarate at 35 degrees C, pH 9.5 were 1.1 and 2.4 mM, respectively. The enzyme activity was strongly inhibited by typical inhibitors of pyridoxal phosphate-dependent enzymes. Possible application of this enzyme for synthesis of enantiomerically pure D-phenylglycine was demonstrated. PMID:9249994

  6. High protein diet induces pericentral glutamate dehydrogenase and ornithine aminotransferase to provide sufficient glutamate for pericentral detoxification of ammonia in rat liver lobules.

    PubMed

    Boon, L; Geerts, W J; Jonker, A; Lamers, W H; Van Noorden, C J

    1999-06-01

    The liver plays a central role in nitrogen metabolism. Nitrogen enters the liver as free ammonia and as amino acids of which glutamine and alanine are the most important precursors. Detoxification of ammonia to urea involves deamination and transamination. By applying quantitative in situ hybridization, we found that mRNA levels of the enzymes involved are mainly expressed in periportal zones of liver lobules. Free ammonia, that is not converted periportally, is efficiently detoxified in the small rim of hepatocytes around the central veins by glutamine synthetase preventing it from entering the systemic circulation. Detoxification of ammonia by glutamine synthetase may be limited due to a shortage of glutamate when the nitrogen load is high. Adaptations in metabolism that prevent release of toxic ammonia from the liver were studied in rats that were fed diets with different amounts of protein, thereby varying the nitrogen load of the liver. We observed that mRNA levels of periportal deaminating and transaminating enzymes increased with the protein content in the diet. Similarly, mRNA levels of pericentral glutamate dehydrogenase and ornithine aminotransferase, the main producers of glutamate in this zone, and pericentral glutamine synthetase all increased with increasing protein levels in the diet. On the basis of these changes in mRNA levels, we conclude that: (a) glutamate is produced pericentrally in sufficient amounts to allow ammonia detoxification by glutamine synthetase and (b) in addition to the catalytic role of ornithine in the periportally localized ornithine cycle, pericentral ornithine degradation provides glutamate for ammonia detoxification.

  7. Characterization of the aspartate transcarbamoylase from Methanococcus jannaschii.

    PubMed

    Hack, E S; Vorobyova, T; Sakash, J B; West, J M; Macol, C P; Hervé, G; Williams, M K; Kantrowitz, E R

    2000-05-26

    The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli. Only the M. jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity. A purification protocol was devised for the Mj-PyrB and M. jannaschii PyrI (Mj-PyrI) gene products. Molecular weight measurements of the Mj-PyrB and Mj-PyrI gene products revealed that the Mj-PyrB gene product is a trimer and the Mj-PyrI gene product is a dimer. Preliminary characterization of the aspartate transcarbamoylase from M. jannaschii cell-free extract revealed that the enzyme has a similar molecular weight to that of the E. coli holoenzyme. Kinetic analysis of the M. jannaschii aspartate transcarbamoylase from the cell-free extract indicates that the enzyme exhibited limited homotropic cooperativity and little if any regulatory properties. The purified Mj-catalytic trimer exhibited hyperbolic kinetics, with an activation energy similar to that observed for the E. coli catalytic trimer. Homology models of the Mj-PyrB and Mj-PyrI gene products were constructed based on the three-dimensional structures of the homologous E. coli proteins. The residues known to be critical for catalysis, regulation, and formation of the quaternary structure from the well characterized E. coli aspartate transcarbamoylase were compared.

  8. Aspartate analysis in formulations using a new enzyme sensor.

    PubMed

    Campanella, L; Aturki, Z; Sammartino, M P; Tomassetti, M

    1995-04-01

    A biosensor has been developed for the purpose of directly analysing aspartate in pharmaceutical formulations and aspartame in sweeteners. This biosensor consists of an ammonia-sensitive gas-diffusion electrode and the enzyme L-aspartase immobilized by means of polyazetidine on a dialysis membrane.

  9. Regulation of N-methyl-D-aspartate receptor expression and N-methyl-D-aspartate-induced cellular response during chronic hypoxia in differentiated rat PC12 cells.

    PubMed

    Kobayashi, S; Millhorn, D E

    2000-01-01

    The purpose of the present study was to examine the effect of chronic hypoxia on N-methyl-D-aspartate-mediated cellular responses in differentiated PC12 cells. PC12 cells were differentiated by treatment with nerve growth factor. Patch-clamp analysis in differentiated PC12 cells showed that extracellularly applied N-methyl-D-aspartate induced an inward current that was abolished by the presence of the N-methyl-D-aspartate receptor antagonist MK-801. Results from Ca(2+) imaging experiments showed that N-methyl-D-aspartate induced an elevation in intracellular free Ca(2+) which was also abolished by MK-801. We also examined the effect of hypoxia on the N-methyl-D-aspartate-induced current in nerve growth factor-treated cells. We found that the N-methyl-D-aspartate-induced inward current and the N-methyl-D-aspartate-induced elevation in intracellular free Ca(2+) were markedly attenuated by chronic hypoxia. We next examined the possibility that the reduced N-methyl-D-aspartate responsiveness was due to down-regulation of N-methyl-D-aspartate receptor levels. Northern blot and immunoblot analyses showed that both messenger RNA and protein levels for N-methyl-D-aspartate receptor subunit 1 were markedly decreased during hypoxia. However, the messenger RNA for N-methyl-D-aspartate receptor subunit 2C was increased, whereas the protein level for subunit 2C did not change. Our results indicate that differentiated PC12 cells express functional N-methyl-D-aspartate receptors and that chronic exposure to hypoxia attenuates the N-methyl-D-aspartate-induced Ca(2+) accumulation in these cells via down-regulation of N-methyl-D-aspartate receptor subunit 1. This mechanism may play an important role in protecting PC12 cells against hypoxic stress. PMID:11113364

  10. Formation of {gamma}-alumina nanorods in presence of alanine

    SciTech Connect

    Dabbagh, Hossein A.; Rasti, Elham; Yalfani, Mohammad S.; Medina, Francesc

    2011-02-15

    Graphical abstract: Nanorod aluminas with a possible hexagonal symmetry, high surface area and relatively narrow pore size distribution were obtained. Research highlights: {yields} Research highlights {yields} Boehmite was prepared using a green sol-gel process in the presence of alanine. {yields} Nanorod aluminas with a high surface area were obtained. {yields} Addition of alanine would shape the size of the holes and crevices. {yields} The morphologies of the nanorods were revealed by transmission electron microscope. -- Abstract: Boehmite and alumina nanostructures were prepared using a simple green sol-gel process in the presence of alanine in water medium at room temperature. The uncalcined (dried at 200 {sup o}C) and the calcined materials (at 500, 600 and 700 {sup o}C for 4 h) were characterized using XRD, TEM, SEM, N{sub 2} physisorption and TGA. Nanorod aluminas with a possible hexagonal symmetry, high surface area and relatively narrow pore size distribution were obtained. The surface area was enhanced and crystallization was retarded as the alanine content increased. The morphologies of the nanoparticles and nanorods were revealed by a transmission electron microscope (TEM).

  11. Spectrophotometric readout for an alanine dosimeter for food irradiation applications

    NASA Astrophysics Data System (ADS)

    Ebraheem, S.; Beshir, W. B.; Eid, S.; Sobhy, R.; Kovács, A.

    2003-06-01

    The alanine-electron spin resonance (EPR) readout system is well known as a reference and transfer dosimetry system for the evaluation of high doses in radiation processing. The high cost of an EPR/alanine dosimetry system is a serious handicap for large-scale routine application in irradiation facilities. In this study, the use of a complex produced by dissolving irradiated L-alanine in 1,4-phenyl diammonium dichloride solution was investigated for dosimetry purposes. This complex—having a purple colour—has an increasing absorbance with increasing dose in the range of 1-20 kGy. The applicability of spectrophotometric evaluation was studied by measuring the absorbance intensity of this complex at 360 and 505 nm, respectively. Fluorimetric evaluation was also investigated by measuring the emission of the complex at 435 nm as a function of dose. The present method is easy for routine application. The effect of the dye concentration as well as the suitable amount of irradiated alanine has been studied. With respect to routine application, the stability of the product complex after its formation was also investigated.

  12. Importance of domain closure for the catalysis and regulation of Escherichia coli aspartate transcarbamoylase.

    PubMed

    Macol, Christine P; Tsuruta, Hiro; Kantrowitz, Evan R

    2002-07-26

    Two hybrid versions of Escherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the

  13. The unresolved puzzle why alanine extensions cause disease.

    PubMed

    Winter, Reno; Liebold, Jens; Schwarz, Elisabeth

    2013-08-01

    The prospective increase in life expectancy will be accompanied by a rise in the number of elderly people who suffer from ill health caused by old age. Many diseases caused by aging are protein misfolding diseases. The molecular mechanisms underlying these disorders receive constant scientific interest. In addition to old age, mutations also cause congenital protein misfolding disorders. Chorea Huntington, one of the most well-known examples, is caused by triplet extensions that can lead to more than 100 glutamines in the N-terminal region of huntingtin, accompanied by huntingtin aggregation. So far, nine disease-associated triplet extensions have also been described for alanine codons. The extensions lead primarily to skeletal malformations. Eight of these proteins represent transcription factors, while the nuclear poly-adenylate binding protein 1, PABPN1, is an RNA binding protein. Additional alanines in PABPN1 lead to the disease oculopharyngeal muscular dystrophy (OPMD). The alanine extension affects the N-terminal domain of the protein, which has been shown to lack tertiary contacts. Biochemical analyses of the N-terminal domain revealed an alanine-dependent fibril formation. However, fibril formation of full-length protein did not recapitulate the findings of the N-terminal domain. Fibril formation of intact PABPN1 was independent of the alanine segment, and the fibrils displayed biochemical properties that were completely different from those of the N-terminal domain. Although intranuclear inclusions have been shown to represent the histochemical hallmark of OPMD, their role in pathogenesis is currently unclear. Several cell culture and animal models have been generated to study the molecular processes involved in OPMD. These studies revealed a number of promising future therapeutic strategies that could one day improve the quality of life for the patients.

  14. Beta-alanine supplementation in high-intensity exercise.

    PubMed

    Harris, Roger C; Sale, Craig

    2012-01-01

    Glycolysis involves the oxidation of two neutral hydroxyl groups on each glycosyl (or glucosyl) unit metabolised, yielding two carboxylic acid groups. During low-intensity exercise these, along with the remainder of the carbon skeleton, are further oxidised to CO(2) and water. But during high-intensity exercise a major portion (and where blood flow is impaired, then most) is accumulated as lactate anions and H(+). The accumulation of H(+) has deleterious effects on muscle function, ultimately impairing force production and contributing to fatigue. Regulation of intracellular pH is achieved over time by export of H(+) out of the muscle, although physicochemical buffers in the muscle provide the first line of defence against H(+) accumulation. In order to be effective during high-intensity exercise, buffers need to be present in high concentrations in muscle and have pK(a)s within the intracellular exercise pH transit range. Carnosine (β-alanyl-L-histidine) is ideal for this role given that it occurs in millimolar concentrations within the skeletal muscle and has a pK(a) of 6.83. Carnosine is a cytoplasmic dipeptide formed by bonding histidine and β-alanine in a reaction catalysed by carnosine synthase, although it is the availability of β-alanine, obtained in small amounts from hepatic synthesis and potentially in greater amounts from the diet that is limiting to synthesis. Increasing muscle carnosine through increased dietary intake of β-alanine will increase the intracellular buffering capacity, which in turn might be expected to increase high-intensity exercise capacity and performance where this is pH limited. In this study we review the role of muscle carnosine as an H(+) buffer, the regulation of muscle carnosine by β-alanine, and the available evidence relating to the effects of β-alanine supplementation on muscle carnosine synthesis and the subsequent effects of this on high-intensity exercise capacity and performance.

  15. Alanine radicals, part 3: properties of the components contributing to the EPR spectrum of X-irradiated alanine dosimeters.

    PubMed

    Malinen, Eirik; Heydari, Mojgan Z; Sagstuen, Einar; Hole, Eli O

    2003-01-01

    The amino acid l-alpha-alanine has attracted considerable interest for use in radiation dosimetry and has been formally accepted as a secondary standard for high-dose and transfer dosimetry. Recent results have shown that the alanine EPR spectrum consists of contributions from three different radicals. A set of benchmark spectra describing the essential spectral features of these three radical components was used for reconstructions of the experimental spectra. In the present work, these basis spectra have been used to investigate the differential effects of variations in radiation doses and microwave power, as well as the dependence upon temperature annealing and UV illumination. The results presented here, based solely on relatively low-energy (60-80 keV) X rays, indicate that the three components behave very similarly with respect to radiation dose at room temperature. However, with respect to the thermal annealing/fading behavior and microwave power saturation properties, the three species behave significantly differently. It is concluded that even if it is now realized that three different radicals contribute to the composite EPR alanine spectrum, this has a minor impact on the established protocols for present-day applications (high-dose) of EPR/alanine dosimetry. However, some care should be exercised when e.g. constructing calibration curves, since fading and power saturation behavior may vary over the dose range in question. New results from UV-illumination experiments suggest a possible procedure for experimental spectral separation of the EPR signals due to the three radicals.

  16. Formation of simple biomolecules from alanine in ocean by impacts

    NASA Astrophysics Data System (ADS)

    Umeda, Y.; Sekine, T.; Furukawa, Y.; Kakegawa, T.; Kobayashi, T.

    2013-12-01

    The biomolecules on the Earth are thought either to have originated from the extraterrestrial parts carried with flying meteorites or to have been formed from the inorganic materials on the Earth through given energy. From the standpoint to address the importance of impact energy, it is required to simulate experimentally the chemical reactions during impacts, because violent impacts may have occurred 3.8-4.0 Gyr ago to create biomolecules initially. It has been demonstrated that shock reactions among ocean (H2O), atmospheric nitrogen, and meteoritic constitution (Fe) can induce locally reduction environment to form simple bioorganic molecules such as ammonia and amino acid (Nakazawa et al., 2005; Furukawa et al., 2009). We need to know possible processes for alanine how chemical reactions proceed during repeated impacts and how complicated biomolecules are formed. Alanine can be formed from glycine (Umeda et al., in preparation). In this study, we carried out shock recovery experiments at pressures of 4.4-5.7 GPa to investigate the chemical reactions of alanine. Experiments were carried out with a propellant gun. Stainless steel containers (30 mm in diameter, 30 mm long) with 13C-labeled alanine aqueous solution immersed in olivine or hematite powders were used as targets. Air gap was present in the sample room (18 mm in diameter, 2 mm thick) behind the sample. The powder, solution, and air represent meteorite, ocean, and atmosphere on early Earth, respectively. Two powders of olivine and hematite help to keep the oxygen fugacity low and high during experiments, respectively in order to investigate the effect of oxygen fugacity on chemical processes of alanine. The recovered containers, after cleaned completely, were immersed into liquid nitrogen to freeze sample solution and then we drilled on the impact surface to extract water-soluble run products using pure water. Thus obtained products were analyzed by LC/MS for four amino acids (glycine, alanine, valine, and

  17. Structure of the Mycobacterium tuberculosis D-Alanine:D-Alanine Ligase, a Target of the Antituberculosis Drug D-Cycloserine

    SciTech Connect

    Bruning, John B.; Murillo, Ana C.; Chacon, Ofelia; Barletta, Raúl G.; Sacchettini, James C.

    2011-09-28

    D-Alanine:D-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two D-alanine (D-Ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. D-Cycloserine (DCS), an analog of D-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 {angstrom}. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for D-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC{sub 50}) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.

  18. Kinetic mechanism and inhibition of Mycobacterium tuberculosis D-alanine:D-alanine ligase by the antibiotic D-cycloserine.

    PubMed

    Prosser, Gareth A; de Carvalho, Luiz Pedro S

    2013-02-01

    D-cycloserine (DCS) is an antibiotic that is currently used in second-line treatment of tuberculosis. DCS is a structural analogue of D-alanine, and targets two enzymes involved in the cytosolic stages of peptidoglycan synthesis: alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). The mechanisms of inhibition of DCS have been well-assessed using Alr and Ddl enzymes from various bacterial species, but little is known regarding the interactions of DCS with the mycobacterial orthologues of these enzymes. We have over-expressed and purified recombinant Mycobacterium tuberculosis Ddl (MtDdl; Rv2981c), and report a kinetic examination of the enzyme with both its native substrate and DCS. MtDdl is activated by K(+), follows an ordered ter ter mechanism and displays distinct affinities for D-Ala at each D-Ala binding site (K(m,D-Ala1) = 0.075 mm, K(m,D-Ala2) = 3.6 mm). ATP is the first substrate to bind and is necessary for subsequent binding of D-alanine or DCS. The pH dependence of MtDdl kinetic parameters indicate that general base chemistry is involved in the catalytic step. DCS was found to competitively inhibit D-Ala binding at both MtDdl D-Ala sites with equal affinity (K(i,DCS1) = 14 μm, K(i,DCS2) = 25 μm); however, each enzyme active site can only accommodate a single DCS molecule at a given time. The pH dependence of K(i,DCS2) revealed a loss of DCS binding affinity at high pH (pK(a) = 7.5), suggesting that DCS binds optimally in the zwitterionic form. The results of this study may assist in the design and development of novel Ddl-specific inhibitors for use as anti-mycobacterial agents.

  19. Aspartate 74 as a primary determinant in acetylcholinesterase governing specificity to cationic organophosphonates.

    PubMed

    Hosea, N A; Radić, Z; Tsigelny, I; Berman, H A; Quinn, D M; Taylor, P

    1996-08-20

    Through site-specific mutagenesis, we examined the determinants on acetylcholinesterase which govern the specificity and reactivity of three classes of substrates: enantiomeric alkyl phosphonates, trifluoromethyl acetophenones, and carboxyl esters. By employing cationic and uncharged pairs of enantiomeric alkyl methylphosphonyl thioates of known absolute stereochemistry, we find that an aspartate residue near the gorge entrance (D74) is responsible for the enhanced reactivity of the cationic organophosphonates. Removal of the charge with the mutation D74N causes a near equal reduction in the reaction rate constants for the Rp and Sp enantiomers and exerts a greater influence on the cationic organophosphonates than on the charged trimethylammonio trifluoromethyl acetophenone and acetylthiocholine. This pattern of reactivity suggests that the orientation of the leaving group for both enantiomers is directed toward the gorge exit and in apposition to Asp 74. Replacement of tryptophan 86 with alanine in the choline subsite also diminishes the reaction rates for cationic organophosphonates, although to a lesser extent than with the D74N mutation, while not affecting the reactions with the uncharged compounds. Hence, reaction with cationic OPs depends to a lesser degree on Trp 86 than on Asp 74. Docking of Sp and Rp cycloheptyl methylphosphonyl thiocholines and thioethylates in AChE as models of the reversible complex and transition state using molecular dynamics affords structural insight into the spatial arrangement of the substituents surrounding phosphorus prior to and during reaction. The leaving group of the Rp and Sp enantiomers, regardless of charge, is directed to the gorge exit and toward Asp 74, an orientation unique to tetrahedral ligands. PMID:8718893

  20. New aspartic proteinase of Ulysses retrotransposon from Drosophila virilis.

    PubMed

    Volkov, D A; Dergousova, N I; Rumsh, L D

    2004-06-01

    This work is focused on the investigation of a proteinase of Ulysses mobile genetic element from Drosophila virilis. The primary structure of this proteinase is suggested based on comparative analysis of amino acid sequences of aspartic proteinases from retroviruses and retrotransposons. The corresponding cDNA fragment has been cloned and expressed in E. coli. The protein accumulated in inclusion bodies. The recombinant protein (12 kD) was subjected to refolding and purified by affinity chromatography on pepstatin-agarose. Proteolytic activity of the protein was determined using oligopeptide substrates melittin and insulin B-chain. It was found that the maximum of the proteolytic activity is displayed at pH 5.5 as for the majority of aspartic proteinases. We observed that hydrolysis of B-chain of insulin was totally inhibited by pepstatin A in the micromolar concentration range. The molecular weight of the monomer of the Ulysses proteinase was determined by MALDI-TOF mass-spectrometry.

  1. Behavior of aspartic acid as a corrosion inhibitor for steel

    SciTech Connect

    Kalota, D.J.; Silverman, D.C. )

    1994-02-01

    Corrosion inhibition of steel by aspartic acid (C[sub 4]H[sub 7]NO[sub 4]), an amino acid of low molecular weight, was found to depend strongly on pH. At a pH less than the ionization constant at [approximately]9.5 to 10 (measured at 25 C), C[sub 4]H[sub 7]NO[sub 4] appeared to accelerate corrosion. Above the pH, it acted as a corrosion inhibitor for steel. A specially constructed potential-pH diagram for iron (Fe) that incorporated C[sub 4]H[sub 7]NO[sub 4] showed the change in behavior was accompanied by the most stable thermodynamic state changing from an iron aspartate complex to iron oxide. Polymerized C[sub 4]H[sub 7]NO[sub 4] (polyaspartic acid) behaved in a similar manner. Some other amino acids of low molecular weight behaved similarly.

  2. Participation of cysteine and cystine in inactivation of tyrosine aminotransferase in rat liver homogenates.

    PubMed Central

    Buckley, W T; Milligan, L P

    1978-01-01

    1. Inactivation of tyrosine aminotransferase was studied in rat liver homogenates. Under an O2 atmosphere with cysteine added, inactivation was rapid after a lag period of approx. 1h, whereas a N2 atmosphere extended the lag period to approx. 3h. 2. Replacement of cysteine with cystine resulted in rapid inactivation both aerobically and anaerobically. 3. Removal of the particulate fraction by centrifuging rat liver homogenates at 13,000g for 9min resulted in an aerobic lag period of 0.5h in the presence of cystine and approx. 3h in the presence of cysteine. 4. It is proposed that the stimulatory effect of cysteine on tyrosine aminotransferase inactivation occurs largely as a result of oxidation to cystine, which appears to be a more directly effective agent. PMID:33669

  3. Study on the EPR/dosimetric properties of some substituted alanines

    NASA Astrophysics Data System (ADS)

    Gancheva, Veselka; Sagstuen, Einar; Yordanov, Nicola D.

    2006-02-01

    Polycrystalline phenyl-alanine and perdeuterated L- α-alanine ( L- α-alanine-d 4) were studied as potential high-energy radiation-sensitive materials (RSM) for solid state/EPR dosimetry. It was found that phenyl-alanine exhibits a linear dose response in the dose region 0.1-17 kGy. However, phenyl-alanine is about 10 times less sensitive to γ-irradiation than standard L- α-alanine irradiated at the same doses. Moreover, the EPR response from phenyl-alanine is unstable and, independent of the absorbed dose, decreases by about 50% within 20 days after irradiation upon storage at room temperature. γ-irradiated polycrystalline perdeuterated L- α-alanine (CD 3CD(NH 2)COOH) has not previously been studied at room temperature by EPR spectroscopy. The first part of the present analysis was with respect to the structure of the EPR spectrum. By spectrum simulations, the presence of at least two radiation induced free radicals, R 1=CH 3C •(H)COOH and R 2=H 3N +-C •(CH 3)COO -, was confirmed very clearly. Both these radicals were suggested previously from EPR and ENDOR studies of standard alanine crystals. The further investigations into the potential use of alanine-d 4 as RSM, after choosing optimal EPR spectrometer settings parameters for this purpose, show that it is ca. two times more sensitive than standard L- α-alanine.

  4. Degradation of glycine and alanine on irradiated quartz.

    PubMed

    Pawlikowski, Maciej; Benko, Aleksandra; Wróbel, Tomasz P

    2013-04-01

    Recent researches suggest participation of minerals in the formation of life under primordial conditions. Among all of the minerals, quartz seems to be one of the most probable to take part in such processes. However, an external source of energy is needed, e.g. electric discharge. A device simulating the proposed conditions was designed and was used to simulate prebiotic conditions. Investigation of processes occurring during the stimulation of quartz with electric discharge was studied by means of Ultraviolet-visible (UV-VIS) spectroscopy, in order to monitor the generation kinetics of free radicals. Additionally, infrared spectroscopy was applied to identify chemical reaction products created in a solution of alanine or glycine, in the presence of quartz treated with electric discharge. Formation of increased amounts of free radicals, compared to experiments performed without quartz and/or amino acid, is reported, along with identification of possible degradation products of alanine. No synthetic reactions were observed.

  5. Vesicular uptake and exocytosis of l-aspartate is independent of sialin

    PubMed Central

    Morland, Cecilie; Nordengen, Kaja; Larsson, Max; Prolo, Laura M.; Farzampour, Zoya; Reimer, Richard J.; Gundersen, Vidar

    2013-01-01

    The mechanism of release and the role of l-aspartate as a central neurotransmitter are controversial. A vesicular release mechanism for l-aspartate has been difficult to prove, as no vesicular l-aspartate transporter was identified until it was found that sialin could transport l-aspartate and l-glutamate when reconstituted into liposomes. We sought to clarify the release mechanism of l-aspartate and the role of sialin in this process by combining l-aspartate uptake studies in isolated synaptic vesicles with immunocyotchemical investigations of hippocampal slices. We found that radiolabeled l-aspartate was taken up into synaptic vesicles. The vesicular l-aspartate uptake, relative to the l-glutamate uptake, was twice as high in the hippocampus as in the whole brain, the striatum, and the entorhinal and frontal cortices and was not inhibited by l-glutamate. We further show that sialin is not essential for exocytosis of l-aspartate, as there was no difference in ATP-dependent l-aspartate uptake in synaptic vesicles from sialin-knockout and wild-type mice. In addition, expression of sialin in PC12 cells did not result in significant vesicle uptake of l-aspartate, and depolarization-induced depletion of l-aspartate from hippocampal nerve terminals was similar in hippocampal slices from sialin-knockout and wild-type mice. Further, there was no evidence for nonvesicular release of l-aspartate via volume-regulated anion channels or plasma membrane excitatory amino acid transporters. This suggests that l-aspartate is exocytotically released from nerve terminals after vesicular accumulation by a transporter other than sialin.—Morland, C., Nordengen, K., Larsson, M., Prolo, L. M., Farzampour, Z., Reimer, R. J., Gundersen, V. Vesicular uptake and exocytosis of l-aspartate is independent of sialin. PMID:23221336

  6. Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion.

    PubMed

    Sousa, Cristovão M; Biancur, Douglas E; Wang, Xiaoxu; Halbrook, Christopher J; Sherman, Mara H; Zhang, Li; Kremer, Daniel; Hwang, Rosa F; Witkiewicz, Agnes K; Ying, Haoqiang; Asara, John M; Evans, Ronald M; Cantley, Lewis C; Lyssiotis, Costas A; Kimmelman, Alec C

    2016-08-25

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by an intense fibrotic stromal response and deregulated metabolism. The role of the stroma in PDAC biology is complex and it has been shown to play critical roles that differ depending on the biological context. The stromal reaction also impairs the vasculature, leading to a highly hypoxic, nutrient-poor environment. As such, these tumours must alter how they capture and use nutrients to support their metabolic needs. Here we show that stroma-associated pancreatic stellate cells (PSCs) are critical for PDAC metabolism through the secretion of non-essential amino acids (NEAA). Specifically, we uncover a previously undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle, and thus NEAA and lipid biosynthesis. This shift in fuel source decreases the tumour’s dependence on glucose and serum-derived nutrients, which are limited in the pancreatic tumour microenvironment. Moreover, we demonstrate that alanine secretion by PSCs is dependent on PSC autophagy, a process that is stimulated by cancer cells. Thus, our results demonstrate a novel metabolic interaction between PSCs and cancer cells, in which PSC-derived alanine acts as an alternative carbon source. This finding highlights a previously unappreciated metabolic network within pancreatic tumours in which diverse fuel sources are used to promote growth in an austere tumour microenvironment. PMID:27509858

  7. Characterization of psychrophilic alanine racemase from Bacillus psychrosaccharolyticus.

    PubMed

    Okubo, Y; Yokoigawa, K; Esaki, N; Soda, K; Kawai, H

    1999-03-16

    A psychrophilic alanine racemase gene from Bacillus psychrosaccharolyticus was cloned and expressed in Escherichia coli SOLR with a plasmid pYOK3. The gene starting with the unusual initiation codon GTG showed higher preference for codons ending in A or T. The enzyme purified to homogeneity showed the high catalytic activity even at 0 degrees C and was extremely labile over 35 degrees C. The enzyme was found to have a markedly large Km value (5.0 microM) for the pyridoxal 5'-phosphate (PLP) cofactor in comparison with other reported alanine racemases, and was stabilized up to 50 degrees C in the presence of excess amounts of PLP. The low affinity of the enzyme for PLP may be related to the thermolability, and may be related to the high catalytic activity, initiated by the transaldimination reaction, at low temperature. The enzyme has a distinguishing hydrophilic region around the residue no. 150 in the deduced amino acid sequence (383 residues), whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of the region in the three dimensional structure of C atoms of the enzyme was predicted to be in a surface loop surrounding the active site. The region may interact with solvent and reduce the compactness of the active site. PMID:10080917

  8. Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion.

    PubMed

    Sousa, Cristovão M; Biancur, Douglas E; Wang, Xiaoxu; Halbrook, Christopher J; Sherman, Mara H; Zhang, Li; Kremer, Daniel; Hwang, Rosa F; Witkiewicz, Agnes K; Ying, Haoqiang; Asara, John M; Evans, Ronald M; Cantley, Lewis C; Lyssiotis, Costas A; Kimmelman, Alec C

    2016-08-25

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by an intense fibrotic stromal response and deregulated metabolism. The role of the stroma in PDAC biology is complex and it has been shown to play critical roles that differ depending on the biological context. The stromal reaction also impairs the vasculature, leading to a highly hypoxic, nutrient-poor environment. As such, these tumours must alter how they capture and use nutrients to support their metabolic needs. Here we show that stroma-associated pancreatic stellate cells (PSCs) are critical for PDAC metabolism through the secretion of non-essential amino acids (NEAA). Specifically, we uncover a previously undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle, and thus NEAA and lipid biosynthesis. This shift in fuel source decreases the tumour’s dependence on glucose and serum-derived nutrients, which are limited in the pancreatic tumour microenvironment. Moreover, we demonstrate that alanine secretion by PSCs is dependent on PSC autophagy, a process that is stimulated by cancer cells. Thus, our results demonstrate a novel metabolic interaction between PSCs and cancer cells, in which PSC-derived alanine acts as an alternative carbon source. This finding highlights a previously unappreciated metabolic network within pancreatic tumours in which diverse fuel sources are used to promote growth in an austere tumour microenvironment.

  9. Probing alanine transaminase catalysis with hyperpolarized 13CD3-pyruvate

    NASA Astrophysics Data System (ADS)

    Barb, A. W.; Hekmatyar, S. K.; Glushka, J. N.; Prestegard, J. H.

    2013-03-01

    Hyperpolarized metabolites offer a tremendous sensitivity advantage (>104 fold) when measuring flux and enzyme activity in living tissues by magnetic resonance methods. These sensitivity gains can also be applied to mechanistic studies that impose time and metabolite concentration limitations. Here we explore the use of hyperpolarization by dissolution dynamic nuclear polarization (DNP) in mechanistic studies of alanine transaminase (ALT), a well-established biomarker of liver disease and cancer that converts pyruvate to alanine using glutamate as a nitrogen donor. A specific deuterated, 13C-enriched analog of pyruvic acid, 13C3D3-pyruvic acid, is demonstrated to have advantages in terms of detection by both direct 13C observation and indirect observation through methyl protons introduced by ALT-catalyzed H-D exchange. Exchange on injecting hyperpolarized 13C3D3-pyruvate into ALT dissolved in buffered 1H2O, combined with an experimental approach to measure proton incorporation, provided information on mechanistic details of transaminase action on a 1.5 s timescale. ALT introduced, on average, 0.8 new protons into the methyl group of the alanine produced, indicating the presence of an off-pathway enamine intermediate. The opportunities for exploiting mechanism-dependent molecular signatures as well as indirect detection of hyperpolarized 13C3-pyruvate and products in imaging applications are discussed.

  10. Alanine-dependent reactions of 5'-deoxypyridoxal in water.

    PubMed

    Go, Maybelle K; Richard, John P

    2008-12-01

    The non-enzymatic reaction of 5'-deoxypyridoxal (DPL) with l-alanine in water at 25 degrees C was investigated. DPL reacts with alanine to form an imine, which then undergoes deprotonation at the alpha-amino carbon of alanine to form a resonance delocalized DPL-stabilized carbanion. At early reaction times the only detectable products are pyruvate and the dimeric species formed by addition of the alpha-pyridine stabilized carbanion to DPL. No Claisen-type products of addition of the alpha-amino carbanion to DPL, as was previously reported to form from the reaction between DPL and glycine [K. Toth, T.L. Amyes, J.P. Richard, J.P.G. Malthouse, M.E. Ni Beilliu, J. Am. Chem. Soc. 126 (2004) 10538-10539], are observed. The electrophile reacts instead at the alpha-pyridyl carbon. This dimer is in chemical equilibrium with reactants. At longer reaction times about 50% of DPL is converted to 5'-deoxypyridoxamine, the thermodynamically favored product of formal transamination of DPL.

  11. Suspected fusariomycotoxicosis in sandhill cranes (Grus canadensis): clinical and pathological findings.

    USGS Publications Warehouse

    Roffe, Thomas J.; Stroud, Richard K.; Windingstad, Ronald M.

    1989-01-01

    In 1985 and 1986, large-scale natural die-offs of sandhill cranes in Texas were attributed to fusariomycotoxicosis. These birds demonstrated a progressive loss of motor control to the neck, wings, and legs. Based on necropsy and/or histopathology of 31 cranes, the most common lesions involved skeletal muscle and included hemorrhages, granulomatous myositis, thrombosis, and vascular degeneration. Serum chemistry results revealed that levels of creatinine kinase, aspartate aminotransferase, and alanine aminotransferase were above published normals. However, only alanine aminotransferase was higher in clinically affected cranes than in normal cranes collected from the same area.

  12. In Vivo d-Serine Hetero-Exchange through Alanine-Serine-Cysteine (ASC) Transporters Detected by Microelectrode Biosensors

    PubMed Central

    2013-01-01

    d-Serine, a co-agonist of N-methyl d-aspartate (NMDA) receptors, has been implicated in neurological and psychiatric disorders such as cerebral ischemia, lateral amyotrophic sclerosis, or schizophrenia. d-Serine signaling represents an important pharmacological target for treating these diseases; however, the biochemical mechanisms controlling extracellular d-serine levels in vivo are still unclear. d-Serine heteroexchange through small neutral amino acid transporters has been shown in cell cultures and brain slices and could provide a biochemical mechanism for the control of d-serine extracellular concentration in vivo. Alternatively, exocytotic d-serine release has also been proposed. In this study, the dynamics of d-serine release and clearance were explored in vivo on a second-by-second time scale using microelectrode biosensors. The rate of d-serine clearance in the rat frontal cortex after a microionophoretic injection revealed a transporter-mediated uptake mechanism. d-Serine uptake was blocked by small neutral l-amino acids, implicating alanine-serine-cysteine (ASC) transporters, in particular high affinity Asc-1 and low affinity ASCT2 transporters. Interestingly, changes in alanine, serine, or threonine levels resulted in d-serine release through ASC transporters. Asc-1, but not ASCT2, appeared to release d-serine in response to changes in amino acid concentrations. Finally, neuronal silencing by tetrodotoxin increased d-serine extracellular concentration by an ASC-transporter-dependent mechanism. Together, these results indicate that d-serine heteroexchange through ASC transporters is present in vivo and may constitute a key component in the regulation of d-serine extracellular concentration. PMID:23581544

  13. Aspartic proteinases in the digestive tract of marine decapod crustaceans.

    PubMed

    Navarrete del Toro, María de Los Angeles; García-Carreño, Fernando; López, Manuel Díaz; Celis-Guerrero, Laura; Saborowski, Reinhard

    2006-08-01

    Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others. PMID:16788916

  14. Pediatric anti-N methyl D aspartate receptor encephalitis.

    PubMed

    Suri, Vinit; Sharma, Sushma; Gupta, Rohan; Sogani, S K; Mediratta, Sunit; Jadhao, Nilesh

    2013-05-01

    Anti-N Methyl D Aspartate Receptor encephalitis (anti-NMDARE) is a recently defined disease, which is probably more under-recognized than rare. We report a case of anti-NMDARE in a 13-years-old girl, who presented with intractable seizures. To the best of our knowledge, this is the second case of pediatric anti-NMDARE being reported from India. The need for a greater awareness of this disease and the subtle differences in clinical presentation between pediatric and adult patients are highlighted. PMID:24082929

  15. The cytosolic branched-chain aminotransferases of Arabidopsis thaliana influence methionine supply, salvage and glucosinolate metabolism.

    PubMed

    Lächler, Kurt; Imhof, Janet; Reichelt, Michael; Gershenzon, Jonathan; Binder, Stefan

    2015-05-01

    Arabidopsis thaliana possesses six branched-chain aminotransferases (BCAT1-6). Previous studies revealed that some members of this protein family are involved in the biosynthesis of branched-chain amino acids and/or in the Met chain elongation pathway, the initial steps towards the biosynthesis of Met-derived glucosinolates. We now analyzed branched-chain aminotransferase 6 (BCAT6). In vivo GFP-tagging experiments strongly suggest this enzyme to be localized to the cytosol. Substrate specificity assays performed with recombinant enzyme revealed that BCAT6 transaminates Val, Leu and Ile as well as the corresponding 2-oxo acids but also transaminates Met and its cognate ketoacid 4-methyl-2-oxobutanoate. We established single (bcat6-1), double (bcat4-2/bcat6-1) and triple (bcat3-1/bcat4-2/bcat6-1) mutants involving BCAT6 with the latter exhibiting a clear macroscopic phenotype with smaller plants and abnormal leaf morphology. Metabolite profiling of these mutants demonstrated that BCAT6 can contribute to Met chain elongation with the triple mutant line lacking BCAT3, 4 and 6 showing a dramatic reduction of Met-derived glucosinolate species down to 32 and 14% of wild-type levels in plant foliage and seeds, respectively. This drop in glucosinolate levels is accompanied by a 46-fold increase of free Met, demonstrating the important role of the three branched-chain aminotransferases in converting Met to its 2-oxo acid for glucosinolate chain elongation. In addition, we determined the relative amounts of 5'-deoxy-5'-methylthioadenosine, an intermediate of the Met recycling pathway. This metabolite accumulated to relative high amounts in the absence of the cytosolic BCAT4 and BCAT6, suggesting that cytosolic Met salvage also contributes to the biosynthesis of glucosinolates. PMID:25851613

  16. Homology modeling of human kynurenine aminotransferase III and observations on inhibitor binding using molecular docking.

    PubMed

    Nematollahi, Alireza; Church, William B; Nadvi, Naveed A; Gorrell, Mark D; Sun, Guanchen

    2014-01-01

    Kynurenine aminotransferase (KAT) isozymes are responsible for catalyzing the conversion of kynurenine (KYN) to kynurenic acid (KYNA), which is considered to play a key role in central nervous system (CNS) disorders, including schizophrenia. The levels of KYNA in the postmortem prefrontal cortex and in the Cerebrospinal fluid (CSF) of schizophrenics are greater than normal brain. A basic strategy to decrease kynurenic acid levels is to promote the inhibition of the biosynthetic KAT isozymes. As there is no crystallographic model for human kynurenine aminotransferase III (KAT III), therefore, homology modeling has been performed based on the Mus musculus kynurenine aminotransferase III crystal structure (PDB ID: 3E2Y) as a template, and the model of the human KAT III was refined and optimized with molecular dynamics simulations. Further evaluation of the model quality was accomplished by investigating the interaction of KAT III inhibitors with the modeled enzyme. Such interactions were determined employing the AutoDock 4.2 program using the MGLTools 1.5.6 package. The most important interactions for the binding of the inhibitors, which are probably also central components of the active site of KAT III, were identified as Ala134, Tyr135, Lys 280, Lys 288, Thr285 and Arg429, which provide hydrogen bond interactions. Additionally, Tyr135 and Arg429 have good electrostatic interactions with inhibitors consistent with these residues also being essential for inhibition of the enzyme activity. We expect that this model and these docking data will be a useful resource for the rational design of novel drugs for treating neuropathologies.

  17. Homology modeling of human kynurenine aminotransferase III and observations on inhibitor binding using molecular docking.

    PubMed

    Nematollahi, Alireza; Church, William B; Nadvi, Naveed A; Gorrell, Mark D; Sun, Guanchen

    2014-01-01

    Kynurenine aminotransferase (KAT) isozymes are responsible for catalyzing the conversion of kynurenine (KYN) to kynurenic acid (KYNA), which is considered to play a key role in central nervous system (CNS) disorders, including schizophrenia. The levels of KYNA in the postmortem prefrontal cortex and in the Cerebrospinal fluid (CSF) of schizophrenics are greater than normal brain. A basic strategy to decrease kynurenic acid levels is to promote the inhibition of the biosynthetic KAT isozymes. As there is no crystallographic model for human kynurenine aminotransferase III (KAT III), therefore, homology modeling has been performed based on the Mus musculus kynurenine aminotransferase III crystal structure (PDB ID: 3E2Y) as a template, and the model of the human KAT III was refined and optimized with molecular dynamics simulations. Further evaluation of the model quality was accomplished by investigating the interaction of KAT III inhibitors with the modeled enzyme. Such interactions were determined employing the AutoDock 4.2 program using the MGLTools 1.5.6 package. The most important interactions for the binding of the inhibitors, which are probably also central components of the active site of KAT III, were identified as Ala134, Tyr135, Lys 280, Lys 288, Thr285 and Arg429, which provide hydrogen bond interactions. Additionally, Tyr135 and Arg429 have good electrostatic interactions with inhibitors consistent with these residues also being essential for inhibition of the enzyme activity. We expect that this model and these docking data will be a useful resource for the rational design of novel drugs for treating neuropathologies. PMID:24739074

  18. Ergogenic effects of β-alanine and carnosine: proposed future research to quantify their efficacy.

    PubMed

    Caruso, John; Charles, Jessica; Unruh, Kayla; Giebel, Rachel; Learmonth, Lexis; Potter, William

    2012-07-01

    β-alanine is an amino acid that, when combined with histidine, forms the dipeptide carnosine within skeletal muscle. Carnosine and β-alanine each have multiple purposes within the human body; this review focuses on their roles as ergogenic aids to exercise performance and suggests how to best quantify the former's merits as a buffer. Carnosine normally makes a small contribution to a cell's total buffer capacity; yet β-alanine supplementation raises intracellular carnosine concentrations that in turn improve a muscle's ability to buffer protons. Numerous studies assessed the impact of oral β-alanine intake on muscle carnosine levels and exercise performance. β-alanine may best act as an ergogenic aid when metabolic acidosis is the primary factor for compromised exercise performance. Blood lactate kinetics, whereby the concentration of the metabolite is measured as it enters and leaves the vasculature over time, affords the best opportunity to assess the merits of β-alanine supplementation's ergogenic effect. Optimal β-alanine dosages have not been determined for persons of different ages, genders and nutritional/health conditions. Doses as high as 6.4 g day(-1), for ten weeks have been administered to healthy subjects. Paraesthesia is to date the only side effect from oral β-alanine ingestion. The severity and duration of paraesthesia episodes are dose-dependent. It may be unwise for persons with a history of paraesthesia to ingest β-alanine. As for any supplement, caution should be exercised with β-alanine supplementation.

  19. Performance effects of acute β-alanine induced paresthesia in competitive cyclists.

    PubMed

    Bellinger, Phillip M; Minahan, Clare L

    2016-01-01

    β-alanine is a common ingredient in supplements consumed by athletes. Indeed, athletes may believe that the β-alanine induced paresthesia, experienced shortly after ingestion, is associated with its ergogenic effect despite no scientific mechanism supporting this notion. The present study examined changes in cycling performance under conditions of β-alanine induced paresthesia. Eight competitive cyclists (VO2max = 61.8 ± 4.2 mL·kg·min(-1)) performed three practices, one baseline and four experimental trials. The experimental trials comprised a 1-km cycling time trial under four conditions with varying information (i.e., athlete informed β-alanine or placebo) and supplement content (athlete received β-alanine or placebo) delivered to the cyclist: informed β-alanine/received β-alanine, informed placebo/received β-alanine, informed β-alanine/received placebo and informed placebo/received placebo. Questionnaires were undertaken exploring the cyclists' experience of the effects of the experimental conditions. A possibly likely increase in mean power was associated with conditions in which β-alanine was administered (±95% CL: 2.2% ± 4.0%), but these results were inconclusive for performance enhancement (p = 0.32, effect size = 0.18, smallest worthwhile change = 56% beneficial). A possibly harmful effect was observed when cyclists were correctly informed that they had ingested a placebo (-1.0% ± 1.9%). Questionnaire data suggested that β-alanine ingestion resulted in evident sensory side effects and six cyclists reported placebo effects. Acute ingestion of β-alanine is not associated with improved 1-km TT performance in competitive cyclists. These findings are in contrast to the athlete's "belief" as cyclists reported improved energy and the ability to sustain a higher power output under conditions of β-alanine induced paresthesia.

  20. Wheat-germ aspartate transcarbamoylase. Purification and cold-lability.

    PubMed Central

    Grayson, J E; Yon, R J; Butterworth, P J

    1979-01-01

    1. Aspartate transcarbamoylase was purified approx. 3000-fold from wheat (Triticum vulgare) germ in 15-20% yield. The product has a specific activity of 14 mumol/min per mg of protein and is approx. 90% pure. The purification scheme includes the use of biospecific "imphilyte" chromatography as described by Yon [Biochem.J.(1977) 161, 233-237]. The enzyme was passed successively through columns of CPAD [N-(3-carboxypropionyl)aminodecyl]-Sepharose in the absence and presence respectively of the ligands UMP and L-aspartate. In the second passage the enzyme was specifically displaced away from impurities with which it co-migrated in the first passage. These two steps contributed a factor of 80 to the overall purification. 2. The enzyme is slowly inactivated on dilution at 0 degrees C and pH 7.0, the inactivation being partially reversible. A detailed investigation of the temperature- and pH-dependence of the cold-inactivation suggested that it was initiated by the perturbation of the pKa values of groups with a moderately high and positive heat of ionization, which were tentatively identified as histidine residues. These findings support a new concept of cold-lability proposed by Bock, Gilbert & Frieden [Biochem. Biophys. Res. Commun. (1975) 66, 564-569]. PMID:43131

  1. A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing.

    PubMed

    Xie, Jian; Owen, Timothy; Xia, Ke; Callahan, Brian; Wang, Chunyu

    2016-08-31

    Hedgehog (Hh) signaling is driven by the cholesterol-modified Hh ligand, generated by autoprocessing of Hh precursor protein. Two steps in Hh autoprocessing, N-S acyl shift and transesterification, must be coupled for efficient Hh cholesteroylation and downstream signal transduction. In the present study, we show that a conserved aspartate residue, D46 of the Hh autoprocessing domain, coordinates these two catalytic steps. Mutagenesis demonstrated that D46 suppresses non-native Hh precursor autoprocessing and is indispensable for transesterification with cholesterol. NMR measurements indicated that D46 has a pKa of 5.6, ∼2 units above the expected pKa of aspartate, due to a hydrogen-bond between protonated D46 and a catalytic cysteine residue. However, the deprotonated form of D46 side chain is also essential, because a D46N mutation cannot mediate cholesteroylation. On the basis of these data, we propose that the proton shuttling of D46 side chain mechanistically couples the two steps of Hh cholesteroylation. PMID:27529645

  2. The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography.

    PubMed

    Canyuk, B; Focia, P J; Eakin, A E

    2001-03-01

    The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribosyltransferases (HPRTs) was examined by site-directed and saturation mutagenesis, functional analysis, and X-ray crystallography using the HPRT from Trypanosoma cruzi. Alanine substitution (D137A) resulted in a 30-fold decrease of k(cat), suggesting that Asp137 participates in catalysis. Saturation mutagenesis was used to generate a library of mutant HPRTs with random substitutions at position 137, and active enzymes were identified by complementation of a bacterial purine auxotroph. Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate dependence, indicate that glutamic acid or glutamine can replace the wild-type aspartate. However, the catalytic efficiency and pH-rate profile for the structural isosteric mutant, D137N, were similar to the D137A mutant. Crystal structures of four of the mutant enzymes were determined in ternary complex with substrate ligands. Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in addition to protein atoms, that position these side chains within hydrogen bond distance of the bound purine analogue, similar in position to the aspartate in the wild-type structure. The crystal structure of the D137N mutant demonstrates that the Asn137 side chain does not form interactions with the purine substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer. The results from these structural and functional analyses demonstrate that HPRTs do not require a general base at position 137 for catalysis. Instead, hydrogen bonding sufficiently stabilizes the developing partial positive charge at the N7-atom of the purine substrate in the transition-state to promote catalysis.

  3. Use of the entire spectrum of irradiated alanine for dosimetry.

    PubMed

    Dolo, J M; Moignau, F

    2005-02-01

    Alanine is an amino acid commonly used in ESR dosimetry as a reference detector. The classic approach for the measurement of irradiated samples is to determine the amplitude of the central peak of the first derivative spectrum. It is generally considered that this technique represents the best and most reproducible solution for achieving an accurate proportionality between the concentration of free radicals inside the resonant cavity, characterized by the amplitude, and the dose. It is also accepted that this central peak corresponds to the free radical CH3CHCOO-. The hyperfine structure of this radical in the spectrum shows five main peaks with the approximate ratios 1:4:6:4:1 as regards coupling. This paper presents another approach featuring analysis of the entire spectrum: (i) ratios of identified peaks, (ii) ratio variation vs time with regard to several parameters affecting fading. These variations in the alanine spectrum are probably correlated with the variation of the concentrations of different free radical species. These variations and their positions in the spectrum are very important constraints that increase the uncertainty of this type of measurement.

  4. The effect of immunonutrition (glutamine, alanine) on fracture healing

    PubMed Central

    Küçükalp, Abdullah; Durak, Kemal; Bayyurt, Sarp; Sönmez, Gürsel; Bilgen, Muhammed S.

    2014-01-01

    Background There have been various studies related to fracture healing. Glutamine is an amino acid with an important role in many cell and organ functions. This study aimed to make a clinical, radiological, and histopathological evaluation of the effects of glutamine on fracture healing. Methods Twenty rabbits were randomly allocated into two groups of control and immunonutrition. A fracture of the fibula was made to the right hind leg. All rabbits received standard food and water. From post-operative first day for 30 days, the study group received an additional 2 ml/kg/day 20% L-alanine L-glutamine solution via a gastric catheter, and the control group received 2 ml/kg/day isotonic via gastric catheter. At the end of 30 days, the rabbits were sacrificed and the fractures were examined clinically, radiologically, and histopathologically in respect to the degree of union. Results Radiological evaluation of the control group determined a mean score of 2.5 according to the orthopaedists and 2.65 according to the radiologists. In the clinical evaluation, the mean score was 1.875 for the control group and 2.0 for the study group. Histopathological evaluation determined a mean score of 8.5 for the control group and 9.0 for the study group. Conclusion One month after orally administered glutamine–alanine, positive effects were observed on fracture healing radiologically, clinically, and histopathologically, although no statistically significant difference was determined.

  5. Formation of chloroform during chlorination of alanine in drinking water.

    PubMed

    Chu, Wen-Hai; Gao, Nai-Yun; Deng, Yang; Dong, Bing-Zhi

    2009-11-01

    Currently, dissolved nitrogenous organic matters in water, important precursors of disinfection by-products (DBPs), are of significant concern. This study was to explore the formation of chloroform (CF) during chlorination of alanine (Ala), an important nitrogenous organic compound commonly present in water sources. Our results indicated that the CF yield reached a maximum value of 0.143% at the molar ratio of chlorine atom to nitrogen atom (Cl/N)=1.0 over a Cl/N range of 0.2-5.0 (pH=7.0, reaction time=5d, and initial Ala=0.1mM). At an acidic-neutral condition (pH 4-7), the formation of CF was suppressed. However, the highest CF yield (0.227%) occurred at weakly alkaline condition (pH 8.0) (initial Ala=0.1mM, and Cl/N=1.0). The increase of Br(-) in water can increase total trihalomethanes (THMs) and bromo-THMs. However, the bromo-THMs level reached a plateau at Br(-)/Cl>0.04. Finally, based on the computation of frontier electron density and identification and measurement of key intermediates during Ala chlorination, we proposed a formation pathway of CF from Ala chlorination: Ala-->monochloro-N-alanine (MC-N-Ala)-->acetaldehyde (AAld)-->monochloroacetaldehyde acetaldehyde (MCAld)-->dichloroacetaldehyde (DCAld)-->trichloroacetaldehyde (TCAld)-->CF.

  6. Biochemical and Structural Insights into the Aminotransferase CrmG in Caerulomycin Biosynthesis.

    PubMed

    Zhu, Yiguang; Xu, Jinxin; Mei, Xiangui; Feng, Zhan; Zhang, Liping; Zhang, Qingbo; Zhang, Guangtao; Zhu, Weiming; Liu, Jinsong; Zhang, Changsheng

    2016-04-15

    Caerulomycin A (CRM A 1) belongs to a family of natural products containing a 2,2'-bipyridyl ring core structure and is currently under development as a potent novel immunosuppressive agent. Herein, we report the functional characterization, kinetic analysis, substrate specificity, and structure insights of an aminotransferase CrmG in 1 biosynthesis. The aminotransferase CrmG was confirmed to catalyze a key transamination reaction to convert an aldehyde group to an amino group in the 1 biosynthetic pathway, preferring l-glutamate and l-glutamine as the amino donor substrates. The crystal structures of CrmG in complex with the cofactor 5'-pyridoxal phosphate (PLP) or 5'-pyridoxamine phosphate (PMP) or the acceptor substrate were determined to adopt a canonical fold-type I of PLP-dependent enzymes with a unique small additional domain. The structure guided site-directed mutagenesis identified key amino acid residues for substrate binding and catalytic activities, thus providing insights into the transamination mechanism of CrmG.

  7. Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase

    SciTech Connect

    Han,Q.; Gao, Y.; Robinson, H.; Li, J.

    2008-01-01

    Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant a-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate {beta}-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and {alpha}7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.

  8. Point mutations in the tyrosine aminotransferase gene in tyrosinemia type II.

    PubMed

    Natt, E; Kida, K; Odievre, M; Di Rocco, M; Scherer, G

    1992-10-01

    Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disease of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), a 454-amino acid protein encoded by a gene with 12 exons. To identify the causative mutations in five TAT alleles cloned from three RHS patients, chimeric genes constructed from normal and mutant TAT alleles were tested in directing TAT activity in a transient expression assay. DNA sequence analysis of the regions identified as nonfunctional revealed six different point mutations. Three RHS alleles have nonsense mutations at codons 57, 223, and 417, respectively. One "complex" RHS allele carries a GT----GG splice donor mutation in intron 8 together with a Gly----Val substitution at amino acid 362. A new splice acceptor site in intron 2 of the fifth RHS allele leads to a shift in reading frame.

  9. Involvement of alanine racemase in germination of Bacillus cereus spores lacking an intact exosporium.

    PubMed

    Venir, Elena; Del Torre, Manuela; Cunsolo, Vincenzo; Saletti, Rosaria; Musetti, Rita; Stecchini, Mara Lucia

    2014-02-01

    The L-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of D-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting L-alanine to the germination inhibitor D-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of L- to D-alanine. Unlike ATCC 14579 spores, L-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.

  10. Use of alanine-silicone pellets for electron paramagnetic resonance gamma dosimetry

    SciTech Connect

    Flores, J.; Galindo, S. )

    1991-03-01

    Silicone is proposed as an alternative binding substance in the production of D-L alanine pellets used in electron paramagnetic resonance (EPR) dosimetry of gamma rays. The dosimeters are manufactured at room temperature, making the production simple. Examination by EPR silicone-alanine pellets irradiated with 60Co gamma rays in the dose range 10 to 10(6) Gy shows that the proposed silicone binder does not affect typical alanine dose-response curves. Thermal stability of the pellets below 40 degrees C is good, but their pre-dose EPR signal amplitude is slightly higher than for nonirradiated alanine.

  11. Effect of β-alanine supplementation on high-intensity exercise performance.

    PubMed

    Harris, Roger C; Stellingwerff, Trent

    2013-01-01

    Carnosine is a dipeptide of β-alanine and L-histidine found in high concentrations in skeletal muscle. Combined with β-alanine, the pKa of the histidine imidazole ring is raised to ∼6.8, placing it within the muscle intracellular pH high-intensity exercise transit range. Combination with β-alanine renders the dipeptide inert to intracellular enzymic hydrolysis and blocks the histidinyl residue from participation in proteogenesis, thus making it an ideal, stable intracellular buffer. For vegetarians, synthesis is limited by β-alanine availability; for meat-eaters, hepatic synthesis is supplemented with β-alanine from the hydrolysis of dietary carnosine. Direct oral β-alanine supplementation will compensate for low meat and fish intake, significantly raising the muscle carnosine concentration. This is best achieved with a sustained-release formulation of β-alanine to avoid paresthesia symptoms and decreasing urinary spillover. In humans, increased levels of carnosine through β-alanine supplementation have been shown to increase exercise capacity and performance of several types, particularly where the high-intensity exercise range is 1-4 min. β-Alanine supplementation is used by athletes competing in high-intensity track and field cycling, rowing, swimming events and other competitions.

  12. Effect of β-alanine supplementation on high-intensity exercise performance.

    PubMed

    Harris, Roger C; Stellingwerff, Trent

    2013-01-01

    Carnosine is a dipeptide of β-alanine and L-histidine found in high concentrations in skeletal muscle. Combined with β-alanine, the pKa of the histidine imidazole ring is raised to ∼6.8, placing it within the muscle intracellular pH high-intensity exercise transit range. Combination with β-alanine renders the dipeptide inert to intracellular enzymic hydrolysis and blocks the histidinyl residue from participation in proteogenesis, thus making it an ideal, stable intracellular buffer. For vegetarians, synthesis is limited by β-alanine availability; for meat-eaters, hepatic synthesis is supplemented with β-alanine from the hydrolysis of dietary carnosine. Direct oral β-alanine supplementation will compensate for low meat and fish intake, significantly raising the muscle carnosine concentration. This is best achieved with a sustained-release formulation of β-alanine to avoid paresthesia symptoms and decreasing urinary spillover. In humans, increased levels of carnosine through β-alanine supplementation have been shown to increase exercise capacity and performance of several types, particularly where the high-intensity exercise range is 1-4 min. β-Alanine supplementation is used by athletes competing in high-intensity track and field cycling, rowing, swimming events and other competitions. PMID:23899755

  13. Haematology and blood chemistry values for several flamingo species.

    PubMed

    Peinado, V I; Polo, F J; Viscor, G; Palomeque, J

    1992-01-01

    Reference values for some haematological and plasma chemical values in four species of clinically normal adult flamingos were established for use in avian medicine. The following variables were studied in rosy, greater, Chilean and lesser flamingos: haematocrit, haemoglobin concentration, erythrocyte and leucocyte counts, haematimetric indices, erythrocyte dimensions, glucose, urea, uric acid, cholesterol, creatinine, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, phosphokinase, lactic dehydrogenase, total phosphorus, chloride, total plasma protein, albumin, globulins, albumin-globulin ratio, sodium, potassium, calcium, magnesium and osmolality.

  14. Liver enzymes, race, gender and diabetes risk: the Atherosclerosis Risk in Communities (ARIC) Study

    PubMed Central

    Schneider, A. L. C.; Lazo, M.; Ndumele, C. E.; Pankow, J. S.; Coresh, J.; Clark, J. M.; Selvin, E.

    2013-01-01

    Aims To examine the associations of the liver enzymes alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl transferase with diabetes risk and to determine whether associations differ by race and/or gender. We hypothesized that all liver enzymes would be associated with diabetes risk and that associations would differ by race and gender. Methods Prospective cohort of 7495 white and 1842 black participants without diabetes in the Atherosclerosis Risk in Communities Study. Poisson and Cox models adjusted for demographic, socio-behavioural, and metabolic and health-related factors were used. Results During a median of 12 years of follow-up, 2182 incident cases of diabetes occurred. Higher liver enzyme levels were independently associated with diabetes risk: adjusted hazard ratios (95% confidence intervals) were 1.68 (1.49–1.89), 1.16 (1.02–1.31) and 1.95 (1.70–2.24) comparing the highest with the lowest quartiles of alanine aminotransferase, aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT), respectively. gamma-Glutamyl transferase was most strongly related to diabetes risk, even at levels considered within normal range (≤ 60 U/l) in clinical practice. Adjusted incidence rates by quartiles of liver enzymes were similar by gender but higher in black versus white participants. Nonetheless, relative associations of alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transferase (GGT) with diabetes were similar by race (P for interactions > 0.05). Conclusions Compared with aspartate aminotransferase and alanine aminotransferase, gamma-glutamyl transferase was more strongly associated with diabetes risk. Our findings suggest that abnormalities in liver enzymes precede the diagnosis of diabetes by many years and that individuals with elevated liver enzymes, even within the normal range as defined in clinical practice, are at high risk for diabetes. PMID:23510198

  15. Charge dependent photodynamic activity of alanine based zinc phthalocyanines.

    PubMed

    Wang, Ao; Li, Yejing; Zhou, Lin; Yuan, Linxin; Lu, Shan; Lin, Yun; Zhou, Jiahong; Wei, Shaohua

    2014-12-01

    In this paper, to minimize the effects of different structure, three alanine-based zinc phthalocyanines (Pcs) of differing charges were engineered and synthesized with the same basic structure. On this premise, the relationship between nature of charge and photodynamic activity was studied. Besides, further verification and explanation of some inconsistent results were also carried out. The results showed that charge can influence the aggregation state, singlet oxygen generation ability and cellular uptake of Pcs, thereby affecting their photodynamic activity. In addition, the biomolecules inside cells may interact with Pcs of differing charges, which can also influence the aggregation state and singlet oxygen generation of the Pcs, and then influence the relationship between nature of charge and photodynamic activity.

  16. Radiolysis of alanine adsorbed in a clay mineral

    SciTech Connect

    Aguilar-Ovando, Ellen Y.; Negron-Mendoza, Alicia

    2013-07-03

    Optical activity in molecules is a chemical characteristic of living beings. In this work, we examine the hypothesis of the influence of different mineral surfaces on the development of a specific chirality in organic molecules when subjected to conditions simulating the primitive Earth during the period of chemical evolution. By using X-ray diffraction techniques and HPLC/ELSD to analyze aqueous suspensions of amino acids adsorbed on minerals irradiated in different doses with a cobalt-60 gamma source, the experiments attempt to prove the hypothesis that some solid surfaces (like clays and meteorite rocks) may have a concentration capacity and protective role against external sources of ionizing radiation (specifically {gamma}-ray) for some organic compounds (like some amino acids) adsorbed on them. Preliminary results show a slight difference in the adsorption and radiolysis of the D-and L-alanine.

  17. Effects of beta-alanine supplementation on sprint endurance.

    PubMed

    Jagim, Andrew R; Wright, Glenn A; Brice, A Glenn; Doberstein, Scott T

    2013-02-01

    Recent research has shown that beta-alanine (BA) supplementation can increase intramuscular carnosine levels. Carnosine is an intramuscular buffer, and it has been linked to improvements in performance, specifically during bouts of high-intensity exercise that are likely limited by muscle acidosis. Therefore, the purpose of this study was to examine the effect of BA supplementation on sprint endurance at 2 different supramaximal intensities. Twenty-one anaerobically trained (rugby players [n = 4], wrestlers [n = 11], and recreationally strength trained athletes [n = 6]) college-aged men participated in a double-blind, placebo controlled study. The subjects performed an incremental VO2max test and 2 sprint to exhaustion tests set at 115 and 140% of their VO2max on a motorized treadmill before (PRE) and after (POST) a 5-week supplementation period. During this time, the subjects ingested either a BA supplement or placebo (PLA) with meals. The subjects ingested 4 g·d(-1) of BA or PLA during the first week and 6 g·d(-1) the following 4 weeks. Capillary blood samples were taken before and after each sprint to determine blood lactate response to the sprint exercise. No significant group (BA, PLA) × intensity (115%, 140%; p = 0.60), group by time (PRE, POST; p = 0.72), or group × intensity × time (p = 0.74) interactions were observed for time to exhaustion. In addition, similar nonsignificant observations were made for lactate response to the sprints (group × intensity, p = 0.43; group × time, p = 0.33, group × intensity × time, p = 0.56). From the results of this study, it was concluded that beta-alanine supplementation did not have a significant effect on sprint endurance at supramaximal intensities.

  18. Radiation dose measurements with alanine/agarose gel and thin alanine films around a 192Ir brachytherapy source, using ESR spectroscopy.

    PubMed

    Olsson, S; Bergstrand, E S; Carlsson, A K; Hole, E O; Lund, E

    2002-04-21

    Alanine/agarose gel and alanine films in stacks have been used for measurements of absorbed dose around an HDR 192Ir source in a vaginal cylinder-applicator, with and without a 180 degrees tungsten shield. The gel and the films were analysed by means of ESR spectroscopy and calibrated against an ion chamber in a 4 MV photon beam to obtain absolute dose values. The gel serves as both dosimeter and phantom material, and the thin (130 microm) films are used to achieve an improved spatial resolution in the dose estimations. Experimental values were compared with Monte Carlo simulations using two different codes. Results from the measurements generally agree with the simulations to within 5%, for both the alanine/agarose gel and the alanine films.

  19. Efficient L-Alanine Production by a Thermo-Regulated Switch in Escherichia coli.

    PubMed

    Zhou, Li; Deng, Can; Cui, Wen-Jing; Liu, Zhong-Mei; Zhou, Zhe-Min

    2016-01-01

    L-Alanine has important applications in food, pharmaceutical and veterinary and is used as a substrate for production of engineered thermoplastics. Microbial fermentation could reduce the production cost and promote the application of L-alanine. However, the presence of L-alanine significantly inhibit cell growth rate and cause a decrease in the ultimate L-alanine productivity. For efficient L-alanine production, a thermo-regulated genetic switch was designed to dynamically control the expression of L-alanine dehydrogenase (alaD) from Geobacillus stearothermophilus on the Escherichia coli B0016-060BC chromosome. The optimal cultivation conditions for the genetically switched alanine production using B0016-060BC were the following: an aerobic growth phase at 33 °C with a 1-h thermo-induction at 42 °C followed by an oxygen-limited phase at 42 °C. In a bioreactor experiment using the scaled-up conditions optimized in a shake flask, B0016-060BC accumulated 50.3 g biomass/100 g glucose during the aerobic growth phase and 96 g alanine/100 g glucose during the oxygen-limited phase, respectively. The L-alanine titer reached 120.8 g/l with higher overall and oxygen-limited volumetric productivities of 3.09 and 4.18 g/l h, respectively, using glucose as the sole carbon source. Efficient cell growth and L-alanine production were reached separately, by switching cultivation temperature. The results revealed the application of a thermo-regulated strategy for heterologous metabolic production and pointed to strategies for improving L-alanine production.

  20. An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase.

    PubMed

    Yoo, Heejin; Widhalm, Joshua R; Qian, Yichun; Maeda, Hiroshi; Cooper, Bruce R; Jannasch, Amber S; Gonda, Itay; Lewinsohn, Efraim; Rhodes, David; Dudareva, Natalia

    2013-01-01

    Phenylalanine is a vital component of proteins in all living organisms, and in plants is a precursor for thousands of additional metabolites. Animals are incapable of synthesizing phenylalanine and must primarily obtain it directly or indirectly from plants. Although plants can synthesize phenylalanine in plastids through arogenate, the contribution of an alternative pathway via phenylpyruvate, as occurs in most microbes, has not been demonstrated. Here we show that plants also utilize a microbial-like phenylpyruvate pathway to produce phenylalanine, and flux through this route is increased when the entry point to the arogenate pathway is limiting. Unexpectedly, we find the plant phenylpyruvate pathway utilizes a cytosolic aminotransferase that links the coordinated catabolism of tyrosine to serve as the amino donor, thus interconnecting the extra-plastidial metabolism of these amino acids. This discovery uncovers another level of complexity in the plant aromatic amino acid regulatory network, unveiling new targets for metabolic engineering.

  1. Crystallization and preliminary X-ray analysis of phosphoserine aminotransferase from Bacillus circulans subsp. alkalophilus.

    PubMed Central

    Moser, M.; Müller, R.; Battchikova, N.; Koivulehto, M.; Korpela, T.; Jansonius, J. N.

    1996-01-01

    Recombinant phosphoserine aminotransferase (EC 2.6.1.52) from Bacillus circulans subsp. alkalophilus was crystallized at room temperature from 0.1 M sodium acetate buffer, pH 4.6, and 2% PEG 20000, using macroseeding techniques. The crystals diffract X-rays to at least 2.0 A nominal resolution. They belong to space group C2 with unit cell dimensions a = 93.2 A, b = 93.1 A, c = 45.6 A, alpha = 90.0 degrees, beta = 106.8 degrees, gamma = 90.0 degrees. A native data set to 2.3 A has been collected. Assuming an average packing density of the crystals, there is one monomer in the asymmetric unit, resulting in a calculated solvent content of 48.2%. PMID:8819175

  2. Activation of tyrosine aminotransferase expression in fetal liver by 5-azacytidine

    SciTech Connect

    Rothrock, R.; Perry, S.T.; Isham, K.R.; Lee, K.L.; Kenney, F.T.

    1983-06-15

    Rat fetuses of 20 days gestational age were treated in utero with the inhibitor of DNA methylation, 5-azacytidine. The liver enzyme tyrosine aminotransferase, normally expressed at very low levels until several hours after birth, was increased by the drug in the fetal livers after a lag period of about 9 hours, reaching a level 70-fold above control levels 18 hours after treatment. The high levels attained after 5-azacytidine treatment are comparable to those of glucocorticoid-treated adult livers, and were not further increased by administration of hydrocortisone to dams carrying treated fetuses. Cytidine and two other analogs, cytosine arabinoside and 6-azacytidine, were essentially without effect. 15 references, 2 figures, 1 table.

  3. Splicing defect at the ornithine aminotransferase (OAT) locus in gyrate atrophy.

    PubMed

    McClatchey, A I; Kaufman, D L; Berson, E L; Tobin, A J; Shih, V E; Gusella, J F; Ramesh, V

    1990-11-01

    Gyrate atrophy (GA), a recessive eye disease involving progressive vision loss due to chorioretinal degeneration, is associated with the deficiency of the mitochondrial enzyme ornithine aminotransferase (OAT), with consequent hyperornithinemia. We and others have reported a number of missense mutations at the OAT locus which result in GA. Here we report a GA patient of Danish/Swedish ancestry in whom one OAT allele produces an mRNA that is missing a single 96-bp exon relative to the normal mRNA. Polymerase-chain-reaction amplification and sequencing revealed a 9-bp deletion covering the splice acceptor region of exon 5, resulting in the absence of exon 5 sequences from the mRNA with no disruption to the reading frame. This mutation, which was not present in 15 other independent GA patients, adds to the array of allelic heterogeneity observed in GA and represents the first example of a splicing mutation associated with this disorder.

  4. Effect of stanozolol on factors VIII and IX and serum aminotransferases in haemophilia.

    PubMed

    Greer, I A; Greaves, M; Madhok, R; McLoughlin, K; Porter, N; Lowe, G D; Preston, F E; Forbes, C D

    1985-06-24

    The treatment of haemophilia has been dramatically improved since the introduction of factor VIII and IX concentrates, however these concentrates have brought new problems such as hepatitis and A.I.D.S. An oral agent which could raise endogenous levels of factor VIII and IX would be of great benefit. Danazol, an anabolic steroid, has recently been shown to increase levels of factors VIII and IX in haemophilia. We therefore studied the effect of stanozolol, a closely related anabolic steroid, in 15 patients with haemophilia A or Christmas disease over a 2-4 week period. There was no consistent change in factor VIIIc or factor IX, and fibrinolysis was significantly enhanced. No effect was apparent on the incidence of spontaneous bleeds. However serum aminotransferases which were abnormal in 11 of the 15 patients at the start of the study fell significantly with stanozolol therapy. This raises the interesting possibility that anabolic steroids may be beneficial in patients with chronic liver diseases.

  5. Properties of Copolymers of Aspartic Acid and Aliphatic Dicarboxylic Acids Prepared by Reactive Extrusion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspartic acid may be prepared chemically or by the fermentation of carbohydrates. Currently, low molecular weight polyaspartic acids are prepared commercially by heating aspartic acid at high temperatures (greater than 220 degrees C) for several hours in the solid state. In an effort to develop a ...

  6. Structural Analysis of the Ligand-Binding Domain of the Aspartate Receptor Tar from Escherichia coli.

    PubMed

    Mise, Takeshi

    2016-07-01

    The Escherichia coli cell-surface aspartate receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni(2+). These signals are transmitted from the extracellular region of Tar to the cytoplasmic region via the transmembrane domain. The mechanism by which extracellular signals are transmitted into the cell through conformational changes in Tar is predicted to involve a piston displacement of one of the α4 helices of the homodimer. To understand the molecular mechanisms underlying the induction of Tar activity by an attractant, the three-dimensional structures of the E. coli Tar periplasmic domain with and without bound aspartate, Asp-Tar and apo-Tar, respectively, were determined. Of the two ligand-binding sites, only one site was occupied, and it clearly showed the electron density of an aspartate. The slight changes in conformation and the electrostatic surface potential around the aspartate-binding site were observed. In addition, the presence of an aspartate stabilized residues Phe-150' and Arg-73. A pistonlike displacement of helix α4b' was also induced by aspartate binding as predicted by the piston model. Taken together, these small changes might be related to the induction of Tar activity and might disturb binding of the second aspartate to the second binding site in E. coli. PMID:27292793

  7. Energetics and dynamics of the fragmentation reactions of protonated peptides containing methionine sulfoxide or aspartic acid via energy- and time-resolved surface induced dissociation.

    PubMed

    Lioe, Hadi; Laskin, Julia; Reid, Gavin E; O'Hair, Richard A J

    2007-10-25

    The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64 Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility in these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (nonmobile proton conditions) to lysine (partially mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFECs) reveal that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1-2 orders of magnitude lower than nonselective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to nonselective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these processes

  8. Energetics and Dynamics of the Fragmentation Reactions of Protonated Peptides Containing Methionine Sulfoxide or Aspartic Acid via Energy- and Time-Resolved Surface Induced Dissociation

    SciTech Connect

    Lioe, Hadi; Laskin, Julia; Reid, Gavin E.; O'Hair, Richard Aj

    2007-10-25

    The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility on these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (non-mobile proton conditions) to lysine (partially-mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFEC) reveals that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1–2 orders of magnitude lower than non-selective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to non-selective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these

  9. Regulation of the ald gene encoding alanine dehydrogenase by AldR in Mycobacterium smegmatis.

    PubMed

    Jeong, Ji-A; Baek, Eun-Young; Kim, Si Wouk; Choi, Jong-Soon; Oh, Jeong-Il

    2013-08-01

    The regulatory gene aldR was identified 95 bp upstream of the ald gene encoding L-alanine dehydrogenase in Mycobacterium smegmatis. The AldR protein shows sequence similarity to the regulatory proteins of the Lrp/AsnC family. Using an aldR deletion mutant, we demonstrated that AldR serves as both activator and repressor for the regulation of ald gene expression, depending on the presence or absence of L-alanine. The purified AldR protein exists as a homodimer in the absence of L-alanine, while it adopts the quaternary structure of a homohexamer in the presence of L-alanine. The binding affinity of AldR for the ald control region was shown to be increased significantly by L-alanine. Two AldR binding sites (O1 and O2) with the consensus sequence GA-N₂-ATC-N₂-TC and one putative AldR binding site with the sequence GA-N₂-GTT-N₂-TC were identified upstream of the ald gene. Alanine and cysteine were demonstrated to be the effector molecules directly involved in the induction of ald expression. The cellular level of L-alanine was shown to be increased in M. smegmatis cells grown under hypoxic conditions, and the hypoxic induction of ald expression appears to be mediated by AldR, which senses the intracellular level of alanine.

  10. Polymerization of alanine in the presence of a non-swelling montmorillonite

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.; Lahav, N.

    1977-01-01

    Alanine, starting from alanine-adenylate, has been polymerized in the presence of non-swelling Al-montmorillonite. The yield of polymerization is much lower than that obtained in the presence of swelling Na-montmorillonite. The possibility that the changing interlayer spacing in Na-montmorillonite might be responsible for its catalytic properties, is discussed.

  11. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...)(4), (c)(4) (where N = 100). The requirement of 40 CFR 721.91(a)(4) that the amount of the substance... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical...

  12. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...)(4), (c)(4) (where N = 100). The requirement of 40 CFR 721.91(a)(4) that the amount of the substance... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical...

  13. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...)(4), (c)(4) (where N = 100). The requirement of 40 CFR 721.91(a)(4) that the amount of the substance... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical...

  14. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...)(4), (c)(4) (where N = 100). The requirement of 40 CFR 721.91(a)(4) that the amount of the substance... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical...

  15. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...)(4), (c)(4) (where N = 100). The requirement of 40 CFR 721.91(a)(4) that the amount of the substance... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical...

  16. How similar is the electronic structures of β-lactam and alanine?

    NASA Astrophysics Data System (ADS)

    Chatterjee, Subhojyoti; Ahmed, Marawan; Wang, Feng

    2016-02-01

    The C1s spectra of β-lactam i.e. 2-azetidinone (C3H5NO), a drug and L-alanine (C3H7NO2), an amino acid, exhibit striking similarities, which may be responsible for the competition between 2-azetidinone and the alanyl-alanine moiety in biochemistry. The present study is to reveal the degree of similarities and differences between their electronic structures of the two model molecular pairs. It is found that the similarities in C1s and inner valence binding energy spectra are due to their bonding connections but other properties such as ring structure (in 2-azetidinone) and chiral carbon (alanine) can be very different. Further, the inner valence region of ionization potential greater than 18 eV for 2-azetidinone and alanine is also significantly similar. Finally the strained lactam ring exhibits more chemical reactivity measured at all non-hydrogen atoms by Fukui functions with respect to alanine.

  17. Thermal decomposition behavior of potassium and sodium jarosite synthesized in the presence of methylamine and alanine

    SciTech Connect

    J. Michelle Kotler; Nancy W. Hinman; C. Doc Richardson; Jill R. Scott

    2010-10-01

    Biomolecules, methylamine and alanine, found associated with natural jarosite samples peaked the interest of astrobiologists and planetary geologists. How the biomolecules are associated with jarosite remains unclear although the mechanism could be important for detecting biosignatures in the rock record on Earth and other planets. A series of thermal gravimetric experiments using synthetic K-jarosite and Na-jarosite were conducted to determine if thermal analysis could differentiate physical mixtures of alanine and methylamine with jarosite from samples where the methylamine or alanine was incorporated into the synthesis procedure. Physical mixtures and synthetic experiments with methylamine and alanine could be differentiated from one another and from the standards by thermal analysis for both the K-jarosite and Na-jarosite end-member suites. Changes included shifts in on-set temperatures, total temperature changes from on-set to final, and the presence of indicator peaks for methylamine and alanine in the physical mixture experiments.

  18. The effect of portacaval anastomosis on the expression of glutamine synthetase and ornithine aminotransferase in perivenous hepatocytes.

    PubMed

    da Silva, Robin; Levillain, Oliver; Brosnan, John T; Araneda, Silvia; Brosnan, Margaret E

    2013-05-01

    There is functional zonation of metabolism across the liver acinus, with glutamine synthetase restricted to a narrow band of cells around the terminal hepatic venules. Portacaval anastomosis, where there is a major rerouting of portal blood flow from the portal vein directly to the vena cava bypassing the liver, has been reported to result in a marked decrease in the activity of glutamine synthetase. It is not known whether this represents a loss of perivenous hepatocytes or whether there is a specific loss of glutamine synthetase. To answer this question, we have determined the activity of glutamine synthetase and another enzyme from the perivenous compartment, ornithine aminotransferase, as well as the immunochemical localization of both glutamine synthetase and ornithine aminotransferase in rats with a portacaval shunt. The portacaval shunt caused a marked decrease in glutamine synthetase activity and an increase in ornithine aminotransferase activity. Immunohistochemical analysis showed that the glutamine synthetase and ornithine aminotransferase proteins maintained their location in the perivenous cells. These results indicate that there is no generalized loss of perivenous hepatocytes, but rather, there is a significant alteration in the expression of these proteins and hence metabolism in this cell population. PMID:23656379

  19. The effect of portacaval anastomosis on the expression of glutamine synthetase and ornithine aminotransferase in perivenous hepatocytes.

    PubMed

    da Silva, Robin; Levillain, Oliver; Brosnan, John T; Araneda, Silvia; Brosnan, Margaret E

    2013-05-01

    There is functional zonation of metabolism across the liver acinus, with glutamine synthetase restricted to a narrow band of cells around the terminal hepatic venules. Portacaval anastomosis, where there is a major rerouting of portal blood flow from the portal vein directly to the vena cava bypassing the liver, has been reported to result in a marked decrease in the activity of glutamine synthetase. It is not known whether this represents a loss of perivenous hepatocytes or whether there is a specific loss of glutamine synthetase. To answer this question, we have determined the activity of glutamine synthetase and another enzyme from the perivenous compartment, ornithine aminotransferase, as well as the immunochemical localization of both glutamine synthetase and ornithine aminotransferase in rats with a portacaval shunt. The portacaval shunt caused a marked decrease in glutamine synthetase activity and an increase in ornithine aminotransferase activity. Immunohistochemical analysis showed that the glutamine synthetase and ornithine aminotransferase proteins maintained their location in the perivenous cells. These results indicate that there is no generalized loss of perivenous hepatocytes, but rather, there is a significant alteration in the expression of these proteins and hence metabolism in this cell population.

  20. Age-Related Changes in D-Aspartate Oxidase Promoter Methylation Control Extracellular D-Aspartate Levels and Prevent Precocious Cell Death during Brain Aging.

    PubMed

    Punzo, Daniela; Errico, Francesco; Cristino, Luigia; Sacchi, Silvia; Keller, Simona; Belardo, Carmela; Luongo, Livio; Nuzzo, Tommaso; Imperatore, Roberta; Florio, Ermanno; De Novellis, Vito; Affinito, Ornella; Migliarini, Sara; Maddaloni, Giacomo; Sisalli, Maria Josè; Pasqualetti, Massimo; Pollegioni, Loredano; Maione, Sabatino; Chiariotti, Lorenzo; Usiello, Alessandro

    2016-03-01

    The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that d-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of d-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate. PMID:26961959

  1. Calibration of helical tomotherapy machine using EPR/alanine dosimetry

    SciTech Connect

    Perichon, Nicolas; Garcia, Tristan; Francois, Pascal; Lourenco, Valerie; Lesven, Caroline; Bordy, Jean-Marc

    2011-03-15

    Purpose: Current codes of practice for clinical reference dosimetry of high-energy photon beams in conventional radiotherapy recommend using a 10x10 cm{sup 2} square field, with the detector at a reference depth of 10 cm in water and 100 cm source to surface distance (SSD) (AAPM TG-51) or 100 cm source-to-axis distance (SAD) (IAEA TRS-398). However, the maximum field size of a helical tomotherapy (HT) machine is 40x5 cm{sup 2} defined at 85 cm SAD. These nonstandard conditions prevent a direct implementation of these protocols. The purpose of this study is twofold: To check the absorbed dose in water and dose rate calibration of a tomotherapy unit as well as the accuracy of the tomotherapy treatment planning system (TPS) calculations for a specific test case. Method: Both topics are based on the use of electron paramagnetic resonance (EPR) using alanine as transfer dosimeter between the Laboratoire National Henri Becquerel (LNHB) {sup 60}Co-{gamma}-ray reference beam and the Institut Curie's HT beam. Irradiations performed in the LNHB reference {sup 60}Co-{gamma}-ray beam allowed setting up the calibration method, which was then implemented and tested at the LNHB 6 MV linac x-ray beam, resulting in a deviation of 1.6% (at a 1% standard uncertainty) relative to the reference value determined with the standard IAEA TRS-398 protocol. Results: HT beam dose rate estimation shows a difference of 2% with the value stated by the manufacturer at a 2% standard uncertainty. A 4% deviation between measured dose and the calculation from the tomotherapy TPS was found. The latter was originated by an inadequate representation of the phantom CT-scan values and, consequently, mass densities within the phantom. This difference has been explained by the mass density values given by the CT-scan and used by the TPS which were not the true ones. Once corrected using Monte Carlo N-Particle simulations to validate the accuracy of this process, the difference between corrected TPS

  2. N-methyl-D-aspartate promotes the survival of cerebellar granule cells in culture.

    PubMed

    Balázs, R; Jørgensen, O S; Hack, N

    1988-11-01

    Our previous studies on the survival-promoting influence of elevated concentrations of extracellular K+ ([K+]e) on cultured cerebellar granule cells led to the proposal that depolarization in vitro mimics the effect of the earliest afferent inputs received by the granule cells in vivo. This, in turn, might be mediated through the stimulation of excitatory amino acid receptors, in particular the N-methyl-D-aspartate-preferring subtype gating ion channels which are also permeable to Ca2+. Here we report that N-methyl-D-aspartate indeed has a dramatic effect on the survival in culture of cells derived from dissociated cerebella of 7-8-day-old rats and cultured in media containing 'low' [K+]e (5-15 mM). In addition to the visual inspection of the cultures, the effect of N-methyl-D-aspartate was quantitatively evaluated, using estimates related to the number of viable cells (determination of DNA and of reduction rate of a tetrazolium salt). Furthermore, proteins which are relatively enriched in either nerve cells (neuronal cell adhesion molecule, D3-protein and synaptin) or in glia (glutamine synthetase) were also measured. The findings showed that the rescue of cells by N-methyl-D-aspartate involved primarily nerve cells and that the survival requirement for N-methyl-D-aspartate, as for high K+, developed between 2 and 4 days in vitro. The effect depended on both the concentration of N-methyl-D-aspartate and the degree of depolarization of the cells: both the potency and the efficacy of N-methyl-D-aspartate were increased as [K+]e was raised from 5 to 15 mM, at which range K+ on its own has little if any influence on granule cell survival. These characteristics are consistent with the voltage-dependence of ion conductance through the N-methyl-D-aspartate receptor-linked channel. The most pronounced effect of N-methyl-D-aspartate was obtained in the presence of 15 mM K+, when cell survival approached that obtained in 'control' cultures (grown in 25 mM K

  3. Alanine racemase mutants of Mycobacterium tuberculosis require D-alanine for growth and are defective for survival in macrophages and mice.

    PubMed

    Awasthy, Disha; Bharath, Sowmya; Subbulakshmi, Venkita; Sharma, Umender

    2012-02-01

    Alanine racemase (Alr) is an essential enzyme in most bacteria; however, some species (e.g. Listeria monocytogenes) can utilize d-amino acid transaminase (Dat) to generate d-alanine, which renders Alr non-essential. In addition to the conflicting reports on gene knockout of alr in Mycobacterium smegmatis, a recent study concluded that depletion of Alr does not affect the growth of M. smegmatis. In order to get an unambiguous answer on the essentiality of Alr in Mycobacterium tuberculosis and validate it as a drug target in vitro and in vivo, we have inactivated the alr gene of M. tuberculosis and found that it was not possible to generate an alr knockout in the absence of a complementing gene copy or d-alanine in the growth medium. The growth kinetics of the alr mutant revealed that M. tuberculosis requires very low amounts of d-alanine (5-10 µg ml(-1)) for optimum growth. Survival kinetics of the mutant in the absence of d-alanine indicated that depletion of this amino acid results in rapid loss of viability. The alr mutant was found to be defective for growth in macrophages. Analysis of phenotype in mice suggested that non-availability of d-alanine in mice leads to clearance of bacteria followed by stabilization of bacterial number in lungs and spleen. Additionally, reversal of d-cycloserine inhibition in the presence of d-alanine in M. tuberculosis suggested that Alr is the primary target of d-cycloserine. Thus, Alr of M. tuberculosis is a valid drug target and inhibition of Alr alone should result in loss of viability in vitro and in vivo.

  4. Compound-specific nitrogen isotope analysis of D-alanine, L-alanine, and valine: application of diastereomer separation to delta15N and microbial peptidoglycan studies.

    PubMed

    Takano, Yoshinori; Chikaraishi, Yoshito; Ogawa, Nanako O; Kitazato, Hiroshi; Ohkouchi, Naohiko

    2009-01-01

    We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (R)-(-)-2-butanol or (S)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of D- and L-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; Enterococcus faecalis, Staphylococcus aureus, Staphylococcus staphylolyticus, Lactobacillus acidophilus, Bacillus subtilis, Micrococcus luteus, and Streptomyces sp.), natural whole bacterial cells (Bacillus subtilis var. natto), (pseudo)-peptidoglycan from archaea (Methanobacterium sp.), and cell wall from eukaryota (Saccharomyces cerevisiae). We observed statistically significant differences in nitrogen isotopic compositions; e.g., delta15N ( per thousand vs air) in Staphylococcus staphylolyticus for d-alanine (19.2 +/- 0.5 per thousand, n = 4) and L-alanine (21.3 +/- 0.8 per thousand, n = 4) and in Bacillus subtilis for D-alanine (6.2 +/- 0.2 per thousand, n = 3) and L-alanine (8.2 +/- 0.4 per thousand, n = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in 15N-depleted D-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers.

  5. Mycobacterium smegmatis L-alanine dehydrogenase (Ald) is required for proficient utilization of alanine as a sole nitrogen source and sustained anaerobic growth.

    PubMed

    Feng, Zhengyu; Cáceres, Nancy E; Sarath, Gautam; Barletta, Raúl G

    2002-09-01

    NAD(H)-dependent L-alanine dehydrogenase (EC 1.4.1.1) (Ald) catalyzes the oxidative deamination of L-alanine and the reductive amination of pyruvate. To assess the physiological role of Ald in Mycobacterium smegmatis, we cloned the ald gene, identified its promoter, determined the protein expression levels, and analyzed the combined effects of nutrient supplementation, oxygen availability, and growth stage on enzyme activity. High Ald activities were observed in cells grown in the presence of L- or D-alanine regardless of the oxygen availability and growth stage. In exponentially growing cells under aerobic conditions, supplementation with alanine resulted in a 25- to 50-fold increase in the enzyme activity. In the absence of alanine supplementation, 23-fold-higher Ald activities were observed in cells grown exponentially under anaerobic conditions. Furthermore, M. smegmatis ald null mutants were constructed by targeted disruption and were shown to lack any detectable Ald activity. In contrast, the glycine dehydrogenase (EC 1.4.1.10) (Gdh) activity in mutant cells remained at wild-type levels, indicating that another enzyme protein is responsible for the physiologically relevant reductive amination of glyoxylate. The ald mutants grew poorly in minimal medium with L-alanine as the sole nitrogen source, reaching a saturation density 100-fold less than that of the wild-type strain. Likewise, mutants grew to a saturation density 10-fold less than that of the wild-type strain under anaerobic conditions. In summary, the phenotypes displayed by the M. smegmatis ald mutants suggest that Ald plays an important role in both alanine utilization and anaerobic growth.

  6. New paradigm for allosteric regulation of Escherichia coli aspartate transcarbamoylase.

    PubMed

    Cockrell, Gregory M; Zheng, Yunan; Guo, Wenyue; Peterson, Alexis W; Truong, Jennifer K; Kantrowitz, Evan R

    2013-11-12

    For nearly 60 years, the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and five X-ray structures determined in the absence and presence of a Mg(2+) concentration within the physiological range. In the presence of 2 mM divalent cations (Mg(2+), Ca(2+), Zn(2+)), CTP does not significantly inhibit the enzyme, while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor of ATCase in vivo is the combination of CTP, UTP, and a divalent cation, and the actual allosteric activator is a divalent cation with ATP or ATP and GTP. The structural data reveals that two NTPs can bind to each allosteric site with a divalent cation acting as a bridge between the triphosphates. Thus, the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal that the catalytic chains undergo essentially no alternations; however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal region of the regulatory chains exists in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed.

  7. A single amino acid change (substitution of the conserved Glu-590 with alanine) in the C-terminal domain of rat liver carnitine palmitoyltransferase I increases its malonyl-CoA sensitivity close to that observed with the muscle isoform of the enzyme.

    PubMed

    Napal, Laura; Dai, Jia; Treber, Michelle; Haro, Diego; Marrero, Pedro F; Woldegiorgis, Gebre

    2003-09-01

    Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of l-carnitine. To determine the role of the highly conserved C-terminal glutamate residue, Glu-590, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, lysine, glutamine, and aspartate. Substitution of Glu-590 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild-type enzyme, resulted in complete loss in the activity of the liver isoform of CPTI (L-CPTI). A change of Glu-590 to alanine, glutamine, and lysine caused a significant 9- to 16-fold increase in malonyl-CoA sensitivity but only a partial decrease in catalytic activity. Substitution of Glu-590 with neutral uncharged residues (alanine and glutamine) and/or a basic positively charged residue (lysine) significantly increased L-CPTI malonyl-CoA sensitivity to the level observed with the muscle isoform of the enzyme, suggesting the importance of neutral and/or positive charges in the switch of the kinetic properties of L-CPTI to the muscle isoform of CPTI. Since a conservative substitution of Glu-590 to aspartate but not glutamine resulted in complete loss in activity, we suggest that the longer side chain of glutamate is essential for catalysis and malonyl-CoA sensitivity. This is the first demonstration whereby a single residue mutation in the C-terminal region of the liver isoform of CPTI resulted in a change of its kinetic properties close to that observed with the muscle isoform of the enzyme and provides the rationale for the high malonyl-CoA sensitivity of muscle CPTI compared with the liver isoform of the enzyme. PMID:12826662

  8. Tb(3+)-triggered luminescence in a supramolecular gel and its use as a fluorescent chemoprobe for proteins containing alanine.

    PubMed

    Jung, Sung Ho; Kim, Ka Young; Woo, Dong Kyun; Lee, Shim Sung; Jung, Jong Hwa

    2014-11-01

    A tetracarboxylic acid-appended thiacalix[4]arene-based ligand with Tb(3+) formed a supramolecular gel which showed novel fluorogenic sensor capability for probing alanine and proteins containing alanine.

  9. Energy landscapes and global thermodynamics for alanine peptides

    NASA Astrophysics Data System (ADS)

    Somani, Sandeep; Wales, David J.

    2013-09-01

    We compare different approaches for computing the thermodynamics of biomolecular systems. Techniques based on parallel replicas evolving via molecular dynamics or Monte Carlo simulations produce overlapping histograms for the densities of states. In contrast, energy landscape methods employ a superposition partition function constructed from local minima of the potential energy surface. The latter approach is particularly powerful for systems exhibiting broken ergodicity, and it is usually implemented using a harmonic normal mode approximation, which has not been extensively tested for biomolecules. The present contribution compares these alternative approaches for small alanine peptides modelled using the CHARMM and AMBER force fields. Densities of states produced from canonical sampling using multiple temperature replicas provide accurate reference data to evaluate the effect of the harmonic normal mode approximation in the superposition calculations. This benchmarking lays foundations for the application of energy landscape methods to larger biomolecules. It will also provide well characterised model systems for developing enhanced sampling methods, and for the treatment of anharmonicity corresponding to individual local minima.

  10. Alanine synthesis from glyceraldehyde and ammonium ion in aqueous solution

    NASA Technical Reports Server (NTRS)

    Weber, A. L.

    1985-01-01

    The formation of alanine (ala) form C(14)-glyceraldehyde and ammonium phosphate in the presence or absence of a thiol is reported. At ambient temperature, ala synthesis was six times more rapid in the presence of 3-mercaptopropionic acid than in its absence (0.6 and 0.1 percent, respectively, after 60 days). Similarly, the presence of another thiol, N-acetylcysteinate, increased the production of ala, as well as of lactate. The reaction pathway of thiol-catalyzed synthesis of ala, with the lactic acid formed in a bypath, is suggested. In this, dehydration of glyceraldehyde is followed by the formation of hemithioacetal. In the presence of ammonia, an imine is formed, which eventually yields ala. This pathway is consistent with the observation that the rate ratio of ala/lactate remains constant throughout the process. The fact that the reaction takes place under anaerobic conditions in the presence of H2O and with the low concentrations of simple substrates and catalysts makes it an attractive model prebiotic reaction in the process of molecular evolution.

  11. Folding simulations of alanine-based peptides with lysine residues.

    PubMed Central

    Sung, S S

    1995-01-01

    The folding of short alanine-based peptides with different numbers of lysine residues is simulated at constant temperature (274 K) using the rigid-element Monte Carlo method. The solvent-referenced potential has prevented the multiple-minima problem in helix folding. From various initial structures, the peptides with three lysine residues fold into helix-dominated conformations with the calculated average helicity in the range of 60-80%. The peptide with six lysine residues shows only 8-14% helicity. These results agree well with experimental observations. The intramolecular electrostatic interaction of the charged lysine side chains and their electrostatic hydration destabilize the helical conformations of the peptide with six lysine residues, whereas these effects on the peptides with three lysine residues are small. The simulations provide insight into the helix-folding mechanism, including the beta-bend intermediate in helix initiation, the (i, i + 3) hydrogen bonds, the asymmetrical helix propagation, and the asymmetrical helicities in the N- and C-terminal regions. These findings are consistent with previous studies. PMID:7756550

  12. Ontogenetic trends in aspartic acid racemization and amino acid composition within modern and fossil shells of the bivalve Arctica

    NASA Astrophysics Data System (ADS)

    Goodfriend, Glenn A.; Weidman, Christopher R.

    2001-06-01

    Ontogenetic trends (umbo to growth edge of shell) in aspartic acid (Asp) racemization and amino acid composition and their evolution over time are examined in serial samples of annual growth bands from a time-series of three live-collected and two fossil (ca. 500 and 1000 y BP) shells of the long-lived bivalve Arctica islandica. The rate of Asp racemization is shown to be higher in the umbonal portion of the shells (laid down when the clams are young) but constant from a biological age of 10 to 20 y to more than 100 y. Corresponding changes are also seen in amino acid composition and concentration: with increasing biological age of the clam: total amino acid concentration increases substantially, the acidic amino acids Asp, glutamic acid, and alanine decrease in relative concentration (mole-percent) and more basic amino acids including tyrosine, phenylalanine, and lysine increase in relative concentration. These ontogenetic trends are generally retained in the fossil shells. These trends may reflect changing protein composition related to changes in growth rate. Clams grow considerably faster in their youth than when they are older, as indicated by changes in the annual growth increments. Production of more acidic proteins, which play a role in crystal growth, may be favored during the phase of faster growth, whereas more structural proteins, perhaps enhancing structural strength of the shell, may be favored during later growth. These ontogenetic differences in protein composition affect the observed rates of racemization of the protein pool. Some weak diagenetic trends in amino acid composition and abundance may be represented in the time series of shells. These results emphasize the importance of standardization of the location from which samples are taken from shells for dating by amino acid racemization analysis.

  13. Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin

    SciTech Connect

    Otto, H.; Marti, T.; Holz, M.; Mogi, T.; Lindau, M.; Khorana, H.G.; Heyn, M.P. )

    1989-12-01

    Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, (H+). In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition. The decay rate of N is proportional to (H+). By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time (log (tau)) linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.

  14. Position of the Third Na+ Site in the Aspartate Transporter GltPh and the Human Glutamate Transporter, EAAT1

    PubMed Central

    Bastug, Turgut; Heinzelmann, Germano; Kuyucak, Serdar; Salim, Marietta; Vandenberg, Robert J.; Ryan, Renae M.

    2012-01-01

    Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na+ ions, one H+ and the counter-transport of one K+ ion. Transport by an archaeal homologue of the human glutamate transporters, GltPh, whose three dimensional structure is known is also coupled to three Na+ ions but only two Na+ ion binding sites have been observed in the crystal structure of GltPh. In order to fully utilize the GltPh structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of GltPh and accurately determine the number and location of Na+ ions coupled to transport. Several sites have been proposed for the binding of a third Na+ ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for GltPh and reveal a new site for the third Na+ ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in GltPh, and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na+ compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na+ ion in GltPh and EAAT1. PMID:22427946

  15. Observing N-Acetyl Aspartate via Both Its N-Acetyl and Its Strongly Coupled Aspartate Groups in in VivoProton Magnetic Resonance Spectroscopy

    NASA Astrophysics Data System (ADS)

    Wilman, Alan H.; Allen, Peter S.

    1996-12-01

    The ∼2.6 ppm aspartate multiplet ofN-acetyl aspartate (NAA) is considered a potential source of additional information onN-acetyl aspartatein vivo.Because the aspartate multiplet is the AB part of a strongly coupled ABX system it gives rise, as is shown in the analysis presented, to a significant field-strength dependence in the echo-time-dependent modulations of the response to typical spatial-localization sequences. The echo-time dependence of this response is developed analytically, not only for the STEAM and the PRESS localization sequences, but also for a spin-echo sequence. It is then verified experimentally at 2.35 T. The field-strength dependence of the response is demonstrated by evaluating the changes in the echo-time-dependent responses to each of the three sequences at field strengths of 1.5, 2.35, and 4.0 T. By means of these results, the preferred sequence (PRESS) can be optimized for the NAA aspartate multiplet at each field strength, as is illustrated with the human brain spectra obtainedin vivoat 1.5 T. Thesein vivospectra compare the optimal, long TE timing (163 ms) with a suboptimal TE (70 ms), for the observation of the ∼2.6 ppm aspartate resonances of NAA.

  16. Clinical experience with insulin detemir, biphasic insulin aspart and insulin aspart in people with type 2 diabetes: Results from the Hyderabad cohort of the A1chieve study

    PubMed Central

    Santosh, R.; Mehrotra, Ravi; Sastry, N. G.

    2013-01-01

    Background: The A1chieve, a multicentric (28 countries), 24-week, non-interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with T2DM (n = 66,726) in routine clinical care across four continents. Materials and Methods: Data was collected at baseline, at 12 weeks and at 24 weeks. This short communication presents the results for patients enrolled from Hyderabad, India. Results: A total of 1249 patients were enrolled in the study. Four different insulin analogue regimens were used in the study. Patients had started on or were switched to biphasic insulin aspart (n = 893), insulin detemir (n = 158), insulin aspart (n = 124), basal insulin plus insulin aspart (n = 19) and other insulin combinations (n = 54). At baseline glycaemic control was poor for both insulin naïve (mean HbA1c: 9.0%) and insulin user (mean HbA1c: 9.5%) groups. After 24 weeks of treatment, both the groups showed improvement in HbA1c (insulin naïve: −0.9%, insulin users: −1.1%). SADRs including major hypoglycaemic events or episodes did not occur in any of the study patients. Conclusion: Starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia. PMID:24404501

  17. Identification and metabolic role of the mitochondrial aspartate-glutamate transporter in Saccharomyces cerevisiae.

    PubMed

    Cavero, S; Vozza, A; del Arco, A; Palmieri, L; Villa, A; Blanco, E; Runswick, M J; Walker, J E; Cerdán, S; Palmieri, F; Satrústegui, J

    2003-11-01

    The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspartate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identified Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomyces cerevisiae. On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast. PMID:14622413

  18. A Deficiency in Aspartate Biosynthesis in Lactococcus lactis subsp. lactis C2 Causes Slow Milk Coagulation†

    PubMed Central

    Wang, Hua; Yu, Weizhu; Coolbear, Tim; O’Sullivan, Dan; McKay, Larry L.

    1998-01-01

    A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc−) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis. PMID:9572935

  19. Alanine-EPR as a transfer standard dosimetry system for low energy X radiation

    NASA Astrophysics Data System (ADS)

    Khoury, H. J.; da Silva, E. J.; Mehta, K.; de Barros, V. S.; Asfora, V. K.; Guzzo, P. L.; Parker, A. G.

    2015-11-01

    The purpose of this paper is to evaluate the use of alanine-EPR as a transfer standard dosimetry system for low energy X radiation, such as that in RS-2400, which operates in the range from 25 to 150 kV and 2 to 45 mA. Two types of alanine dosimeters were investigated. One is a commercial alanine pellets from Aérial-Centre de Ressources Technologiques, France and one was prepared in our laboratory (LMRI-DEN/UFPE). The EPR spectra of the irradiated dosimeters were recorded in the Nuclear Energy Department of UFPE, using a Bruker EMX10 EPR spectrometer operating in the X-band. The alanine-EPR dosimetry system was calibrated in the range of 20-220 Gy in this X-ray field, against an ionization chamber calibrated at the relevant X-ray energy with traceability to PTB. The results showed that both alanine dosimeters presented a linear dose response the same sensitivity, when the EPR signal was normalized to alanine mass. The total uncertainty in the measured dose was estimated to be about 3%. The results indicate that it is possible to use the alanine-EPR dosimetry system for validation of a low-energy X ray irradiator, such as RS-2400.

  20. Revised mechanism of D-alanine incorporation into cell wall polymers in Gram-positive bacteria.

    PubMed

    Reichmann, Nathalie T; Cassona, Carolina Picarra; Gründling, Angelika

    2013-09-01

    Teichoic acids (TAs) are important for growth, biofilm formation, adhesion and virulence of Gram-positive bacterial pathogens. The chemical structures of the TAs vary between bacteria, though they typically consist of zwitterionic polymers that are anchored to either the peptidoglycan layer as in the case of wall teichoic acid (WTA) or the cell membrane and named lipoteichoic acid (LTA). The polymers are modified with D-alanines and a lack of this decoration leads to increased susceptibility to cationic antimicrobial peptides. Four proteins, DltA-D, are essential for the incorporation of d-alanines into cell wall polymers and it has been established that DltA transfers D-alanines in the cytoplasm of the cell onto the carrier protein DltC. However, two conflicting models have been proposed for the remainder of the mechanism. Using a cellular protein localization and membrane topology analysis, we show here that DltC does not traverse the membrane and that DltD is anchored to the outside of the cell. These data are in agreement with the originally proposed model for D-alanine incorporation through a process that has been proposed to proceed via a D-alanine undecaprenyl phosphate membrane intermediate. Furthermore, we found that WTA isolated from a Staphylococcus aureus strain lacking LTA contains only a small amount of D-alanine, indicating that LTA has a role, either direct or indirect, in the efficient D-alanine incorporation into WTA in living cells.

  1. Importance of intrahepatic mechanisms to gluconeogenesis from alanine during exercise and recovery

    SciTech Connect

    Wasserman, D.H.; Williams, P.E.; Lacy, D.B.; Green, D.R.; Cherrington, A.D.

    1988-04-01

    These studies were performed to assess the importance of intrahepatic mechanisms to gluconeogenesis in the dog during 150 min of treadmill exercise and 90 min of recovery. Sampling catheters were implanted in an artery and portal and hepatic veins 16 days before experimentation. Infusions of (U-/sup 14/C)alanine, (3-/sup 3/H)glucose, and indocyanine green were used to assess gluconeogenesis. During exercise, a decline in arterial and portal vein plasma alanine and in hepatic blood flow led to a decrease in hepatic alanine delivery. During recovery, hepatic blood flow was restored to basal, causing an increase in hepatic alanine delivery beyond exercise rates but still below resting rates. Hepatic fractional alanine extraction increased from 0.26 +/- 0.02 at rest to 0.64 +/- 0.03 during exercise and remained elevated during recovery. Net hepatic alanine uptake was 2.5 +/- 0.2 mumol.kg-1.min-1 at rest and remained unchanged during exercise but was increased during recovery. The conversion rate of (/sup 14/C)alanine to glucose had increased by 248 +/- 38% by 150 min of exercise and had increased further during recovery. The efficiency with which alanine was channeled into glucose in the liver was accelerated to a rate of 338 +/- 55% above basal by 150 min of exercise but declined slightly during recovery. In conclusion, 1) gluconeogenesis from alanine is accelerated during exercise, due to an increase in the hepatic fractional extraction of the amino acid and through intrahepatic mechanisms that more efficiently channel it into glucose.

  2. Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.

    PubMed

    Kim, Seryoung; Ihara, Kohei; Katsube, Satoshi; Hori, Hatsuhiro; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

    2015-08-01

    We previously reported that the alaE gene of Escherichia coli encodes the l-alanine exporter AlaE. The objective of this study was to elucidate the mechanism of the AlaE exporter. The minimum inhibitory concentration of l-alanine and l-alanyl-l-alanine in alaE-deficient l-alanine-nonmetabolizing cells MLA301ΔalaE was 4- and >4000-fold lower, respectively, than in the alaE-positive parent cells MLA301, suggesting that AlaE functions as an efflux pump to avoid a toxic-level accumulation of intracellular l-alanine and its derivatives. Furthermore, the growth of the alaE-deficient mutant derived from the l-alanine-metabolizing strain was strongly inhibited in the presence of a physiological level of l-alanyl-l-alanine. Intact MLA301ΔalaE and MLA301ΔalaE/pAlaE cells producing plasmid-borne AlaE, accumulated approximately 200% and 50%, respectively, of the [(3) H]l-alanine detected in MLA301 cells, suggesting that AlaE exports l-alanine. When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 μmol/L l-alanine, energy-dependent [(3) H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [(3) H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force. Based on these results, physiological roles of the l-alanine exporter are discussed.

  3. Applicability of EPR/alanine dosimetry for quality assurance in proton eye radiotherapy.

    PubMed

    Michalec, B; Mierzwinska, G; Ptaszkiewicz, M; Sowa, U; Stolarczyk, L; Weber, A

    2014-06-01

    A new quality assurance and quality control method for proton eye radiotherapy based on electron paramagnetic resonance (EPR)/alanine dosimetry has been developed. It is based on Spread-Out Bragg Peak entrance dose measurement with alanine detectors. The entrance dose is well correlated with the dose at the facility isocenter, where, during the therapeutic irradiation, the tumour is placed. The unique alanine detector features namely keeping the dose record in a form of stable radiation-induced free radicals trapped in the material structure, and the non-destructive read-out makes this type of detector a good candidate for additional documentation of the patient's exposure over the therapy course.

  4. Progress towards an alanine/ESR therapy level reference dosimetry service at NPL.

    PubMed

    Sharpe, P H; Rajendran, K; Sephton, J P

    1996-01-01

    This paper describes work being carried out at the National Physical Laboratory towards the establishment of an alanine reference dosimetry service for radiotherapy applications. A precision fused quartz holder has been constructed to allow precise positioning of alanine dosimeters in the ESR cavity. A novel method of signal analysis based on spectrum fitting has been developed to minimize the effect of baseline distortions. Data are also presented on the relative response of alanine to 60Co gamma rays and high energy photons (4-12 MeV).

  5. Optical and Spectral Studies on β Alanine Metal Halide Hybrid Crystals

    NASA Astrophysics Data System (ADS)

    Sweetlin, M. Daniel; Selvarajan, P.; Perumal, S.; Ramalingom, S.

    2011-10-01

    We have synthesized and grown β alanine metal halide hybrid crystals viz. β alanine cadmium chloride (BACC), an amino acid transition metal halide complex crystal and β alanine potassium chloride (BAPC), an amino acid alkali metal halide complex crystal by slow evaporation method. The grown crystals were found to be transparent and have well defined morphology. The optical characteristics of the grown crystals were carried out with the help of UV-Vis Spectroscopy. The optical transmittances of the spectrums show that BAPC is more transparent than BACC. The Photoluminescence of the materials were determined by the Photoluminescent Spectroscopy

  6. Treponema denticola cystalysin exhibits significant alanine racemase activity accompanied by transamination: mechanistic implications.

    PubMed Central

    Bertoldi, Mariarita; Cellini, Barbara; Paiardini, Alessandro; Di Salvo, Martino; Borri Voltattorni, Carla

    2003-01-01

    To obtain information on the reaction specificity of cystalysin from the spirochaete bacterium Treponema denticola, the interaction with L- and D-alanine has been investigated. Binding of both alanine enantiomers leads to the appearance of an external aldimine absorbing at 429 nm and of a band absorbing at 498 nm, indicative of a quinonoid species. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The steady-state kinetic parameters for racemization, k (cat) and K (m), for L-alanine are 1.05+/-0.03 s(-1) and 10+/-1 mM respectively, whereas those for D-alanine are 1.4+/-0.1 s(-1) and 10+/-1 mM. During the reaction of cystalysin with L- or D-alanine, a time-dependent loss of beta-elimination activity occurs concomitantly with the conversion of the pyridoxal 5'-phosphate (PLP) coenzyme into pyridoxamine 5'-phosphate (PMP). The catalytic efficiency of the half-transamination of L-alanine is found to be 5.3x10(-5) mM(-1) x s(-1), 5-fold higher when compared with that of D-alanine. The partition ratio between racemization and half-transamination reactions is 2.3x10(3) for L-alanine and 1.4x10(4) for D-alanine. The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with p K values of 6.5-6.6, which must be unprotonated for catalysis. Addition of pyruvate converts the PMP form of the enzyme back into the PLP form and causes the concomitant recovery of beta-elimination activity. In contrast with other PLP enzymes studied so far, but similar to alanine racemases, the apoform of the enzyme abstracted tritium from C4' of both (4' S)- and (4' R)-[4'-(3)H]PMP in the presence of pyruvate. Together with molecular modelling of the putative binding sites of L- and D-alanine at the active site of the enzyme, the implications of these studies for the mechanisms of the side reactions catalysed by cystalysin are discussed. PMID:12519070

  7. Pharmacology of triheteromeric N-Methyl-D-Aspartate Receptors.

    PubMed

    Cheriyan, John; Balsara, Rashna D; Hansen, Kasper B; Castellino, Francis J

    2016-03-23

    The N-Methyl-D-Aspartate Receptors (NMDARs) are heteromeric cation channels involved in learning, memory, and synaptic plasticity, and their dysregulation leads to various neurodegenerative disorders. Recent evidence has shown that apart from the GluN1/GluN2A and GluN1/GluN2B diheteromeric ion channels, the NMDAR also exists as a GluN1/GluN2A/GluN2B triheteromeric channel that occupies the majority of the synaptic space. These GluN1/GluN2A/GluN2B triheteromers exhibit pharmacological and electrophysiological properties that are distinct from the GluN1/GluN2A and GluN1/GluN2B diheteromeric subtypes. However, these receptors have not been characterized with regards to their inhibition by conantokins, as well as their allosteric modulation by polyamines and extracellular protons. Here, we show that the GluN1/GluN2A/GluN2B triheteromeric channels showed less sensitivity to GluN2B-specific conantokin (con)-G and con-RlB, and subunit non-specific con-T, compared to the GluN2A-specific inhibitor TCN-201. Also, spermine modulation of GluN1/GluN2A/GluN2B triheteromers switched its nature from potentiation to inhibition in a pH dependent manner, and was 2.5-fold slower compared to the GluN1/GluN2B diheteromeric channels. Unraveling the distinctive functional attributes of the GluN1/GluN2A/GluN2B triheteromers is physiologically relevant since they form an integral part of the synapse, which will aid in understanding spermine/pH-dependent potentiation of these receptors in pathological settings. PMID:26917100

  8. An aspartate ring at the TolC tunnel entrance determines ion selectivity and presents a target for blocking by large cations.

    PubMed

    Andersen, Christian; Koronakis, Eva; Hughes, Colin; Koronakis, Vassilis

    2002-06-01

    The TolC protein of Escherichia coli comprises an outer membrane beta-barrel channel and a contiguous alpha-helical tunnel spanning the periplasm, providing an exit duct for protein export and multidrug efflux. It forms a single transmembrane pore that is open to the outside of the cell but constricted at the peri-plasmic tunnel entrance. This sole constriction is lined by a ring of six aspartate residues, two in each of the three identical monomers. When these were replaced by alanines, the resulting TolC(DADA) protein reconstituted normally in black lipid membranes but showed altered electrophysiological characteristics. In particular, it had lost the strong pH dependence of the wild type and had switched ion selectivity from cations to anions. The function of wild-type TolC as a membrane pore was severely inhibited by divalent and trivalent cations entering the channel tunnel from the channel ("extracurricular") side. Divalent cations bound reversibly to effect complete blocking of the transmembrane ion flux. Trivalent cations were more potent. Hexamminecobalt bound at nanomolar concentrations allowed visualization of single blocking events, whereas the smaller Cr(3+) cation bound irreversibly and could also access the cation binding site via the tunnel entrance. The inhibitory cations had no effect on the mutant TolC(DADA), supporting the view that the aspartate ring is the cation binding site. The electronegative entrance is widely conserved throughout the TolC family, which is essential for efflux and export my Gram-negative bacteria, suggesting that it could present a general target for drugs.

  9. Mechanism of adenylate kinase. Demonstration of a functional relationship between aspartate 93 and Mg2+ by site-directed mutagenesis and proton, phosphorus-31, and magnesium-25 NMR.

    PubMed

    Yan, H G; Tsai, M D

    1991-06-01

    Earlier magnetic resonance studies suggested no direct interaction between Mg2+ ions and adenylate kinase (AK) in the AK.MgATP (adenosine 5'-triphosphate) complex. However, recent NMR studies concluded that the carboxylate of aspartate 119 accepts a hydrogen bond from a water ligand of the bound Mg2+ ion in the muscle AK.MgATP complex [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. On the other hand, in the 2.6-A crystal structure of the yeast AK.MgAP5A [P1,P5-bis(5'-adenosyl)pentaphosphate] complex, the Mg2+ ion is in proximity to aspartate 93 [Egner, U., Tomasselli, A.G., & Schulz, G.E. (1987) J. Mol. Biol. 195, 649-658]. Substitution of Asp-93 with alanine resulted in no change in dissociation constants, 4-fold increases in Km, and a 650-fold decrease in kcat. Notable changes have been observed in the chemical shifts of the aromatic protons of histidine 36 and a few other aromatic residues. However, the results of detailed analyses of the free enzymes and the AK.MgAP5A complexes by one- and two-dimensional NMR suggested that the changes are due to localized perturbations. Thus it is concluded that Asp-93 stabilizes the transition state by ca. 3.9 kcal/mol. The next question is how. Since proton NMR results indicated that binding of Mg2+ to the AK.AP5A complex induces some changes in the proton NMR signals of WT but not those of D93A, the functional role of Asp-93 should be in binding to Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Molecular basis of ornithine aminotransferase deficiency in B-6-responsive and -nonresponsive forms of gyrate atrophy

    SciTech Connect

    Ramesh, V.; McClatchey, A.I.; Ramesh, N.; Benoit, L.A.; Berson, E.L.; Shih, V.E.; Gusella, J.F. )

    1988-06-01

    Gyrate atrophy (GA), a recessive eye disease involving progressive loss of vision due to chorioretinal degeneration, is associated with a deficiency of the mitochondrial enzyme ornithine aminotransferase with consequent hyperornithinemia. Genetic heterogeneity of GA has been suggested by the demonstration that administration of pyridoxine to increase the level of pyridoxal phosphate, a cofactor of OATase, reduces hyperornithinemia in a subset of patients. The authors have cloned and sequences cDNAs for OATase from two GA patients, one responsive and one nonresponsive to pyridoxine treatment. The respective cDNAs contained different single missense mutations, which were sufficient to eliminate OATase activity when each cDNA was tested in a eukaryotic expression system. However, like the enzyme in fibroblasts from the pyridoxine-responsive patient, OATase encoded by the corresponding cDNA from this individual showed a significant increase in activity when assayed in the presence of an increased pyridoxal phosphate concentration. These data firmly establish that both pyridoxine responsive and nonresponsive forms of GA result from mutations in the OATase structural gene. Moreover, they provide a molecular characterization of the primary lesion in a pyridoxine-responsive genetic disorder.

  11. Structural Insight into the Inhibition of Human Kynurenine Aminotransferase I/Glutamine Transaminase K

    SciTech Connect

    Han, Q.; Robinson, H; Cai, T; Tagle, D; Li, J

    2009-01-01

    Human kynurenine aminotransferase I (hKAT I) catalyzes the formation of kynurenic acid, a neuroactive compound. Here, we report three high-resolution crystal structures (1.50-1.55 A) of hKAT I that are in complex with glycerol and each of two inhibitors of hKAT I: indole-3-acetic acid (IAC) and Tris. Because Tris is able to occupy the substrate binding position, we speculate that this may be the basis for hKAT I inhibition. Furthermore, the hKAT/IAC complex structure reveals that the binding moieties of the inhibitor are its indole ring and a carboxyl group. Six chemicals with both binding moieties were tested for their ability to inhibit hKAT I activity; 3-indolepropionic acid and dl-indole-3-lactic acid demonstrated the highest level of inhibition, and as they cannot be considered as substrates of the enzyme, these two inhibitors are promising candidates for future study. Perhaps even more significantly, we report the discovery of two different ligands located simultaneously in the hKAT I active center for the first time.

  12. Design and mechanism of tetrahydrothiophene-based γ-aminobutyric acid aminotransferase inactivators.

    PubMed

    Le, Hoang V; Hawker, Dustin D; Wu, Rui; Doud, Emma; Widom, Julia; Sanishvili, Ruslan; Liu, Dali; Kelleher, Neil L; Silverman, Richard B

    2015-04-01

    Low levels of γ-aminobutyric acid (GABA), one of two major neurotransmitters that regulate brain neuronal activity, are associated with many neurological disorders, such as epilepsy, Parkinson's disease, Alzheimer's disease, Huntington's disease, and cocaine addiction. One of the main methods to raise the GABA level in human brain is to use small molecules that cross the blood-brain barrier and inhibit the activity of γ-aminobutyric acid aminotransferase (GABA-AT), the enzyme that degrades GABA. We have designed a series of conformationally restricted tetrahydrothiophene-based GABA analogues with a properly positioned leaving group that could facilitate a ring-opening mechanism, leading to inactivation of GABA-AT. One compound in the series is 8 times more efficient an inactivator of GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. Our mechanistic studies show that the compound inactivates GABA-AT by a new mechanism. The metabolite resulting from inactivation does not covalently bind to amino acid residues of GABA-AT but stays in the active site via H-bonding interactions with Arg-192, a π-π interaction with Phe-189, and a weak nonbonded S···O═C interaction with Glu-270, thereby inactivating the enzyme.

  13. Effects of self-association of ornithine aminotransferase on its physicochemical characteristics

    SciTech Connect

    Boernke, W.E.; Stevens, F.J.; Peraino, C.

    1981-01-01

    Previous work in this laboratory has shown that the molecular weight of ornithine aminotransferase (OAT) is concentration dependent. In the present study this property of OAT was further characterized by using sedimentation equilibrium centrifugation to determine the molecular weight of OAT in a range of enzyme concentrations. It was shown that OAT aggregates in a two-stage process as its concentration increases. The first stage involves the association of enzymatically active monomers into trimers, with association of the trimmers into higher order aggregates occurring in the second stage. Decreasing the pH or raising the ionic strength enhanced aggregation, while raising the pH inhibits aggregation; however, the two-stage nature of the aggregation process was not affected by changes in pH and ionic strength. Kinetic analyses of purified enzyme showed that aggregatio results in an increase in the K/sub m/ for both substrates with the V/sub max/ remaining constant, indicating that aggregation of monomers sterically hinders substrate binding. Increased K/sub m/ values were also obtained for OAT sequestered in mitochondia from rats fed a high-protein diet to increase mitochondrial OAT levels. The higher K/sub m/ values suggest that the elevation of OAT in vivo is accompanied by aggregation of the enzyme within the mitochondrion. We propose that the aggregation-dependent increase of K/sub m/ in vivo has adaptive value in that it spares ornithine for use in the urea cycle.

  14. The rice FISH BONE gene encodes a tryptophan aminotransferase, which affects pleiotropic auxin-related processes.

    PubMed

    Yoshikawa, Takanori; Ito, Momoyo; Sumikura, Tsuyoshi; Nakayama, Akira; Nishimura, Takeshi; Kitano, Hidemi; Yamaguchi, Isomaro; Koshiba, Tomokazu; Hibara, Ken-Ichiro; Nagato, Yasuo; Itoh, Jun-Ichi

    2014-06-01

    Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole-3-pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole-3-acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin-related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.

  15. A missense mutation in kynurenine aminotransferase-1 in spontaneously hypertensive rats.

    PubMed

    Kwok, John B J; Kapoor, Ranjna; Gotoda, Takanari; Iwamoto, Yasuhiko; Iizuka, Yoko; Yamada, Nobuhiro; Isaacs, Kim E; Kushwaha, Virag V; Church, W Bret; Schofield, Peter R; Kapoor, Vimal

    2002-09-27

    Spontaneously hypertensive rats (SHR) are the most extensively used animal model for genetic hypertension, increased stroke damage, and insulin resistance syndromes; however, the identification of target genes has proved difficult. SHR show elevated sympathetic nerve activity, and stimulation of the central blood pressure control centers with glutamate or nicotine results in exaggerated blood pressure responses, effects that appear to be genetically determined. Kynurenic acid, a competitive glutamate antagonist and a non-competitive nicotinic antagonist, can be synthesized in the brain by the enzyme kynurenine aminotransferase-1 (KAT-1). We have previously shown that KAT-1 activity is significantly reduced in SHR compared with normotensive Wistar Kyoto rats (WKY). Here we show that KAT-1 contains a missense mutation, E61G, in all the strains of SHR examined but not in any of the WKY or outbred strains. Previous studies on F2 rats from a cross of stroke-prone SHR and WKY have shown a suggestive level of linkage between elevated blood pressure and the KAT-1 locus on chromosome 3. In addition, the mutant enzyme expressed in Escherichia coli displays altered kinetics. This mutation may explain the enhanced sensitivity to glutamate and nicotine seen in SHR that may be related to an underlying mechanism of hypertension and increased sensitivity to stroke. PMID:12145272

  16. Bicyclic γ-amino acids as inhibitors of γ-aminobutyrate aminotransferase.

    PubMed

    Pinto, Andrea; Tamborini, Lucia; Pennacchietti, Eugenia; Coluccia, Antonio; Silvestri, Romano; Cullia, Gregorio; De Micheli, Carlo; Conti, Paola; De Biase, Daniela

    2016-01-01

    The γ-aminobutyrate (GABA)-degradative enzyme GABA aminotransferase (GABA-AT) is regarded as an attractive target to control GABA levels in the central nervous system: this has important implications in the treatment of several neurological disorders and drug dependencies. We have investigated the ability of newly synthesized compounds to act as GABA-AT inhibitors. These compounds have a unique bicyclic structure: the carbocyclic ring bears the GABA skeleton, while the fused 3-Br-isoxazoline ring contains an electrophilic warhead susceptible of nucleophilic attack by an active site residue of the target enzyme. Out of the four compounds tested, only the one named (+)-3 was found to significantly inhibit mammalian GABA-AT in vitro. Docking studies, performed on the available structures of GABA-AT, support the experimental findings: out of the four tested compounds, only (+)-3 suitably orients the electrophilic 3-Br-isoxazoline warhead towards the active site nucleophilic residue Lys329, thereby explaining the irreversible inhibition of GABA-AT observed experimentally. PMID:25807299

  17. The rice FISH BONE gene encodes a tryptophan aminotransferase, which affects pleiotropic auxin-related processes.

    PubMed

    Yoshikawa, Takanori; Ito, Momoyo; Sumikura, Tsuyoshi; Nakayama, Akira; Nishimura, Takeshi; Kitano, Hidemi; Yamaguchi, Isomaro; Koshiba, Tomokazu; Hibara, Ken-Ichiro; Nagato, Yasuo; Itoh, Jun-Ichi

    2014-06-01

    Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole-3-pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole-3-acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin-related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated. PMID:24654985

  18. Solvation Free Energies of Alanine Peptides: The Effect of Flexibility

    SciTech Connect

    Kokubo, Hironori; Harris, Robert C.; Asthagiri, Dilip; Pettitt, Bernard M.

    2013-12-03

    The electrostatic (?Gel), cavity-formation (?Gvdw), and total (?G) solvation free energies for 10 alanine peptides ranging in length (n) from 1 to 10 monomers were calculated. The free energies were computed both with xed, extended conformations of the peptides and again for some of the peptides without constraints. The solvation free energies, ?Gel, ?Gvdw, and ?G, were found to be linear in n, with the slopes of the best-fit lines being gamma_el, gamma_vdw, and gamma, respectively. Both gamma_el and gamma were negative for fixed and flexible peptides, and gamma_vdw was negative for fixed peptides. That gamma_vdw was negative was surprising, as experimental data on alkanes, theoretical models, and MD computations on small molecules and model systems generally suggest that gamma_vdw should be positive. A negative gamma_vdw seemingly contradicts the notion that ?Gvdw drives the initial collapse of the protein when it folds by favoring conformations with small surface areas, but when we computed ?Gvdw for the flexible peptides, thereby allowing the peptides to assume natural ensembles of more compact conformations, gamma-vdw was positive. Because most proteins do not assume extended conformations, a ?Gvdw that increases with increasing surface area may be typical for globular proteins. An alternative hypothesis is that the collapse is driven by intramolecular interactions. We show that the intramolecular van der Waal's interaction energy is more favorable for the flexible than for the extended peptides, seemingly favoring this hypothesis, but the large fluctuations in this energy may make attributing the collapse of the peptide to this intramolecular energy difficult.

  19. Rapid Ti(III) reduction of perchlorate in the presence of beta-alanine: kinetics, pH effect, complex formation, and beta-alanine effect.

    PubMed

    Wang, Chao; Huang, Zhengdao; Lippincott, Lee; Meng, Xiaoguang

    2010-03-15

    Ti(III) reduction of perchlorate might be a useful method for the treatment of highly perchlorate-contaminated water. Though the reaction rate was usually low, we observed that beta-alanine (HOOCCH(2)CH(2)NH(2)) could significantly promote the reaction. A complete (>99.9%) perchlorate removal was obtained in a solution containing [ClO(4)(-)]=1.0mM, [Ti(III)]=40 mM, and [beta-alanine]=120 mM after 2.5h of reaction under 50 degrees C. The effects of both pH and complex formation on the reaction were then studied. The results showed that without beta-alanine the optimal pH was 2.3. When pH increased from 1.6 to 2.3, the reduction rate increased remarkably. In the pH range >2.3, however, the reduction was significantly inhibited, attributed to the formation of Ti(III) precipitate. The presence of beta-alanine at a molar ratio of [beta-alanine]:[Ti(III)]=3:1 significantly increased the reduction rate of perchlorate even at near neutral pH. This is because beta-alanine formed complexes with Ti(III), which greatly improved the total soluble [Ti(III)] in the pH range between 3.5 and 6. The findings may lead to the development of rapid treatment methods for intermittent and small stream of highly perchlorate-contaminated water, which are resulted from the manufacturing, storage, handling, use and/or disposal of large quantities of perchlorate salts. PMID:19864064

  20. Rapid Ti(III) reduction of perchlorate in the presence of beta-alanine: kinetics, pH effect, complex formation, and beta-alanine effect.

    PubMed

    Wang, Chao; Huang, Zhengdao; Lippincott, Lee; Meng, Xiaoguang

    2010-03-15

    Ti(III) reduction of perchlorate might be a useful method for the treatment of highly perchlorate-contaminated water. Though the reaction rate was usually low, we observed that beta-alanine (HOOCCH(2)CH(2)NH(2)) could significantly promote the reaction. A complete (>99.9%) perchlorate removal was obtained in a solution containing [ClO(4)(-)]=1.0mM, [Ti(III)]=40 mM, and [beta-alanine]=120 mM after 2.5h of reaction under 50 degrees C. The effects of both pH and complex formation on the reaction were then studied. The results showed that without beta-alanine the optimal pH was 2.3. When pH increased from 1.6 to 2.3, the reduction rate increased remarkably. In the pH range >2.3, however, the reduction was significantly inhibited, attributed to the formation of Ti(III) precipitate. The presence of beta-alanine at a molar ratio of [beta-alanine]:[Ti(III)]=3:1 significantly increased the reduction rate of perchlorate even at near neutral pH. This is because beta-alanine formed complexes with Ti(III), which greatly improved the total soluble [Ti(III)] in the pH range between 3.5 and 6. The findings may lead to the development of rapid treatment methods for intermittent and small stream of highly perchlorate-contaminated water, which are resulted from the manufacturing, storage, handling, use and/or disposal of large quantities of perchlorate salts.

  1. Concordance of collagen-based radiocarbon and aspartic-acid racemization ages.

    PubMed

    Bada, J L; Schroeder, R A; Protsch, R; Berger, R

    1974-03-01

    By determining the extent of racemization of aspartic acid in a well-dated bone, it is possible to calculate the in situ first-order rate constant for the interconversion of the L and D enantiomers of aspartic acid. Collagen-based radiocarbon-dated bones are shown to be suitable samples for use in "calibrating" the racemization reaction. Once the aspartic-acid racemization reaction has been "calibrated" for a site, the reaction can be used to date other bones from the deposit. Ages deduced by this method are in good agreement with radiocarbon ages. These results provide evidence that the aspartic-acid racemization reaction is an important chronological tool for dating bones either too old or too small for radiocarbon dating. As an example of the potential application of the technique for dating fossil man, a piece of Rhodesian Man from Broken Hill, Zambia, was analyzed and tentatively assigned an age of about 110,000 years.

  2. N-Methyl-D-Aspartate Receptor Activation May Contribute to Glufosinate Neurotoxicity

    EPA Science Inventory

    N-Methyl-D-aspartate Receptor Activation May Contribute to Glufosinate Neurotoxicity Glufosinate (GLF) at high levels in mammals causes convulsions through a mechanism that is not completely understood. The structural similarity of GLF to glutamate (GLU) implicates the glutamate...

  3. Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells

    PubMed Central

    Sullivan, Lucas B.; Gui, Dan Y.; Hosios, Aaron M.; Bush, Lauren N.; Freinkman, Elizaveta; Vander Heiden, Matthew G.

    2015-01-01

    Summary Mitochondrial respiration is important for cell proliferation, however the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis. PMID:26232225

  4. Atomic resolution crystal structure of Sapp2p, a secreted aspartic protease from Candida parapsilosis.

    PubMed

    Dostál, Jiří; Pecina, Adam; Hrušková-Heidingsfeldová, Olga; Marečková, Lucie; Pichová, Iva; Řezáčová, Pavlina; Lepšík, Martin; Brynda, Jiří

    2015-12-01

    The virulence of the Candida pathogens is enhanced by the production of secreted aspartic proteases, which therefore represent possible targets for drug design. Here, the crystal structure of the secreted aspartic protease Sapp2p from Candida parapsilosis was determined. Sapp2p was isolated from its natural source and crystallized in complex with pepstatin A, a classical aspartic protease inhibitor. The atomic resolution of 0.83 Å allowed the protonation states of the active-site residues to be inferred. A detailed comparison of the structure of Sapp2p with the structure of Sapp1p, the most abundant C. parapsilosis secreted aspartic protease, was performed. The analysis, which included advanced quantum-chemical interaction-energy calculations, uncovered molecular details that allowed the experimentally observed equipotent inhibition of both isoenzymes by pepstatin A to be rationalized.

  5. Chiral Asymmetric Structures in Aspartic Acid and Valine Crystals Assessed by Atomic Force Microscopy.

    PubMed

    Teschke, Omar; Soares, David Mendez

    2016-03-29

    Structures of crystallized deposits formed by the molecular self-assembly of aspartic acid and valine on silicon substrates were imaged by atomic force microscopy. Images of d- and l-aspartic acid crystal surfaces showing extended molecularly flat sheets or regions separated by single molecule thick steps are presented. Distinct orientation surfaces were imaged, which, combined with the single molecule step size, defines the geometry of the crystal. However, single molecule step growth also reveals the crystal chirality, i.e., growth orientations. The imaged ordered lattice of aspartic acid (asp) and valine (val) mostly revealed periodicities corresponding to bulk terminations, but a previously unreported molecular hexagonal lattice configuration was observed for both l-asp and l-val but not for d-asp or d-val. Atomic force microscopy can then be used to identify the different chiral forms of aspartic acid and valine crystals.

  6. Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells.

    PubMed

    Sullivan, Lucas B; Gui, Dan Y; Hosios, Aaron M; Bush, Lauren N; Freinkman, Elizaveta; Vander Heiden, Matthew G

    2015-07-30

    Mitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis.

  7. Determination of the carbon, hydrogen and nitrogen contents of alanine and their uncertainties using the certified reference material L-alanine (NMIJ CRM 6011-a).

    PubMed

    Itoh, Nobuyasu; Sato, Ayako; Yamazaki, Taichi; Numata, Masahiko; Takatsu, Akiko

    2013-01-01

    The carbon, hydrogen, and nitrogen (CHN) contents of alanine and their uncertainties were estimated using a CHN analyzer and the certified reference material (CRM) L-alanine. The CHN contents and their uncertainties, as measured using the single-point calibration method, were 40.36 ± 0.20% for C, 7.86 ± 0.13% for H, and 15.66 ± 0.09% for N; the results obtained using the bracket calibration method were also comparable. The method described in this study is reasonable, convenient, and meets the general requirement of having uncertainties ≤ 0.4%.

  8. Aspartic Peptidases of Human Pathogenic Trypanosomatids: Perspectives and Trends for Chemotherapy

    PubMed Central

    Santos, L.O.; Garcia-Gomes, A.S.; Catanho, M.; Sodré, C.L.; Santos, A.L.S.; Branquinha, M.H.; d’Avila-Levy, C.M.

    2013-01-01

    Aspartic peptidases are proteolytic enzymes present in many organisms like vertebrates, plants, fungi, protozoa and in some retroviruses such as human immunodeficiency virus (HIV). These enzymes are involved in important metabolic processes in microorganisms/virus and play major roles in infectious diseases. Although few studies have been performed in order to identify and characterize aspartic peptidase in trypanosomatids, which include the etiologic agents of leishmaniasis, Chagas’ disease and sleeping sickness, some beneficial properties of aspartic peptidase inhibitors have been described on fundamental biological events of these pathogenic agents. In this context, aspartic peptidase inhibitors (PIs) used in the current chemotherapy against HIV (e.g., amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) were able to inhibit the aspartic peptidase activity produced by different species of Leishmania. Moreover, the treatment of Leishmania promastigotes with HIV PIs induced several perturbations on the parasite homeostasis, including loss of the motility and arrest of proliferation/growth. The HIV PIs also induced an increase in the level of reactive oxygen species and the appearance of irreversible morphological alterations, triggering parasite death pathways such as programed cell death (apoptosis) and uncontrolled autophagy. The blockage of physiological parasite events as well as the induction of death pathways culminated in its incapacity to adhere, survive and escape of phagocytic cells. Collectively, these results support the data showing that parasites treated with HIV PIs have a significant reduction in the ability to cause in vivo infection. Similarly, the treatment of Trypanosoma cruzi cells with pepstatin A showed a significant inhibition on both aspartic peptidase activity and growth as well as promoted several and irreversible morphological changes. These studies indicate that aspartic peptidases can be promising targets in

  9. The standard enthalpies of formation of crystalline N-(carboxymethyl)aspartic acid and its aqueous solutions

    NASA Astrophysics Data System (ADS)

    Lytkin, A. I.; Chernyavskaya, N. V.; Volkov, A. V.; Nikol'Skii, V. M.

    2007-07-01

    The energy of combustion of N-(carboxymethyl)aspartic acid (CMAA) was determined by bomb calorimetry in oxygen. The standard enthalpies of combustion and formation of crystalline N-(carboxymethyl)aspartic acid were calculated. The heat effects of solution of crystalline CMAA in water and a solution of sodium hydroxide were measured at 298.15 K by direct calorimetry. The standard enthalpies of formation of CMAA and its dissociation products in aqueous solution were determined.

  10. Effect of beta-alanine supplementation on repeated sprint performance during the Loughborough Intermittent Shuttle Test.

    PubMed

    Saunders, Bryan; Sale, Craig; Harris, Roger C; Sunderland, Caroline

    2012-07-01

    The aim of this study was to examine the effect of β-alanine supplementation on repeated sprint performance during an intermittent exercise protocol designed to replicate games play. Sixteen elite and twenty non-elite game players performed the Loughborough Intermittent Shuttle Test (LIST) on two separate occasions. Trials were separated by 4 weeks of supplementation with either β-alanine (BA) or maltodextrin (MD). There was no deterioration in sprint times from Set 1 to Set 6 of the LIST in either group prior to supplementation (elite: P=0.92; non-elite: P=0.12). Neither BA nor MD supplementation affected sprint times. Blood lactate concentrations were elevated during exercise in both groups, with no effect of supplementation. β-Alanine supplementation did not significantly improve sprint performance during the LIST. Neither group showed a performance decrement prior to supplementation, which might have masked any benefit from increased muscle buffering capacity due to β-alanine supplementation.

  11. Enzymatic determination of carbon-14 labeled L-alanine in biological samples

    SciTech Connect

    Serra, F.; Palou, A.; Pons, A.

    1987-07-15

    A method for determination of L-alanine-specific radioactivity in biological samples is presented. This method is based on the specific enzymatic transformation of L-alanine to pyruvic acid hydrazone catalyzed by the enzyme L-alanine dehydrogenase, formation of the pyruvic acid 2,4-dinitrophenylhydrazone derivative, and quantitative trapping in Amberlite XAD-7 columns, followed by radioactivity counting of the lipophilic eluate. No interferences from other UC-labeled materials such as D-glucose, glycerol, L-lactate, L-serine, L-glutamate, L-phenylalanine, glycine, L-leucine, and L-arginine were observed. This inexpensive and high-speed method is applicable to the simultaneous determination of L-alanine-specific radioactivity for a large number of samples.

  12. Titration of Alanine Monitored by NMR Spectroscopy: A Biochemistry Laboratory Experiment

    ERIC Educational Resources Information Center

    Waller, Francis J.; And Others

    1977-01-01

    The experiment described here involves simultaneous monitoring of pH and NMR chemical shifts during an aqueous titration of alpha- and beta-alanine. This experiment is designed for use in an undergraduate biochemistry course. (MR)

  13. Alanine blends for ESR measurements of thermal neutron fluence in a mixed radiation field.

    PubMed

    Marrale, M; Brai, M; Gennaro, G; Triolo, A; Bartolotta, A; D'Oca, M C; Rosi, G

    2007-01-01

    In this paper, the results of a study on the electron spin resonance (ESR) dosimetry to measure thermal neutron fluence in a mixed radiation field (neutron and photons) are presented. The ESR responses of alanine dosemeters with different additives are compared. In particular, the (10)B-acid boric and the Gd-oxide were chosen to enhance the sensitivity of alanine dosemeters to thermal neutrons. Irradiations were carried out inside the thermal column of the TAPIRO reactor of the ENEA center, Casaccia Rome. The main results are a greater neutron sensitivity and a smaller lowest detectable fluence for the dosemeters with gadolinium than for dosemeters of alanine with (10)B, which is well known to be much more sensitive to thermal neutrons than simple alanine.

  14. An automated system for the measurement of alanine/EPR dosimeters

    PubMed

    Sharpe; Sephton

    2000-05-01

    NPL for several years has offered mailed reference dosimetry services based on alanine/EPR dosimeters, both at industrial and therapy dose levels. Compared to other methods of reference dosimetry, operator involvement in alanine/EPR has been found to be relatively high, and contributes significantly to the overall economics of the process. Commercially available sample changers are not suitable for high accuracy applications, and it has proved necessary to develop a dedicated automation system to handle NPL alanine dosimeter pellets. In this paper we describe an automatic sample changer for placing and retrieving alanine pellets into and out of the cavity of a standard research grade EPR spectrometer. Up to 32 pellets can be held in each removable sample tray. The sample changer software has been interfaced into the spectrometer control software to enable complete automation of the measurement process, including the optimization of spectrometer settings and rotation of the sample within the cavity.

  15. Alanine as an end product during fermentation of monosaccharides by Clostridium strain P2.

    PubMed

    Orlygsson, J; Anderson, R; Svensson, B H

    1995-11-01

    The thermophilic Clostridium P2 was isolated from a semi-continuously fed reactor with high ammonium concentration. This bacterium formed substantial amounts of L-alanine as a major fermentation product from glucose, fructose and mannose. Low amounts of acetate, butyrate, carbon dioxide and hydrogen were also formed. A high partial pressure of hydrogen inhibited the degradation of the monosaccharides, whereas hydrogen removal, in the form of methanogenesis was found to be stimulatory. However, the amount of alanine produced per mole of hexose degraded did not change. Hexose degradation and alanine production were favoured by high ammonium concentrations. Nuclear magnetic resonance spectroscopy studies provided strong evidence that an active Embden-Meyerhof-Parnas pathway existed and that alanine was produced via an amination of pyruvate.

  16. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the glutamate-1-semialdehyde aminotransferase from Bacillus subtilis

    SciTech Connect

    Lv, Xinhuai; Fan, Jun; Ge, Honghua; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun Niu, Liwen

    2006-05-01

    Crystals of glutamate-1-semialdehyde aminotransferase (GSAT) from B. subtilis were obtained and diffraction data were collected to 2.0 Å resolution. 5-Aminolevulinic acid (ALA) is the first committed universal precursor in the tetrapyrrole-biosynthesis pathway. Plants, algae and many other bacteria synthesize ALA from glutamate by a C5 pathway in which the carbon skeleton of glutamate is converted into ALA by a series of enzymes. Glutamate-1-semialdehyde aminotransferase (GSAT) is the last enzyme in this pathway. The gene that codes for GSAT was amplified from the cDNA library of Bacillus subtilis and overexpressed in Escherichia coli strain BL21(DE3). The protein was purified and crystallized. Well diffracting single crystals were obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray diffraction studies yielded excellent diffraction data to a resolution of 2.0 Å.

  17. Purification, crystallization and preliminary X-ray crystallographic analysis of branched-chain aminotransferase from Deinococcus radiodurans

    SciTech Connect

    Chen, Chung-Der; Huang, Tien-Feng; Lin, Chih-Hao; Guan, Hong-Hsiang; Hsieh, Yin-Cheng; Lin, Yi-Hung; Huang, Yen-Chieh; Liu, Ming-Yih; Chang, Wen-Chang; Chen, Chun-Jung

    2007-06-01

    The crystallization of branched-chain aminotransferase from D. radiodurans is described. The branched-chain amino-acid aminotransferase (BCAT), which requires pyridoxal 5′-phosphate (PLP) as a cofactor, is a key enzyme in the biosynthetic pathway of the hydrophobic amino acids leucine, isoleucine and valine. DrBCAT from Deinococcus radiodurans, which has a molecular weight of 40.9 kDa, was crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction data to 2.50 Å resolution from a DrBCAT crystal, the crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 56.37, b = 90.70, c = 155.47 Å. Preliminary analysis indicates the presence of two DrBCAT molecules in the asymmetric unit, with a solvent content of 47.52%.

  18. Repeated Supramaximal Exercise-Induced Oxidative Stress: Effect of β-Alanine Plus Creatine Supplementation

    PubMed Central

    Belviranli, Muaz; Okudan, Nilsel; Revan, Serkan; Balci, Serdar; Gokbel, Hakki

    2016-01-01

    Background: Carnosine is a dipeptide formed from the β-alanine and histidine amino acids and found in mainly in the brain and muscle, especially fast twitch muscle. Carnosine and creatine has an antioxidant effect and carnosine accounts for about 10% of the muscle's ability to buffer the H+ ions produced by exercise. Objectives: The aim of the study was to investigate the effects of beta alanine and/or creatine supplementation on oxidant and antioxidant status during repeated Wingate tests (WTs). Patients and Methods: Forty four sedentary males participated in the study. Participants performed three 30s WTs with 2 minutes rest between exercise bouts. After the first exercise session, the subjects were assigned to one of four groups: Placebo, Creatine, Beta-alanine and Beta-alanine plus creatine. Participants ingested twice per day for 22 consecutive days, then four times per day for the following 6 days. After the supplementation period the second exercise session was applied. Blood samples were taken before and immediately after the each exercise session for the analysis of oxidative stress and antioxidant markers. Results: Malondialdehyde levels and superoxide dismutase activities were affected by neither supplementation nor exercise. During the pre-supplementation session, protein carbonyl reduced and oxidized glutathione (GSH and GSSG) levels increased immediately after the exercise. However, during the post-supplementation session GSH and GSSG levels increased in beta-alanine and beta-alanine plus creatine groups immediately after the exercise compared to pre-exercise. In addition, during the post-supplementation session total antioxidant capacity increased in beta-alanine group immediately after the exercise. Conclusions: Beta-alanine supplementation has limited antioxidant effect during the repeated WTs. PMID:27217925

  19. Prothrombin time as an index of mortality in kwashiorkor.

    PubMed

    Akinyinka, O O; Falade, A G; Ogbechie, C O

    1990-03-01

    Prothrombin time, serum albumin, aminotransferases and liver size were evaluated in 40 consecutive cases of kwashiorkor. Eleven (27.5%) of the 40 patients died. Eight out of the 11 patients who died had a prolonged prothrombin time of more than 3 s above the control compared to only 4 out of the 29 who survived (p = 0.005). Mean serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) albumin, globulin and liver size were abnormal but similar in both groups. These results may indicate a predictive mortality value of prothrombin time in kwashiorkor.

  20. [Alanine solution as enzyme reaction buffer used in A to O blood group conversion].

    PubMed

    Li, Su-Bo; Zhang, Xue; Zhang, Yin-Ze; Tan, Ying-Xia; Bao, Guo-Qiang; Wang, Ying-Li; Ji, Shou-Ping; Gong, Feng; Gao, Hong-Wei

    2014-06-01

    The aim of this study was to investigate the effect of alanine solution as α-N-acetylgalactosaminidase enzyme reaction buffer on the enzymatic activity of A antigen. The binding ability of α-N-acetylgalactosaminidase with RBC in different reaction buffer such as alanine solution, glycine solution, normal saline (0.9% NaCl), PBS, PCS was detected by Western blot. The results showed that the efficiency of A to O conversion in alanine solution was similar to that in glycine solution, and Western blot confirmed that most of enzymes blinded with RBC in glycine or alanine solution, but few enzymes blinded with RBC in PBS, PCS or normal saline. The evidences indicated that binding of enzyme with RBC was a key element for A to O blood group conversion, while the binding ability of α-N-acetylgalactosaminidase with RBC in alanine or glycine solution was similar. It is concluded that alanine solution can be used as enzyme reaction buffer in A to O blood group conversion. In this buffer, the α-N-acetylgalactosaminidase is closely blinded with RBC and α-N-acetylgalactosaminidase plays efficient enzymatic activity of A antigen.

  1. EPR dosimetry of radiotherapy photon beams in inhomogeneous media using alanine films

    NASA Astrophysics Data System (ADS)

    Helge Østerås, Bjørn; Olaug Hole, Eli; Rune Olsen, Dag; Malinen, Eirik

    2006-12-01

    In the current work, EPR (electron paramagnetic resonance) dosimetry using alanine films (134 µm thick) was utilized for dose measurements in inhomogeneous phantoms irradiated with radiotherapy photon beams. The main phantom material was PMMA, while either Styrofoam or aluminium was introduced as an inhomogeneity. The phantoms were irradiated to a maximum dose of about 30 Gy with 6 or 15 MV photons. The performance of the alanine film dosimeters was investigated and compared to results from ion chamber dosimetry, Monte Carlo simulations and radiotherapy treatment planning calculations. It was found that the alanine film dosimeters had a linear dose response above approximately 5 Gy, while a background signal obscured the response at lower dose levels. For doses between 5 and 60 Gy, the standard deviation of single alanine film dose estimates was about 2%. The alanine film dose estimates yielded results comparable to those from the Monte Carlo simulations and the ion chamber measurements, with absolute differences between estimates in the order of 1 15%. The treatment planning calculations exhibited limited applicability. The current work shows that alanine film dosimetry is a method suitable for estimating radiotherapeutical doses and for dose measurements in inhomogeneous media.

  2. EPR dosimetry of radiotherapy photon beams in inhomogeneous media using alanine films.

    PubMed

    Osterås, Bjørn Helge; Hole, Eli Olaug; Olsen, Dag Rune; Malinen, Eirik

    2006-12-21

    In the current work, EPR (electron paramagnetic resonance) dosimetry using alanine films (134 microm thick) was utilized for dose measurements in inhomogeneous phantoms irradiated with radiotherapy photon beams. The main phantom material was PMMA, while either Styrofoam or aluminium was introduced as an inhomogeneity. The phantoms were irradiated to a maximum dose of about 30 Gy with 6 or 15 MV photons. The performance of the alanine film dosimeters was investigated and compared to results from ion chamber dosimetry, Monte Carlo simulations and radiotherapy treatment planning calculations. It was found that the alanine film dosimeters had a linear dose response above approximately 5 Gy, while a background signal obscured the response at lower dose levels. For doses between 5 and 60 Gy, the standard deviation of single alanine film dose estimates was about 2%. The alanine film dose estimates yielded results comparable to those from the Monte Carlo simulations and the ion chamber measurements, with absolute differences between estimates in the order of 1-15%. The treatment planning calculations exhibited limited applicability. The current work shows that alanine film dosimetry is a method suitable for estimating radiotherapeutical doses and for dose measurements in inhomogeneous media. PMID:17148820

  3. Relaxed evolution in the tyrosine aminotransferase gene tat in old world fruit bats (Chiroptera: Pteropodidae).

    PubMed

    Shen, Bin; Fang, Tao; Yang, Tianxiao; Jones, Gareth; Irwin, David M; Zhang, Shuyi

    2014-01-01

    Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.

  4. Participation of Ets transcription factors in the glucocorticoid response of the rat tyrosine aminotransferase gene.

    PubMed Central

    Espinás, M L; Roux, J; Ghysdael, J; Pictet, R; Grange, T

    1994-01-01

    We have previously shown that two remote glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase (TAT) gene contain multiple binding sites for several transcription factor families, including the glucocorticoid receptor (GR). We report here the identification of two novel binding sites for members of the Ets family of transcription factors in one of these GRUs. One of these binding sites overlaps the major GR-binding site (GRBS), whereas the other is located in its vicinity. Inactivation of the latter binding site leads to a twofold reduction of the glucocorticoid response, whereas inactivation of the site overlapping the GRBS has no detectable effect. In vivo footprinting analysis reveals that the active site is occupied in a glucocorticoid-independent manner, in a TAT-expressing cell line, even though it is located at a position where there is a glucocorticoid-dependent alteration of the nucleosomal structure. This same site is not occupied in a cell line that does not express TAT but expresses Ets-related DNA-binding activities, suggesting the existence of an inhibitory effect of chromatin structure at a hierarchical level above the nucleosome. The inactive Ets-binding site that overlaps the GRBS is not occupied even in TAT-expressing cells. However, this same overlapping site can confer Ets-dependent stimulation of both basal and glucocorticoid-induced levels when it is isolated from the GRU and duplicated. Ets-1 expression in COS cells mimics the activity of the Ets-related activities present in hepatoma cells. These Ets-binding sites could participate in the integration of the glucocorticoid response of the TAT gene with signal transduction pathways triggered by other nonsteroidal extracellular stimuli. Images PMID:7910945

  5. Auxin and Tryptophan Homeostasis Are Facilitated by the ISS1/VAS1 Aromatic Aminotransferase in Arabidopsis

    PubMed Central

    Pieck, Michael; Yuan, Youxi; Godfrey, Jason; Fisher, Christopher; Zolj, Sanda; Vaughan, Dylan; Thomas, Nicholas; Wu, Connie; Ramos, Julian; Lee, Norman; Normanly, Jennifer; Celenza, John L.

    2015-01-01

    Indole-3-acetic acid (IAA) plays a critical role in regulating numerous aspects of plant growth and development. While there is much genetic support for tryptophan-dependent (Trp-D) IAA synthesis pathways, there is little genetic evidence for tryptophan-independent (Trp-I) IAA synthesis pathways. Using Arabidopsis, we identified two mutant alleles of ISS1 (Indole Severe Sensitive) that display indole-dependent IAA overproduction phenotypes including leaf epinasty and adventitious rooting. Stable isotope labeling showed that iss1, but not WT, uses primarily Trp-I IAA synthesis when grown on indole-supplemented medium. In contrast, both iss1 and WT use primarily Trp-D IAA synthesis when grown on unsupplemented medium. iss1 seedlings produce 8-fold higher levels of IAA when grown on indole and surprisingly have a 174-fold increase in Trp. These findings indicate that the iss1 mutant’s increase in Trp-I IAA synthesis is due to a loss of Trp catabolism. ISS1 was identified as At1g80360, a predicted aromatic aminotransferase, and in vitro and in vivo analysis confirmed this activity. At1g80360 was previously shown to primarily carry out the conversion of indole-3-pyruvic acid to Trp as an IAA homeostatic mechanism in young seedlings. Our results suggest that in addition to this activity, in more mature plants ISS1 has a role in Trp catabolism and possibly in the metabolism of other aromatic amino acids. We postulate that this loss of Trp catabolism impacts the use of Trp-D and/or Trp-I IAA synthesis pathways. PMID:26163189

  6. Relaxed Evolution in the Tyrosine Aminotransferase Gene Tat in Old World Fruit Bats (Chiroptera: Pteropodidae)

    PubMed Central

    Shen, Bin; Fang, Tao; Yang, Tianxiao; Jones, Gareth; Irwin, David M.; Zhang, Shuyi

    2014-01-01

    Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet. PMID:24824435

  7. Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae

    PubMed Central

    Kingsbury, Joanne M.; Sen, Neelam D.; Cardenas, Maria E.

    2015-01-01

    The conserved target of rapamycin complex 1 (TORC1) integrates nutrient signals to orchestrate cell growth and proliferation. Leucine availability is conveyed to control TORC1 activity via the leu-tRNA synthetase/EGOC-GTPase module in yeast and mammals, but the mechanisms sensing leucine remain only partially understood. We show here that both leucine and its α-ketoacid metabolite, α-ketoisocaproate, effectively activate the yeast TORC1 kinase via both EGOC GTPase-dependent and -independent mechanisms. Leucine and α-ketoisocaproate are interconverted by ubiquitous branched-chain aminotransferases (BCAT), which in yeast are represented by the mitochondrial and cytosolic enzymes Bat1 and Bat2, respectively. BCAT yeast mutants exhibit severely compromised TORC1 activity, which is partially restored by expression of Bat1 active site mutants, implicating both catalytic and structural roles of BCATs in TORC1 control. We find that Bat1 interacts with branched-chain amino acid metabolic enzymes and, in a leucine-dependent fashion, with the tricarboxylic acid (TCA)-cycle enzyme aconitase. BCAT mutation perturbed TCA-cycle intermediate levels, consistent with a TCA-cycle block, and resulted in low ATP levels, activation of AMPK, and TORC1 inhibition. We propose the biosynthetic capacity of BCAT and its role in forming multicomplex metabolons connecting branched-chain amino acids and TCA-cycle metabolism governs TCA-cycle flux to activate TORC1 signaling. Because mammalian mitochondrial BCAT is known to form a supramolecular branched-chain α-keto acid dehydrogenase enzyme complex that links leucine metabolism to the TCA-cycle, these findings establish a precedent for understanding TORC1 signaling in mammals. PMID:26659116

  8. Novel and recurrent tyrosine aminotransferase gene mutations in tyrosinemia type II.

    PubMed

    Hühn, R; Stoermer, H; Klingele, B; Bausch, E; Fois, A; Farnetani, M; Di Rocco, M; Boué, J; Kirk, J M; Coleman, R; Scherer, G

    1998-03-01

    Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disorder of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT). We have previously described one deletion and six different point mutations in four RHS patients. We have now analyzed the TAT genes in a further seven unrelated RHS families from Italy, France, the United Kingdom, and the United States. We have established PCR conditions for the amplification of all twelve TAT exons and have screened the products for mutations by direct sequence analysis or by first performing single-strand conformation polymorphism analysis. We have thus identified the presumably pathological mutations in eight RHS alleles, including two nonsense mutations (R57X, E411X) and four amino acid substitutions (R119W, L201R, R433Q, R433W). Only the R57X mutation, which was found in one Scottish and two Italian families, has been previously reported in another Italian family. Haplotype analysis indicates that this mutation, which involves a CpG dinucleotide hot spot, has a common origin in the three Italian families but arose independently in the Scottish family. Two polymorphisms have also been detected, viz., a protein polymorphism, P15S, and a silent substitution S103S (TCG-->TCA). Expression of R433Q and R433W demonstrate reduced activity of the mutant proteins. In all, twelve different TAT gene mutations have now been identified in tyrosinemia type II.

  9. Inhibition of kynurenine aminotransferase II reduces activity of midbrain dopamine neurons.

    PubMed

    Linderholm, Klas R; Alm, Maximilian Tufvesson; Larsson, Markus K; Olsson, Sara K; Goiny, Michel; Hajos, Mihaly; Erhardt, Sophie; Engberg, Göran

    2016-03-01

    Kynurenic acid (KYNA), a neuroactive metabolite of tryptophan, is elevated in the brain of patients with psychotic disorders. Therefore, lowering brain KYNA levels might be a novel approach in the treatment of psychotic disorders. The present in vivo electrophysiological study aimed to investigate the effect of an inhibitor of kynurenine aminotransferase (KAT) II, the primary enzyme for KYNA synthesis, on dopamine (DA) neurons in the ventral tegmental area (VTA). Acute administration of the KAT II inhibitor PF-04859989 (5 or 10 mg/kg) was associated with a short-onset, time-dependent decrease in firing rate and burst activity of DA neurons, both parameters reaching a 50% reduction within 45 min. Furthermore, PF-04859989 reduced the number of spontaneously active DA cells as measured 4-6 after administration. Pretreatment with d-cycloserine (30 mg/kg) or CGP-52432 (10 mg/kg) prevented the inhibitory action of PF-04859989 (5 mg/kg) on firing rate and burst firing activity. In contrast, pretreatment with methyllycaconitine (MLA, 4 mg/kg) did not change the response, whereas picrotoxin (4.5 mg/kg) partially prevented the inhibitory effects of PF-04859989 (5 mg/kg, i.v.). Our results show that a specific inhibition of KAT II is associated with a marked reduction in VTA DA firing activity. This effect appears to be specifically executed by NMDA-receptors and mediated indirectly via a GABA(B)-receptor-induced disinhibition of DA neurons. Our findings are in line with the view that endogenous KYNA, by modulation of the NMDA-receptor, exerts important physiological roles in the brain.

  10. SdrI, a serine-aspartate repeat protein identified in Staphylococcus saprophyticus strain 7108, is a collagen-binding protein.

    PubMed

    Sakinc, Türkan; Kleine, Britta; Gatermann, Sören G

    2006-08-01

    A gene encoding a serine-aspartate repeat protein of Staphylococcus saprophyticus, an important cause of urinary tract infections in young women, has been cloned and sequenced. In contrast to other SD repeat proteins, SdrI carries 21 additional N-terminal repeats with a consensus sequence of (P/A)ATKE(K/E)A(A/V)(T/I)(A/T/S)EE and has the longest SD(AD)(1-5) repetitive region (854 amino acids) described so far. This highly repetitive sequence contains only the amino acids serine, asparagine, and a distinctly greater amount of alanine (37%) than all other known SD repeat proteins (2.3 to 4.4%). In addition, it is a collagen-binding protein of S. saprophyticus and the second example in this organism of a surface protein carrying the LPXTG motif. We constructed an isogenic sdrI knockout mutant that showed decreased binding to immobilized collagen compared with wild-type S. saprophyticus strain 7108. Binding could be reconstituted by complementation. Collagen binding is specifically caused by SdrI, and the recently described UafA protein, the only LPXTG-containing protein in the genome sequence of the type strain, is not involved in this trait. Our experiments suggest that, as in other staphylococci, the presence of different LPXTG-anchored cell wall proteins is common in S. saprophyticus and support the notion that the presence of matrix-binding surface proteins is common in staphylococci.

  11. Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase.

    PubMed Central

    Elliott, T; Avissar, Y J; Rhie, G E; Beale, S I

    1990-01-01

    Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected. Images PMID:2254275

  12. Transport of L-glutamine, L-alanine, L-arginine and L-histidine by the neuron-specific Slc38a8 (SNAT8) in CNS.

    PubMed

    Hägglund, Maria G A; Hellsten, Sofie V; Bagchi, Sonchita; Philippot, Gaëtan; Löfqvist, Erik; Nilsson, Victor C O; Almkvist, Ingrid; Karlsson, Edvin; Sreedharan, Smitha; Tafreshiha, Atieh; Fredriksson, Robert

    2015-03-27

    Glutamine transporters are important for regulating levels of glutamate and GABA in the brain. To date, six members of the SLC38 family (SNATs) have been characterized and functionally subdivided them into System A (SNAT1, SNAT2 and SNAT4) and System N (SNAT3, SNAT5 and SNAT7). Here we present the first functional characterization of SLC38A8, one of the previous orphan transporters from the family, and we suggest that the encoded protein should be named SNAT8 to adhere with the SNAT nomenclature. We show that SLC38A8 has preference for transporting L-glutamine, L-alanine, L-arginine, L-histidine and L-aspartate using a Na+-dependent transport mechanism and that the functional characteristics of SNAT8 have highest similarity to the known System A transporters. We also provide a comprehensive central nervous system expression profile in mouse brain for the Slc38a8 gene and the SNAT8 protein. We show that Slc38a8 (SNAT8) is expressed in all neurons, both excitatory and inhibitory, in mouse brain using in situ hybridization and immunohistochemistry. Furthermore, proximity ligation assay shows highly similar subcellular expression of SNAT7 and SNAT8. In conclusion, the neuronal SLC38A8 has a broad amino acid transport profile and is the first identified neuronal System A transporter. This suggests a key role of SNAT8 in the glutamine/glutamate (GABA) cycle in the brain.

  13. β-alanine improves punch force and frequency in amateur boxers during a simulated contest.

    PubMed

    Donovan, Tim; Ballam, Tim; Morton, James P; Close, Graeme L

    2012-10-01

    The aim of this study was to test the hypothesis that ß-alanine supplementation improves punch power and frequency in amateur boxers during a simulated contest. Sixteen amateur boxers (each approximately 6 yr experience) were assigned to ß-alanine (n = 8; 1.5 g 4 times/d for 4 wk) or placebo supplementation (n = 8) after initially being assessed for baseline punch performance. Before and after the supplementation period, all boxers completed a simulated contest consisting of 3 × 3-min rounds (interspersed with 60-s rests) on a punching bag (with a force transducer attached). Each round involved performing 2 min 50 s standardized punching (standardized jab, cross combination) based on notation analysis, whereas the last 10 s involved maximal-output punching (standardized jab, cross combination), during which time punch force and frequency were recorded. Postcontest blood lactate was significantly increased in the ß-alanine group (presupplementation 9.5 ± 0.9 mmol/L, postsupplementation 12.6 ± 0.5 mmol/L, p < .05), whereas the placebo group showed no change (presupplementation 8 ± 2.8 mmol/L, postsupplementation 7.0 ± 2.7 mmol/L; p > .05). During the 10-s maximal-output punching, changes in mean punch force (ß-alanine 20 ± 1.01 kg, placebo 1 ± 1 kg) and punch frequency (ß-alanine 5 ± 4, placebo -2 ± 3) were greater (p < .05) in the ß-alanine-supplemented group. The authors conclude that ß-alanine supplementation improves punching performance in amateur boxers and suggest that this supplementation protocol may also prove ergogenic for other combat-related sports.

  14. Effects of β-alanine supplementation on exercise performance: a meta-analysis.

    PubMed

    Hobson, R M; Saunders, B; Ball, G; Harris, R C; Sale, C

    2012-07-01

    Due to the well-defined role of β-alanine as a substrate of carnosine (a major contributor to H+ buffering during high-intensity exercise), β-alanine is fast becoming a popular ergogenic aid to sports performance. There have been several recent qualitative review articles published on the topic, and here we present a preliminary quantitative review of the literature through a meta-analysis. A comprehensive search of the literature was employed to identify all studies suitable for inclusion in the analysis; strict exclusion criteria were also applied. Fifteen published manuscripts were included in the analysis, which reported the results of 57 measures within 23 exercise tests, using 18 supplementation regimes and a total of 360 participants [174, β-alanine supplementation group (BA) and 186, placebo supplementation group (Pla)]. BA improved (P=0.002) the outcome of exercise measures to a greater extent than Pla [median effect size (IQR): BA 0.374 (0.140-0.747), Pla 0.108 (-0.019 to 0.487)]. Some of that effect might be explained by the improvement (P=0.013) in exercise capacity with BA compared to Pla; no improvement was seen for exercise performance (P=0.204). In line with the purported mechanisms for an ergogenic effect of β-alanine supplementation, exercise lasting 60-240 s was improved (P=0.001) in BA compared to Pla, as was exercise of >240 s (P=0.046). In contrast, there was no benefit of β-alanine on exercise lasting <60 s (P=0.312). The median effect of β-alanine supplementation is a 2.85% (-0.37 to 10.49%) improvement in the outcome of an exercise measure, when a median total of 179 g of β-alanine is supplemented.

  15. FTIR spectra and conformational structure of deutero-β-alanine isolated in argon matrices

    NASA Astrophysics Data System (ADS)

    Stepanian, Stepan G.; Ivanov, Alexander Yu; Adamowicz, Ludwik

    2016-02-01

    Low temperature FTIR spectra of β-alanine-d3 isolated in argon matrices are used to determine the conformational composition of this compound. UV irradiation of the matrix samples is found to change the relative populations of the β-alanine-d3 conformers. The populations of conformers I and II with an Nsbnd D⋯O intramolecular H-bond decrease after the UV irradiation while the populations of conformer V with an N⋯Dsbnd O H-bond and conformer IV which has no intramolecular H-bonds increase. This behavior of the β-alanine-d3 conformers are used to separate the bands of the different conformers. The analysis of the experimental FTIR spectra is based on the calculated harmonic B3LYP/6-311++G(df,pd) frequencies and on the MP2/aug-cc-pVDZ frequencies calculated with a method that includes anharmonic effects. Polynomial scaling of the calculated frequencies is used to achieve better agreement with the experimental data. The observation of the wide band of the OD stretching vibration at 2201 cm-1 is a direct evidence of the presence of the β-alanine-d3 conformer V in the Ar matrix. In total ten bands of conformer V are detected. The influence of the matrix environment on the structures and the IR spectra of the β-alanine and β-alanine-d3 conformers is investigated. This involves performing calculations of the β-alanine conformers embedded in argon clusters containing from 163 to 166 argon atoms using the M06-2X and B3LYP(GD3BJ) density-functional methods. Good agreement between the calculated and the experimental matrix splitting is demonstrated.

  16. Detection of D-aspartate in tau proteins associated with Alzheimer paired helical filaments.

    PubMed

    Kenessey, A; Yen, S H; Liu, W K; Yang, X R; Dunlop, D S

    1995-03-27

    Paired helical filaments (PHF) characteristic of Alzheimer neurofibrillary lesions are known to contain a modified form of microtubule associated protein tau. These proteins, PHF-tau, differ from normal tau in the extent and the site of phosphorylation. To determine whether PHF-tau, tau proteins from normal adult brains (N-tau), tau proteins from Alzheimer brains not associated with PHF (A-tau), and tau proteins from fetal brains (F-tau) differ in racemization, these proteins were compared for their D-aspartate content. The results demonstrated that PHF-tau contain more D-aspartate than N-tau, A-tau and F-tau. The average percentage D-aspartate for these proteins, after a correction for background, are 4.9%, 2.8%, 1.6%, and 1% for PHF-tau, N-tau, A-tau and F-tau, respectively. It remains to be determined if the increase in D-aspartate is a consequence of PHF formation. It is also unknown if the change in D-aspartate content in PHF-tau is associated with phosphorylation, which alters the susceptibility of tau to proteolysis.

  17. Efficient aspartic acid production by a psychrophile-based simple biocatalyst.

    PubMed

    Tajima, Takahisa; Hamada, Mai; Nakashimada, Yutaka; Kato, Junichi

    2015-10-01

    We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds. PMID:26254042

  18. Biodegradation and Osteosarcoma Cell Cultivation on Poly(aspartic acid) Based Hydrogels.

    PubMed

    Juriga, Dávid; Nagy, Krisztina; Jedlovszky-Hajdú, Angéla; Perczel-Kovách, Katalin; Chen, Yong Mei; Varga, Gábor; Zrínyi, Miklós

    2016-09-14

    Development of novel biodegradable and biocompatible scaffold materials with optimal characteristics is important for both preclinical and clinical applications. The aim of the present study was to analyze the biodegradability of poly(aspartic acid)-based hydrogels, and to test their usability as scaffolds for MG-63 osteoblast-like cells. Poly(aspartic acid) was fabricated from poly(succinimide) and hydrogels were prepared using natural amines as cross-linkers (diaminobutane and cystamine). Disulfide bridges were cleaved to thiol groups and the polymer backbone was further modified with RGD sequence. Biodegradability of the hydrogels was evaluated by experiments on the base of enzymes and cell culture medium. Poly(aspartic acid) hydrogels possessing only disulfide bridges as cross-links proved to be degradable by collagenase I. The MG-63 cells showed healthy, fibroblast-like morphology on the double cross-linked and RGD modified hydrogels. Thiolated poly(aspartic acid) based hydrogels provide ideal conditions for adhesion, survival, proliferation, and migration of osteoblast-like cells. The highest viability was found on the thiolated PASP gels while the RGD motif had influence on compacted cluster formation of the cells. These biodegradable and biocompatible poly(aspartic acid)-based hydrogels are promising scaffolds for cell cultivation. PMID:27541725

  19. Interaction between L-aspartate and the brucite [Mg(OH)2]-water interface

    NASA Astrophysics Data System (ADS)

    Estrada, Charlene F.; Sverjensky, Dimitri A.; Pelletier, Manuel; Razafitianamaharavo, Angélina; Hazen, Robert M.

    2015-04-01

    The interaction of biomolecules at the mineral-water interface could have played a prominent role in the emergence of more complex organic species in life's origins. Serpentinite-hosted hydrothermal vents may have acted as a suitable environment for this process to occur, although little is known about biomolecule-mineral interactions in this system. We used batch adsorption experiments and surface complexation modeling to study the interaction of L-aspartate onto a thermodynamically stable product of serpentinization, brucite [Mg(OH)2], over a wide range of initial aspartate concentrations at four ionic strengths governed by [Mg2+] and [Ca2+]. We observed that up to 1.0 μmol of aspartate adsorbed per m2 of brucite at pH ∼ 10.2 and low Mg2+ concentrations (0.7 × 10-3 M), but surface adsorption decreased at high Mg2+ concentrations (5.8 × 10-3 M). At high Ca2+ concentrations (4.0 × 10-3 M), aspartate surface adsorption doubled (to 2.0 μmol m-2), with Ca2+ adsorption at 29.6 μmol m-2. We used the extended triple-layer model (ETLM) to construct a quantitative thermodynamic model of the adsorption data. We proposed three surface reactions involving the adsorption of aspartate (HAsp-) and/or Ca2+ onto brucite:

  20. Purification and characterization of alanine dehydrogenase from a cyanobacterium, Phormidium lapideum.

    PubMed

    Sawa, Y; Tani, M; Murata, K; Shibata, H; Ochiai, H

    1994-11-01

    Alanine dehydrogenase (AlaDH) was purified to homogeneity from cell-free extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The molecular mass of the native enzyme was 240 kDa, and SDS-PAGE revealed a minimum molecular mass of 41 kDa, suggesting a six-subunit structure. The NH2 terminal amino acid residues of the purified AlaDH revealed marked similarity with that of other AlaDHs. The enzyme was highly specific for L-alanine and NAD+, but showed relatively low amino-acceptor specificity. The pH optimum was 8.4 for reductive amination of pyruvate and 9.2 for oxidative deamination of L-alanine. The Km values were 5.0 mM for L-alanine and 0.04 mM for NAD+, 0.33 mM for pyruvate, 60.6 mM for NH4+ (pH 8.7), and 0.02 mM for NADH. Various L-amino acids including alanine, serine, threonine, and aromatic amino acids, inhibited the aminating reaction. The enzyme was inactivated upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The copresence of NADH and pyruvate largely protected the enzyme against the inactivation by PLP. PMID:7896761

  1. Theoretical and experimental study of valence photoelectron spectrum of D,L-alanine amino acid.

    PubMed

    Farrokhpour, H; Fathi, F; De Brito, A Naves

    2012-07-01

    In this work, the He-I (21.218 eV) photoelectron spectrum of D,L-alanine in the gas phase is revisited experimentally and theoretically. To support the experiment, the high level ab initio calculations were used to calculate and assign the photoelectron spectra of the four most stable conformers of gaseous alanine, carefully. The symmetry adapted cluster/configuration interaction (SAC-CI) method based on single and double excitation operators (SD-R) and its more accurate version, termed general-R, was used to separately calculate the energies and intensities of the ionization bands of the L- and D-alanine conformers. The intensities of ionization bands were calculated based on the monopole approximation. Also, natural bonding orbital (NBO) calculations were employed for better spectral band assignment. The relative electronic energy, Gibbs free energy, and Boltzmann population ratio of the conformers were calculated at the experimental temperature (403 K) using several theoretical methods. The theoretical photoelectron spectrum of alanine was calculated by summing over the spectra of individual D and L conformers weighted by different population ratios. Finally, the population ratio of the four most stable conformers of alanine was estimated from the experimental photoelectron spectrum using theoretical calculations for the first time.

  2. Perturbation correction for alanine dosimeters in different phantom materials in high-energy photon beams.

    PubMed

    von Voigts-Rhetz, P; Anton, M; Vorwerk, H; Zink, K

    2016-02-01

    In modern radiotherapy the verification of complex treatments plans is often performed in inhomogeneous or even anthropomorphic phantoms. For dose verification small detectors are necessary and therefore alanine detectors are most suitable. Though the response of alanine for a wide range of clinical photon energies in water is well know, the knowledge about the influence of the surrounding phantom material on the response of alanine is sparse. Therefore we investigated the influence of twenty different surrounding/phantom materials for alanine dosimeters in clinical photon fields via Monte Carlo simulations. The relative electron density of the used materials was in the range [Formula: see text] up to 1.69, covering almost all materials appearing in inhomogeneous or anthropomorphic phantoms used in radiotherapy. The investigations were performed for three different clinical photon spectra ranging from 6 to 25 MV-X and Co-60 and as a result a perturbation correction [Formula: see text] depending on the environmental material was established. The Monte Carlo simulation show, that there is only a small dependence of [Formula: see text] on the phantom material and the photon energy, which is below  ±0.6%. The results confirm the good suitability of alanine detectors for in-vivo dosimetry.

  3. Perturbation correction for alanine dosimeters in different phantom materials in high-energy photon beams

    NASA Astrophysics Data System (ADS)

    von Voigts-Rhetz, P.; Anton, M.; Vorwerk, H.; Zink, K.

    2016-02-01

    In modern radiotherapy the verification of complex treatments plans is often performed in inhomogeneous or even anthropomorphic phantoms. For dose verification small detectors are necessary and therefore alanine detectors are most suitable. Though the response of alanine for a wide range of clinical photon energies in water is well know, the knowledge about the influence of the surrounding phantom material on the response of alanine is sparse. Therefore we investigated the influence of twenty different surrounding/phantom materials for alanine dosimeters in clinical photon fields via Monte Carlo simulations. The relative electron density of the used materials was in the range {{n}e}/{{n}e,\\text{w}}=0.20 up to 1.69, covering almost all materials appearing in inhomogeneous or anthropomorphic phantoms used in radiotherapy. The investigations were performed for three different clinical photon spectra ranging from 6 to 25 MV-X and Co-60 and as a result a perturbation correction {{k}\\text{env}} depending on the environmental material was established. The Monte Carlo simulation show, that there is only a small dependence of {{k}\\text{env}} on the phantom material and the photon energy, which is below  ±0.6%. The results confirm the good suitability of alanine detectors for in-vivo dosimetry.

  4. Effect of 10 week beta-alanine supplementation on competition and training performance in elite swimmers.

    PubMed

    Chung, Weiliang; Shaw, Greg; Anderson, Megan E; Pyne, David B; Saunders, Philo U; Bishop, David J; Burke, Louise M

    2012-10-09

    Although some laboratory-based studies show an ergogenic effect with beta-alanine supplementation, there is a lack of field-based research in training and competition settings. Elite/Sub-elite swimmers (n = 23 males and 18 females, age = 21.7 ± 2.8 years; mean ± SD) were supplemented with either beta-alanine (4 weeks loading phase of 4.8 g/day and 3.2 g/day thereafter) or placebo for 10 weeks. Competition performance times were log-transformed, then evaluated before (National Championships) and after (international or national selection meet) supplementation. Swimmers also completed three standardized training sets at baseline, 4 and 10 weeks of supplementation. Capillary blood was analyzed for pH, bicarbonate and lactate concentration in both competition and training. There was an unclear effect (0.4%; ± 0.8%, mean, ± 90% confidence limits) of beta-alanine on competition performance compared to placebo with no meaningful changes in blood chemistry. While there was a transient improvement on training performance after 4 weeks with beta-alanine (-1.3%; ± 1.0%), there was an unclear effect at ten weeks (-0.2%; ± 1.5%) and no meaningful changes in blood chemistry. Beta-alanine supplementation appears to have minimal effect on swimming performance in non-laboratory controlled real-world training and competition settings.

  5. Conformational composition and population analysis of β-alanine isolated in solid parahydrogen

    NASA Astrophysics Data System (ADS)

    Angel Wong, Y. T.; Toh, Shin Y.; Djuricanin, Pavle; Momose, Takamasa

    2015-04-01

    The conformational composition and the change in conformational ratio induced by UV irradiation of β-alanine have been investigated using solid parahydrogen FT-IR matrix isolation spectroscopy for the first time. In order to assign the observed spectra, the vibrational wavenumbers and intensities of the eleven lowest energy β-alanine conformers were calculated at the B3LYP/aug-cc-pVTZ level of theory. In-situ UV photo-irradiation of β-alanine in solid parahydrogen was used to assist the spectral assignment. Out of the eleven lowest energy conformers, conformers I, II, III, IV, and VII were identified in the solid parahydrogen matrix, with conformer III observed in a matrix environment for the first time. Argon matrix FT-IR spectra of β-alanine were also recorded for comparison and only four conformers, conformers I, II, IV and VII, were found, as reported previously. Conformational changes to higher energy structures were observed when β-alanine was irradiated with UV radiation. These changes were more pronounced in parahydrogen matrices than in argon matrices, indicating the usefulness of solid parahydrogen matrix isolation spectroscopy for the conformational study of amino acids.

  6. UV-induced isomerization of β-alanine isolated in argon matrices

    NASA Astrophysics Data System (ADS)

    Stepanian, Stepan G.; Ivanov, Alexander Yu.; Smyrnova, Daryna A.; Adamowicz, Ludwik

    2012-10-01

    We have employed low-temperature matrix-isolation FTIR spectroscopy, the density functional theory and ab initio calculations at the MP2 and CCSD(T) levels of theory to determine the conformational composition of the simplest β-amino acid, β-alanine. UV irradiation and thermal annealing of the samples together with the FTIR spectra of deuterated β-alanine were used to separate bands of different conformers. A detailed study of the potential energy surface of β-alanine obtained at the MP2/aug-cc-pVDZ level of theory reveals twenty β-alanine conformers, but only five of them may exist in matrices due to their sufficiently high relative stabilities and low energy barriers separating them from each other. An analysis of the FTIR spectra allows us to confirm the presence of four β-alanine conformers in argon matrices with certainty. Two of them, conformers I and II, have an Nsbnd H⋯O intramolecular H-bond, the third, conformer V, has an N⋯Hsbnd O H-bond, and the fourth, conformer IV, has no intramolecular H-bonds. The relative populations of the conformers determined using the relative Gibbs free energies calculated at the CCSD(T)/CBS level of theory at 420 K are 48.1%, 23.7%, 16.8% and 3.2% for the conformers I, II, IV, and V, respectively. Some trace amount of conformer VII was also detected.

  7. Biochemical characterization of alanine racemase--a spore protein produced by Bacillus anthracis.

    PubMed

    Kanodia, Shivani; Agarwal, Shivangi; Singh, Priyanka; Agarwal, Shivani; Singh, Preeti; Bhatnagar, Rakesh

    2009-01-31

    Alanine racemase catalyzes the interconversion of L-alanine and D-alanine and plays a crucial role in spore germination and cell wall biosynthesis. In this study, alanine racemase produced by Bacillus anthracis was expressed and purified as a monomer in Escherichia coli and the importance of lysine 41 in the cofactor binding octapeptide and tyrosine 270 in catalysis was evaluated. The native enzyme exhibited an apparent K(m) of 3 mM for L-alanine, and a V(max) of 295 micromoles/min/mg, with the optimum activity occurring at 37 degrees C and a pH of 8-9. The activity observed in the absence of exogenous pyridoxal 5'-phosphate suggested that the cofactor is bound to the enzyme. Additionally, the UV-visible absorption spectra indicated that the activity was pH independece, of VV-visible absorption spectra suggests that the bound PLP exists as a protonated Schiff's base. Furthermore, the loss of activity observed in the apoenzyme suggested that bound PLP is required for catalysis. Finally, the enzyme followed non-competitive and mixed inhibition kinetics for hydroxylamine and propionate with a K(i) of 160 microM and 30 mM, respectively. [BMB reports 2009; 42(1): 47-52]. PMID:19192393

  8. Role of Alanine Dehydrogenase of Mycobacterium tuberculosis during Recovery from Hypoxic Nonreplicating Persistence.

    PubMed

    Giffin, Michelle M; Shi, Lanbo; Gennaro, Maria L; Sohaskey, Charles D

    2016-01-01

    Mycobacterium tuberculosis can maintain a nonreplicating persistent state in the host for decades, but must maintain the ability to efficiently reactivate and produce active disease to survive and spread in a population. Among the enzymes expressed during this dormancy is alanine dehydrogenase, which converts pyruvate to alanine, and glyoxylate to glycine concurrent with the oxidation of NADH to NAD. It is involved in the metabolic remodeling of M. tuberculosis through its possible interactions with both the glyoxylate and methylcitrate cycle. Both mRNA levels and enzymatic activities of isocitrate lyase, the first enzyme of the glyoxylate cycle, and alanine dehydrogenase increased during entry into nonreplicating persistence, while the gene and activity for the second enzyme of the glyoxylate cycle, malate synthase were not. This could suggest a shift in carbon flow away from the glyoxylate cycle and instead through alanine dehydrogenase. Expression of ald was also induced in vitro by other persistence-inducing stresses such as nitric oxide, and was expressed at high levels in vivo during the initial lung infection in mice. Enzyme activity was maintained during extended hypoxia even after transcription levels decreased. An ald knockout mutant of M. tuberculosis showed no reduction in anaerobic survival in vitro, but resulted in a significant lag in the resumption of growth after reoxygenation. During reactivation the ald mutant had an altered NADH/NAD ratio, and alanine dehydrogenase is proposed to maintain the optimal NADH/NAD ratio during anaerobiosis in preparation of eventual regrowth, and during the initial response during reoxygenation. PMID:27203084

  9. ald of Mycobacterium tuberculosis encodes both the alanine dehydrogenase and the putative glycine dehydrogenase.

    PubMed

    Giffin, Michelle M; Modesti, Lucia; Raab, Ronald W; Wayne, Lawrence G; Sohaskey, Charles D

    2012-03-01

    The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tu