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Sample records for alanine dehydrogenase aladh

  1. 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

  2. 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

  3. Determination of Ammonium Ion Using a Reagentless Amperometric Biosensor Based on Immobilized Alanine Dehydrogenase

    PubMed Central

    Tan, Ling Ling; Musa, Ahmad; Lee, Yook Heng

    2011-01-01

    The use of the enzyme alanine dehydrogenase (AlaDH) for the determination of ammonium ion (NH4+) usually requires the addition of pyruvate substrate and reduced nicotinamide adenine dinucleotide (NADH) simultaneously to effect the reaction. This addition of reagents is inconvenient when an enzyme biosensor based on AlaDH is used. To resolve the problem, a novel reagentless amperometric biosensor using a stacked methacrylic membrane system coated onto a screen-printed carbon paste electrode (SPE) for NH4+ ion determination is described. A mixture of pyruvate and NADH was immobilized in low molecular weight poly(2-hydroxyethyl methacrylate) (pHEMA) membrane, which was then deposited over a photocured pHEMA membrane (photoHEMA) containing alanine dehydrogenase (AlaDH) enzyme. Due to the enzymatic reaction of AlaDH and the pyruvate substrate, NH4+ was consumed in the process and thus the signal from the electrocatalytic oxidation of NADH at an applied potential of +0.55 V was proportional to the NH4+ ion concentration under optimal conditions. The stacked methacrylate membranes responded rapidly and linearly to changes in NH4+ ion concentrations between 10–100 mM, with a detection limit of 0.18 mM NH4+ ion. The reproducibility of the amperometrical NH4+ biosensor yielded low relative standard deviations between 1.4–4.9%. The stacked membrane biosensor has been successfully applied to the determination of NH4+ ion in spiked river water samples without pretreatment. A good correlation was found between the analytical results for NH4+ obtained from the biosensor and the Nessler spectrophotometric method. PMID:22163699

  4. GlnR negatively regulates the transcription of the alanine dehydrogenase encoding gene ald in Amycolatopsis mediterranei U32 under nitrogen limited conditions via specific binding to its major transcription initiation site.

    PubMed

    Wang, Ying; Li, Chen; Duan, Na; Li, Bin; Ding, Xiao-Ming; Yao, Yu-Feng; Hu, Jun; Zhao, Guo-Ping; Wang, Jin

    2014-01-01

    Ammonium assimilation is catalyzed by two enzymatic pathways, i.e., glutamine synthetase/glutamate synthase (GS/GOGAT) and alanine dehydrogenase (AlaDH) in Amycolatopsis mediterranei U32. Under nitrogen-rich conditions, the AlaDH pathway is the major route for ammonium assimilation, while the GS/GOGAT pathway takes over when the extracellular nitrogen supply is limited. The global nitrogen regulator GlnR was previously characterized to activate the transcription of the GS encoding gene glnA in response to nitrogen limitation and is demonstrated in this study as a repressor for the transcription of the AlaDH encoding gene ald, whose regulation is consistent with the switch of the ammonium assimilation pathways from AlaDH to GS/GOGAT responding to nitrogen limitation. Three transcription initiation sites (TISs) of ald were determined with primer extension assay, among which transcription from aldP2 contributed the major transcripts under nitrogen-rich conditions but was repressed to an undetectable level in response to nitrogen limitation. Through DNase I footprinting assay, two separate regions were found to be protected by GlnR within ald promoter, within which three GlnR binding sites (a1, b1 sites in region I and a2 site in region II) were defined. Interestingly, the major TIS aldP2 is located in the middle of a2 site within region II. Therefore, one may easily conclude that GlnR represses the transcription of ald via specific binding to the GlnR binding sites, which obviously blocks the transcription initiation from aldP2 and therefore reduces ald transcripts.

  5. GlnR Negatively Regulates the Transcription of the Alanine Dehydrogenase Encoding Gene ald in Amycolatopsis mediterranei U32 under Nitrogen Limited Conditions via Specific Binding to Its Major Transcription Initiation Site

    PubMed Central

    Li, Bin; Ding, Xiao-Ming; Yao, Yu-Feng; Hu, Jun; Zhao, Guo-Ping; Wang, Jin

    2014-01-01

    Ammonium assimilation is catalyzed by two enzymatic pathways, i.e., glutamine synthetase/glutamate synthase (GS/GOGAT) and alanine dehydrogenase (AlaDH) in Amycolatopsis mediterranei U32. Under nitrogen-rich conditions, the AlaDH pathway is the major route for ammonium assimilation, while the GS/GOGAT pathway takes over when the extracellular nitrogen supply is limited. The global nitrogen regulator GlnR was previously characterized to activate the transcription of the GS encoding gene glnA in response to nitrogen limitation and is demonstrated in this study as a repressor for the transcription of the AlaDH encoding gene ald, whose regulation is consistent with the switch of the ammonium assimilation pathways from AlaDH to GS/GOGAT responding to nitrogen limitation. Three transcription initiation sites (TISs) of ald were determined with primer extension assay, among which transcription from aldP2 contributed the major transcripts under nitrogen-rich conditions but was repressed to an undetectable level in response to nitrogen limitation. Through DNase I footprinting assay, two separate regions were found to be protected by GlnR within ald promoter, within which three GlnR binding sites (a1, b1 sites in region I and a2 site in region II) were defined. Interestingly, the major TIS aldP2 is located in the middle of a2 site within region II. Therefore, one may easily conclude that GlnR represses the transcription of ald via specific binding to the GlnR binding sites, which obviously blocks the transcription initiation from aldP2 and therefore reduces ald transcripts. PMID:25144373

  6. 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. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown.

  7. 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

  8. Role of Alanine Dehydrogenase of Mycobacterium tuberculosis during Recovery from Hypoxic Nonreplicating Persistence

    PubMed Central

    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. 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. 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.

  11. 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

  12. 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.

  13. Methylmalonic semialdehyde dehydrogenase deficiency: demonstration of defective valine and beta-alanine metabolism and reduced malonic semialdehyde dehydrogenase activity in cultured fibroblasts

    SciTech Connect

    Gray, R.G.; Pollitt, R.J.; Webley, J.

    1987-08-01

    Intact cultured fibroblasts from a child with a new metabolic disorder, thought to be due to a deficiency of methylmalonic semialdehyde dehydrogenase, produced labeled CO/sub 2/ normally from (1-/sup 14/C)valine but not from (2-/sup 14/C)valine. CO/sub 2/ production from labeled beta-alanine was also much reduced, confirming the suspicion that malonic semialdehyde dehydrogenase is also deficient in this condition. An assay for malonic semialdehyde dehydrogenase in cell homogenates showed low activity but it was impossible to assess the degree of reduction.

  14. Design and development of new class of Mycobacterium tuberculosisl-alanine dehydrogenase inhibitors.

    PubMed

    Reshma, Rudraraju Srilakshmi; Saxena, Shalini; Bobesh, Karyakulam Andrews; Jeankumar, Variam Ullas; Gunda, Saritha; Yogeeswari, Perumal; Sriram, Dharmarajan

    2016-09-15

    Mycobacterium tuberculosisl-alanine dehydrogenase (MTB l-AlaDH) is one of the important drug targets for treating latent/persistent tuberculosis. In this study we used crystal structure of the MTB l-AlaDH bound with cofactor NAD(+) as a structural framework for virtual screening of our in-house database to identified new classes of l-AlaDH inhibitor. We identified azetidine-2,4-dicarboxamide derivative as one of the potent inhibitor with IC50 of 9.22±0.72μM. Further lead optimization by synthesis leads to compound 1-(isonicotinamido)-N(2),N(4)-bis(benzo[d]thiazol-2-yl)azetidine-2,4-dicarboxamide (18) with l-AlaDH IC50 of 3.83±0.12μM, 2.0log reduction in nutrient starved dormant MTB model and MIC of 11.81μM in actively replicative MTB. PMID:27477207

  15. 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

  16. 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

  17. Domain Motions and Functionally-Key Residues of l-Alanine Dehydrogenase Revealed by an Elastic Network Model

    PubMed Central

    Li, Xing-Yuan; Zhang, Jing-Chao; Zhu, Yan-Ying; Su, Ji-Guo

    2015-01-01

    Mycobacterium tuberculosis l-alanine dehydrogenase (l-MtAlaDH) plays an important role in catalyzing l-alanine to ammonia and pyruvate, which has been considered to be a potential target for tuberculosis treatment. In the present work, the functional domain motions encoded in the structure of l-MtAlaDH were investigated by using the Gaussian network model (GNM) and the anisotropy network model (ANM). The slowest modes for the open-apo and closed-holo structures of the enzyme show that the domain motions have a common hinge axis centered in residues Met133 and Met301. Accompanying the conformational transition, both the 1,4-dihydronicotinamide adenine dinucleotide (NAD)-binding domain (NBD) and the substrate-binding domain (SBD) move in a highly coupled way. The first three slowest modes of ANM exhibit the open-closed, rotation and twist motions of l-MtAlaDH, respectively. The calculation of the fast modes reveals the residues responsible for the stability of the protein, and some of them are involved in the interaction with the ligand. Then, the functionally-important residues relevant to the binding of the ligand were identified by using a thermodynamic method. Our computational results are consistent with the experimental data, which will help us to understand the physical mechanism for the function of l-MtAlaDH. PMID:26690143

  18. Use of primary deuterium and /sup 15/N isotope effects to deduce the relative rates of steps in the mechanisms of alanine and glutamate dehydrogenases

    SciTech Connect

    Weiss, P.M.; Chen, C.Y.; Cleland, W.W.; Cook, P.F.

    1988-06-28

    The authors have used deuterium and /sup 15/N isotope effects to study the relative rates of the steps in the mechanisms of alanine and glutamate dehydrogenases. The proposed chemical mechanisms for these enzymes involve carbinolamine formation, imine formation, and reduction of the imine to the amino acid. These steps are almost equally rate limiting for V/K/sub ammonia/ with alanine dehydrogenase, while with glutamate dehydrogenase carbinolamine formation, imine formation, and release of glutamate after hydride transfer provide most of the rate limitation of V/K/sub ammonia/. Release of oxidized nucleotide is largely rate limiting for V/sub max/ for both enzymes. When ..beta..-hydroxypyruvate replaces pyruvate, or 3-acetylpyridine NADH (Acpyr-NADH) or thio-NADH replaces NADH with alanine dehydrogenase, nucleotide release no longer limits V/sub max/, and hydride transfer becomes more rate limiting. With glutamate dehydrogenase, replacement of ..cap alpha..-ketoglutarate by ..cap alpha..-ketovalerate makes hydride transfer more rate limiting. Use of Acpyr-NADPH has a minimal effect with ..cap alpha..-ketoglutarate but causes an 8-fold decrease in V/sub max/ with ..cap alpha..-ketovalerate, with hydride transfer the major rate-limiting step. In contrast, thio-NADPH with either ..cap alpha..-keto acid causes carbinolamide formation to become almost completely rate limiting. These studies show the power of multiple isotope effects in deducing details of the chemistry and changes in rate-limiting step(s) in complicated reaction mechanisms such as those of alanine and glutamate dehydrogenases.

  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. NAD(+)-aminoaldehyde dehydrogenase candidates for 4-aminobutyrate (GABA) and β-alanine production during terminal oxidation of polyamines in apple fruit.

    PubMed

    Zarei, Adel; Trobacher, Christopher P; Shelp, Barry J

    2015-09-14

    The last step of polyamine catabolism involves the oxidation of 3-aminopropanal or 4-aminobutanal via aminoaldehyde dehydrogenase. In this study, two apple (Malus x domestica) AMADH genes were selected (MdAMADH1 and MdAMADH2) as candidates for encoding 4-aminobutanal dehydrogenase activity. Maximal activity and catalytic efficiency were obtained with NAD(+) and 3-aminopropanal, followed by 4-aminobutanal, at pH 9.8. NAD(+) reduction was accompanied by the production of GABA and β-alanine, respectively, when 4-aminobutanal and 3-aminopropanal were utilized as substrates. MdAMADH2 was peroxisomal and MdAMADH1 cytosolic. These findings shed light on the potential role of apple AMADHs in 4-aminobutyrate and β-alanine production.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a type of ...

  6. 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.

  7. 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.

  8. Prolonged L-alanine exposure induces changes in metabolism, Ca(2+) handling and desensitization of insulin secretion in clonal pancreatic beta-cells.

    PubMed

    McClenaghan, Neville H; Scullion, Siobhan M; Mion, Brian; Hewage, Chandralal; Malthouse, J Paul G; Flatt, Peter R; Newsholme, Philip; Brennan, Lorraine

    2009-02-01

    Acute insulin-releasing actions of amino acids have been studied in detail, but comparatively little is known about the beta-cell effects of long-term exposure to amino acids. The present study examined the effects of prolonged exposure of beta-cells to the metabolizable amino acid L-alanine. Basal insulin release or cellular insulin content were not significantly altered by alanine culture, but acute alanine-induced insulin secretion was suppressed by 74% (P<0.001). Acute stimulation of insulin secretion with glucose, KCl or KIC (2-oxoisocaproic acid) following alanine culture was not affected. Acute alanine exposure evoked strong cellular depolarization after control culture, whereas AUC (area under the curve) analysis revealed significant (P<0.01) suppression of this action after culture with alanine. Compared with control cells, prior exposure to alanine also markedly decreased (P<0.01) the acute elevation of [Ca(2+)](i) (intracellular [Ca(2+)]) induced by acute alanine exposure. These diminished stimulatory responses were partially restored after 18 h of culture in the absence of alanine, indicating reversible amino-acid-induced desensitization. (13)C NMR spectra revealed that alanine culture increased glutamate labelling at position C4 (by 60%; P<0.01), as a result of an increase in the singlet peak, indicating increased flux through pyruvate dehydrogenase. Consistent with this, protein expression of the pyruvate dehydrogenase kinases PDK2 and PDK4 was significantly reduced. This was accompanied by a decrease in cellular ATP (P<0.05), consistent with diminished insulin-releasing actions of this amino acid. Collectively, these results illustrate the phenomenon of beta-cell desensitization by amino acids, indicating that prolonged exposure to alanine can induce reversible alterations to metabolic flux, Ca(2+) handling and insulin secretion. PMID:18702613

  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. Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.

    PubMed

    Yamamoto, Shogo; Gunji, Wataru; Suzuki, Hiroaki; Toda, Hiroshi; Suda, Masako; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

    2012-06-01

    We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159-165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD(+) ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.

  11. 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.

  12. The physiological role of liver alcohol dehydrogenase.

    PubMed

    Krebs, H A; Perkins, J R

    1970-07-01

    1. Yeast alcohol dehydrogenase was used to determine ethanol in the portal and hepatic veins and in the contents of the alimentary canal of rats given a diet free from ethanol. Measurable amounts of a substance behaving like ethanol were found. Its rate of interaction with yeast alcohol dehydrogenase and its volatility indicate that the substance measured was in fact ethanol. 2. The mean alcohol concentration in the portal blood of normal rats was 0.045mm. In the hepatic vein, inferior vena cava and aorta it was about 15 times lower. 3. The contents of all sections of the alimentary canal contained measurable amounts of ethanol. The highest values (average 3.7mm) were found in the stomach. 4. Infusion of pyrazole (an inhibitor of alcohol dehydrogenase) raised the alcohol concentration in the portal vein 10-fold and almost removed the difference between portal and hepatic venous blood. 5. Addition of antibiotics to the food diminished the ethanol concentration of the portal blood to less than one-quarter and that of the stomach contents to less than one-fortieth. 6. The concentration of alcohol in the alimentary canal and in the portal blood of germ-free rats was much decreased, to less than one-tenth in the alimentary canal and to one-third in the portal blood, but detectable quantities remained. These are likely to arise from acetaldehyde formed by the normal pathways of degradation of threonine, deoxyribose phosphate and beta-alanine. 7. The results indicate that significant amounts of alcohol are normally formed in the gastro-intestinal tract. The alcohol is absorbed into the circulation and almost quantitatively removed by the liver. Thus the function, or a major function, of liver alcohol dehydrogenase is the detoxication of ethanol normally present. 8. The alcohol concentration in the stomach of alloxan-diabetic rats was increased about 8-fold. 9. The activity of liver alcohol dehydrogenase is generally lower in carnivores than in herbivores and omnivores

  13. 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.

  14. 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.

  15. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  16. 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.

  17. 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...

  18. 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.

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. 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.

  5. 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.

  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. 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...

  8. 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...

  9. 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.

  10. 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...

  11. 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...

  12. 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

  13. Enzymatic syntheses of (1-(C-11))pyruvic acid and L-(1-(C-11))lactic acid via DL-(1-(C-11))alanine

    SciTech Connect

    Ropchan, J.R.; Barrio, J.R.

    1984-01-01

    L-(1-(C-11)) Lactic acid was prepared in three steps using a remote, semi-automated procedure: (1) production of DL-(1-(C-11)) alanine (2) enzymatic conversion of DL (1-(C-11)) alanine to (1-(C-11)) pyruvate and (3) enzymatic transformation of (1-(C-11)) pyruvate to L-(1-(C-11)) lactic acid. DL-(1-(C-11)) Alanine was synthesized from NCA C-11 HCN using a modification of the Bucherer-Strecker reaction. The DL-isomers were converted to (1-(C-11)) pyruvate by passage through (1) immobilized D-amino acid oxidase enzyme column followed by (2) immobilized L-alanine dehydrogenase (l-ADH) enzyme column. (1-(C-11)) Pyruvate was then transformed to L-(1-(C-11)) lactic acid by elution through a L-lactic dehydrogenase enzyme column. These enzyme columns are reusable beyond three months, give high radiochemical purity (>98%), eliminate the possibility of protein contamination, assure sterile, pyrogen-free products and allow rapid separation and quantitative conversion of DL-isomers to the desired products. Typically the synthesis required 30-40 min after cyclotron production of NCA C-11 HCN with radiochemical yields of 15-25 mCi (23%) of L-(1-(C-11)) lactic acid and 20-35 mCi (33%) of (l-(C-11)) pyruvic acid starting with 250-400 mCi of C-11 HCN. Also 10-20 mCi (19%) of L-(1-(C-11)) alanine was produced by resin separation (AG50W-X8), H/sup +/ form of (1-(C-11)) pyruvate and L-(1-(C-11)) alanine following elution through D-AAO enzyme column. The radiochemical purities of (1-(C-11)) pyruvic acid, L-(1-(C-11)) lactic acid and L-(1-(C-11)) alanine were verified routinely by reversed-phase HPLC.

  14. Weak BMAA toxicity compares with that of the dietary supplement β-alanine.

    PubMed

    Lee, Moonhee; McGeer, Patrick L

    2012-07-01

    β-N-methylamino-L-alanine (BMAA) is routinely described in the literature as a potent neurotoxin and as a possible cause of neurodegenerative disorders of aging such as Alzheimer's disease, amyotrophic lateral sclerosis, and the amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC) syndrome of Guam. To test for the toxicity of BMAA against human neurons, we chose 3 standard human neuronal cell lines for examination and compared the toxicity with the muscle-building nutritional supplement β-alanine, glutamic acid, and the established excitotoxins kainic acid, quisqualic acid, ibotenic acid, domoic acid, and quinolinic acid. Neurotoxicity was measured by the standard lactic dehydrogenase release assay after 5-day incubation of NT-2, SK-N-MC, and SH-SY5Y cells with BMAA and the comparative substances. The ED(50) of BMAA, corresponding to 50% death of neurons, varied from 1430 to 1604 μM while that of the nutritional supplement β-alanine was almost as low, varying from 1945 to 2134 μM. The ED(50) for glutamic acid and the 5 established excitotoxins was 200- to 360-fold lower, varying from 44 to 70 μM. These in vitro data are in accord with previously published in vivo data on BMAA toxicity in which mice showed no pathological effects from oral consumption of 500 mg/kg/day for more than 10 weeks. Because there are no known natural sources of BMAA that would make consumption of such amounts possible, and because the toxicity observed was in the same range as the nutritional supplement β-alanine, the hypothesis that BMAA is an environmental hazard and a contributor to degenerative neurological diseases becomes untenable.

  15. 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 +/.

  16. 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.

  17. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  18. Post-translational Introduction of D-Alanine into Ribosomally Synthesized Peptides by the Dehydroalanine Reductase NpnJ.

    PubMed

    Yang, Xiao; van der Donk, Wilfred A

    2015-10-01

    Ribosomally synthesized peptides are generally limited to L-amino acid building blocks. Given the advantageous properties of peptides containing D-amino acids such as stabilization of certain turns and against proteolytic degradation, methods to introduce D-stereocenters are valuable. Here we report the first in vitro reconstitution and characterization of a dehydrogenase that carries out the asymmetric reduction of dehydroalanine. NpnJA reduces dehydroalanine to D-Ala using NAPDH as cosubstrate. The enzyme displays high substrate tolerance allowing introduction of D-Ala into a range of non-native substrates. In addition to the in vitro reactions, we describe five examples of using Escherichia coli as biosynthetic host for D-alanine introduction into ribosomal peptides. A deuterium-label-based coupled-enzyme assay was used to rapidly determine the stereochemistry of the newly installed alanine.

  19. 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.

  20. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... control the activity of the complex: pyruvate dehydrogenase phosphatase turns on (activates) the complex, while pyruvate dehydrogenase ... binding protein (the PDHX gene), and pyruvate dehydrogenase phosphatase (the PDP1 gene) have been identified in people ...

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. Biosynthesis of prodigiosin by non-proliferating wild-type Serratia marcescens and mutants deficient in catabolism of alanine, histidine, and proline.

    PubMed

    Lim, D V; Qadri, S M; Nichols, C; Williams, R P

    1977-01-01

    Mutants of Serratia marcescens Nima, designated as Aut, Hut, or Put, did not utilize L-alanine, L-histidine, or L-proline, respectively, as a sole carbon source but did utilize other amino acids or glycerol as carbon sources. The bacteria were permeable to alanine, histidine, and proline but lacked the enzymes responsible for degradation of these amino acids. The Aut mutant contained no L-alanine dehydrogenase activity, whereas the Hut and Put mutants contained only 7 and 4% of the histidase and proline oxidase activities, respectively, found in the wild-type strain. Rates of oxygen uptake and protein synthesis were significantly lower when the mutants were incubated in the presence of amino acids they could not degrade. Studies of L-[14C]alanine, L-[14C]histidine, and L-[14C]proline incorporation into prodigiosin synthesized by these mutants and the wild-type strain revealed that proline was incorporated intact, whereas all of alanine except the carboxyl group was incorporated into the pigment molecule. Histidine did not enter prodigiosin directly. These data suggested that the presence of unique biosynthetic pathways, independent of primary metabolism, leads to formation of prodigiosin from specific amino acids.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex

    SciTech Connect

    Park, Yun-Hee; Patel, Mulchand S.

    2010-05-07

    Unlike pyruvate dehydrogenase complexes (PDCs) from prokaryotes, PDCs from higher eukaryotes have an additional structural component, E3-binding protein (BP), for binding of dihydrolipoamide dehydrogenase (E3) in the complex. Based on the 3D structure of the subcomplex of human (h) E3 with the di-domain (L3S1) of hBP, the amino acid residues (H348, D413, Y438, and R447) of hE3 for binding to hBP were substituted singly by alanine or other residues. These substitutions did not have large effects on hE3 activity when measured in its free form. However, when these hE3 mutants were reconstituted in the complex, the PDC activity was significantly reduced to 9% for Y438A, 20% for Y438H, and 18% for D413A. The binding of hE3 mutants with L3S1 determined by isothermal titration calorimetry revealed that the binding affinities of the Y438A, Y438H, and D413A mutants to L3S1 were severely reduced (1019-, 607-, and 402-fold, respectively). Unlike wild-type hE3 the binding of the Y438A mutant to L3S1 was accompanied by an unfavorable enthalpy change and a large positive entropy change. These results indicate that hE3-Y438 and hE3-D413 play important roles in binding of hE3 to hBP.

  13. 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

  14. 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.

  15. Alcohol Dehydrogenase from Methylobacterium organophilum

    PubMed Central

    Wolf, H. J.; Hanson, R. S.

    1978-01-01

    The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10−5 M for methanol and 8.2 × 10−5 M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum. Images PMID:80974

  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. Adaptation of methods for glutamate dehydrogenase and alcohol dehydrogenase activities to a centrifugal analyser: assessment of their clinical use in anoxic states of the liver.

    PubMed Central

    Shephard, M D; Penberthy, L A; Berry, M N

    1987-01-01

    Sensitive, precise, and rapid methods for the measurement of alcohol dehydrogenase (ADH) and glutamate dehydrogenase (GDH) were developed on the Cobas Bio centrifugal analyser. The optimal pH for ADH in caucasians was 9.8. Non-linearity of ADH enzyme activity was observed when samples were diluted in saline; linearity was restored when inactivated serum was used as diluent. ADH was shown to be a sensitive index of liver anoxia due to cardiorespiratory disturbance (clinical sensitivity 90%) and generalised anoxia. GDH exhibited sensitivity equal to that of alanine aminotransferase (ALT) but was inferior to gamma-glutamyltransferase (GGT) in the detection of specific liver disease. Both ADH and GDH were sensitive indicators of alcoholic liver disease. PMID:2890662

  18. 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.

  19. Purification, characterization, and cDNA cloning of opine dehydrogenases from the polychaete rockworm Marphysa sanguinea.

    PubMed

    Endo, Noriyuki; Kan-no, Nobuhiro; Nagahisa, Eizoh

    2007-06-01

    Alanopine dehydrogenase (AlDH) and three isoforms of strombine/alanopine dehydrogenase (St/AlDH) were purified from muscle tissue of the polychaete rockworm Marphysa sanguinea. The four enzymes, which can be distinguished by the isoelectric point, are monomeric 42 kDa proteins, possess similar pH-activity profiles, and display specificity for pyruvate and NAD(H). The three isoforms of St/AlDH show equivalent Km and Vmax for glycine and L-alanine and for D-strombine and meso-alanopine. Free amino acid levels in the muscle and D-strombine accumulation in vivo during muscle activity suggest that St/AlDHs function physiologically as StDH. AlDH shows specificity for L-alanine and meso-alanopine, but not for glycine or D-strombine. The amino acid sequences of AlDH and one of the St/AlDH isoforms were determined by a combination of amino acid sequence analysis and cDNA cloning. St/AlDH cDNA consisted of 1586 bp nucleotides that encode a 399-residue protein (43,346.70 Da), and AlDH cDNA consisted of 1587 bp nucleotides that encode a 399-residue protein (43,886.68 Da). The two amino acid sequences deduced from the cDNA displayed 67% amino acid identity, with greatest similarity to that of tauropine dehydrogenase from the polychaete Arabella iricolor. PMID:17350870

  20. 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

  1. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  2. 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.

  3. Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology.

    PubMed

    Satomura, Takenori; Sakuraba, Haruhiko; Suye, Shin-Ichiro; Ohshima, Toshihisa

    2015-11-01

    Dye-linked D-amino acid dehydrogenases (Dye-DADHs) catalyze the dehydrogenation of free D-amino acids in the presence of an artificial electron acceptor. Although Dye-DADHs functioning in catabolism of L-alanine and as primary enzymes in electron transport chains are widely distributed in mesophilic Gram-negative bacteria, biochemical and biotechnological information on these enzymes remains scanty. This is in large part due to their instability after isolation. On the other hand, in the last decade, several novel types of Dye-DADH have been found in thermophilic bacteria and hyperthermophilic archaea, where they contribute not only to L-alanine catabolism but also to the catabolism of other amino acids, including D-arginine and L-hydroxyproline. In this minireview, we summarize recent developments in our understanding of the biochemical characteristics of Dye-DADHs and their specific application to electrochemical biosensors. PMID:26362681

  4. Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology.

    PubMed

    Satomura, Takenori; Sakuraba, Haruhiko; Suye, Shin-Ichiro; Ohshima, Toshihisa

    2015-11-01

    Dye-linked D-amino acid dehydrogenases (Dye-DADHs) catalyze the dehydrogenation of free D-amino acids in the presence of an artificial electron acceptor. Although Dye-DADHs functioning in catabolism of L-alanine and as primary enzymes in electron transport chains are widely distributed in mesophilic Gram-negative bacteria, biochemical and biotechnological information on these enzymes remains scanty. This is in large part due to their instability after isolation. On the other hand, in the last decade, several novel types of Dye-DADH have been found in thermophilic bacteria and hyperthermophilic archaea, where they contribute not only to L-alanine catabolism but also to the catabolism of other amino acids, including D-arginine and L-hydroxyproline. In this minireview, we summarize recent developments in our understanding of the biochemical characteristics of Dye-DADHs and their specific application to electrochemical biosensors.

  5. Characterization of testis-specific isoenzyme of human pyruvate dehydrogenase.

    PubMed

    Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

    2006-04-01

    Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1. PMID:16436377

  6. Genetics Home Reference: lactate dehydrogenase deficiency

    MedlinePlus

    ... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase

    PubMed Central

    Wu, Gang; Fiser, András; ter Kuile, Benno; Šali, Andrej; Müller, Miklós

    1999-01-01

    Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently. PMID:10339579

  13. Sorbitol dehydrogenase is a zinc enzyme.

    PubMed Central

    Jeffery, J; Chesters, J; Mills, C; Sadler, P J; Jörnvall, H

    1984-01-01

    Evidence is given that tetrameric sorbitol dehydrogenase from sheep liver contains one zinc atom per subunit, most probably located at the active site, and no other specifically bound zinc or iron atom. In alcohol dehydrogenases that are structurally related to sorbitol dehydrogenase, more than one zinc atom per subunit can complicate investigations of zinc atom function. Therefore, sorbitol dehydrogenase will be particularly valuable for defining the precise roles of zinc in alcohol and polyol dehydrogenases, and for establishing correlations of structure and function with other important zinc-containing proteins. PMID:6370679

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. [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.

  20. 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.

  1. 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.

  2. [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.

  3. 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

  4. 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).

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Methemoglobin reduction mediated by D-amino acid dehydrogenase in Propsilocerus akamusi (Tokunaga) larvae.

    PubMed

    Kobori, Hiroki; Tanigawa, Minoru; Maeda, Shintaro; Hori, Hiroshi; Yubisui, Toshitsugu; Nagata, Yoko

    2015-06-01

    A methemoglobin (metHb) reduction system is required for aerobic respiration. In humans, Fe(III)-heme-bearing metHb (the oxidized form of hemoglobin), which cannot bind oxygen, is converted to Fe(II)-heme-bearing oxyhemoglobin (oxyHb, the reduced form), which can bind oxygen, in a system comprising NADH, NADH-cytochrome b5 reductase, and cytochrome b5. However, the mechanism of metHb reduction in organisms that inhabit oxygen-deficient environments is unknown. In the coelomic fluid of the larvae of Propsilocerus akamusi, which inhabit a microaerobic environment, we found that metHb was reduced by D-alanine. We purified an FAD-containing enzyme, D-amino acid dehydrogenase (DAD), and component V hemoglobin from the larvae. Using the purified components and spectrophotometric analyses, we showed a novel function of DAD: DAD-mediation of P. akamusi component V metHb reduction with using D-alanine as an electron donor. P. akamusi larvae possess this D-alanine-DAD metHb reduction system in addition to a previously discovered NADH-NADH-cytochrome b5 reductase system. This is the first report of the presence of DAD in a multicellular organism. The molecular mass of DAD was estimated to be 45 kDa. The optimal pH and temperature of the enzyme were 7.4 and 20 °C, respectively, and the optimal substrate was D-alanine. The enzyme activity was inhibited by benzoate and sulfhydryl-binding reagents. PMID:25896287

  16. Isocitrate dehydrogenases and oxoglutarate dehydrogenase activities of baker's yeast grown in a variety of hypoxic conditions.

    PubMed

    Machado, A; Nuñez de Castro, I; Mayor, F

    1975-02-28

    The activities of isocitrate dehydrogenase (NAD), isocitrate dehydrogenase (NADP) and oxoglutarate dehydrogenase have been investigated in Saccharomyces cerevisiae grown in a variety of aerobic and hypoxic conditions, the latter including oxygen deprivation, high glucose concentration, addition of inhibitors of mitochondrial protein synthesis, respiratory inhibition by azide, and impaired respiration mutants. All hypoxic conditions led to a marked decrease of oxoglutarate dehydrogenase and significant decreases of the two isocitrate dehydrogenases. According to its kinetic properties, the NAD-isocitrate dehydrogenase will not be operative in hypoxia "in vivo". From these and other related facts it is concluded that hypoxic conditions in yeast generally lead to a splitting of the tricarboxylic acid cycle and that glutamate synthesis in these conditions takes place through the coupling of the NADP-linked isocitrate and glutamate dehydrogenases.

  17. [The PQQ-dehydrogenases. A novel example of bacterial quinoproteins].

    PubMed

    Flores-Encarnación, Marcos; Sánchez-Cuevas, Mariano; Ortiz-Gutiérrez, Felipe

    2004-01-01

    The word "quinoprotein" describes four groups of different enzymes which have cofactors containing o-quinones. Pyrrolo-quinoline quinone (PQQ) is not covalently attached. PQQ is the cofactor of several quinoprotein bacterial dehydrogenases including glucose dehydrogenase (G-DH), alcohol dehydrogenase (A-DH) and aldehyde dehydrogenase (AL-DH). These dehydrogenases are located in the periplasm of Gram-negative bacteria. This report summarises the structural properties of quinoprotein dehydrogenases, such as the biological functions and biotechnological aspects more important.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  4. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  5. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  6. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  7. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  8. Formate dehydrogenase from Pseudomonas oxalaticus.

    PubMed

    Müller, U; Willnow, P; Ruschig, U; Höpner, T

    1978-02-01

    Formate dehydrogenase (EC 1.2.1.2) from Pseudomonas oxalaticus has been isolated and characterized. The enzyme (molecular weight 315000) is a complex flavoprotein containing 2 FMN, 18--25 non-heme iron atoms and 15--20 acid-labile sulphides. In the last step of the purification, a sucrose gradient centrifugation, a second catalytically active species has been found apparently originating from a dissociation of the enzyme into two equal subunits. The enzyme is specific toward its natural substrate formate. It transfers electrons to NAD+, oxygen, ferricyanide, and a lot of nonphysiological acceptors (dyes). In addition electrons are transferred from NADH to these acceptors. The (reversible) removal of FMN requires a reduction step. Reincorporation has been followed by the reappearance of the reactivity against formate and by fluorescence titration. The deflavo enzyme also binds FAD and riboflavin. The resulting enzyme species show characteristic catalytic abilities. Activity against formate is peculiar to the FMN species. PMID:631130

  9. Opine dehydrogenases in marine invertebrates.

    PubMed

    Harcet, Matija; Perina, Drago; Pleše, Bruna

    2013-10-01

    It is well known today that opine production anaerobic pathways are analogs to the classical glycolytic pathway (lactate production pathway). These pathways, catalyzed by a group of enzymes called opine dehydrogenases (OpDHs), ensure continuous flux of glycolysis and a constant supply of ATP by maintaining the NADH/NAD(+) ratio during exercise and hypoxia, thus regulating the cytosolic redox balance in glycolysis under anoxia. OpDHs are distributed in a wide range of marine invertebrate phyla, including sponges (Porifera). Phylogenetic analyses supported with enzymatic assays strongly indicate that sponge OpDHs constitute an enzyme class unrelated to other OpDHs. Therefore, OpDHs in marine invertebrates are divided into two groups, a mollusk/annelid type and a sponge type, which belongs to the OCD/mu-crystallin family.

  10. Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus.

    PubMed Central

    Gillooly, D J; Robertson, A G; Fewson, C A

    1998-01-01

    The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases. PMID:9494109

  11. Vertebrate Acyl CoA synthetase family member 4 (ACSF4-U26) is a β-alanine-activating enzyme homologous to bacterial non-ribosomal peptide synthetase.

    PubMed

    Drozak, Jakub; Veiga-da-Cunha, Maria; Kadziolka, Beata; Van Schaftingen, Emile

    2014-03-01

    Mammalian ACSF4-U26 (Acyl CoA synthetase family member 4), a protein of unknown function, comprises a putative adenylation domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site, and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 show greater identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was not formed in a point mutant lacking the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was almost specific for β-alanine, and a KM of ~ 5 μm was calculated for this reaction. The loaded enzyme was used to study the formation of a potential end product. Among the 20 standard amino acids, only cysteine stimulated unloading of the enzyme. This effect was mimicked by cysteamine and dithiothreitol, and was unaffected by absence of the PQQDH-related domain, suggesting that β-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification.

  12. Shikimate dehydrogenase from Pinu sylvestris L. needles

    SciTech Connect

    Osipov, V.I.; Shein, I.V.

    1986-07-10

    Shikimate dehydrogenase was isolated by extraction from pine needles and partially purified by fractionation with ammonium sulfate. In conifers, in contrast to other plants, all three isoenzymes of shikimate dehydrogenase exhibit activity not only with NADP/sup +/, but also with NAD/sup +/. The values of K/sub m/ for shikimate, when NADP/sup +/ and NAD/sup +/ are used as cofactors, are 0.22 and 1.13 mM, respectively. The enzyme is maximally active at pH 10 with both cofactors. It is suggested that NAD-dependent shikimate dehydrogenase catalyzes the initial reaction of the alternative pathway of the conversion of shikimic acid to hydroxybenzoic acid. The peculiarities of the organization and regulation of the initial reactions of the shikimate pathway in conifers and in plants with shikimate dehydrogenase absolutely specific for NADP are discussed.

  13. 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.

  14. 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.

  15. Phosphorylation site on yeast pyruvate dehydrogenase complex

    SciTech Connect

    Uhlinger, D.J.

    1986-01-01

    The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the /sup 32/P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. Residues that influence coenzyme preference in the aldehyde dehydrogenases.

    PubMed

    González-Segura, Lilian; Riveros-Rosas, Héctor; Julián-Sánchez, Adriana; Muñoz-Clares, Rosario A

    2015-06-01

    To find out the residues that influence the coenzyme preference of aldehyde dehydrogenases (ALDHs), we reviewed, analyzed and correlated data from their known crystal structures and amino-acid sequences with their published kinetic parameters for NAD(P)(+). We found that the conformation of the Rossmann-fold loops participating in binding the adenosine ribose is very conserved among ALDHs, so that coenzyme specificity is mainly determined by the nature of the residue at position 195 (human ALDH2 numbering). Enzymes with glutamate or proline at 195 prefer NAD(+) because the side-chains of these residues electrostatically and/or sterically repel the 2'-phosphate group of NADP(+). But contrary to the conformational rigidity of proline, the conformational flexibility of glutamate may allow NADP(+)-binding in some enzymes by moving the carboxyl group away from the 2'-phosphate group, which is possible if a small neutral residue is located at position 224, and favored if the residue at position 53 interacts with Glu195 in a NADP(+)-compatible conformation. Of the residues found at position 195, only glutamate interacts with the NAD(+)-adenosine ribose; glutamine and histidine cannot since their side-chain points are opposite to the ribose, probably because the absence of the electrostatic attraction by the conserved nearby Lys192, or its electrostatic repulsion, respectively. The shorter side-chains of other residues-aspartate, serine, threonine, alanine, valine, leucine, or isoleucine-are distant from the ribose but leave room for binding the 2'-phosphate group. Generally, enzymes having a residue different from Glu bind NAD(+) with less affinity, but they can also bind NADP(+) even sometimes with higher affinity than NAD(+), as do enzymes containing Thr/Ser/Gln195. Coenzyme preference is a variable feature within many ALDH families, consistent with being mainly dependent on a single residue that apparently has no other structural or functional roles, and therefore can

  4. The snakehead Channa asiatica accumulates alanine during aerial exposure, but is incapable of sustaining locomotory activities on land through partial amino acid catabolism.

    PubMed

    Chew, Shit F; Wong, Mei Y; Tam, Wai L; Ip, Yuen K

    2003-02-01

    The freshwater snakehead Channa asiatica is an obligatory air-breather that resides in slow-flowing streams and in crevices near riverbanks in Southern China. In its natural habitat, it may encounter bouts of aerial exposure during the dry seasons. In the laboratory, the ammonia excretion rate of C. asiatica exposed to terrestrial conditions in a 12 h:12 h dark:light regime was one quarter that of the submerged control. Consequently, the ammonia contents in the muscle, liver and plasma increased significantly, and C. asiatica was able to tolerate quite high levels of ammonia in its tissues. Urea was not the major product of ammonia detoxification in C. asiatica, which apparently did not possess a functioning ornithine urea cycle. Rather, alanine increased fourfold to 12.6 micromol g(-1) in the muscle after 48 h of aerial exposure. This is the highest level known in adult teleosts exposed to air or an ammonia-loading situation. The accumulated alanine could account for 70% of the deficit in ammonia excretion during this period, indicating that partial amino acid catabolism had occurred. This would allow the utilization of certain amino acids as energy sources and, at the same time, maintain the new steady state levels of ammonia in various tissues, preventing them from rising further. There was a reduction in the aminating activity of glutamate dehydrogenase from the muscle and liver of specimens exposed to terrestrial conditions. Such a phenomenon has not been reported before and could, presumably, facilitate the entry of alpha-ketoglutarate into the Krebs cycle instead of its amination to glutamate, as has been suggested elsewhere. However, in contrast to mudskippers, C. asiatica was apparently unable to reduce the rates of proteolysis and amino acid catabolism, because the reduction in nitrogenous excretion during 48 h of aerial exposure was completely balanced by nitrogenous accumulation in the body. Alanine accumulation also occurred in specimens exposed to

  5. Carbohydrate metabolism during prolonged exercise and recovery: interactions between pyruvate dehydrogenase, fatty acids, and amino acids.

    PubMed

    Mourtzakis, Marina; Saltin, Bengt; Graham, Terry; Pilegaard, Henriette

    2006-06-01

    During prolonged exercise, carbohydrate oxidation may result from decreased pyruvate production and increased fatty acid supply and ultimately lead to reduced pyruvate dehydrogenase (PDH) activity. Pyruvate also interacts with the amino acids alanine, glutamine, and glutamate, whereby the decline in pyruvate production could affect tricarboxycylic acid cycle flux as well as gluconeogenesis. To enhance our understanding of these interactions, we studied the time course of changes in substrate utilization in six men who cycled at 44+/-1% peak oxygen consumption (mean+/-SE) until exhaustion (exhaustion at 3 h 23 min+/-11 min). Femoral arterial and venous blood, blood flow measurements, and muscle samples were obtained hourly during exercise and recovery (3 h). Carbohydrate oxidation peaked at 30 min of exercise and subsequently decreased for the remainder of the exercise bout (P<0.05). PDH activity peaked at 2 h of exercise, whereas pyruvate production peaked at 1 h of exercise and was reduced (approximately 30%) thereafter, suggesting that pyruvate availability primarily accounted for reduced carbohydrate oxidation. Increased free fatty acid uptake (P<0.05) was also associated with decreasing PDH activity (P<0.05) and increased PDH kinase 4 mRNA (P<0.05) during exercise and recovery. At 1 h of exercise, pyruvate production was greatest and was closely linked to glutamate, which was the predominant amino acid taken up during exercise and recovery. Alanine and glutamine were also associated with pyruvate metabolism, and they comprised approximately 68% of total amino-acid release during exercise and recovery. Thus reduced pyruvate production was primarily associated with reduced carbohydrate oxidation, whereas the greatest production of pyruvate was related to glutamate, glutamine, and alanine metabolism in early exercise. PMID:16424076

  6. Conserved catalytic residues of the ALDH1L1 aldehyde dehydrogenase domain control binding and discharging of the coenzyme.

    PubMed

    Tsybovsky, Yaroslav; Krupenko, Sergey A

    2011-07-01

    The C-terminal domain (C(t)-FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP(+)-dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several C(t)-FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP(+), Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP(+) but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type C(t)-FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP(+) with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a long-range communication between the catalytic center and the coenzyme-binding domain and points toward an α-helix involved in the adenine moiety binding as a participant of this communication.

  7. Benzene toxicity: emphasis on cytosolic dihydrodiol dehydrogenases

    SciTech Connect

    Bolcsak, L.E.

    1982-01-01

    Blood dyscrasias such as leukopenia and anemia have been clearly identified as consequences of chronic benzene exposure. The metabolites, phenol, catechol, and hydroquinone produced inhibition of /sup 59/Fe uptake in mice which followed the same time course as that produced by benzene. The inhibitor of benzene oxidation, 3-amino-1,2,4-triazole, mitigated the inhibitory effects of benzene and phenol only. These data support the contention that benzene toxicity is mediated by a metabolite and suggest that the toxicity of phenol is a consequence of its metabolism to hydroquinone and that the route of metabolism to catechol may also contribute to the production of toxic metabolite(s). The properties of mouse liver cytosolic dihydrodiol dehydrogenases were examined. These enzymes catalyze the NADP/sup +/-dependent oxidation of trans-1,2-dihydro-1,2-dihydroxybenzene (BDD) to catechol, a possible toxic metabolite of benzene produced via this metabolic route. Four distinct dihydrodiol dehydrogenases (DD1, DD2, DD3, and DD4) were purified to apparent homogeneity as judged by SDS polyacrylamide gel electrophoresis and isoelectric focusing. DD1 appeared to be identical to the major ketone reductase and 17..beta..-hydroxysteroid dehydrogenase activity in the liver. DD2 exhibited aldehyde reductase activity. DD3 and DD4 oxidized 17..beta..-hydroxysteroids, but no carbonyl reductase activity was detected. These relationships between BDD dehydrogenases and carbonyl reductase and/or 17..beta..-hydroxysteroid dehydrogenase activities were supported by several lines of evidence.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Mechanism of hyperinsulinism in short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency involves activation of glutamate dehydrogenase.

    PubMed

    Li, Changhong; Chen, Pan; Palladino, Andrew; Narayan, Srinivas; Russell, Laurie K; Sayed, Samir; Xiong, Guoxiang; Chen, Jie; Stokes, David; Butt, Yasmeen M; Jones, Patricia M; Collins, Heather W; Cohen, Noam A; Cohen, Akiva S; Nissim, Itzhak; Smith, Thomas J; Strauss, Arnold W; Matschinsky, Franz M; Bennett, Michael J; Stanley, Charles A

    2010-10-01

    The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh(-/-)). The hadh(-/-) mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh(-/-) mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh(-/-) mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh(-/-) islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh(-/-) islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh(-/-) islets also have increased [U-(14)C]glutamine oxidation. In contrast, hadh(-/-) mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh(-/-) islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh(-/-) islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.

  14. 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.

  15. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed Central

    Kelly, N; Delaney, M; O'Carra, P

    1978-01-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  16. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed

    Kelly, N; Delaney, M; O'Carra, P

    1978-06-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  17. Molybdopterin cofactor from Methanobacterium formicicum formate dehydrogenase.

    PubMed Central

    May, H D; Schauer, N L; Ferry, J G

    1986-01-01

    The molybdopterin cofactor from the formate dehydrogenase of Methanobacterium formicicum was studied. The cofactor was released by guanidine denaturation of homogeneous enzyme, which also released greater than 80% of the molybdenum present in the enzyme. The anoxically isolated cofactor was nonfluorescent, but after exposure to air it fluoresced with spectra similar to those of described molybdopterin cofactors. Aerobic release from acid-denatured formate dehydrogenase in the presence of I2 and potassium iodide produced a mixture of fluorescent products. Alkaline permanganate oxidation of the mixture yielded pterin-6-carboxylic acid as the only detectable fluorescent product. The results showed that the cofactor from formate dehydrogenase contained a pterin nucleus with a 6-alkyl side chain of unknown structure. Covalently bound phosphate was also present. The isolated cofactor was unable to complement the cofactor-deficient nitrate reductase of the Neurospora crassa nit-1 mutant. PMID:3700335

  18. NAD + -dependent Formate Dehydrogenase from Plants

    PubMed Central

    Alekseeva, A.A.; Savin, S.S.; Tishkov, V.I.

    2011-01-01

    NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) widely occurs in nature. FDH consists of two identical subunits and contains neither prosthetic groups nor metal ions. This type of FDH was found in different microorganisms (including pathogenic ones), such as bacteria, yeasts, fungi, and plants. As opposed to microbiological FDHs functioning in cytoplasm, plant FDHs localize in mitochondria. Formate dehydrogenase activity was first discovered as early as in 1921 in plant; however, until the past decade FDHs from plants had been considerably less studied than the enzymes from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehydrogenases. PMID:22649703

  19. The conserved Glu-60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis

    PubMed Central

    Kleifeld, Oded; Shi, Shu Ping; Zarivach, Raz; Eisenstein, Miriam; Sagi, Irit

    2003-01-01

    Glu-60 of the zinc-dependent Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a strictly conserved residue in all members of the alcohol dehydrogenase (ADH) family. Unlike most other ADHs, the crystal structures of TbADH and its analogs, ADH from Clostridium beijerinckii (CbADH), exhibit a unique zinc coordination environment in which this conserved residue is directly coordinated to the catalytic zinc ion in the native form of the enzymes. To explore the role of Glu-60 in TbADH catalysis, we have replaced it by alanine (E60A-TbADH) and aspartate (E60D-TbADH). Steady-state kinetic measurements show that the catalytic efficiency of these mutants is only four- and eightfold, respectively, lower than that of wild-type TbADH. We applied X-ray absorption fine-structure (EXAFS) and near-UV circular dichroism to characterize the local environment around the catalytic zinc ion in the variant enzymes in their native, cofactor-bound, and inhibited forms. We show that the catalytic zinc site in the studied complexes of the variant enzymes exhibits minor changes relative to the analogous complexes of wild-type TbADH. These moderate changes in the kinetic parameters and in the zinc ion environment imply that the Glu-60 in TbADH does not remain bound to the catalytic zinc ion during catalysis. Furthermore, our results suggest that a water molecule replaces this residue during substrate turnover. PMID:12592017

  20. Targeting Tumor Metabolism for Cancer Treatment: Is Pyruvate Dehydrogenase Kinases (PDKs) a Viable Anticancer Target?

    PubMed Central

    Zhang, Wen; Zhang, Shao-Lin; Hu, Xiaohui; Tam, Kin Yip

    2015-01-01

    Cancer remains a lethal threat to global lives. Development of novel anticancer therapeutics is still a challenge to scientists in the field of biomedicine. In cancer cells, the metabolic features are significantly different from those of normal ones, which are hallmarks of several malignancies. Recent studies brought atypical cellular metabolism, such as aerobic glycolysis or the Warburg effect, into the scientific limelight. Targeting these altered metabolic pathways in cancer cells presents a promising therapeutic strategy. Pyruvate dehydrogenase kinases (PDKs), key enzymes in the pathway of glucose metabolism, could inactivate the pyruvate dehydrogenase complex (PDC) by phosphorylating it and preserving the substrates pyruvate, lactate and alanine for gluconeogenesis. Overexpression of PDKs could block the oxidative decarboxylation of pyruvate to satisfy high oxygen demand in cancer cells, while inhibition of PDKs could upregulate the activity of PDC and rectify the balance between the demand and supply of oxygen, which could lead to cancer cell death. Thus, inhibitors targeting PDKs represent a promising strategy for cancer treatment by acting on glycolytic tumors while showing minimal side effects on the oxidative healthy organs. This review considers the role of PDKs as regulator of PDC that catalyzes the oxidative decarboxylation of pyruvate in mitochondrion. It is concluded that PDKs are solid therapeutic targets. Inhibition of PDKs could be an attractive therapeutic approach for the development of anti-cancer drugs. PMID:26681918

  1. Production and characterization of L-fucose dehydrogenase from newly isolated Acinetobacter sp. strain SA-134.

    PubMed

    Ohshiro, Takashi; Morita, Noriyuki

    2014-01-01

    Microorganisms producing L-fucose dehydrogenase were screened from soil samples, and one of the isolated bacterial strains SA-134 was identified as Acinetobacter sp. by 16S rDNA gene analysis. The strain grew well utilizing L-fucose as a sole source of carbon, but all other monosaccharides tested such as D-glucose and D-arabinose did not support the growth of the strain in the absence of L-fucose. D-Arabinose inhibited the growth even in the culture medium containing L-fucose. Although the strain grew on some organic acids and amino acids such as citric acid and L-alanine as sole sources of carbon, the enzyme was produced only in the presence of L-fucose. The fucose dehydrogenase was purified to apparently homogeneity from the strain, and the native enzyme was a monomer of 25 kD. L-Fucose and D-arabinose were good substrates for the enzyme, but L-galactose was a poor substrate. The enzyme acted on both NAD(+) and NADP(+) in the similar manner.

  2. Determination of the anti-inflammatory and cytoprotective effects of l-glutamine and l-alanine, or dipeptide, supplementation in rats submitted to resistance exercise.

    PubMed

    Raizel, Raquel; Leite, Jaqueline Santos Moreira; Hypólito, Thaís Menezes; Coqueiro, Audrey Yule; Newsholme, Philip; Cruzat, Vinicius Fernandes; Tirapegui, Julio

    2016-08-01

    We evaluated the effects of chronic oral supplementation with l-glutamine and l-alanine in their free form or as the dipeptide l-alanyl-l-glutamine (DIP) on muscle damage, inflammation and cytoprotection, in rats submitted to progressive resistance exercise (RE). Wistar rats (n 8/group) were submitted to 8-week RE, which consisted of climbing a ladder with progressive loads. In the final 21 d before euthanasia, supplements were delivered in a 4 % solution in drinking water. Glutamine, creatine kinase (CK), lactate dehydrogenase (LDH), TNF-α, specific IL (IL-1β, IL-6 and IL-10) and monocyte chemoattractant protein-1 (MCP-1) levels were evaluated in plasma. The concentrations of glutamine, TNF-α, IL-6 and IL-10, as well as NF-κB activation, were determined in extensor digitorum longus (EDL) skeletal muscle. HSP70 level was assayed in EDL and peripheral blood mononuclear cells (PBMC). RE reduced glutamine concentration in plasma and EDL (P<0·05 v. sedentary group). However, l-glutamine supplements (l-alanine plus l-glutamine (GLN+ALA) and DIP groups) restored glutamine levels in plasma (by 40 and 58 %, respectively) and muscle (by 93 and 105 %, respectively). GLN+ALA and DIP groups also exhibited increased level of HSP70 in EDL and PBMC, consistent with the reduction of NF-κB p65 activation and cytokines in EDL. Muscle protection was also indicated by attenuation in plasma levels of CK, LDH, TNF-α and IL-1β, as well as an increase in IL-6, IL-10 and MCP-1. Our study demonstrates that chronic oral l-glutamine treatment (given with l-alanine or as dipeptide) following progressive RE induces cyprotective effects mediated by HSP70-associated responses to muscle damage and inflammation. PMID:27215379

  3. Determination of the anti-inflammatory and cytoprotective effects of l-glutamine and l-alanine, or dipeptide, supplementation in rats submitted to resistance exercise.

    PubMed

    Raizel, Raquel; Leite, Jaqueline Santos Moreira; Hypólito, Thaís Menezes; Coqueiro, Audrey Yule; Newsholme, Philip; Cruzat, Vinicius Fernandes; Tirapegui, Julio

    2016-08-01

    We evaluated the effects of chronic oral supplementation with l-glutamine and l-alanine in their free form or as the dipeptide l-alanyl-l-glutamine (DIP) on muscle damage, inflammation and cytoprotection, in rats submitted to progressive resistance exercise (RE). Wistar rats (n 8/group) were submitted to 8-week RE, which consisted of climbing a ladder with progressive loads. In the final 21 d before euthanasia, supplements were delivered in a 4 % solution in drinking water. Glutamine, creatine kinase (CK), lactate dehydrogenase (LDH), TNF-α, specific IL (IL-1β, IL-6 and IL-10) and monocyte chemoattractant protein-1 (MCP-1) levels were evaluated in plasma. The concentrations of glutamine, TNF-α, IL-6 and IL-10, as well as NF-κB activation, were determined in extensor digitorum longus (EDL) skeletal muscle. HSP70 level was assayed in EDL and peripheral blood mononuclear cells (PBMC). RE reduced glutamine concentration in plasma and EDL (P<0·05 v. sedentary group). However, l-glutamine supplements (l-alanine plus l-glutamine (GLN+ALA) and DIP groups) restored glutamine levels in plasma (by 40 and 58 %, respectively) and muscle (by 93 and 105 %, respectively). GLN+ALA and DIP groups also exhibited increased level of HSP70 in EDL and PBMC, consistent with the reduction of NF-κB p65 activation and cytokines in EDL. Muscle protection was also indicated by attenuation in plasma levels of CK, LDH, TNF-α and IL-1β, as well as an increase in IL-6, IL-10 and MCP-1. Our study demonstrates that chronic oral l-glutamine treatment (given with l-alanine or as dipeptide) following progressive RE induces cyprotective effects mediated by HSP70-associated responses to muscle damage and inflammation.

  4. 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.

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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.

  11. 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.

  12. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems.

  13. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems. PMID:27459246

  14. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the activity of the lactic dehydrogenase enzyme in serum. Increased levels of lactic dehydrogenase...

  15. 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

  16. 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.

  17. 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.

  18. Effects of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity.

    PubMed

    Li, Sha; Gan, Li-Qin; Li, Shu-Ke; Zheng, Jie-Cong; Xu, Dong-Ping; Li, Hua-Bin

    2014-01-01

    Various alcoholic beverages containing different concentrations of ethanol are widely consumed, and excessive alcohol consumption may result in serious health problems. The consumption of alcoholic beverages is often accompanied by non-alcoholic beverages, such as herbal infusions, tea and carbonated beverages to relieve drunk symptoms. The aim of this study was to supply new information on the effects of these beverages on alcohol metabolism for nutritionists and the general public, in order to reduce problems associated with excessive alcohol consumption. The effects of 57 kinds of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity were evaluated. Generally, the effects of these beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity are very different. The results suggested that some beverages should not be drank after excessive alcohol consumption, and several beverages may be potential dietary supplements for the prevention and treatment of problems related to excessive alcohol consumption.

  19. Multiple retinoid dehydrogenases in testes cytosol from alcohol dehydrogenase negative or positive deermice.

    PubMed

    Posch, K C; Napoli, J L

    1992-05-28

    Retinoic acid syntheses from retinol by cytosol from testes of alcohol dehydrogenase negative or positive deermice were similar in specific activity and in their insensitivity to 1 M ethanol or 100 mM 4-methylpyrazole. Anion-exchange followed by size-exclusion chromatography revealed multiple and similarly migrating peaks in each cytosol that had both retinol and retinal dehydrogenase activities. Thus, the effects of ethanol on testes cannot be caused by direct inhibition of cytosolic retinoic acid synthesis because retinoid dehydrogenases distinct from mouse class A2 alcohol dehydrogenases, which corresponds to human class I, occurred in testes and they were not inhibited by ethanol. These data also demonstrate the occurrence of multiple cytosolic retinoic acid synthesis activities and indicate that the two reactions of cytosolic retinoic acid synthesis, retinol and retinal dehydrogenation, may be catalyzed by enzymes that occur as complexes. PMID:1599517

  20. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5560 Lactic dehydrogenase immunological test system. (a) Identification. A lactic dehydrogenase... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactic dehydrogenase immunological test...

  1. Properties of formate dehydrogenase in Methanobacterium formicicum.

    PubMed Central

    Schauer, N L; Ferry, J G

    1982-01-01

    Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm. Images PMID:7061389

  2. Characterization of xylitol dehydrogenase from Debaryomyces hansenii

    SciTech Connect

    Girio, F.M.; Amaral-Collaco, M.T.; Pelica, F.

    1996-01-01

    The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells of Debaryomyces hansenii was partially purified in two chromatographic steps, and characterization studies were carried out in order to investigate the role of the xylitol dehydrogenase-catalyzed step in the regulation of D-xylose metabolism. The enzyme was most active at pH 9.0-9.5, and exhibited a broad polyol specificity. The Michaelis constants for xylitol and NAD{sup +} were 16.5 and 0.55 mM, respectively. Ca{sup 2+}, Mg{sup 2+}, and Mn{sup 2+} did not affect the enzyme activity. Conversely, Zn{sup 2+}, Cd{sup 2+}, and Co{sup 2+} strongly inhibited the enzyme activity. It was concluded that NAD{sup +}-xylitol dehydrogenase from D. hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and K{sub m} value for xylitol, and therefore should be named L-iditol:NAD{sup +}-5-oxidoreductase (EC 1.1.1.14). The reason D. hansenii is a good xylitol producer is not because of its value of K for xylitol, which is low enough to assure its fast oxidation by NAD{sup +}-xylitol dehydrogenase. However, a higher K{sub m} value of xylitol dehydrogenase for NAD{sup +} compared to the K{sub m} values of other xylose-fermenting yeasts may be responsible for the higher xylitol yields. 22 refs., 4 figs., 2 tabs.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. A β-Alanine Catabolism Pathway Containing a Highly Promiscuous ω-Transaminase in the 12-Aminododecanate-Degrading Pseudomonas sp. Strain AAC

    PubMed Central

    Wilding, Matthew; Peat, Thomas S.; Newman, Janet

    2016-01-01

    ABSTRACT We previously isolated the transaminase KES23458 from Pseudomonas sp. strain AAC as a promising biocatalyst for the production of 12-aminododecanoic acid, a constituent building block of nylon-12. Here, we report the subsequent characterization of this transaminase. It exhibits activity with a broad substrate range which includes α-, β-, and ω-amino acids, as well as α,ω-diamines and a number of other industrially relevant compounds. It is therefore a prospective candidate for the biosynthesis of a range of polyamide monomers. The crystal structure of KES23458 revealed that the protein forms a dimer containing a large active site pocket and unusual phosphorylated histidine residues. To infer the physiological role of the transaminase, we expressed, purified, and characterized a dehydrogenase from the same operon, KES23460. Unlike the transaminase, the dehydrogenase was shown to be quite selective, catalyzing the oxidation of malonic acid semialdehyde, formed from β-alanine transamination via KES23458. In keeping with previous reports, the dehydrogenase was shown to catalyze both a coenzyme A (CoA)-dependent reaction to form acetyl-CoA and a significantly slower CoA-independent reaction to form acetate. These findings support the original functional assignment of KES23458 as a β-alanine transaminase. However, a seemingly well-adapted active site and promiscuity toward unnatural compounds, such as 12-aminododecanoic acid, suggest that this enzyme could perform multiple functions for Pseudomonas sp. strain AAC. IMPORTANCE We describe the characterization of an industrially relevant transaminase able to metabolize 12-aminododecanoic acid, a constituent building block of the widely used polymer nylon-12, and we report the biochemical and structural characterization of the transaminase protein. A physiological role for this highly promiscuous enzyme is proposed based on the characterization of a related gene from the host organism. Molecular dynamics

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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).

  14. 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

  15. 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

  16. Substrate specificity, substrate channeling, and allostery in BphJ: an acylating aldehyde dehydrogenase associated with the pyruvate aldolase BphI.

    PubMed

    Baker, Perrin; Carere, Jason; Seah, Stephen Y K

    2012-06-01

    BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-β-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling. PMID:22574886

  17. 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

  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. 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%.

  2. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

    Peafowl, like other vertebrates, contain multiple forms of lactate dehydrogenase. The electrophoretic properties of the peafowl isoenzymes are unusual in that the isoenzyme from heart tissue can be either more or less anodic than that of muscle, depending on the pH. This finding focuses attention on the problem of isoenzyme identification. It is suggested that isoenzymes be identified on the basis of properties that are chemically and biologically more significant than electrophoretic mobility.

  3. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

    Peafowl, like other vertebrates, contain multiple forms of lactate dehydrogenase. The electrophoretic properties of the peafowl isoenzymes are unusual in that the isoenzyme from heart tissue can be either more or less anodic than that of muscle, depending on the pH. This finding focuses attention on the problem of isoenzyme identification. It is suggested that isoenzymes be identified on the basis of properties that are chemically and biologically more significant than electrophoretic mobility. PMID:5917779

  4. Succinate dehydrogenase-deficient gastrointestinal stromal tumors

    PubMed Central

    Wang, Ya-Mei; Gu, Meng-Li; Ji, Feng

    2015-01-01

    Most gastrointestinal stromal tumors (GISTs) are characterized by KIT or platelet-derived growth factor alpha (PDGFRA) activating mutations. However, there are still 10%-15% of GISTs lacking KIT and PDGFRA mutations, called wild-type GISTs (WT GISTs). Among these so-called WT GISTs, a small subset is associated with succinate dehydrogenase (SDH) deficiency, known as SDH-deficient GISTs. In addition, GISTs that occur in Carney triad and Carney-Stratakis syndrome represent specific examples of SDH-deficient GISTs. SDH-deficient GISTs locate exclusively in the stomach, showing predilection for children and young adults with female preponderance. The tumor generally pursues an indolent course and exhibits primary resistance to imatinib therapy in most cases. Loss of succinate dehydrogenase subunit B expression and overexpression of insulin-like growth factor 1 receptor (IGF1R) are common features of SDH-deficient GISTs. In WT GISTs without succinate dehydrogenase activity, upregulation of hypoxia-inducible factor 1α may lead to increased growth signaling through IGF1R and vascular endothelial growth factor receptor (VEGFR). As a result, IGF1R and VEGFR are promising to be the novel therapeutic targets of GISTs. This review will update the current knowledge on characteristics of SDH-deficient GISTs and further discuss the possible mechanisms of tumorigenesis and clinical management of SDH-deficient GISTs. PMID:25741136

  5. Prenatal presentation of pyruvate dehydrogenase complex deficiency.

    PubMed

    Natarajan, Niranjana; Tully, Hannah M; Chapman, Teresa

    2016-08-01

    We present the case of a female infant referred for prenatal MR evaluation of ventriculomegaly, which had been attributed by the referring obstetrician to aqueductal stenosis. Fetal MR confirmed ventriculomegaly but also demonstrated cerebral volume loss and white matter abnormalities. After birth, the infant developed persistent lactic acidosis. A diagnosis of pyruvate dehydrogenase complex deficiency was made on the basis of metabolic and molecular genetic studies. Ventriculomegaly is a common referral reason for fetal MR, yet there are few published reports of the radiographic findings that accompany inborn errors of metabolism, one potentially under-recognized cause of enlarged ventricles. This case contributes to this small body of literature on the imaging features of pyruvate dehydrogenase complex deficiency by describing pre- and postnatal MR findings and key clinical details. Our report emphasizes the necessity of considering pyruvate dehydrogenase complex deficiency and other metabolic disorders as potential etiologies for fetal ventriculomegaly since prompt diagnosis may allow for early initiation of treatment and improve outcome. PMID:27026023

  6. Dihydrodiol dehydrogenase and polycyclic aromatic hydrocarbon metabolism

    SciTech Connect

    Smithgall, T.E.

    1986-01-01

    Carcinogenic activation of polycyclic aromatic hydrocarbons by microsomal monoxygenases proceeds through trans-dihydrodiol metabolites to diol-epoxide ultimate carcinogens. This thesis directly investigated the role of dihydrodiol dehydrogenase, a cytosolic NAD(P)-linked oxidoreductase, in the detoxification of polycyclic aromatic trans-dihydrodiols. A wide variety of non-K-region trans-dihydrodiols were synthesized and shown to be substrates for the homogeneous rat liver dehydrogenase, including several potent proximate carcinogens derived from 7,12-dimethylbenz(a)anthracene, 5-methylchrysene, and benzo(a)pyrene. Since microsomal activation of polycyclic aromatic hydrocarbons is highly stereospecific, the stereochemical course of enzymatic trans-dihydrodiol oxidation was monitored using circular dichroism spectropolarimetry. The major product formed from the dehydrogenase-catalyzed oxidation of the trans-1,2-dihydrodiol of naphthalene was characterized using UV, IR, NMR, and mass spectroscopy, and appears to be 4-hydroxy-1,2-naphthoquinone. Mass spectral analysis suggests that an analogous hydroxylated o-quinone is formed as the major product of benzo(a)pyrene-7,8-dihydrodiol oxidation. Enzymatic oxidation of trans-dihydrodiols was shown to be potently inhibited by all of the major classes of the nonsteroidal antiinflammatory drugs. Enhancement of trans-dihydrodiol proximate carcinogen oxidation may protect against possible adverse effects of the aspirin-like drugs, and help maintain the balance between activation and detoxification of polycyclic aromatic hydrocarbons.

  7. Relationships within the aldehyde dehydrogenase extended family.

    PubMed

    Perozich, J; Nicholas, H; Wang, B C; Lindahl, R; Hempel, J

    1999-01-01

    One hundred-forty-five full-length aldehyde dehydrogenase-related sequences were aligned to determine relationships within the aldehyde dehydrogenase (ALDH) extended family. The alignment reveals only four invariant residues: two glycines, a phenylalanine involved in NAD binding, and a glutamic acid that coordinates the nicotinamide ribose in certain E-NAD binary complex crystal structures, but which may also serve as a general base for the catalytic reaction. The cysteine that provides the catalytic thiol and its closest neighbor in space, an asparagine residue, are conserved in all ALDHs with demonstrated dehydrogenase activity. Sixteen residues are conserved in at least 95% of the sequences; 12 of these cluster into seven sequence motifs conserved in almost all ALDHs. These motifs cluster around the active site of the enzyme. Phylogenetic analysis of these ALDHs indicates at least 13 ALDH families, most of which have previously been identified but not grouped separately by alignment. ALDHs cluster into two main trunks of the phylogenetic tree. The largest, the "Class 3" trunk, contains mostly substrate-specific ALDH families, as well as the class 3 ALDH family itself. The other trunk, the "Class 1/2" trunk, contains mostly variable substrate ALDH families, including the class 1 and 2 ALDH families. Divergence of the substrate-specific ALDHs occurred earlier than the division between ALDHs with broad substrate specificities. A site on the World Wide Web has also been devoted to this alignment project.

  8. 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

  9. 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.

  10. 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)

  11. 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.

  12. 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.

  13. 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.

  14. Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition.

    PubMed Central

    Koenig, K; Andreesen, J R

    1990-01-01

    The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with [185W]tungstate. This labeling was used as a reporter to purify both labile proteins. The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps. Both radioactive proteins were separated and purified to homogeneity. Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively. With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis. Molybdate was absolutely required for incorporation of 185W, formation of cross-reacting material, and enzymatic activity. The latter parameters showed a perfect correlation. This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase. The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000. The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme. The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined. The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Fu1 was catalyzed by two different dehydrogenases. Images PMID:2170335

  15. 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

  16. [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.

  17. 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.

  18. 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

  19. Assignment of the human dihydropyrimidine dehydrogenase gene (DPYD) to chromosome region 1p22 by fluorescence in situ hybridization

    SciTech Connect

    Takai, Setsuo; Fernandez-Salguero, Pedro; Kimura, Shioko

    1994-12-01

    Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the three-step pathway of uracil and thymine catabolism leading to the formation of {beta}-alanine and {beta}-aminobutyric acid, respectively. Several studies have demonstrated the importance of DPD in cancer patients, particularly in those lacking or having only low levels of activity. Patients exhibiting severe toxicity when administered 5-fluorouracil were shown to have low DPD activity. Studies of affected families demonstrated that the deficiency was inherited in an autosomal recessive pattern. DPD deficiency is one of several inherited disorders of pyrimidine metabolism, clinically termed thymine-uracil-uria. 14 refs., 1 fig.

  20. Structural Studies of the Final Enzyme in the alpha-Aminoadipate Pathway-Saccharopine Dehydrogenase from Saccharomyces cerevisiae

    SciTech Connect

    Burk,D.; Hwang, J.; Kwok, E.; Marrone, L.; Goodfellow, V.; Dmitrienko, G.; Berghuis, A.

    2007-01-01

    The 1.64 Angstroms structure of the apoenzyme form of saccharopine dehydrogenase (SDH) from Saccharomyces cerevisiae shows the enzyme to be composed of two domains with similar dinucleotide binding folds with a deep cleft at the interface. The structure reveals homology to alanine dehydrogense, despite low primary sequence similarity. A model of the ternary complex of SDH, NAD, and saccharopine identifies residues Lys77 and Glu122 as potentially important for substrate binding and/or catalysis, consistent with a proton shuttle mechanism. Furthermore, the model suggests that a conformational change is required for catalysis and that residues Lys99 and Asp281 may be instrumental in mediating this change. Analysis of the crystal structure in the context of other homologous enzymes from pathogenic fungi and human sources sheds light into the suitability of SDH as a target for antimicrobial drug development.

  1. β-Alanine Supplementation Does Not Augment the Skeletal Muscle Adaptive Response to 6 Weeks of Sprint Interval Training.

    PubMed

    Cochran, Andrew J R; Percival, Michael E; Thompson, Sara; Gillen, Jenna B; MacInnis, Martin J; Potter, Murray A; Tarnopolsky, Mark A; Gibala, Martin J

    2015-12-01

    Sprint interval training (SIT), repeated bouts of high-intensity exercise, improves skeletal muscle oxidative capacity and exercise performance. β-alanine (β-ALA) supplementation has been shown to enhance exercise performance, which led us to hypothesize that chronic β-ALA supplementation would augment work capacity during SIT and augment training-induced adaptations in skeletal muscle and performance. Twenty-four active but untrained men (23 ± 2 yr; VO2peak = 50 ± 6 mL · kg(-1) · min(-1)) ingested 3.2 g/day of β-ALA or a placebo (PLA) for a total of 10 weeks (n = 12 per group). Following 4 weeks of baseline supplementation, participants completed a 6-week SIT intervention. Each of 3 weekly sessions consisted of 4-6 Wingate tests, i.e., 30-s bouts of maximal cycling, interspersed with 4 min of recovery. Before and after the 6-week SIT program, participants completed a 250-kJ time trial and a repeated sprint test. Biopsies (v. lateralis) revealed that skeletal muscle carnosine content increased by 33% and 52%, respectively, after 4 and 10 weeks of β-ALA supplementation, but was unchanged in PLA. Total work performed during each training session was similar across treatments. SIT increased markers of mitochondrial content, including cytochome c oxidase (40%) and β-hydroxyacyl-CoA dehydrogenase maximal activities (19%), as well as VO2peak (9%), repeated-sprint capacity (5%), and 250-kJ time trial performance (13%), but there were no differences between treatments for any measure (p < .01, main effects for time; p > .05, interaction effects). The training stimulus may have overwhelmed any potential influence of β-ALA, or the supplementation protocol was insufficient to alter the variables to a detectable extent.

  2. 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

  3. A comparison of potato and vertebrate lactate dehydrogenases.

    PubMed Central

    Poerio, E; Davies, D D

    1980-01-01

    A 2000-fold purification of L(+)-lactate dehydrogenase from potatoes is reported. Five isoenzymes of lactate dehydrogenase can be detected in crude extracts of potato, and three of these are present in the purified preparation. The enzyme (mol.wt. 150 000), which is composed of four subunits (mol.wt. 37 500), is active with the same oxo acids and hydroxy acids that have been reported as substrates with the same oxo acids and hydroxy acids that have been reported as substrates for vertebrate lactate dehydrogenases. These similarities between potato and vertebrate lactate dehydrogenases contrast sharply with some other reports on potato lactate dehydrogenase. These discrepancies are discussed in relation to the proposition that vertebrate and potato lactate dehydrogenases share a common evolutionary origin. PMID:7236200

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Partial Similarities Between Yeast and Liver Alcohol Dehydrogenases

    PubMed Central

    Jörnvall, Hans

    1973-01-01

    The primary structure of about half of the protein chain of yeast alcohol dehydrogenase has been determined and compared with the amino-acid sequences of other dehydrogenases. The enzyme is found to be distantly related to horse-liver alcohol dehydrogenase, although these two proteins have different quaternary structures and subunit sizes. Some regions show no significant similarities, but long segments within the N-terminal parts of the molecules are homologous, suggesting a common and important function for these segments. Ancestral connections between some different dehydrogenases can be concluded and the degree of evolutionary changes may be estimated. PMID:4599620

  9. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  10. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  11. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  12. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary...

  13. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary...

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. Phosphorylation-dephosphorylation of yeast pyruvate dehydrogenase

    SciTech Connect

    Uhlinger, D.J.; Reed, L.J.

    1986-05-01

    Pyruvate dehydrogenase complex (PDC) was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase (PDH) kinase activity was detected at any stage of the purification. However, the purified PDC was phosphorylated and inactivated by purified PDH kinase from bovine kidney mitochondria, Mg/sup 2 +/, and (..gamma..-/sup 32/P)ATP. The protein-bound radioactivity was localized in the PDH ..cap alpha.. subunit. The phosphorylated, inactivated PDC was dephosphorylated and reactivated with purified bovine PDH phosphatase, Mg/sup 2 +/, and Ca/sup 2 +/. From a tryptic digest of phosphorylated yeast PDC a radioactive peptide was isolated by anion and reverse phase HPLC. The sequence of this tetradecapeptide is Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphopeptide derived from the ..cap alpha.. subunit of bovine kidney and heart PDH: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

  3. Transcriptional regulation of pyruvate dehydrogenase kinase.

    PubMed

    Jeong, Ji Yun; Jeoung, Nam Ho; Park, Keun-Gyu; Lee, In-Kyu

    2012-10-01

    The pyruvate dehydrogenase complex (PDC) activity is crucial to maintains blood glucose and ATP levels, which largely depends on the phosphorylation status by pyruvate dehydrogenase kinase (PDK) isoenzymes. Although it has been reported that PDC is phosphorylated and inactivated by PDK2 and PDK4 in metabolically active tissues including liver, skeletal muscle, heart, and kidney during starvation and diabetes, the precise mechanisms by which expression of PDK2 and PDK4 are transcriptionally regulated still remains unclear. Insulin represses the expression of PDK2 and PDK4 via phosphorylation of FOXO through PI3K/Akt signaling pathway. Several nuclear hormone receptors activated due to fasting or increased fat supply, including peroxisome proliferator-activated receptors, glucocorticoid receptors, estrogen-related receptors, and thyroid hormone receptors, also participate in the up-regulation of PDK2 and PDK4; however, the endogenous ligands that bind those nuclear receptors have not been identified. It has been recently suggested that growth hormone, adiponectin, epinephrine, and rosiglitazone also control the expression of PDK4 in tissue-specific manners. In this review, we discuss several factors involved in the expressional regulation of PDK2 and PDK4, and introduce current studies aimed at providing a better understanding of the molecular mechanisms that underlie the development of metabolic diseases such as diabetes. PMID:23130316

  4. Mechanism of mercurial inhibition of sodium-coupled alanine uptake in liver plasma membrane vesicles from Raja erinacea

    SciTech Connect

    Sellinger, M.; Ballatori, N.; Boyer, J.L. )

    1991-02-01

    In mammalian hepatocytes the L-alanine carrier contains a sulfhydryl group that is essential for its activity and is inhibited by mercurials. In hepatocytes of the evolutionarily primitive little skate (Raja erinacea), HgCl2 inhibits Na(+)-dependent alanine uptake and Na+/K(+)-ATPase and increase K+ permeability. To distinguish between direct effects of HgCl2 on the Na(+)-alanine cotransporter and indirect effects on membrane permeability, (3H)alanine transport was studied in plasma membrane vesicles. (3H)Alanine uptake was stimulated by an out-to-in Na+ but not K+ gradient and was saturable confirming the presence of Na(+)-alanine cotransport in liver plasma membranes from this species. Preincubation of the vesicles with HgCl2 for 5 min reduced initial rates of Na(+)-dependent but not Na(+)-independent alanine uptake in a dose-dependent manner (10-200 microM). In the presence of equal concentrations of NaCl or KCl inside and outside of the vesicles, 75 microM HgCl2 directly inhibited sodium-dependent alanine-(3H)alanine exchange, demonstrating that HgCl2 directly affected the alanine cotransporter. Inhibition of Na(+)-dependent alanine uptake by 30 microM HgCl2 was reversed by dithiothreitol (1 mM). HgCl2 (10-30 microM) also increased initial rates of 22Na uptake (at 5 sec), whereas 22Na uptake rates were decreased at HgCl2 concentrations greater than 50 microM. Higher concentrations of HgCl2 (100-200 microM) produced nonspecific effects on vesicle integrity. These studies indicate that HgCl2 inhibits Na(+)-dependent alanine uptake in skate hepatocytes by three different concentration-dependent mechanisms: direct interaction with the transporters, dissipation of the driving force (Na+ gradient), and loss of membrane integrity.

  5. A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:d-alanine ligase of Escherichia coli.

    PubMed Central

    Fan, C; Moews, P C; Shi, Y; Walsh, C T; Knox, J R

    1995-01-01

    Examination of x-ray crystallographic structures shows the tertiary structure of D-alanine:D-alanine ligase (EC 6.3.2.4). a bacterial cell wall synthesizing enzyme, is similar to that of glutathione synthetase (EC 6.32.3) despite low sequence homology. Both Escherichia coli enzymes, which convert ATP to ADP during ligation to produce peptide products, are made of three domains, each folded around a 4-to 6-stranded beta-sheet core. Sandwiched between the beta-sheets of the C-terminal and central domains of each enzyme is a nonclassical ATP-binding site that contains a common set of spatially equivalent amino acids. In each enzyme, two loops are proposed to exhibit a required flexibility that allows entry of ATP and substrates, provides protection of the acylphosphate intermediate and tetrahedral adduct from hydrolysis during catalysis, and then permits release of products. PMID:7862655

  6. On the roles of the alanine and serine in the β-sheet structure of fibroin.

    PubMed

    Carrascoza Mayen, Juan Francisco; Lupan, Alexandru; Cosar, Ciprian; Kun, Attila-Zsolt; Silaghi-Dumitrescu, Radu

    2015-02-01

    In its silk II form, fibroin is almost exclusively formed from layers of β-sheets, rich in glycine, alanine and serine. Reported here are computational results on fibroin models at semi-empirical, DFT levels of theory and molecular dynamics (MD) for (Gly)10, (Gly-Ala)5 and (Gly-Ser)5 decapeptides. While alanine and serine introduce steric repulsions, the alanine side-chain adds to the rigidity of the sheet, allowing it to maintain a properly pleated structure even in a single β-sheet, and thus avoiding two alternative conformations which would interfere with the formation of the multi-layer pleated-sheet structure. The role of the serine is proposed to involve modulation of the hydrophobicity in order to construct the supramolecular assembly as opposed to random precipitation due to hydrophobicity.

  7. Antimicrobial activity of antihypertensive food-derived peptides and selected alanine analogues.

    PubMed

    McClean, Stephen; Beggs, Louise B; Welch, Robert W

    2014-03-01

    This study evaluated four food-derived peptides with known antihypertensive activities for antimicrobial activity against pathogenic microorganisms, and assessed structure-function relationships using alanine analogues. The peptides (EVSLNSGYY, barley; PGTAVFK, soybean; TTMPLW, α-casein; VHLPP, α-zein) and the six alanine substitution peptides of PGTAVFK were synthesised, characterised and evaluated for antimicrobial activity using the bacteria, Escherichia coli, Staphylococcus aureus, and Micrococcus luteus and the yeast, Candida albicans. The peptides TTMPLW and PGTAVFK inhibited growth of all four microorganisms tested, with activities of a similar order of magnitude to ampicillin and ethanol controls. EVSLNSGYY inhibited the growth of the bacteria, but VHLPP showed no antimicrobial activity. The alanine analogue, PGAAVFK showed the highest overall antimicrobial activity and PGTAVFA showed no activity; overall, the activities of the analogues were consistent with their structures. Some peptides with antihypertensive activity also show antimicrobial activity, suggesting that food-derived peptides may exert beneficial effects via a number of mechanisms.

  8. A photoactivable amino acid based on a novel functional coumarin-6-yl-alanine.

    PubMed

    Fonseca, Andrea S C; Gonçalves, M Sameiro T; Costa, Susana P G

    2012-12-01

    A novel fluorescent amino acid, L-4-chloromethylcoumarin-6-yl-alanine, was obtained from tyrosine by a Pechmann reaction. The assembly of the heterocyclic ring at the tyrosine side chain could be achieved before or after incorporation of tyrosine into a dipeptide, and amino acid and dipeptide ester conjugates were obtained by coupling to a model N-protected alanine. The behaviour of one of the fluorescent conjugates towards irradiation was studied in a photochemical reactor at different wavelengths (254, 300, 350 and 419 nm). The photoreaction course in methanol/HEPES buffer solution (80:20) was followed by HPLC/UV monitoring. It was found that the novel unnatural amino acid could act as a fluorescent label, due to its fluorescence properties, and, more importantly, as a photoactivable unit, due to the short irradiation times necessary to cleave the ester bond between the model amino acid and the coumarin-6-yl-alanine.

  9. Nucleation kinetics, growth and studies of β-alanine single crystals.

    PubMed

    Shanthi, D; Selvarajan, P; HemaDurga, K K; Lincy Mary Ponmani, S

    2013-06-01

    Solubility and metastable zone width for the re-crystallized salt of β-alanine was determined. Induction period measurement for the selected supersaturation ratios at room temperature (31 °C) was carried out for supersaturated aqueous solutions of β-alanine and it is noticed that induction period decreases with increase of supersaturation ratio. The nucleation parameters such as Gibbs free energy change, radius and number of molecules of the critical nucleus, interfacial tension and the nucleation rate have been evaluated by classical nucleation theory. Single crystals of β-alanine were grown using the optimized nucleation parameters by solution method and grown crystals have been subjected to various studies like XRD studies, FTIR, optical, thermal and SHG studies.

  10. Combined TL and 10B-alanine ESR dosimetry for BNCT.

    PubMed

    Bartolotta, A; D'Oca, M C; Lo Giudice, B; Brai, M; Borio, R; Forini, N; Salvadori, P; Manera, S

    2004-01-01

    The dosimetric technique described in this paper is based on electron spin resonance (ESR) detectors using an alanine-boric compound acid enriched with (10)B, and beryllium oxide thermoluminescent (TL) detectors; with this combined dosimetry, it is possible to discriminate the doses due to thermal neutrons and gamma radiation in a mixed field. Irradiations were carried out inside the thermal column of a TRIGA MARK II water-pool-type research nuclear reactor, also used for Boron Neutron Capture therapy (BNCT) applications, with thermal neutron fluence from 10(9) to 10(14) nth cm(-2). The ESR dosemeters using the alanine-boron compound indicated ESR signals about 30-fold stronger than those using only alanine. Moreover, a negligible correction for the gamma contribution, measured with TL detectors, almost insensitive to thermal neutrons, was necessary. Therefore, a simultaneous analysis of our TL and ESR detectors allows discrimination between thermal neutron and gamma doses, as required in BNCT.

  11. Nucleation kinetics, growth and studies of β-alanine single crystals

    NASA Astrophysics Data System (ADS)

    Shanthi, D.; Selvarajan, P.; HemaDurga, K. K.; Lincy Mary Ponmani, S.

    2013-06-01

    Solubility and metastable zone width for the re-crystallized salt of β-alanine was determined. Induction period measurement for the selected supersaturation ratios at room temperature (31 °C) was carried out for supersaturated aqueous solutions of β-alanine and it is noticed that induction period decreases with increase of supersaturation ratio. The nucleation parameters such as Gibbs free energy change, radius and number of molecules of the critical nucleus, interfacial tension and the nucleation rate have been evaluated by classical nucleation theory. Single crystals of β-alanine were grown using the optimized nucleation parameters by solution method and grown crystals have been subjected to various studies like XRD studies, FTIR, optical, thermal and SHG studies.

  12. Combined TL and 10B-alanine ESR dosimetry for BNCT.

    PubMed

    Bartolotta, A; D'Oca, M C; Lo Giudice, B; Brai, M; Borio, R; Forini, N; Salvadori, P; Manera, S

    2004-01-01

    The dosimetric technique described in this paper is based on electron spin resonance (ESR) detectors using an alanine-boric compound acid enriched with (10)B, and beryllium oxide thermoluminescent (TL) detectors; with this combined dosimetry, it is possible to discriminate the doses due to thermal neutrons and gamma radiation in a mixed field. Irradiations were carried out inside the thermal column of a TRIGA MARK II water-pool-type research nuclear reactor, also used for Boron Neutron Capture therapy (BNCT) applications, with thermal neutron fluence from 10(9) to 10(14) nth cm(-2). The ESR dosemeters using the alanine-boron compound indicated ESR signals about 30-fold stronger than those using only alanine. Moreover, a negligible correction for the gamma contribution, measured with TL detectors, almost insensitive to thermal neutrons, was necessary. Therefore, a simultaneous analysis of our TL and ESR detectors allows discrimination between thermal neutron and gamma doses, as required in BNCT. PMID:15353720

  13. NADP-dehydrogenases from pepper fruits: effect of maturation.

    PubMed

    Mateos, Rosa M; Bonilla-Valverde, Daniel; del Río, Luis A; Palma, José M; Corpas, Francisco J

    2009-02-01

    NADPH is an important molecule in the redox balance of the cell. Pepper fruits are the second worldwide consumable vegetables and exhibit different phenotypes after maturation. In this paper, two pepper cultivars were studied: Vergasa whose fruits shift from green to red after maturation, and Biela that shifts to yellow. Using fresh fruits from the same plants of the two cultivars at distinct maturation stages, the activity and gene expression of the main NADPH-generating dehydrogenases was studied. The activity analysis of the main NADP-dehydrogenases, glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-isocitrate dehydrogenase (NADP-ICDH) and NADP-malic enzyme (NADP-ME), showed that, except for the G6PDH, all the activities were enhanced (54-100%) in the mature pepper fruits from both cultivars (red or yellow) with respect to green pepper fruits. The content of NADPH and NADP in the mature fruits of both cultivars showed a noteworthy increase with respect to green fruits. For the transcript analysis, a partial cDNA of each NADP-dehydrogenase was obtained, and the NADP-ME was the only NADP-dehydrogenase that showed a significant induction. The increase in the content of NADPH in mature fruits because of the enhanced activity of NADP-dehydrogenases suggests that these NADPH-generating enzymes could be involved in the maturation of pepper fruits.

  14. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Sorbitol dehydrogenase test system. 862.1670 Section 862.1670 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1670 Sorbitol dehydrogenase...

  15. Saturation transfer difference NMR studies on substrates and inhibitors of succinic semialdehyde dehydrogenases

    SciTech Connect

    Jaeger, Martin Rothacker, Boris; Ilg, Thomas

    2008-08-01

    Saturation transfer difference (STD) NMR experiments on Escherichia coli and Drosophila melanogaster succinic semialdehyde dehydrogenase (SSADH, EC1.2.1.24) suggest that only the aldehyde forms and not the gem-diol forms of the specific substrate succinic semialdehyde (SSA), of selected aldehyde substrates, and of the inhibitor 3-tolualdehyde bind to these enzymes. Site-directed mutagenesis of the active site cysteine311 to alanine in D. melanogaster SSADH leads to an inactive product binding both SSA aldehyde and gem-diol. Thus, the residue cysteine311 is crucial for their discrimination. STD experiments on SSADH and NAD{sup +}/NADP{sup +} indicate differential affinity in agreement with the respective cosubstrate properties. Epitope mapping by STD points to a strong interaction of the NAD{sup +}/NADP{sup +} adenine H2 proton with SSADH. Adenine H8, nicotinamide H2, H4, and H6 also show STD signals. Saturation transfer to the ribose moieties is limited to the anomeric protons of E. coli SSADH suggesting that the NAD{sup +}/NADP{sup +} adenine and nicotinamide, but not the ribose moieties are important for the binding of the coenzymes.

  16. Purification and properties of NADP-dependent glutamate dehydrogenase from Ruminococcus flavefaciens FD-1.

    PubMed Central

    Duncan, P A; White, B A; Mackie, R I

    1992-01-01

    Glutamate dehydrogenase (GDH) (L-glutamate:NADP+ oxidoreductase, deaminating, EC 1.4.1.4) from the cellulolytic ruminal bacterium Ruminococcus flavefaciens has been purified and characterized. The native enzyme and subunit are 280 and 48 kDa, respectively, suggesting that the native enzyme is a hexamer. The enzyme requires 0.5 M KCl for optimal activity and has a pH optimum of 6.9 to 7.0. The Kms for ammonia, alpha-ketoglutarate, and glutamate are 19, 0.41, and 62 mM, respectively. The sigmoidal NADPH saturation curve revealed positive cooperativity for the binding of this coenzyme. The first residue in the N-terminal amino acid sequence from R. flavefaciens GDH was alanine, suggesting that the protein may be modified posttranslationally. Comparison of the N-terminal sequence with those of Escherichia coli, Salmonella typhimurium, and Clostridium symbiosum revealed only 39% amino acid homologies. The GDH from R. flavefaciens was unique in that its specific activity was highest during ammonia-limited growth but was not affected by ammonia shock treatment (20 mM). Images PMID:1335719

  17. The energy dependence of lithium formate and alanine EPR dosimeters for medium energy x rays

    SciTech Connect

    Waldeland, Einar; Hole, Eli Olaug; Sagstuen, Einar; Malinen, Eirik

    2010-07-15

    Purpose: To perform a systematic investigation of the energy dependence of alanine and lilthium formate EPR dosimeters for medium energy x rays. Methods: Lithium formate and alanine EPR dosimeters were exposed to eight different x-ray beam qualities, with nominal potentials ranging from 50 to 200 kV. Following ionometry based on standards of absorbed dose to water, the dosimeters were given two different doses of approximately 3 and 6 Gy for each radiation quality, with three dosimeters for each dose. A reference series was also irradiated to three different dose levels at a {sup 60}Co unit. The dose to water energy response, that is, the dosimeter reading per absorbed dose to water relative to that for {sup 60}Co {gamma}-rays, was estimated for each beam quality. In addition, the energy response was calculated by Monte Carlo simulations and compared to the experimental energy response. Results: The experimental energy response estimates ranged from 0.89 to 0.94 and from 0.68 to 0.90 for lithium formate and alanine, respectively. The uncertainties in the experimental energy response estimates were typically 3%. The relative effectiveness, that is, the ratio of the experimental energy response to that following Monte Carlo simulations was, on average, 0.96 and 0.94 for lithium formate and alanine, respectively. Conclusions: This work shows that lithium formate dosimeters are less dependent on x-ray energy than alanine. Furthermore, as the relative effectiveness for both lithium formate and alanine were systematically less than unity, the yield of radiation-induced radicals is decreased following x-irradiation compared to irradiation with {sup 60}Co {gamma}-rays.

  18. Relative response of the alanine dosimeter to medium energy x-rays.

    PubMed

    Anton, M; Büermann, L

    2015-08-01

    The response of the alanine dosimeter to kilovoltage x-rays with respect to the dose to water was measured, relative to the response to Co-60 radiation.Two series of x-ray qualities were investigated, one ranging from 30 kV to 100 kV tube voltage (TW series), the other one ranging from 70 kV to 280 kV (TH series). Due to the use of the water calorimeter as a primary standard, the uncertainty of the delivered dose is significantly lower than for other published data. The alanine response was measured as described in a previous publication (Anton et al 2013 Phys. Med. Biol. 58 3259-82). The uncertainty component due to the alanine measurement and analysis is ⩽0.4%, the major part of the combined uncertainty of the relative response originates from the uncertainty of the delivered dose. The relative uncertainties of the relative response vary from ⩽2% for the TW series to ⩽1.1% for the TH series.Different from the behaviour of the alanine dosimeter for megavoltage x-rays or electrons, the relative response drops significantly from unity for Co-60 radiation to less than 64% for the TW quality with a tube voltage of 30 kV. In order to reproduce this behaviour through Monte Carlo simulations, not only the ratio of the absorbed dose to alanine to the absorbed dose to water has to be known, but also the intrinsic efficiency, i.e. the dependence of the number of free radicals generated per unit of absorbed dose on the photon energy. This quantity is not yet accessible for the TW series.For a possible use of the alanine dosimeter for kilovoltage x-rays, for example in electronic brachytherapy, users should rely on the measured data for the relative response which have become available with this publication. PMID:26216572

  19. Relative response of the alanine dosimeter to medium energy x-rays

    NASA Astrophysics Data System (ADS)

    Anton, M.; Büermann, L.

    2015-08-01

    The response of the alanine dosimeter to kilovoltage x-rays with respect to the dose to water was measured, relative to the response to Co-60 radiation. Two series of x-ray qualities were investigated, one ranging from 30 kV to 100 kV tube voltage (TW series), the other one ranging from 70 kV to 280 kV (TH series). Due to the use of the water calorimeter as a primary standard, the uncertainty of the delivered dose is significantly lower than for other published data. The alanine response was measured as described in a previous publication (Anton et al 2013 Phys. Med. Biol. 58 3259-82). The uncertainty component due to the alanine measurement and analysis is ⩽0.4%, the major part of the combined uncertainty of the relative response originates from the uncertainty of the delivered dose. The relative uncertainties of the relative response vary from ⩽2% for the TW series to ⩽1.1% for the TH series. Different from the behaviour of the alanine dosimeter for megavoltage x-rays or electrons, the relative response drops significantly from unity for Co-60 radiation to less than 64% for the TW quality with a tube voltage of 30 kV. In order to reproduce this behaviour through Monte Carlo simulations, not only the ratio of the absorbed dose to alanine to the absorbed dose to water has to be known, but also the intrinsic efficiency, i.e. the dependence of the number of free radicals generated per unit of absorbed dose on the photon energy. This quantity is not yet accessible for the TW series. For a possible use of the alanine dosimeter for kilovoltage x-rays, for example in electronic brachytherapy, users should rely on the measured data for the relative response which have become available with this publication.

  20. Relative response of the alanine dosimeter to medium energy x-rays.

    PubMed

    Anton, M; Büermann, L

    2015-08-01

    The response of the alanine dosimeter to kilovoltage x-rays with respect to the dose to water was measured, relative to the response to Co-60 radiation.Two series of x-ray qualities were investigated, one ranging from 30 kV to 100 kV tube voltage (TW series), the other one ranging from 70 kV to 280 kV (TH series). Due to the use of the water calorimeter as a primary standard, the uncertainty of the delivered dose is significantly lower than for other published data. The alanine response was measured as described in a previous publication (Anton et al 2013 Phys. Med. Biol. 58 3259-82). The uncertainty component due to the alanine measurement and analysis is ⩽0.4%, the major part of the combined uncertainty of the relative response originates from the uncertainty of the delivered dose. The relative uncertainties of the relative response vary from ⩽2% for the TW series to ⩽1.1% for the TH series.Different from the behaviour of the alanine dosimeter for megavoltage x-rays or electrons, the relative response drops significantly from unity for Co-60 radiation to less than 64% for the TW quality with a tube voltage of 30 kV. In order to reproduce this behaviour through Monte Carlo simulations, not only the ratio of the absorbed dose to alanine to the absorbed dose to water has to be known, but also the intrinsic efficiency, i.e. the dependence of the number of free radicals generated per unit of absorbed dose on the photon energy. This quantity is not yet accessible for the TW series.For a possible use of the alanine dosimeter for kilovoltage x-rays, for example in electronic brachytherapy, users should rely on the measured data for the relative response which have become available with this publication.

  1. The effect of β-alanine supplementation on cycling time trials of different length.

    PubMed

    Bellinger, Phillip M; Minahan, Clare L

    2016-10-01

    The varying results reported in response to β-alanine supplementation may be related to the duration and nature of the exercise protocol employed. We investigated the effects of β-alanine supplementation on a wide range of cycling performance tests in order to produce a clear concise set of criteria for its efficacy. Fourteen trained cyclists (Age = 24.8 ± 6.7 years; VO2max = 65.4 ± 10.2 mL·kg·min(-1)) participated in this placebo-controlled, double-blind study. Prior to supplementation, subjects completed two (familiarization and baseline) supramaximal cycling bouts until exhaustion (120% pre-supplementation VO2max) and two 1-, 4- and 10-km cycling time trial (TT). Subjects then supplemented orally for 4 weeks with 6.4 g/d placebo or β-alanine and repeated the battery of performance tests. Blood lactate was measured pre-exercise, post-exercise and 5  min post-exercise. β-alanine supplementation elicited significant increases in time to exhaustion (TTE) (17.6 ± 11.5 s; p = 0.013, effect compared with placebo) and was likely to be beneficial to 4-km TT performance time (-7.8 ± 8.1 s; 94% likelihood), despite not being statistically different (p = 0.060). Performance times in the 1- and 10-km TT were not affected by treatment. For the highly trained cyclists in the current study, β-alanine supplementation significantly extended supramaximal cycling TTE and may have provided a worthwhile improvement to 4-km TT performance. However, 1- and 10-km cycling TT performance appears to be unaffected by β-alanine supplementation.

  2. Conformations of Diphosphopyridine Coenzymes upon Binding to Dehydrogenases

    PubMed Central

    Lee, Chi-Yu; Eichner, Ronald D.; Kaplan, Nathan O.

    1973-01-01

    The binding of oxidized as well as reduced coenzyme to some dehydrogenases has been studied under different concentration ratios and temperatures by nuclear magnetic resonance spectroscopy. A significant difference in the spectral behavior between DPN+ and DPNH upon binding is interpreted in terms of fast and slow on-off rates relative to the nuclear magnetic resonance time scale in the binding of these two coenzymes. Significant downfield shifts of DPN+ were observed upon binding, comparable in magnitude to those expected upon opening (destacking) of the coenzymes in the case of chicken-muscle and lobster-tail lactate dehydrogenase (EC 1.1.1.27) and yeast alchol dehydrogenase (EC 1.1.1.1.). A preliminary survey of several other dehydrogenases is consistent with these findings. In the case of 3-phosphoglyceraldehyde dehydrogenase, there is a possibility that the coenzyme exists in the folded form. PMID:4351183

  3. On the fragmentation of biomolecules: Fragmentation of alanine dipeptide along the polypeptide chain

    SciTech Connect

    Solov'yov, I. A. Yakubovich, A. V.; Solov'yov, A. V.; Greiner, W.

    2006-09-15

    The interaction potential between amino acids in alanine dipeptide has been studied for the first time taking into account exact molecular geometry. Ab initio calculation has been performed in the framework of density functional theory taking into account all electrons in the system. The fragmentation of dipeptide along the polypeptide chain, as well as the interaction between alanines, has been considered. The energy of the system has been analyzed as a function of the distance between fragments for all possible dipeptide fragmentation channels. Analysis of the energy barriers makes it possible to estimate the characteristic fragmentation times and to determine the degree of applicability of classical electrodynamics for describing the system energy.

  4. Effect of β-alanine treatment on mitochondrial taurine level and 5-taurinomethyluridine content

    PubMed Central

    2010-01-01

    Background The β-amino acid, taurine, is a nutritional requirement in some species. In these species, the depletion of intracellular stores of taurine leads to the development of severe organ dysfunction. The basis underlying these defects is poorly understood, although there is some suggestion that oxidative stress may contribute to the abnormalities. Recent studies indicate that taurine is required for normal mitochondrial protein synthesis and normal electron transport chain activity; it is known that defects in these events can lead to severe mitochondrial oxidative stress. The present study examines the effect of taurine deficiency on the first step of mitochondrial protein synthesis regulation by taurine, namely, the formation of taurinomethyluridine containing tRNA. Methods Isolated rat cardiomyocytes were rendered taurine deficient by incubation with medium containing the taurine transport inhibitor, β-alanine. The time course of cellular and mitochondrial taurine depletion was measured. The primer extension method was employed to evaluate the effect of β-alanine treatment on taurinomethyluridine content of tRNALeu. The protein levels of ND6 were also determined by Western blot analysis. Results β-alanine caused a time-dependent decrease in cellular taurine content, which were reduced in half after 48 hrs of incubation. The amount of taurine in the mitochondria was considerably less than that in the cytosol and was unaffected by β-alanine treatment. Approximately 70% of the tRNALeu in the untreated cell lacked taurinomethyluridine and these levels were unchanged following β-alanine treatment. Protein content of ND6, however, was significantly reduced after 48 hours incubation with β-alanine. Conclusions The taurine levels of the cytosol and the mitochondria are not directly coupled. The β-alanine-mediated reduction in taurine levels is too small to affect taurinomethyluridine levels. Nonetheless, it interferes with mitochondrial protein synthesis

  5. Steric effect exerted by the proline residue on the antecedent alanine residue.

    PubMed

    Siemión, I Z; Sobczyk, K; Nawrocka, E

    1982-05-01

    Five model tetrapeptides: Ala-Ala-Ala-Ala, Pro-Ala-Ala-Ala, Ala-Pro-Ala-Ala, Ala-Ala-Pro-Ala and Ala-Ala-Ala-Pro, were synthesized and measured in D2O by 13 C-n.m.r. spectroscopy. The spectra analysis led us to the conclusion that for each model (irrespective of pD) in conformational equilibrium, the predominant conformation is the one in which side methyl of alanine preceding proline residue eclipses alanine carbonyl group. The influence of pD changes in cis-trans isomerism of Ala-Pro amide bond was also investigated. PMID:7118413

  6. [Leucine and alanine aminopeptidase activity in the organs of cattle, sheep and swine].

    PubMed

    Goranov, Kh

    1982-01-01

    Studied was the activity of leucine-aminopeptidase and alanine-aminopeptidase in fresh tissue homogenates of liver, spleen, kidney, heart, pancreas, femoral muscle, stomach (rumen), small intestine, and lung taken from 8 cattle, sheep, and pigs. Both enzymes showed ubiquity. Leucine-aminopeptidase exhibited highest activity in the spleen of pigs and the kidney of sheep and cattle. The kidneys of all investigated animal species showed 10 to 15 times higher alanine-aminopeptidase activity than the remaining organs. This pointed to the relative ubiquity of the enzyme with special reference to kidneys.

  7. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

  8. Interactions between heparinoids and alcohol dehydrogenase.

    PubMed

    Paulíková, H; Valusová, E; Antalík, M

    1997-07-01

    The interaction between polysulfated polysaecharides (low-molecular-weight heparin LMWH, dextran sulfate DS and pentosan sulfate PS) and yeast alcohol dehydrogenase (YADH) was investigated. The fluorescence and UV spectra of YADH after adding the tested polysaccharides have confirmed the interaction between the enzyme and these compounds. Kinetic studies have shown that LMWH, DS and PS are inhibitors of YADH (mixed type with respect to NAD). The most potent inhibitor is PS (ID50=37.5 ng/ml, Ki=0.6 muM). The inhibition effect depends on the ionic strength (the inhibition decreased by about 50% in the presence of 100 mM Na2SO4) and pH value (the inhibition decreased at pH>7). The results indicate that the inhibition effect of these polyanions is caused by their electrostatic interactions with the NAD-binding region of YADH.

  9. The Aldehyde Dehydrogenase Gene Superfamily Resource Center

    PubMed Central

    2009-01-01

    The website http://www.aldh.org is a publicly available database for nomenclature and functional and molecular sequence information for members of the aldehyde dehydrogenase (ALDH) gene superfamily for animals, plants, fungi and bacteria. The site has organised gene-specific records. It provides synopses of ALDH gene records, marries trivial terms to correct nomenclature and links global accession identifiers with source data. Server-side alignment software characterises the integrity of each sequence relative to the latest genomic assembly and provides identifier-specific detail reports, including a graphical presentation of the transcript's exon - intron structure, its size, coding sequence, genomic strand and locus. Also included are a summary of substrates, inhibitors and enzyme kinetics. The site provides reference lists and is designed to facilitate data mining by interested investigators. PMID:20038501

  10. Mitochondrial aldehyde dehydrogenase and cardiac diseases

    PubMed Central

    Chen, Che-Hong; Sun, Lihan; Mochly-Rosen, Daria

    2010-01-01

    Numerous conditions promote oxidative stress, leading to the build-up of reactive aldehydes that cause cell damage and contribute to cardiac diseases. Aldehyde dehydrogenases (ALDHs) are important enzymes that eliminate toxic aldehydes by catalysing their oxidation to non-reactive acids. The review will discuss evidence indicating a role for a specific ALDH enzyme, the mitochondrial ALDH2, in combating oxidative stress by reducing the cellular ‘aldehydic load’. Epidemiological studies in humans carrying an inactive ALDH2, genetic models in mice with altered ALDH2 levels, and small molecule activators of ALDH2 all highlight the role of ALDH2 in cardioprotection and suggest a promising new direction in cardiovascular research and the development of new treatments for cardiovascular diseases. PMID:20558439

  11. Untangling the glutamate dehydrogenase allosteric nightmare.

    PubMed

    Smith, Thomas J; Stanley, Charles A

    2008-11-01

    Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the 'missing link' in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion.

  12. Fast internal dynamics in alcohol dehydrogenase.

    PubMed

    Monkenbusch, M; Stadler, A; Biehl, R; Ollivier, J; Zamponi, M; Richter, D

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains. PMID:26298156

  13. NADH electrochemical sensor coupled with dehydrogenase enzymes

    SciTech Connect

    Yamanaka, Hideko; Mascini, Marco )

    1992-06-01

    A graphite electrode assembled in a flow cell has shown to be a good detector for NADH. Current is linearly dependent on concentration in the range 10{sup {minus}7}-10{sup {minus}3} M without any mediator at the potential applied of 300 mV vs Ag/AgCl. Lactate and alcohol dehydrogenases were immobilized near to the electrode surface or in a reactor to obtain an NADH-based biosensor for lactate or ethanol. With lactate the authors succeeded to obtain a response only if the reactor was used and for alcohol a current proportional to the concentration was obtained either if the enzyme was immobilized in a membrane and placed near the electrode surface or when the enzyme was immobilized in a reactor form. By FIA procedures fast responses and recoveries were obtained, but with a short linear range.

  14. Crystal structure of Arabidopsis thaliana cytokinin dehydrogenase

    SciTech Connect

    Bae, Euiyoung; Bingman, Craig A.; Bitto, Eduard; Aceti, David J.; Phillips, Jr., George N.

    2008-08-13

    Since first discovered in Zea mays, cytokinin dehydrogenase (CKX) genes have been identified in many plants including rice and Arabidopsis thaliana, which possesses CKX homologues (AtCKX1-AtCKX7). So far, the three-dimensional structure of only Z. mays CKX (ZmCKX1) has been determined. The crystal structures of ZmCKX1 have been solved in the native state and in complex with reaction products and a slowly reacting substrate. The structures revealed four glycosylated asparagine residues and a histidine residue covalently linked to FAD. Combined with the structural information, recent biochemical analyses of ZmCKX1 concluded that the final products of the reaction, adenine and a side chain aldehyde, are formed by nonenzymatic hydrolytic cleavage of cytokinin imine products resulting directly from CKX catalysis. Here, we report the crystal structure of AtCKX7 (gene locus At5g21482.1, UniProt code Q9FUJ1).

  15. Fast internal dynamics in alcohol dehydrogenase

    SciTech Connect

    Monkenbusch, M.; Stadler, A. Biehl, R.; Richter, D.; Ollivier, J.; Zamponi, M.

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D{sub 2}O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

  16. Betaine aldehyde dehydrogenase isozymes of spinach

    SciTech Connect

    Hanson, A.D.; Weretilnyk, E.A.; Weigel, P.

    1986-04-01

    Betaine is synthesized in spinach chloroplasts via the pathway Choline ..-->.. Betaine Aldehyde ..-->.. Betaine; the second step is catalyzed by betaine aldehyde dehydrogenase (BADH). The subcellular distribution of BADH was determined in leaf protoplast lysates; BADH isozymes were separated by 6-9% native PAGE. The chloroplast stromal fraction contains a single BADH isozyme (number1) that accounts for > 80% of the total protoplast activity; the extrachloroplastic fraction has a minor isozyme (number2) which migrates more slowly than number1. Both isozymes appear specific for betaine aldehyde, are more active with NAD than NADP, and show a ca. 3-fold activity increase in salinized leaves. The phenotype of a natural variant of isozyme number1 suggests that the enzyme is a dimer.

  17. Structure-Function Relationships in Lactate Dehydrogenase

    PubMed Central

    Adams, Margaret J.; Buehner, Manfred; Chandrasekhar, K.; Ford, Geoffrey C.; Hackert, Marvin L.; Liljas, Anders; Rossmann, Michael G.; Smiley, Ira E.; Allison, William S.; Everse, Johannes; Kaplan, Nathan O.; Taylor, Susan S.

    1973-01-01

    The binding of coenzyme and substrate are considered in relation to the known primary and tertiary structure of lactate dehydrogenase (EC 1.1.1.27). The adenine binds in a hydrophobic crevice, and the two coenzyme phosphates are oriented by interactions with the protein. The positively charged guanidinium group of arginine 101 then folds over the negatively charged phosphates, collapsing the loop region over the active center and positioning the unreactive B side of the nicotinamide in a hydrophobic protein environment. Collapse of the loop also introduces various charged groups into the vicinity of the substrate binding site. The substrate is situated between histidine 195 and the C4 position on the nicotinamide ring, and is partially oriented by interactions between its carboxyl group and arginine 171. The spatial arrangements of these groups may provide the specificity for the L-isomer of lactate. PMID:4146647

  18. Molybdenum and tungsten-dependent formate dehydrogenases.

    PubMed

    Maia, Luisa B; Moura, José J G; Moura, Isabel

    2015-03-01

    The prokaryotic formate metabolism is considerably diversified. Prokaryotes use formate in the C1 metabolism, but also evolved to exploit the low reduction potential of formate to derive energy, by coupling its oxidation to the reduction of numerous electron acceptors. To fulfil these varied physiological roles, different types of formate dehydrogenase (FDH) enzymes have evolved to catalyse the reversible 2-electron oxidation of formate to carbon dioxide. This review will highlight our present knowledge about the diverse physiological roles of FDH in prokaryotes, their modular structural organisation and active site structures and the mechanistic strategies followed to accomplish the formate oxidation. In addition, the ability of FDH to catalyse the reverse reaction of carbon dioxide reduction, a potentially relevant reaction for carbon dioxide sequestration, will also be addressed.

  19. Cloning and sequencing of the gene encoding the 72-kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti.

    PubMed

    Inoue, T; Sunagawa, M; Mori, A; Imai, C; Fukuda, M; Takagi, M; Yano, K

    1989-06-01

    A genomic library of Acetobacter aceti DNA was constructed by using a broad-host-range cosmid vector. Complementation of a spontaneous alcohol dehydrogenase-deficient mutant resulted in the isolation of a plasmid designated pAA701. Subcloning and deletion analysis of pAA701 limited the region that complemented the deficiency in alcohol dehydrogenase activity of the mutant. The nucleotide sequence of this region was determined and showed that this region contained the full structural gene for the 72-kilodalton dehydrogenase subunit of the alcohol dehydrogenase enzyme complex. The predicted amino acid sequence of the gene showed homology with sequences of methanol dehydrogenase structural genes of Paracoccus denitrificans and Methylobacterium organophilum.

  20. Expression of rat liver Na+/L-alanine co-transport in Xenopus laevis oocytes. Effect of glucagon in vivo.

    PubMed Central

    Palacin, M; Werner, A; Dittmer, J; Murer, H; Biber, J

    1990-01-01

    Poly(A)+ RNA (mRNA) isolated from rat liver was injected into Xenopus laevis oocytes, and expression of Na+/L-alanine transport was assayed by measuring Na(+)-dependent uptake of L-[3H]alanine. Expression of Na+/L-alanine transport was detected 3-7 days after mRNA injection, and was due to an increment of the Na(+)-dependent component. After injection of 40 ng of total mRNA, Na(+)-dependent uptake of L-alanine was 2.5-fold higher than in water-injected oocytes. In contrast with Na+/L-alanine transport by water-injected oocytes, expressed Na+/L-alanine transport was inhibited by N-methylaminoisobutyric acid, was inhibited by an extracellular pH of 6.5 and was saturated at approx. 1 mM-L-alanine. After sucrose-density-gradient fractionation, highest expression of Na+/L-alanine uptake was observed with mRNA of 1.9-2.5 kb in length. Compared with mRNA isolated from control rats, mRNA isolated from glucagon-treated rats showed a approx. 2-fold higher expression of Na+/L-alanine transport. The results demonstrate that both liver Na+/L-alanine transport systems (A and ASC) can be expressed in X. laevis oocytes. Furthermore, the data obtained with mRNA isolated from glucagon-treated rats suggest that glucagon regulates liver Na+/L-alanine transport (at least in part) via the availability of the corresponding mRNA. Images Fig. 6. PMID:2396979

  1. Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci.

    PubMed

    Pavlova, Sylvia I; Jin, Ling; Gasparovich, Stephen R; Tao, Lin

    2013-07-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci.

  2. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes.

  3. Pyruvate Dehydrogenase Complex from Chloroplasts of Pisum sativum L 1

    PubMed Central

    Williams, Michael; Randall, Douglas D.

    1979-01-01

    Pyruvate dehydrogenase complex is associated with intact chloroplasts and mitochondria of 9-day-old Pisum sativum L. seedlings. The ratio of the mitochondrial complex to the chloroplast complex activities is about 3 to 1. Maximal rates observed for chloroplast pyruvate dehydrogenase complex activity ranged from 6 to 9 micromoles of NADH produced per milligram of chlorophyll per hour. Osmotic rupture of pea chloroplasts released 88% of the complex activity, indicating that chloroplast pyruvate dehydrogenase complex is a stromal complex. The pH optimum for chloroplast pyruvate dehydrogenase complex was between 7.8 and 8.2, whereas the mitochondrial pyruvate dehydrogenase complex had a pH optimum between 7.3 and 7.7. Chloroplast pyruvate dehydrogenase complex activity was specific for pyruvate, dependent upon coenzyme A and NAD and partially dependent upon Mg2+ and thiamine pyrophosphate. Chloroplast-associated pyruvate dehydrogenase complex provides a direct link between pyruvate metabolism and chloroplast fatty acid biosynthesis by providing the substrate, acetyl-CoA, necessary for membrane development in young plants. Images PMID:16661100

  4. Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids.

    PubMed

    Reid, E E; Thompson, P; Lyttle, C R; Dennis, D T

    1977-05-01

    The pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria was purified 23-fold by high speed centrifugation and glycerol gradient fractionation. The complex had a s(20,w) of 47.5S but this is a minimal value since the complex is unstable. The complex is specific for NAD(+) and pyruvate; NADP(+) and other keto acids give no reaction. Mg(2+), thiamine pyrophosphate, and cysteine are also required for maximal activity. The pH optimum for the complex was between 6.5 and 7.5.Continuous sucrose density gradients were used to separate castor bean (Ricinus communis L.) endosperm proplastids from mitochondria. Pyruvate dehydrogenase complex activity was found to be coincident with the proplastid peak on all of the gradients. Some separation of proplastids and mitochondria could be achieved by differential centrifugation and the ratios of the activities of the pyruvate dehydrogenase complex to succinic dehydrogenase and acetyl-CoA carboxylase to succinic dehydrogenase were consistent with both the pyruvate dehydrogenase complex and acetyl-CoA carboxylase being present in the proplastid. The proplastid fraction has to be treated with a detergent, Triton X-100, before maximal activity of the pyruvate dehydrogenase complex activity is expressed, indicating that it is bound in the organelle. The complex had a sharp pH optimum of 7.5. The complex required added Mg(2+), cysteine, and thiamine pyrophosphate for maximal activity but thiamine pyrophosphate was inhibitory at higher concentrations.

  5. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. PMID:25542170

  6. Probing the interaction of the amino acid alanine with the surface of ZnO(1010).

    PubMed

    Gao, Y K; Traeger, F; Shekhah, O; Idriss, H; Wöll, C

    2009-10-01

    The adsorption modes and stability of the amino acid alanine (NH(2)-CH(CH(3))-COOH) have been studied on the nonpolar single crystal surface of zinc oxide, ZnO(1010), experimentally by X-ray photoelectron spectroscopy (XPS) and computationally using density functional theory (DFT). Deposition at 200 K was found to lead to the formation of multilayers identified by an XPS N1s peak at 401.7 eV assigned to the NH(3)(+) group, a fingerprint of the zwitterionic structure of alanine in the solid state. Heating to 300 K resulted in the removal of most of the multilayers with the remaining surface coverage estimated to 0.4 with respect to Zn cations. At this temperature most of the alanine molecules are found to be deprotonated (dissociated), yielding a carboxylate species (NH(2)-CH(CH(3))-COO(-) (a) + OH (s); where O is surface oxygen, (a) for adsorbed and (s) for surface species). Further heating of the surface resulted in a gradual decrease of the surface coverage and by 500 K a large fraction of adsorbed alanine molecules have desorbed from the surface. Total energy DFT computations of different adsorbate species identified two stable dissociative adsorption modes: bidentate and monodentate. The bidentate species with adsorption energy of 1.75 eV was found to be more stable than the monodentate species by about 0.7 eV.

  7. AlaScan: A Graphical User Interface for Alanine Scanning Free-Energy Calculations.

    PubMed

    Ramadoss, Vijayaraj; Dehez, François; Chipot, Christophe

    2016-06-27

    Computation of the free-energy changes that underlie molecular recognition and association has gained significant importance due to its considerable potential in drug discovery. The massive increase of computational power in recent years substantiates the application of more accurate theoretical methods for the calculation of binding free energies. The impact of such advances is the application of parent approaches, like computational alanine scanning, to investigate in silico the effect of amino-acid replacement in protein-ligand and protein-protein complexes, or probe the thermostability of individual proteins. Because human effort represents a significant cost that precludes the routine use of this form of free-energy calculations, minimizing manual intervention constitutes a stringent prerequisite for any such systematic computation. With this objective in mind, we propose a new plug-in, referred to as AlaScan, developed within the popular visualization program VMD to automate the major steps in alanine-scanning calculations, employing free-energy perturbation as implemented in the widely used molecular dynamics code NAMD. The AlaScan plug-in can be utilized upstream, to prepare input files for selected alanine mutations. It can also be utilized downstream to perform the analysis of different alanine-scanning calculations and to report the free-energy estimates in a user-friendly graphical user interface, allowing favorable mutations to be identified at a glance. The plug-in also assists the end-user in assessing the reliability of the calculation through rapid visual inspection.

  8. Partial enzymatic elimination and quantification of sarcosine from alanine using liquid chromatography-tandem mass spectrometry.

    PubMed

    Burton, Casey; Gamagedara, Sanjeewa; Ma, Yinfa

    2013-04-01

    Since sarcosine and D,L-alanine co-elute on reversed-phase high-performance liquid chromatography (HPLC) columns and the tandem mass spectrometer cannot differentiate them due to equivalent parent and fragment ions, derivatization is often required for analysis of sarcosine in LC/MS systems. This study offers an alternative to derivatization by employing partial elimination of sarcosine by enzymatic oxidation. The decrease in apparent concentration from the traditionally merged sarcosine-alanine peak associated with the enzymatic elimination has been shown to be proportional to the total sarcosine present (R(2) = 0.9999), allowing for determinations of urinary sarcosine. Sarcosine oxidase was shown to eliminate only sarcosine in the presence of D,L-alanine, and was consequently used as the selective enzyme. This newly developed technique has a method detection limit of 1 μg/L (parts per billion) with a linear range of 3 ppb-1 mg/L (parts per million) in urine matrices. The method was further validated through spiked recoveries of real urine samples, as well as the analysis of 35 real urine samples. The average recoveries for low, middle, and high sarcosine concentration spikes were 111.7, 90.8, and 90.1 %, respectively. In conclusion, this simple enzymatic approach coupled with HPLC/MS/MS is able to resolve sarcosine from D,L-alanine leading to underivatized quantification of sarcosine.

  9. EPR study of light illumination effects on radicals in gamma-irradiated ?-alanine

    NASA Astrophysics Data System (ADS)

    Ciesielski, B.; Schultka, K.; Penkowski, M.; Sagstuen, E.

    2004-05-01

    Exposure of γ-irradiated L-alanine samples to sunlight and to light from a regular, fluorescent lamp resulted in significant changes in their EPR resonance patterns, both to spectral shapes and intensities. The experimental EPR spectra were numerically decomposed into three components reflecting contributions of three different radicals (R1-R3) generated by ionizing radiation in alanine. The light exposure caused a decay of the measured EPR signal intensity. For similar light intensities and exposure times the decay was much more pronounced in samples illuminated by sunlight than in samples illuminated by the fluorescent lamp. In both cases light-induced decay of R1 radicals was observed. Sunlight illumination resulted in a moderate decay of R2 radicals and in a doubling of the R3 radical population. On the other hand, fluorescent light caused a significant increase of R2 radicals and did not change the amount of R3 radicals. A quantitative analysis of the variations of the three radical contributions to the total EPR spectra upon fluorescent light exposure suggests a net R1→R2 free radical transformation. These effects of light on the alanine dosimetric signal should be taken into account in dosimetry protocols, assuring protection of alanine dosimeters from extended exposure to light.

  10. 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.

  11. High-velocity intermittent running: effects of beta-alanine supplementation.

    PubMed

    Smith-Ryan, Abbie E; Fukuda, David H; Stout, Jeffrey R; Kendall, Kristina L

    2012-10-01

    The use of β-alanine in sport is widespread. However, the effects across all sport activities are inconclusive. The purpose of this study was to evaluate the effects of β-alanine supplementation on high-intensity running performance and critical velocity (CV) and anaerobic running capacity (ARC). Fifty recreationally trained men were randomly assigned, in a double-blind fashion, to a β-alanine group (BA, 2 × 800 mg tablets, 3 times daily; CarnoSyn; n = 26) or placebo group (PL, 2 × 800 mg maltodextrin tablets, 3 times daily; n = 24). A graded exercise test (GXT) was performed to establish peak velocity (PV). Three high-speed runs to exhaustion were performed at 110, 100, and 90% of PV, with 15 minutes of rest between bouts. The distances achieved were plotted over the time to exhaustion (TTE). Linear regression was used to determine the slope (CV) and y-intercept (ARC) of these relationships to assess aerobic and anaerobic performances, respectively. There were no significant treatment effects (p > 0.05) on CV or ARC for either men or women. Additionally, no TTE effects were evident for bouts at 90-110%PV lasting 1.95-5.06 minutes. There seems to be no ergogenic effect of β-alanine supplementation on CV, ARC, or high-intensity running lasting approximately 2-5 minutes in either men or women in the current study.

  12. Uncertainties in alanine/ESR dosimetry at the Physikalisch-Technische Bundesanstalt.

    PubMed

    Anton, Mathias

    2006-11-01

    In radiation therapy, the effect of ionizing radiation is quantified in terms of the absorbed dose to water. Dosimetry with alanine and readout via electron spin resonance (ESR) is a method which is used as a secondary standard by several national metrology institutions. The advantages of the method are the good water-equivalence of the probes, their small size and the very weak dependence of the response on the radiation quality for MV x-rays and high-energy electrons used in radiation therapy. For radiation therapy, a small uncertainty of the applied dose is required. The present publication describes the determination of the uncertainty budget for the alanine/ESR dosimetry system of the Physikalisch-Technische Bundesanstalt (PTB), which relies on the use of a reference sample. A method is also presented which allows a reduction of the influence of fading or other changes of the ESR amplitude of irradiated alanine probes with time. If certain conditions are met which are described in detail, a relative uncertainty of less than 0.5% can be reached for probes irradiated with (60)Co in the 5-25 Gy dose range, including the uncertainty of the primary standard. First results for dose values between 2 Gy and 10 Gy are presented as well. From the high accuracy achievable with alanine dosimetry, we conclude that this method has great potential to solve measurement problems for modern methods of radiation therapy such as intensity modulated radiation therapy (IMRT) or tomotherapy.

  13. Mechanism of inactivation of alanine racemase by beta, beta, beta-trifluoroalanine

    SciTech Connect

    Faraci, W.S.; Walsh, C.T.

    1989-01-24

    The alanine racemases are a group of PLP-dependent bacterial enzymes that catalyze the racemization of alanine, providing D-alanine for cell wall synthesis. Inactivation of the alanine racemases from the Gram-negative organism Salmonella typhimurium and Gram-positive organism Bacillus stearothermophilus with beta, beta, beta-trifluoroalanine has been studied. The inactivation occurs with the same rate constant as that for formation of a broad 460-490-nm chromophore. Loss of two fluoride ions per mole of inactivated enzyme and retention of (1-/sup 14/C)trifluoroalanine label accompany inhibition, suggesting a monofluoro enzyme adduct. Partial denaturation (1 M guanidine) leads to rapid return of the initial 420-nm chromophore, followed by a slower (t1/2 approximately 30 min-1 h) loss of the fluoride ion and /sup 14/CO/sub 2/ release. At this point, reduction by NaB/sub 3/H/sub 4/ and tryptic digestion yield a single radiolabeled peptide. Purification and sequencing of the peptide reveals that lysine-38 is covalently attached to the PLP cofactor. A mechanism for enzyme inactivation by trifluoroalanine is proposed and contrasted with earlier results on monohaloalanines, in which nucleophilic attack of released aminoacrylate on the PLP aldimine leads to enzyme inactivation. For trifluoroalanine inactivation, nucleophilic attack of lysine-38 on the electrophilic beta-difluoro-alpha, beta-unsaturated imine provides an alternative mode of inhibition for these enzymes.

  14. Probing the interaction of the amino acid alanine with the surface of ZnO(1010).

    PubMed

    Gao, Y K; Traeger, F; Shekhah, O; Idriss, H; Wöll, C

    2009-10-01

    The adsorption modes and stability of the amino acid alanine (NH(2)-CH(CH(3))-COOH) have been studied on the nonpolar single crystal surface of zinc oxide, ZnO(1010), experimentally by X-ray photoelectron spectroscopy (XPS) and computationally using density functional theory (DFT). Deposition at 200 K was found to lead to the formation of multilayers identified by an XPS N1s peak at 401.7 eV assigned to the NH(3)(+) group, a fingerprint of the zwitterionic structure of alanine in the solid state. Heating to 300 K resulted in the removal of most of the multilayers with the remaining surface coverage estimated to 0.4 with respect to Zn cations. At this temperature most of the alanine molecules are found to be deprotonated (dissociated), yielding a carboxylate species (NH(2)-CH(CH(3))-COO(-) (a) + OH (s); where O is surface oxygen, (a) for adsorbed and (s) for surface species). Further heating of the surface resulted in a gradual decrease of the surface coverage and by 500 K a large fraction of adsorbed alanine molecules have desorbed from the surface. Total energy DFT computations of different adsorbate species identified two stable dissociative adsorption modes: bidentate and monodentate. The bidentate species with adsorption energy of 1.75 eV was found to be more stable than the monodentate species by about 0.7 eV. PMID:19596338

  15. 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.

  16. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  17. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  18. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  19. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  20. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  1. Cadmium inhibition of L-alanine transport into renal brush border membrane vesicles isolated from the winter flounder (Pseudopleuronectes americanus)

    SciTech Connect

    Bevan, C.; Kinne-Saffran, E.; Foulkes, E.C.; Kinne, R.K. )

    1989-12-01

    Using isolated brush border membrane vesicles from the kidney of the winter flounder (Pseudopleuronectes americanus), we have studied the effect of cadmium on L-alanine transport. Pretreatment of vesicles with 0.1 mM Cd{sup 2+} resulted in inhibition of L-alanine uptake in the presence of a NaCl (but not KCl) gradient. Inhibition was due to a specific interaction with the sodium-alanine cotransport system and not a change in the driving forces for alanine transport, since Cd{sup 2+} did not affect sodium-dependent D-glucose uptake. The effect of Cd{sup 2+} on Na{sup +}-alanine cotransport showed mixed-type inhibition which is only partially reversible by EDTA. Cd{sup 2+} uptake itself was shown to be time and temperature dependent, resulting in binding to both sides of the membrane. No direct correlation was possible between inhibition of L-alanine transport and the amount of Cd{sup 2+} taken up by the membranes. Nevertheless, the striking time dependence of the effect of Cd{sup 2+} on sodium-dependent L-alanine uptake and the inability of EDTA to reverse the inhibitory action of Cd{sup 2+} suggest that Cd{sup 2+} inhibits Na+-alanine cotransport at the cytoplasmic side of the membrane.

  2. Knockout of the alanine racemase gene in Aeromonas hydrophila HBNUAh01 results in cell wall damage and enhanced membrane permeability.

    PubMed

    Liu, Dong; Zhang, Lu; Xue, Wen; Wang, Yaping; Ju, Jiansong; Zhao, Baohua

    2015-07-01

    This study focused on the alanine racemase gene (alr-2), which is involved in the synthesis of d-alanine that forms the backbone of the cell wall. A stable alr-2 knockout mutant of Aeromonas hydrophila HBNUAh01 was constructed. When the mutant was supplemented with d-alanine, growth was unaffected; deprivation of d-alanine caused the growth arrest of the starved mutant cells, but not cell lysis. No alanine racemase activity was detected in the culture of the mutant. Additionally, a membrane permeability assay showed increasing damage to the cell wall during d-alanine starvation. No such damage was observed in the wild type during culture. Scanning and transmission electron microscopy analyses revealed deficiencies of the cell envelope and perforation of the cell wall. Leakage of UV-absorbing substances from the mutants was also observed. Thus, the partial viability of the mutants and their independence of d-alanine for growth indicated that inactivation of alr-2 does not impose an auxotrophic requirement for d-alanine.

  3. Biospecific affinity chromatographic purification of octopine dehydrogenase from molluscs.

    PubMed

    Mulcahy, P; Griffin, T; O'Carra, P

    1997-02-01

    The development of a biospecific affinity chromatographic method for the purification of octopine dehydrogenase from molluscs is described. The method utilizes immobilized NAD+ derivatives in conjunction with soluble specific substrates to promote binding. Using this method, octopine dehydrogenase has been purified to electrophoretic homogeneity in a single chromatographic step from three different marine invertebrate sources [the queen scallop, Chlamys opercularis (adductor muscle), the great scallop, Pecten maximus (adductor muscle), and the squid Loligo vulgaris (mantle muscle)]. However, the system is not applicable to the purification of octopine dehydrogenase from some other marine invertebrate sources investigated (the mussel Mytilus edulis and the topshell Monodonta lineata). PMID:9116492

  4. Role of quinate dehydrogenase in quinic acid metabolism in conifers

    SciTech Connect

    Osipov, V.I.; Shein, I.V.

    1986-08-10

    Quinate dehydrogenase was isolated from young needles of the Siberian larch and partially purified by ammonium sulfate fractionation. It was found that in conifers, in contrast to other plants, quinate dehydrogenase is active both with NAD and with NADP. The values of K/sub m/ for quinate and NADP were 1.8 and 0.18 mM. The enzyme exhibits maximum activity at pH 9.0. It was assumed that NADP-dependent quinate dehydrogenase is responsible for quinic acid synthesis. The special features of the organization and regulation of the initial stages of the shikimate pathway in conifers are discussed.

  5. Ornithine cyclodeaminase/μ-crystallin homolog from the hyperthermophilic archaeon Thermococcus litoralis functions as a novel Δ1-pyrroline-2-carboxylate reductase involved in putative trans-3-hydroxy-l-proline metabolism

    PubMed Central

    Watanabe, Seiya; Tozawa, Yuzuru; Watanabe, Yasuo

    2014-01-01

    l-Ornithine cyclodeaminase (OCD) is involved in l-proline biosynthesis and catalyzes the unique deaminating cyclization of l-ornithine to l-proline via a Δ1-pyrroline-2-carboxyrate (Pyr2C) intermediate. Although this pathway functions in only a few bacteria, many archaea possess OCD-like genes (proteins), among which only AF1665 protein (gene) from Archaeoglobus fulgidus has been characterized as an NAD+-dependent l-alanine dehydrogenase (AfAlaDH). However, the physiological role of OCD-like proteins from archaea has been unclear. Recently, we revealed that Pyr2C reductase, involved in trans-3-hydroxy-l-proline (T3LHyp) metabolism of bacteria, belongs to the OCD protein superfamily and catalyzes only the reduction of Pyr2C to l-proline (no OCD activity) [FEBS Open Bio (2014) 4, 240–250]. In this study, based on bioinformatics analysis, we assumed that the OCD-like gene from Thermococcus litoralis DSM 5473 is related to T3LHyp and/or proline metabolism (TlLhpI). Interestingly, TlLhpI showed three different enzymatic activities: AlaDH; N-methyl-l-alanine dehydrogenase; Pyr2C reductase. Kinetic analysis suggested strongly that Pyr2C is the preferred substrate. In spite of their similar activity, TlLhpI had a poor phylogenetic relationship to the bacterial and mammalian reductases for Pyr2C and formed a close but distinct subfamily to AfAlaDH, indicating convergent evolution. Introduction of several specific amino acid residues for OCD and/or AfAlaDH by site-directed mutagenesis had marked effects on both AlaDH and Pyr2C reductase activities. The OCC_00387 gene, clustered with the TlLhpI gene on the genome, encoded T3LHyp dehydratase, homologous to the bacterial and mammalian enzymes. To our knowledge, this is the first report of T3LHyp metabolism from archaea. PMID:25161870

  6. Glutamate Racemase Is the Primary Target of β-Chloro-d-Alanine in Mycobacterium tuberculosis

    PubMed Central

    Rodenburg, Anne; Khoury, Hania; de Chiara, Cesira; Howell, Steve; Snijders, Ambrosius P.

    2016-01-01

    The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except in Mycobacterium tuberculosis, where it has so far been underexploited. d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within the d-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies of M. tuberculosis. During our studies on alternative inhibitors of the d-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro–d-alanine (BCDA) is a very poor inhibitor of recombinant M. tuberculosis Alr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit the d-alanine pathway in intact cells, consistent with its poor in vitro activity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI in M. tuberculosis and thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development. PMID:27480853

  7. Persistent GABAA/C responses to gabazine, taurine and beta-alanine in rat hypoglossal motoneurons.

    PubMed

    Chesnoy-Marchais, D

    2016-08-25

    In hypoglossal motoneurons, a sustained anionic current, sensitive to a blocker of ρ-containing GABA receptors, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) and insensitive to bicuculline, was previously shown to be activated by gabazine. In order to better characterize the receptors involved, the sensitivity of this atypical response to pentobarbital (30μM), allopregnanolone (0.3μM) and midazolam (0.5μM) was first investigated. Pentobarbital potentiated the response, whereas the steroid and the benzodiazepine were ineffective. The results indicate the involvement of hybrid heteromeric receptors, including at least a GABA receptor ρ subunit and a γ subunit, accounting for the pentobarbital-sensitivity. The effects of the endogenous β amino acids, taurine and β-alanine, which are released under various pathological conditions and show neuroprotective properties, were then studied. In the presence of the glycine receptor blocker strychnine (1μM), both taurine (0.3-1mM) and β-alanine (0.3mM) activated sustained anionic currents, which were partly blocked by TPMPA (100μM). Thus, both β amino acids activated ρ-containing GABA receptors in hypoglossal motoneurons. Bicuculline (20μM) reduced responses to taurine and β-alanine, but small sustained responses persisted in the presence of both strychnine and bicuculline. Responses to β-alanine were slightly increased by allopregnanolone, indicating a contribution of the bicuculline- and neurosteroid-sensitive GABAA receptors underlying tonic inhibition in these motoneurons. Since sustained activation of anionic channels inhibits most mature principal neurons, the ρ-containing GABA receptors permanently activated by taurine and β-alanine might contribute to some of their neuroprotective properties under damaging overexcitatory situations. PMID:27246441

  8. A preliminary optimization of alanine blends for ESR dosimetry in a mixed n-γ field: Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Hoseininaveh, M.; Ranjbar, A. H.

    2016-04-01

    In this study, a preliminary work on the enhancement of ESR response of several arrangements of alanine and boron compounds, exposed to a thermal neutron beam, is presented using FLUKA code. A multi-layer dosimeter consist of consecutive layers of alanine and boron compounds showed that the amount of energy deposited in the alanine layers is maximized when their thickness is 5 μm and the thickness of boron compound layers are between 2 and 3 μm. Furthermore, the optimum number of 10B layers in the dosimeter was found to be 35 layers. Moreover, the alanine samples consisting of small spherical grains of boron compounds, arranged regularly in the middle plane of the dosimeters, exposed to a thermal neutron beam, were modeled. The dependence of energy deposition in the alanine material on the size of grains, and on their composition were also studied, as well.

  9. Characterization of the metabolic effect of β-alanine on markers of oxidative metabolism and mitochondrial biogenesis in skeletal muscle

    PubMed Central

    Sunderland, Kyle L.; Kuennen, Matthew R.; Vaughan, Roger A.

    2016-01-01

    [Purpose] β-alanine is a common component of numerous sports supplements purported to improve athletic performance through enhanced carnosine biosynthesis and related intracellular buffering. To date, the effects of β-alanine on oxidative metabolism remain largely unexplored. This work investigated the effects of β-alanine on the expression of proteins which regulate cellular energetics. [Methods] C2C12 myocytes were cultured and differentiated under standard conditions followed by treatment with either β-alanine or isonitrogenous non-metabolizable control D-alanine at 800μM for 24 hours. Metabolic gene and protein expression were quantified by qRT-PCR and immunoblotting, respectively. Glucose uptake and oxygen consumption were measured via fluorescence using commercially available kits. [Results] β-alanine-treated myotubes displayed significantly elevated markers of improved oxidative metabolism including elevated peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and mitochondrial transcription factor a (TFAM) which led to increased mitochondrial content (evidenced by concurrent increases in cytochrome c content). Additionally, β-alanine-treated cells exhibited significantly increased oxygen consumption compared to control in a PPARβ/δ-dependent manner. β-alanine significantly enhanced expression of myocyte enhancer factor 2 (MEF-2) leading to increased glucose transporter 4 (GLUT4) content. [Conclusion] β-alanine appears to increase cellular oxygen consumption as well as the expression of several cellular proteins associated with improved oxidative metabolism, suggesting β-alanine supplementation may provide additional metabolic benefit (although these observations require in vivo experimental verification). PMID:27508152

  10. 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.

  11. ALDEHYDE DEHYDROGENASES EXPRESSION DURING POSTNATAL DEVELOPMENT: LIVER VS. LUNG

    EPA Science Inventory

    Aldehydes are highly reactive molecules present in the environment, and can be produced during biotransformation of xenobiotics. Although the lung can be a major target for aldehyde toxicity, development of aldehyde dehydrogenases (ALDHs), which detoxify aldehydes, in lung has be...

  12. A novel glutamate dehydrogenase from bovine brain: purification and characterization.

    PubMed

    Lee, J; Kim, S W; Cho, S W

    1995-08-01

    A soluble form of novel glutamate dehydrogenase has been purified from bovine brain. The preparation was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and composed of six identical subunits having a subunit size of 57,500 Da. The biochemical properties of glutamate dehydrogenase such as N-terminal amino acids sequences, kinetic parameters, amino acids analysis, and optimum pH were examined in both reductive amination of alpha-ketoglutarate and oxidative deamination of glutamate. N-terminal amino acid sequences of the bovine brain enzyme showed the significant differences in the first 5 amino acids compared to other glutamate dehydrogenases from various sources. These results indicate that glutamate dehydrogenase isolated from bovine brain is a novel polypeptide.

  13. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  14. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  15. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  16. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  17. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  18. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  19. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  20. Genetics Home Reference: 3-beta-hydroxysteroid dehydrogenase deficiency

    MedlinePlus

    ... not by hormone test. Clin Endocrinol (Oxf). 2003 Mar;58(3):323-31. Citation on PubMed Pang S, ... dehydrogenase deficiency. Endocrinol Metab Clin North Am. 2001 Mar;30(1):81-99, vi-vii. Review. Citation ...

  1. Mammalian class IV alcohol dehydrogenase (stomach alcohol dehydrogenase): structure, origin, and correlation with enzymology.

    PubMed Central

    Parés, X; Cederlund, E; Moreno, A; Hjelmqvist, L; Farrés, J; Jörnvall, H

    1994-01-01

    The structure of a mammalian class IV alcohol dehydrogenase has been determined by peptide analysis of the protein isolated from rat stomach. The structure indicates that the enzyme constitutes a separate alcohol dehydrogenase class, in agreement with the distinct enzymatic properties; the class IV enzyme is somewhat closer to class I (the "classical" liver alcohol dehydrogenase; approximately 68% residue identities) than to the other classes (II, III, and V; approximately 60% residue identities), suggesting that class IV might have originated through duplication of an early vertebrate class I gene. The activity of the class IV protein toward ethanol is even higher than that of the classical liver enzyme. Both Km and kcat values are high, the latter being the highest of any class characterized so far. Structurally, these properties are correlated with replacements at the active site, affecting both substrate and coenzyme binding. In particular, Ala-294 (instead of valine) results in increased space in the middle section of the substrate cleft, Gly-47 (instead of a basic residue) results in decreased charge interactions with the coenzyme pyrophosphate, and Tyr-363 (instead of a basic residue) may also affect coenzyme binding. In combination, these exchanges are compatible with a promotion of the off dissociation and an increased turnover rate. In contrast, residues at the inner part of the substrate cleft are bulky, accounting for low activity toward secondary alcohols and cyclohexanol. Exchanges at positions 259-261 involve minor shifts in glycine residues at a reverse turn in the coenzyme-binding fold. Clearly, class IV is distinct in structure, ethanol turnover, stomach expression, and possible emergence from class I. PMID:8127901

  2. Elusive transition state of alcohol dehydrogenase unveiled

    PubMed Central

    Roston, Daniel; Kohen, Amnon

    2010-01-01

    For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (> 1) 2° kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (< 1), this KIE eludes the traditional interpretation of 2° KIEs. It does, however, enable the development of a comprehensive model for the “tunneling ready state” (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate. PMID:20457944

  3. Optimization of adsorptive immobilization of alcohol dehydrogenases.

    PubMed

    Trivedi, Archana; Heinemann, Matthias; Spiess, Antje C; Daussmann, Thomas; Büchs, Jochen

    2005-04-01

    In this work, a systematic examination of various parameters of adsorptive immobilization of alcohol dehydrogenases (ADHs) on solid support is performed and the impact of these parameters on immobilization efficiency is studied. Depending on the source of the enzymes, these parameters differently influence the immobilization efficiency, expressed in terms of residual activity and protein loading. Residual activity of 79% was achieved with ADH from bakers' yeast (YADH) after optimizing the immobilization parameters. A step-wise drying process has been found to be more effective than one-step drying. A hypothesis of deactivation through bubble nucleation during drying of the enzyme/glass bead suspension at low drying pressure (<45 kPa) is experimentally verified. In the case of ADH from Lactobacillus brevis (LBADH), >300% residual activity was found after drying. Hyperactivation of the enzyme is probably caused by structural changes in the enzyme molecule during the drying process. ADH from Thermoanaerobacter species (ADH T) is found to be stable under drying conditions (>15 kPa) in contrast to LBADH and YADH.

  4. SAXS fingerprints of aldehyde dehydrogenase oligomers

    PubMed Central

    Tanner, John J.

    2015-01-01

    Enzymes of the aldehyde dehydrogenase (ALDH) superfamily catalyze the nicotinamide adenine dinucleotide-dependent oxidation of aldehydes to carboxylic acids. ALDHs are important in detoxification of aldehydes, amino acid metabolism, embryogenesis and development, neurotransmission, oxidative stress, and cancer. Mutations in genes encoding ALDHs cause metabolic disorders, including alcohol flush reaction (ALDH2), Sjögren–Larsson syndrome (ALDH3A2), hyperprolinemia type II (ALDH4A1), γ-hydroxybutyric aciduria (ALDH5A1), methylmalonic aciduria (ALDH6A1), pyridoxine dependent epilepsy (ALDH7A1), and hyperammonemia (ALDH18A1). We previously reported crystal structures and small-angle X-ray scattering (SAXS) analyses of ALDHs exhibiting dimeric, tetrameric, and hexameric oligomeric states (Luo et al., Biochemistry 54 (2015) 5513–5522; Luo et al., J. Mol. Biol. 425 (2013) 3106–3120). Herein I provide the SAXS curves, radii of gyration, and distance distribution functions for the three types of ALDH oligomer. The SAXS curves and associated analysis provide diagnostic fingerprints that allow rapid identification of the type of ALDH oligomer that is present in solution. The data sets provided here serve as a benchmark for characterizing oligomerization of ALDHs. PMID:26693506

  5. Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities

    PubMed Central

    Chen, Che-Hong; Ferreira, Julio Cesar Batista; Gross, Eric R.; Mochly-Rosen, Daria

    2014-01-01

    A family of detoxifying enzymes called aldehyde dehydrogenases (ALDHs) has been a subject of recent interest, as its role in detoxifying aldehydes that accumulate through metabolism and to which we are exposed from the environment has been elucidated. Although the human genome has 19 ALDH genes, one ALDH emerges as a particularly important enzyme in a variety of human pathologies. This ALDH, ALDH2, is located in the mitochondrial matrix with much known about its role in ethanol metabolism. Less known is a new body of research to be discussed in this review, suggesting that ALDH2 dysfunction may contribute to a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer. Recent studies suggest that ALDH2 dysfunction is also associated with Fanconi anemia, pain, osteoporosis, and the process of aging. Furthermore, an ALDH2 inactivating mutation (termed ALDH2*2) is the most common single point mutation in humans, and epidemiological studies suggest a correlation between this inactivating mutation and increased propensity for common human pathologies. These data together with studies in animal models and the use of new pharmacological tools that activate ALDH2 depict a new picture related to ALDH2 as a critical health-promoting enzyme. PMID:24382882

  6. Targeting isocitrate dehydrogenase (IDH) in cancer.

    PubMed

    Fujii, Takeo; Khawaja, Muhammad Rizwan; DiNardo, Courtney D; Atkins, Johnique T; Janku, Filip

    2016-05-01

    Isocitrate dehydrogenase (IDH) is an essential enzyme for cellular respiration in the tricarboxylic acid (TCA) cycle. Recurrent mutations in IDH1 or IDH2 are prevalent in several cancers including glioma, acute myeloid leukemia (AML), cholangiocarcinoma and chondrosarcoma. The mutated IDH1 and IDH2 proteins have a gain-of-function, neomorphic activity, catalyzing the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG) by NADPH. Cancer-associated IDH mutations block normal cellular differentiation and promote tumorigenesis via the abnormal production of the oncometabolite 2-HG. High levels of 2-HG have been shown to inhibit α-KG dependent dioxygenases, including histone and deoxyribonucleic acid (DNA) demethylases, which play a key role in regulating the epigenetic state of cells. Current targeted inhibitors of IDH1 (AG120, IDH305), IDH2 (AG221), and pan-IDH1/2 (AG881) selectively inhibit mutant IDH protein and induce cell differentiation in in vitro and in vivo models. Preliminary results from phase I clinical trials with IDH inhibitors in patients with advanced hematologic malignancies have demonstrated an objective response rate ranging from 31% to 40% with durable responses (>1 year) observed. Furthermore, the IDH inhibitors have demonstrated early signals of activity in solid tumors with IDH mutations, including cholangiocarcinomas and low grade gliomas. PMID:27355333

  7. SAXS fingerprints of aldehyde dehydrogenase oligomers.

    PubMed

    Tanner, John J

    2015-12-01

    Enzymes of the aldehyde dehydrogenase (ALDH) superfamily catalyze the nicotinamide adenine dinucleotide-dependent oxidation of aldehydes to carboxylic acids. ALDHs are important in detoxification of aldehydes, amino acid metabolism, embryogenesis and development, neurotransmission, oxidative stress, and cancer. Mutations in genes encoding ALDHs cause metabolic disorders, including alcohol flush reaction (ALDH2), Sjögren-Larsson syndrome (ALDH3A2), hyperprolinemia type II (ALDH4A1), γ-hydroxybutyric aciduria (ALDH5A1), methylmalonic aciduria (ALDH6A1), pyridoxine dependent epilepsy (ALDH7A1), and hyperammonemia (ALDH18A1). We previously reported crystal structures and small-angle X-ray scattering (SAXS) analyses of ALDHs exhibiting dimeric, tetrameric, and hexameric oligomeric states (Luo et al., Biochemistry 54 (2015) 5513-5522; Luo et al., J. Mol. Biol. 425 (2013) 3106-3120). Herein I provide the SAXS curves, radii of gyration, and distance distribution functions for the three types of ALDH oligomer. The SAXS curves and associated analysis provide diagnostic fingerprints that allow rapid identification of the type of ALDH oligomer that is present in solution. The data sets provided here serve as a benchmark for characterizing oligomerization of ALDHs. PMID:26693506

  8. Malic dehydrogenase locus of Paramecium tetraurelia.

    PubMed

    Williams, T J; Smith-Sonneborn, J

    1980-04-01

    A search was undertaken for naturally occurring genetic markers for use in clonal aging studies of Paramecium tetraurelia. Clonal age is defined as the number of cell divisions since the last sexual process. Autogamy (self-fertilization) is a sexual process which can occur in aging lines, resulting in homozygosity and initiation of the next generation. Such "illicit" autogamies must be detected and eliminated from the aged clone. With codominant alleles, heterozygous aging lines can be established which will express a phenotype distinguishable from that of either parental type and autogamy can then be monitored by the appearance of either segregant homozygous phenotype. However, very few codominant alleles are available in this species. Electrophoretic mobilities of malic dehydrogenase (MDH) were assayed in 11 stocks of Paramecium tetraurelia by polyacrylamide gel electrophoresis. Nine stocks showed a single-banded "stock 51" type, while stock 174 and stock 29 each exhibited unique mobility. Crosses between stock 51 and the deviant stocks revealed distinct three-banded patterns indicative of heterozygosity of the F1 generation. In the autogamous F2 generation, 1:1 segregation of the parental types were recovered. The pattern of inheritance is consistent with codominant alleles and Mendelian inheritance. These naturally occurring biochemical markers are stable with increasing clonal age and are therefore useful genetic markers for studies of cellular aging. PMID:6934772

  9. Lactic dehydrogenase and cancer: an overview.

    PubMed

    Gallo, Monica; Sapio, Luigi; Spina, Annamaria; Naviglio, Daniele; Calogero, Armando; Naviglio, Silvio

    2015-01-01

    Despite the intense scientific efforts made, there are still many tumors that are difficult to treat and the percentage of patient survival in the long-term is still too low. Thus, new approaches to the treatment of cancer are needed. Cancer is a highly heterogeneous and complex disease, whose development requires a reorganization of cell metabolism. Most tumor cells downregulate mitochondrial oxidative phosphorylation and increase the rate of glucose consumption and lactate release, independently of oxygen availability (Warburg effect). This metabolic rewiring is largely believed to favour tumor growth and survival, although the underlying molecular mechanisms are not completely understood. Importantly, the correlation between the aerobic glycolysis and cancer is widely regarded as a useful biochemical basis for the development of novel anticancer strategies. Among the enzymes involved in glycolysis, lactate dehydrogenase (LDH) is emerging as a very attractive target for possible pharmacological approaches in cancer therapy. This review addresses the state of the art and the perspectives concerning LDH both as a useful diagnostic marker and a relevant molecular target in cancer therapy and management.

  10. Succinate Dehydrogenase Loss in Familial Paraganglioma: Biochemistry, Genetics, and Epigenetics

    PubMed Central

    Her, Yeng F.; Maher, L. James

    2015-01-01

    It is counterintuitive that metabolic defects reducing ATP production can cause, rather than protect from, cancer. Yet this is precisely the case for familial paraganglioma, a form of neuroendocrine malignancy caused by loss of succinate dehydrogenase in the tricarboxylic acid cycle. Here we review biochemical, genetic, and epigenetic considerations in succinate dehydrogenase loss and present leading models and mysteries associated with this fascinating and important tumor. PMID:26294907

  11. Isotope labeling studies on the formation of multiple addition products of alanine in the pyrolysis residue of glucose/alanine mixtures by high-resolution ESI-TOF-MS.

    PubMed

    Chu, Fong Lam; Sleno, Lekha; Yaylayan, Varoujan A

    2011-11-01

    Pyrolysis was used as a microscale sample preparation tool to generate glucose/alanine reaction products to minimize the use of expensive labeled precursors in isotope labeling studies. The residue remaining after the pyrolysis at 250 °C was analyzed by electrospray time-of-flight mass spectrometry (ESI-TOF-MS). It was observed that a peak at m/z 199.1445 in the ESI-TOF-MS spectrum appeared only when the model system contained at least 2-fold excess alanine. The accurate mass determination indeed indicated the presence of two nitrogen atoms in the molecular formula (C(10)H(18)N(2)O(2)). To verify the origin of the carbon atoms in this unknown compound, model studies with [(13)U(6)]glucose, [(13)C-1]alanine, [(13)C-2]alanine, [(13)C-3]alanine, and [(15)N]alanine were also performed. Glucose furnished six carbon atoms, and alanine provides four carbon (2 × C-2 and 2 × C-3) and two nitrogen atoms. When commercially available fructosylalanine (N-attached to C-1) was reacted with only 1 mol of alanine, a peak at m/z 199.1445 was once again observed. In addition, when 3-deoxyglucosone (3-DG) was reacted with a 2-fold excess of alanine, a peak at m/z 199.1433 was also generated, confirming the points of attachment of the two amino acids at C-1 and C-2 atoms of 3-DG. These studies have indicated that amino acids can undergo multiple addition reactions with 1,2-dicarbonyl compounds such as 3-deoxyglucosone and eventually form a tetrahydropyrazine moiety.

  12. Synthesis and evaluation of 18F labeled alanine derivatives as potential tumor imaging agents

    PubMed Central

    Wang, Limin; Zha, Zhihao; Qu, Wenchao; Qiao, Hongwen; Lieberman, Brian P.; Plössl, Karl; Kung, Hank F.

    2012-01-01

    Introduction This paper reports the synthesis and labeling of 18F alanine derivatives. We also investigate their biological characteristics as potential tumor imaging agents mediated by alanine-serine-cysteine preferring (ASC) transporter system. Methods Three new 18F alanine derivatives were prepared from corresponding tosylate-precursors through a two-step labelling reaction. In vitro uptake studies to evaluate and to compare these three analogs were carried out in 9L glioma and PC-3 prostate cancer cell lines. Potential transport mechanisms, protein incorporation and stability of 3-(1-[18F]fluoromethyl)-L-alanine (L[18F]FMA) were investigated in 9L glioma cells. Its biodistribution was determined in a rat-bearing 9L tumor model. PET imaging studies were performed on rat bearing 9L glioma tumors and transgenic mouse carrying spontaneous generated M/tomND tumor (mammary gland adenocarcinoma). Results New 18F alanine derivatives were prepared with 7–34% uncorrected radiochemical yields, excellent enantiomeric purity (>99%) and good radiochemical purity (>99%). In vitro uptake of the L-[18F]FMA in 9L glioma and PC-3 prostate cancer cells was higher than those observed for other two alanine derivatives and [18F]FDG in first 1 h. Inhibition of cell uptake studies suggested that L-[18F]FMA uptake in 9L glioma was predominantly via transport system ASC. After entering into cells, L-[18F]FMA remained stable and was not incorporated into protein within 2 h. In vivo biodistribution studies demonstrated that L-[18F]FMA had relatively high uptake in liver and kidney. Tumor uptake was fast, reaching a maximum within 30 min. The tumor-to-muscle, tumor-to-blood and tumor-to-brain ratios at 60 min post injection were 2.2, 1.9 and 3.0, respectively. In PET imaging studies, tumors were visualized with L-[18F]FMA in both 9L rat and transgenic mouse. Conclusion L-[18F]FMA showed promising properties as a PET imaging agent for up-regulated ASC transporter associated with tumor

  13. The alanine detector in BNCT dosimetry: Dose response in thermal and epithermal neutron fields

    SciTech Connect

    Schmitz, T.; Bassler, N.; Blaickner, M.; Ziegner, M.; Hsiao, M. C.; Liu, Y. H.; Koivunoro, H.; Auterinen, I.; Serén, T.; Kotiluoto, P.; Palmans, H.; Sharpe, P.; Langguth, P.; Hampel, G.

    2015-01-15

    Purpose: The response of alanine solid state dosimeters to ionizing radiation strongly depends on particle type and energy. Due to nuclear interactions, neutron fields usually also consist of secondary particles such as photons and protons of diverse energies. Various experiments have been carried out in three different neutron beams to explore the alanine dose response behavior and to validate model predictions. Additionally, application in medical neutron fields for boron neutron capture therapy is discussed. Methods: Alanine detectors have been irradiated in the thermal neutron field of the research reactor TRIGA Mainz, Germany, in five experimental conditions, generating different secondary particle spectra. Further irradiations have been made in the epithermal neutron beams at the research reactors FiR 1 in Helsinki, Finland, and Tsing Hua open pool reactor in HsinChu, Taiwan ROC. Readout has been performed with electron spin resonance spectrometry with reference to an absorbed dose standard in a {sup 60}Co gamma ray beam. Absorbed doses and dose components have been calculated using the Monte Carlo codes FLUKA and MCNP. The relative effectiveness (RE), linking absorbed dose and detector response, has been calculated using the Hansen and Olsen alanine response model. Results: The measured dose response of the alanine detector in the different experiments has been evaluated and compared to model predictions. Therefore, a relative effectiveness has been calculated for each dose component, accounting for its dependence on particle type and energy. Agreement within 5% between model and measurement has been achieved for most irradiated detectors. Significant differences have been observed in response behavior between thermal and epithermal neutron fields, especially regarding dose composition and depth dose curves. The calculated dose components could be verified with the experimental results in the different primary and secondary particle fields. Conclusions: The

  14. Mutation in a D-alanine-D-alanine ligase of Azospirillum brasilense Cd results in an overproduction of exopolysaccharides and a decreased tolerance to saline stress.

    PubMed

    Jofré, Edgardo; Fischer, Sonia; Príncipe, Analía; Castro, Marina; Ferrari, Walter; Lagares, Antonio; Mori, Gladys

    2009-01-01

    Bacteria of the genus Azospirillum are free-living nitrogen-fixing, rhizobacteria that are found in close association with plant roots, where they exert beneficial effects on plant growth and yield in many crops of agronomic importance. Unlike other bacteria, little is known about the genetics and biochemistry of exopolysaccharides in Azospirillum brasilense. In an attempt to characterize genes associated with exopolysaccharides production, we generated an A. brasilense Cd Tn5 mutant that showed exopolysaccharides overproduction, decreased tolerance to saline conditions, altered cell morphology, and increased sensitivity to detergents. Genetic characterization showed that the Tn5 was inserted within a ddlB gene encoding for a d-alanine-d-alanine ligase, and located upstream of the ftsQAZ gene cluster responsible for cell division in different bacteria. Heterologous complementation of the ddlB Tn5 mutant restored the exopolysaccharides production to wild-type levels and the ability to grow in the presence of detergents, but not the morphology and growth characteristics of the wild-type bacteria, suggesting a polar effect of Tn5 on the fts genes. This result and the construction of a nonpolar ddlB mutant provide solid evidence of the presence of transcriptional coupling between a gene associated with peptidoglycan biosynthesis and the fts genes required to control cell division.

  15. The structure of the quinoprotein alcohol dehydrogenase of Acetobacter aceti modelled on that of methanol dehydrogenase from Methylobacterium extorquens.

    PubMed

    Cozier, G E; Giles, I G; Anthony, C

    1995-06-01

    The 1.94 A structure of methanol dehydrogenase has been used to provide a model structure for part of a membrane quinohaemoprotein alcohol dehydrogenase. The basic superbarrel structure and the active-site region are retained, indicating essentially similar mechanisms of action, but there are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site.

  16. The behaviour of alanine dosimeters at temperatures between 100 and 300 K

    NASA Astrophysics Data System (ADS)

    Sharpe, P. H. G.; Sephton, J. P.; Gouldstone, C. A.

    2009-07-01

    A cryostat has been constructed to enable irradiations in a MDS Nordion Gammacell 220 irradiator to be carried out at selected temperatures between 100 and 300 K. The principle of operation and the performance of this cryostat are described and results are given of a study into the behaviour of alanine dosimeters at cryogenic temperatures. This work extends previously published data to the region between solid CO 2 and liquid N 2 temperatures and has demonstrated complex dose-dependent behaviour. A sharp discontinuity in the effect of temperature on alanine dosimeter response has been found in the region between 150 and 180 K, with no further influence of irradiation temperature on response observed below this point.

  17. Crystallization and preliminary X-ray data analysis of β-alanine synthase from Drosophila melanogaster

    SciTech Connect

    Lundgren, Stina; Andersen, Birgit; Piškur, Jure; Dobritzsch, Doreen

    2007-10-01

    β-Alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine. Crystals of the recombinant enzyme from D. melanogaster belong to space group C2. Diffraction data to 3.3 Å resolution were collected and analyzed. β-Alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine, which represents the main clearance route for the widely used anticancer drug 5-fluorouracil. Crystals of the recombinant enzyme from Drosophila melanogaster, which is closely related to the human enzyme, were obtained by the hanging-drop vapour-diffusion method. They diffracted to 3.3 Å at a synchrotron-radiation source, belong to space group C2 (unit-cell parameters a = 278.9, b = 95.0, c = 199.3 Å, β = 125.8°) and contain 8–10 molecules per asymmetric unit.

  18. Chiral effects on helicity studied via the energy landscape of short (D, L)-alanine peptides.

    PubMed

    Neelamraju, Sridhar; Oakley, Mark T; Johnston, Roy L

    2015-10-28

    The homochirality of natural amino acids facilitates the formation of regular secondary structures such as α-helices and β-sheets. Here, we study the relationship between chirality and backbone structure for the example of hexa-alanine. The most stable stereoisomers are identified through global optimisation. Further, the energy landscape, a database of connected low-energy local minima and transition points, is constructed for various neutral and zwitterionic stereoisomers of hexa-alanine. Three order parameters for partial helicity are applied and metric disconnectivity graphs are presented with partial helicity as a metric. We also apply the Zimm-Bragg model to derive average partial helicities for Ace-(L-Ala)6-NHMe, Ace-(D-Ala-L-Ala)3-NHMe, and Ace-(L-Ala)3-(D-Ala)3-NHMe from the database of local minima and compare with previous studies.

  19. Chiral effects on helicity studied via the energy landscape of short (d, l)-alanine peptides

    NASA Astrophysics Data System (ADS)

    Neelamraju, Sridhar; Oakley, Mark T.; Johnston, Roy L.

    2015-10-01

    The homochirality of natural amino acids facilitates the formation of regular secondary structures such as α-helices and β-sheets. Here, we study the relationship between chirality and backbone structure for the example of hexa-alanine. The most stable stereoisomers are identified through global optimisation. Further, the energy landscape, a database of connected low-energy local minima and transition points, is constructed for various neutral and zwitterionic stereoisomers of hexa-alanine. Three order parameters for partial helicity are applied and metric disconnectivity graphs are presented with partial helicity as a metric. We also apply the Zimm-Bragg model to derive average partial helicities for Ace-(l-Ala)6-NHMe, Ace-(d-Ala-l-Ala)3-NHMe, and Ace-(l-Ala)3-(d-Ala)3-NHMe from the database of local minima and compare with previous studies.

  20. Different hydroxyl radical scavenging activity of water-soluble beta-alanine C60 adducts.

    PubMed

    Sun, Tao; Jia, Zhishen; Xu, Zhude

    2004-04-01

    Three C(60) derivatives [C(60) (NHCH(2)CH(2)COONa)(n)(H)(n)], n=1, 5, 9] (A, B, C) with different additional number of beta-alanine were synthesized by the control of relative amount of C(60) and beta-alanine added. Hydroxyl radical scavenging activity of the adducts was evaluated in a copper-catalyzed Haber-Weiss reaction by chemiluminescence technology. The 50% inhibition concentrations (IC(50)'s) of A, B, and C were 147.2 micromol/L, 76.3 micromol/L, and 96.2 micromol/L, respectively. The difference should be closely related to the numbers of residual C=C bonds in C(60), steric effect and electron-withstanding effect of amino group especially.

  1. Formation of homochiral glycine/Cu(111) quantum corral array realized using alanine nuclei

    NASA Astrophysics Data System (ADS)

    Nakamura, Miki; Huang, Hui; Kanazawa, Ken; Taninaka, Atsushi; Yoshida, Shoji; Takeuchi, Osamu; Shigekawa, Hidemi

    2015-08-01

    Glycine has enantiomeric isomers on a Cu(111) surface through the dissociation of hydrogen from the carboxyl group and forms an array of quantum corrals of ∼1.3 nm diameter. Stable homo-chiral glycinate trimers are formed in the first step, which subsequently form a network with a hexagonal arrangement. However, domains with R- or S-chirality coexist with the same probability. On the other hand, α-alanine has D- and L-chirality in nature and forms a similar quantum corral array on Cu(111) with R- and S-chirality, respectively. Here, by using α-alanine molecules as nuclei, the chirality of glycine molecules was controlled and a homochiral quantum corral array was successfully formed, which indicates the possibility that the optical isomers can be separated through a method such as preferential crystallization.

  2. Unusual hydroxyl migration in the fragmentation of β-alanine dication in the gas phase.

    PubMed

    Piekarski, Dariusz Grzegorz; Delaunay, Rudy; Maclot, Sylvain; Adoui, Lamri; Martín, Fernando; Alcamí, Manuel; Huber, Bernd A; Rousseau, Patrick; Domaracka, Alicja; Díaz-Tendero, Sergio

    2015-07-14

    We present a combined experimental and theoretical study of the fragmentation of doubly positively charged β-alanine molecules in the gas phase. The dissociation of the produced dicationic molecules, induced by low-energy ion collisions, is analysed by coincidence mass spectrometric techniques; the coupling with ab initio molecular dynamics simulations allows rationalisation of the experimental observations. The present strategy gives deeper insights into the chemical mechanisms of multiply charged amino acids in the gas phase. In the case of the β-alanine dication, in addition to the expected Coulomb explosion and hydrogen migration processes, we have found evidence of hydroxyl-group migration, which leads to unusual fragmentation products, such as hydroxymethyl cation, and is necessary to explain some of the observed dominant channels.

  3. Response of the alanine/ESR dosimeter to radiation from an Ir-192 HDR brachytherapy source.

    PubMed

    Anton, M; Hackel, T; Zink, K; von Voigts-Rhetz, P; Selbach, H-J

    2015-01-01

    The response of the alanine dosimeter to radiation from an Ir-192 source with respect to the absorbed dose to water, relative to Co-60 radiation, was determined experimentally as well as by Monte Carlo simulations. The experimental and Monte Carlo results for the response agree well within the limits of uncertainty. The relative response decreases with an increasing distance between the measurement volume and the source from approximately 98% at a 1 cm distance to 96% at 5 cm. The present data are more accurate, but agree well with data published by Schaeken et al (2011 Phys. Med. Biol. 56 6625-34). The decrease of the relative response with an increasing distance that had already been observed by these authors is confirmed. In the appendix, the properties of the alanine dosimeter with respect to volume and sensitivity corrections are investigated. The inhomogeneous distribution of the detection probability that was taken into account for the analysis was determined experimentally.

  4. Response of the alanine/ESR dosimeter to radiation from an Ir-192 HDR brachytherapy source

    NASA Astrophysics Data System (ADS)

    Anton, M.; Hackel, T.; Zink, K.; von Voigts-Rhetz, P.; Selbach, H.-J.

    2015-01-01

    The response of the alanine dosimeter to radiation from an Ir-192 source with respect to the absorbed dose to water, relative to Co-60 radiation, was determined experimentally as well as by Monte Carlo simulations. The experimental and Monte Carlo results for the response agree well within the limits of uncertainty. The relative response decreases with an increasing distance between the measurement volume and the source from approximately 98% at a 1 cm distance to 96% at 5 cm. The present data are more accurate, but agree well with data published by Schaeken et al (2011 Phys. Med. Biol. 56 6625-34). The decrease of the relative response with an increasing distance that had already been observed by these authors is confirmed. In the appendix, the properties of the alanine dosimeter with respect to volume and sensitivity corrections are investigated. The inhomogeneous distribution of the detection probability that was taken into account for the analysis was determined experimentally.

  5. L-alanine and inosine enhancement of glucose triggering in Bacillus megaterium spores.

    PubMed

    Bédard, J; Lefebvre, G M

    1989-08-01

    Both rate and extent of germination of Bacillus megaterium 14581 (ATCC) spores are considerably augmented when L-alanine and inosine are added to the glucose commonly used as triggering agent for this strain. This enhancement does not arise from heterogeneity in germination requirements of the dormant spore, but is rather a consequence of the combined action of glucose and either or both of the added reagents on a sizeable fraction of spores unable to germinate in glucose alone. Nearly half of the spores that eventually germinate in the mixture of germinants used are either triggered by glucose or are sensitized by it to subsequent triggering by L-alanine and inosine in the first 10 s of imbibition. For a good number of these spores, then, triggering consists of a sequence of separable events. PMID:2510916

  6. Adsorption of di-l-alanine on Cu(110) investigated with scanning tunneling microscopy [rapid communication

    NASA Astrophysics Data System (ADS)

    Stensgaard, I.

    2003-11-01

    Sub-monolayer growth of a small chiral peptide, di- L-alanine, on Cu(1 1 0) was investigated by variable temperature scanning tunneling microscopy (STM). At low coverage and for temperatures above ≈-220 K the molecules nucleate along the [ 3¯ 3 2] direction to form short, mainly one-dimensional islands. An increase in coverage leads to the formation of [ 3¯ 3 2]-directed, elongated islands. Images with sub-molecular resolution reveal that the orientation of the molecules within one particular island depends on the deposition temperature. At higher coverage, up to one monolayer, the islands coalesce, giving rise to phase boundaries between domains of opposite orientation. An atomic-scale model for di- L-alanine on Cu(1 1 0) is presented.

  7. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    SciTech Connect

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.; Kakar, S. N.; Stevens, F. J.; Donnelly, M. I.; Nebraska Wesleyan Univ.

    1995-09-10

    Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the native enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant

  8. Yeast Alcohol Dehydrogenase Structure and Catalysis

    PubMed Central

    2015-01-01

    Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1 is a homotetramer of subunits with 347 amino acid residues. A structure for ADH1 was determined by X-ray crystallography at 2.4 Å resolution. The asymmetric unit contains four different subunits, arranged as similar dimers named AB and CD. The unit cell contains two different tetramers made up of “back-to-back” dimers, AB:AB and CD:CD. The A and C subunits in each dimer are structurally similar, with a closed conformation, bound coenzyme, and the oxygen of 2,2,2-trifluoroethanol ligated to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. In contrast, the B and D subunits have an open conformation with no bound coenzyme, and the catalytic zinc has an alternative, inverted coordination with Cys-43, Cys-153, His-66, and the carboxylate of Glu-67. The asymmetry in the dimeric subunits of the tetramer provides two structures that appear to be relevant for the catalytic mechanism. The alternative coordination of the zinc may represent an intermediate in the mechanism of displacement of the zinc-bound water with alcohol or aldehyde substrates. Substitution of Glu-67 with Gln-67 decreases the catalytic efficiency by 100-fold. Previous studies of structural modeling, evolutionary relationships, substrate specificity, chemical modification, and site-directed mutagenesis are interpreted more fully with the three-dimensional structure. PMID:25157460

  9. Structural Studies of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand S.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Human pyruvate dehydrogenase (E1) catalyzes the irreversible decarboxylation of pyruvate in the presence of Mg(2+) and thiamin pyrophosphate (TPP) followed by the rate-limiting reductive acetylation of the lipoyl moiety linked to dihydrolipoamide acetyltransferase. The three-dimensional structure of human E1 is elucidated using the methods of macromolecular X-ray crystallography. The structure is an alpha, alpha', beta and beta' tetramer with the protein units being in the tetrahedral arrangement. Each 361-residue alpha-subunit and 329-residue beta-subunit is composed of a beta-sheet core surrounded by alpha-helical domains. Each subunit is in extensive contact with all the three subunits involving TPP and magnesium cofactors, and potassium ions. The two binding sites for TPP are at the alpha-beta' and alpha'-beta interfaces, each involving a magnesium ion and Phe6l, His63, Tyr89, and Met200 from the alpha-subunit (or alpha'-subunit), and Met81 Phe85, His128 from the beta-subunit (or beta'-subunit). K+ ions are nestled between two beta-sheets and the end of an alpha-helix in each beta-subunit, where they are coordinated by four carbonyl oxygen groups from Ile12, Ala160, Asp163, and Asnl65, and a water molecule. The catalytic C2 carbon of thiazolium ring in this structure forms a 3.2 A contact with a water molecule involved in a series of H-bonds with other water molecules, and indirectly with amino acids including those involved in the catalysis and regulation of the enzyme.

  10. β-alanine suppresses malignant breast epithelial cell aggressiveness through alterations in metabolism and cellular acidity in vitro

    PubMed Central

    2014-01-01

    Background Deregulated energetics is a property of most cancer cells. This phenomenon, known as the Warburg Effect or aerobic glycolysis, is characterized by increased glucose uptake, lactate export and extracellular acidification, even in the presence of oxygen. β-alanine is a non-essential amino acid that has previously been shown to be metabolized into carnosine, which functions as an intracellular buffer. Because of this buffering capacity, we investigated the effects of β-alanine on the metabolic cancerous phenotype. Methods Non-malignant MCF-10a and malignant MCF-7 breast epithelial cells were treated with β-alanine at 100 mM for 24 hours. Aerobic glycolysis was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate (OCR). mRNA of metabolism-related genes was quantified by qRT-PCR with corresponding protein expression quantified by immunoblotting, or by flow cytometry which was verified by confocal microscopy. Mitochondrial content was quantified using a mitochondria-specific dye and measured by flow cytometry. Results Cells treated with β-alanine displayed significantly suppressed basal and peak ECAR (aerobic glycolysis), with simultaneous increase in glucose transporter 1 (GLUT1). Additionally, cells treated with β-alanine exhibited significantly reduced basal and peak OCR (oxidative metabolism), which was accompanied by reduction in mitochondrial content with subsequent suppression of genes which promote mitochondrial biosynthesis. Suppression of glycolytic and oxidative metabolism by β-alanine resulted in the reduction of total metabolic rate, although cell viability was not affected. Because β-alanine treatment reduces extracellular acidity, a constituent of the invasive microenvironment that promotes progression, we investigated the effect of β-alanine on breast cell viability and migration. β-alanine was shown to reduce both cell migration and proliferation

  11. Photochemical redox reactions of copper(II)-alanine complexes in aqueous solutions.

    PubMed

    Lin, Chen-Jui; Hsu, Chao-Sheng; Wang, Po-Yen; Lin, Yi-Liang; Lo, Yu-Shiu; Wu, Chien-Hou

    2014-05-19

    The photochemical redox reactions of Cu(II)/alanine complexes have been studied in deaerated solutions over an extensive range of pH, Cu(II) concentration, and alanine concentration. Under irradiation, the ligand-to-metal charge transfer results in the reduction of Cu(II) to Cu(I) and the concomitant oxidation of alanine, which produces ammonia and acetaldehyde. Molar absorptivities and quantum yields of photoproducts for Cu(II)/alanine complexes at 313 nm are characterized mainly with the equilibrium Cu(II) speciation where the presence of simultaneously existing Cu(II) species is taken into account. By applying regression analysis, individual Cu(I) quantum yields are determined to be 0.094 ± 0.014 for the 1:1 complex (CuL) and 0.064 ± 0.012 for the 1:2 complex (CuL2). Individual quantum yields of ammonia are 0.055 ± 0.007 for CuL and 0.036 ± 0.005 for CuL2. Individual quantum yields of acetaldehyde are 0.030 ± 0.007 for CuL and 0.024 ± 0.007 for CuL2. CuL always has larger quantum yields than CuL2, which can be attributed to the Cu(II) stabilizing effect of the second ligand. For both CuL and CuL2, the individual quantum yields of Cu(I), ammonia, and acetaldehyde are in the ratio of 1.8:1:0.7. A reaction mechanism for the formation of the observed photoproducts is proposed.

  12. The influence of β-alanine supplementation on markers of exercise-induced oxidative stress.

    PubMed

    Smith-Ryan, Abbie E; Fukuda, David H; Stout, Jeffrey R; Kendall, Kristina L

    2014-01-01

    β-Alanine (BA) has been linked with oxidative protection. This study evaluated antioxidant properties of BA. Twenty-five men consumed BA or placebo for 4 weeks, and completed a 40-min run to induce oxidative stress. Blood draws were taken to measure 8-isoprostane, total antioxidant capacity, superoxide dismutase, and glutathione. BA had no significant influence on reducing exercise-induced oxidative stress. Confidence intervals suggest a reduction in lipid peroxidation. BA supplementation may have little influence as an antioxidant.

  13. Site-directed mutagenesis and /sup 1/H NMR spectroscopy of an interdomain segment in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

    SciTech Connect

    Texter, F.L.; Radford, S.E.; Laue, E.D.; Perham, R.N.; Miles, J.S.; Guest, J.R.

    1988-01-12

    Deletion of two of the three homologous lipoyl domains that form the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain of the Escherichia coli pyruvate dehydrogenase complex can be achieved by in vitro deletion in the structural gene aceF. A site-directed mutagenesis of this shortened aceF gene was carried out to replace the glutamine residue at position 291 (wild-type numbering) with a histidine residue. Residue 291 is near the middle of a long segment (about 30 amino acid residues) of polypeptide chain, rich in alanine, proline, and charged amino acids, that links the remaining lipoyl domain to the dihydrolipoamide dehydrogenase (E3) binding domain in the E2p chain. A fully active enzyme complex was still assembled, and despite the enormous size of the particle, sharp resonances attributable to the single new histidine residue per E2p chain could be detected in the 400-MHz /sup 1/H NMR spectrum of the complex. The sharpness of these resonances, their chemical shifts (7.94 and 7.05 ppm), and the apparent pK/sub a/ (6.4) of the side chain were all consistent with this histidine residue being exposed to solvent in a conformationally flexible region of the E2p polypeptide chain. These experiments provide direct proof for the conformational flexibility of this region of polypeptide chain, which is though to play an important part in the movement of the lipoyl domain required for active site coupling in the enzyme complex. The major sharp resonance (at l.39 ppm) in the 400-MHz /sup 1/H NMR spectrum of the mutated complex, was somewhat smaller than expected, suggesting that the insertion of the histidine residue at position 291 had diminished the flexibility of some at least of the alanine residues in this segment of polypeptide chain.

  14. Monte Carlo Simulation of the Irradiation of Alanine Coated Film Dosimeters with Accelerated Electrons

    NASA Astrophysics Data System (ADS)

    Uribe, R. M.; Salvat, F.; Cleland, M. R.; Berejka, A.

    2009-03-01

    The Monte Carlo code PENELOPE was used to simulate the irradiation of alanine coated film dosimeters with electron beams of energies from 1 to 5 MeV being produced by a high-current industrial electron accelerator. This code includes a geometry package that defines complex quadratic geometries, such as those of the irradiation of products in an irradiation processing facility. In the present case the energy deposited on a water film at the surface of a wood parallelepiped was calculated using the program PENMAIN, which is a generic main program included in the PENELOPE distribution package. The results from the simulation were then compared with measurements performed by irradiating alanine film dosimeters with electrons using a 150 kW Dynamitron™ electron accelerator. The alanine films were placed on top of a set of wooden planks using the same geometrical arrangement as the one used for the simulation. The way the results from the simulation can be correlated with the actual measurements, taking into account the irradiation parameters, is described. An estimation of the percentage difference between measurements and calculations is also presented.

  15. Characterisation of L-alanine and glycine absorption across the gut of an ancient vertebrate.

    PubMed

    Glover, Chris N; Bucking, Carol; Wood, Chris M

    2011-08-01

    This study utilised an in vitro technique to characterise absorption of two amino acids across the intestinal epithelium of Pacific hagfish, Eptatretus stoutii. Uptake of L-alanine and glycine conformed to Michaelis-Menten kinetics. An uptake affinity (K(m); substrate concentration required to attain a 50% uptake saturation) of 7.0 mM and an uptake capacity (J (max)) of 83 nmol cm(-2) h(-1) were described for L-alanine. The K(m) and J(max) for glycine were 2.2 mM and 11.9 nmol cm(-2) h(-1), respectively. Evidence suggested that the pathways of L-alanine and glycine absorption were shared, and sodium dependent. Further analysis indicated that glycine uptake was independent of luminal pH and proline, but a component of uptake was significantly impaired by 100-fold excesses of threonine or asparagine. The presence of a short-term (24 h) exposure to waterborne glycine, similar in nature to that which may be expected to occur when feeding inside an animal carcass, had no significant impact on gastrointestinal glycine uptake. This may indicate a lack of cross talk between absorptive epithelia. These results are the first published data to describe gastrointestinal uptake of an organic nutrient in the oldest extant vertebrate and may provide potential insight into the evolution of nutrient transport systems.

  16. Exogenous alanine and/or glucose plus kanamycin kills antibiotic-resistant bacteria.

    PubMed

    Peng, Bo; Su, Yu-Bin; Li, Hui; Han, Yi; Guo, Chang; Tian, Yao-Mei; Peng, Xuan-Xian

    2015-02-01

    Multidrug-resistant bacteria are an increasingly serious threat to human and animal health. However, novel drugs that can manage infections by multidrug-resistant bacteria have proved elusive. Here we show that glucose and alanine abundances are greatly suppressed in kanamycin-resistant Edwardsiella tarda by GC-MS-based metabolomics. Exogenous alanine or glucose restores susceptibility of multidrug-resistant E. tarda to killing by kanamycin, demonstrating an approach to killing multidrug-resistant bacteria. The mechanism underlying this approach is that exogenous glucose or alanine promotes the TCA cycle by substrate activation, which in turn increases production of NADH and proton motive force and stimulates uptake of antibiotic. Similar results are obtained with other Gram-negative bacteria (Vibrio parahaemolyticus, Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacterium (Staphylococcus aureus), and the results are also reproduced in a mouse model for urinary tract infection. This study establishes a functional metabolomics-based strategy to manage infection by antibiotic-resistant bacteria.

  17. Conformational study of N-methylated alanine peptides and design of Abeta inhibitor.

    PubMed

    Nandel, Fateh S; Jaswal, Radhika R

    2014-02-01

    N-Methylation increases the proteolytic stability of peptides and leads to improved pharmacological and increased nematicidal property against plant pathogens. In this study, the quantum mechanical and molecular dynamic simulation approaches were used to investigate conformational behavior of peptides containing only N-methylated alanine (NMeAla) residues and N-methylated alanine and alanine residues at alternate positions. The amide bond geometry was found to be trans and the poly NMeAla peptides were shown to populate in the helical structure without hydrogen bond with phi, psi values of - 0, 90 degrees stabilized by carbonyl-carbonyl interactions. Molecular dynamic simulations in water/methanol revealed the formation of beta-strand structure, irrespective of the starting geometry due to the interaction of solvent molecules with the carbonyl groups of peptide backbone. Analysis of simulation results as a function of time suggested that the opening of helical structure without hydrogen bond started from C-terminal. Conformational behavior of peptides containing N-MeAla and Ala was used to design Abeta peptide inhibitor and the model tetrapeptide Ac-Ala-NMeAla-Ala-NHMe in the beta-strand structure was shown to interact with the hydrophobic stretch of Abeta15-42 peptide.

  18. VUV photodynamics and chiral asymmetry in the photoionization of gas phase alanine enantiomers.

    PubMed

    Tia, Maurice; Cunha de Miranda, Barbara; Daly, Steven; Gaie-Levrel, François; Garcia, Gustavo A; Nahon, Laurent; Powis, Ivan

    2014-04-17

    The valence shell photoionization of the simplest proteinaceous chiral amino acid, alanine, is investigated over the vacuum ultraviolet region from its ionization threshold up to 18 eV. Tunable and variable polarization synchrotron radiation was coupled to a double imaging photoelectron/photoion coincidence (i(2)PEPICO) spectrometer to produce mass-selected threshold photoelectron spectra and derive the state-selected fragmentation channels. The photoelectron circular dichroism (PECD), an orbital-sensitive, conformer-dependent chiroptical effect, was also recorded at various photon energies and compared to continuum multiple scattering calculations. Two complementary vaporization methods-aerosol thermodesorption and a resistively heated sample oven coupled to an adiabatic expansion-were applied to promote pure enantiomers of alanine into the gas phase, yielding neutral alanine with different internal energy distributions. A comparison of the photoelectron spectroscopy, fragmentation, and dichroism measured for each of the vaporization methods was rationalized in terms of internal energy and conformer populations and supported by theoretical calculations. The analytical potential of the so-called PECD-PICO detection technique-where the electron spectroscopy and circular dichroism can be obtained as a function of mass and ion translational energy-is underlined and applied to characterize the origin of the various species found in the experimental mass spectra. Finally, the PECD findings are discussed within an astrochemical context, and possible implications regarding the origin of biomolecular asymmetry are identified.

  19. Monte Carlo Simulation of the Irradiation of Alanine Coated Film Dosimeters with Accelerated Electrons

    SciTech Connect

    Uribe, R. M.; Salvat, F.; Cleland, M. R.; Berejka, A.

    2009-03-10

    The Monte Carlo code PENELOPE was used to simulate the irradiation of alanine coated film dosimeters with electron beams of energies from 1 to 5 MeV being produced by a high-current industrial electron accelerator. This code includes a geometry package that defines complex quadratic geometries, such as those of the irradiation of products in an irradiation processing facility. In the present case the energy deposited on a water film at the surface of a wood parallelepiped was calculated using the program PENMAIN, which is a generic main program included in the PENELOPE distribution package. The results from the simulation were then compared with measurements performed by irradiating alanine film dosimeters with electrons using a 150 kW Dynamitron electron accelerator. The alanine films were placed on top of a set of wooden planks using the same geometrical arrangement as the one used for the simulation. The way the results from the simulation can be correlated with the actual measurements, taking into account the irradiation parameters, is described. An estimation of the percentage difference between measurements and calculations is also presented.

  20. Survivability and reactivity of glycine and alanine in early oceans: effects of meteorite impacts.

    PubMed

    Umeda, Yuhei; Fukunaga, Nao; Sekine, Toshimori; Furukawa, Yoshihiro; Kakegawa, Takeshi; Kobayashi, Takamichi; Nakazawa, Hiromoto

    2016-01-01

    Prebiotic oceans might have contained abundant amino acids, and were subjected to meteorite impacts, especially during the late heavy bombardment. It is so far unknown how meteorite impacts affected amino acids in the early oceans. Impact experiments were performed under the conditions where glycine was synthesized from carbon, ammonia, and water, using aqueous solutions containing (13)C-labeled glycine and alanine. Selected amino acids and amines in samples were analyzed with liquid chromatography-mass spectrometry (LC/MS). In particular, the (13)C-labeled reaction products were analyzed to distinguish between run products and contaminants. The results revealed that both amino acids survived partially in the early ocean through meteorite impacts, that part of glycine changed into alanine, and that large amounts of methylamine and ethylamine were formed. Fast decarboxylation was confirmed to occur during such impact processes. Furthermore, the formation of n-butylamine, detected only in the samples recovered from the solutions with additional nitrogen and carbon sources of ammonia and benzene, suggests that chemical reactions to form new biomolecules can proceed through marine impacts. Methylamine and ethylamine from glycine and alanine increased considerably in the presence of hematite rather than olivine under similar impact conditions. These results also suggest that amino acids present in early oceans can contribute further to impact-induced reactions, implying that impact energy plays a potential role in the prebiotic formation of various biomolecules, although the reactions are complicated and depend upon the chemical environments as well. PMID:26369758

  1. Survivability and reactivity of glycine and alanine in early oceans: effects of meteorite impacts.

    PubMed

    Umeda, Yuhei; Fukunaga, Nao; Sekine, Toshimori; Furukawa, Yoshihiro; Kakegawa, Takeshi; Kobayashi, Takamichi; Nakazawa, Hiromoto

    2016-01-01

    Prebiotic oceans might have contained abundant amino acids, and were subjected to meteorite impacts, especially during the late heavy bombardment. It is so far unknown how meteorite impacts affected amino acids in the early oceans. Impact experiments were performed under the conditions where glycine was synthesized from carbon, ammonia, and water, using aqueous solutions containing (13)C-labeled glycine and alanine. Selected amino acids and amines in samples were analyzed with liquid chromatography-mass spectrometry (LC/MS). In particular, the (13)C-labeled reaction products were analyzed to distinguish between run products and contaminants. The results revealed that both amino acids survived partially in the early ocean through meteorite impacts, that part of glycine changed into alanine, and that large amounts of methylamine and ethylamine were formed. Fast decarboxylation was confirmed to occur during such impact processes. Furthermore, the formation of n-butylamine, detected only in the samples recovered from the solutions with additional nitrogen and carbon sources of ammonia and benzene, suggests that chemical reactions to form new biomolecules can proceed through marine impacts. Methylamine and ethylamine from glycine and alanine increased considerably in the presence of hematite rather than olivine under similar impact conditions. These results also suggest that amino acids present in early oceans can contribute further to impact-induced reactions, implying that impact energy plays a potential role in the prebiotic formation of various biomolecules, although the reactions are complicated and depend upon the chemical environments as well.

  2. Disruption of NAD+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

    PubMed Central

    Phadke, Manali; Krynetskaia, Natalia; Mishra, Anurag; Barrero, Carlos; Merali, Salim; Gothe, Scott A; Krynetskiy, Evgeny

    2015-01-01

    AIM: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding. RESULTS: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+ (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION: Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners. PMID:26629320

  3. Characterization of the glutamate dehydrogenase activity of Gigantocotyle explanatum and Gastrothylax crumenifer (Trematoda: Digenea).

    PubMed

    Abidi, S M A; Khan, P; Saifullah, M K

    2009-12-01

    Glutamate dehydrogenase (GLDH) (EC 1.4.1.3) is a ubiquitous enzyme, which is present at the protein and carbohydrate metabolism crossroads. The enzyme activity was investigated in biliary and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer, respectively, infecting the Indian water buffalo Bubalus bubalis. The enzyme activity was consistently higher in G. explanatum as compared to G. crumenifer, where NAD(H) was utilized as coenzyme and the pH optima was recorded at 8. The K(m) and V(max) values for α-ketoglutarate were 2.1 mM and 9.09 units in G. explanatum, whereas 3.03 mM and 1.90 units in G. crumenifer, respectively. Among the allosteric modulator nucleotides, AMP, ADP, ATP, GMP, CMP and UMP, only AMP enhanced GLDH activity in G. crumenifer while ADP was stimulatory in G. explanatum. The amino acid leucine stimulated the GLDH activity in both the amphistomes while alanine was stimulatory only in G. crumenifer. Pronounced interspecific differences in response to different metabolic inhibitors like diethyldithiocarbamate, semicarbazide hydrochloride and mercurial ions were also observed. The osmotic stress alters the enzyme activity, particularly in hypertonic saline the GLDH activity increased significantly (p < 0.01) in G. explanatum, while insignificant effects were observed in rumen dwelling G. crumenifer. Histoenzymology revealed region/tissue specific distribution of GLDH with prominent staining in tissues like vitellaria, lymph system and tegument/subtegument, thus showing specific distribution of GLDH indicating differential metabolic state. Such intergeneric differences in GLDH activity could also be a consequence of occupying different microenvironments within the same host.

  4. Polymorphism in supramolecular chiral structures of R- and S-alanine on Cu(1 1 0)

    NASA Astrophysics Data System (ADS)

    Barlow, S. M.; Louafi, S.; Le Roux, D.; Williams, J.; Muryn, C.; Haq, S.; Raval, R.

    2005-10-01

    A comprehensive study of the local and supramolecular adsorption structures created by the chiral R- and S-enantiomers of alanine on the Cu(1 1 0) surface has been conducted using a multi-technique approach, including reflection absorption infrared spectroscopy (RAIRS), X-ray photoelectron spectroscopy (XPS), low energy electron diffraction (LEED) and scanning tunnelling microscopy (STM). Over the entire 300-470 K temperature range studied, the amino acid is found to adsorb as an alaninate species with a local chiral adsorption motif. However, this singular preference of local chemical form contrasts sharply with the supramolecular organisation at the surface where polymorphism is exhibited. This polymorphic behaviour arises from subtle and dynamic changes in the bonding, orientation and adsorption footprints of individual molecules, leading to alterations in the molecule-metal, intermolecular and metal-metal interactions that dictate self-assembly. Thus, at low coverage, a single disordered phase is observed but at higher coverage, three other temperature dependent phases occur. At room temperature, a two-dimensional equivalent of a 'nematic' phase is constructed from short single- and double-chain chiral assemblies that possess a preferred chiral orientation but no long range periodicity. This 'nematic' phase acts as a precursor to a highly ordered chiral supramolecular assembly, created at 430 K, that consists of regular arrays of size- and shape-defined chiral clusters. This phase possesses global organisational chirality with only one chiral domain observed for each enantiomer. For both the 'nematic' and the highly ordered chiral phase, the organisation for the R-enantiomer is the mirror image of that seen for the S-enantiomer, i.e., there is chirality transfer from the nanoscale to the macroscale. By 470 K, both R- and S-alanine form an achirally organised (3 × 2) structure that appears to be the thermodynamically favoured phase for the alanine/Cu(1 1 0

  5. β-alanine supplementation improves tactical performance but not cognitive function in combat soldiers

    PubMed Central

    2014-01-01

    Background There are no known studies that have examined β-alanine supplementation in military personnel. Considering the physiological and potential neurological effects that have been reported during sustained military operations, it appears that β-alanine supplementation may have a potential benefit in maintaining physical and cognitive performance during high-intensity military activity under stressful conditions. The purpose of this study was to examine the effect of 28 days of β-alanine ingestion in military personnel while fatigued on physical and cognitive performance. Methods Twenty soldiers (20.1 ± 0.9 years) from an elite combat unit were randomly assigned to either a β-alanine (BA) or placebo (PL) group. Soldiers were involved in advanced military training, including combat skill development, navigational training, self-defense/hand-to-hand combat and conditioning. All participants performed a 4-km run, 5-countermovement jumps using a linear position transducer, 120-m sprint, a 10-shot shooting protocol with assault rifle, including overcoming a misfire, and a 2-min serial subtraction test to assess cognitive function before (Pre) and after (Post) 28 days of supplementation. Results The training routine resulted in significant increases in 4-km run time for both groups, but no between group differences were seen (p = 0.597). Peak jump power at Post was greater for BA than PL (p = 0.034), while mean jump power for BA at Post was 10.2% greater (p = 0.139) than PL. BA had a significantly greater (p = 0.012) number of shots on target at Post (8.2 ± 1.0) than PL (6.5 ± 2.1), and their target engagement speed at Post was also significantly faster (p = 0.039). No difference in serial subtraction performance was seen between the groups (p = 0.844). Conclusion Results of this study indicate that 4-weeks of β-alanine ingestion in young, healthy soldiers did not impact cognitive performance, but did enhance power

  6. The mechanism of discrimination between oxidized and reduced coenzyme in the aldehyde dehydrogenase domain of Aldh1l1.

    PubMed

    Tsybovsky, Yaroslav; Malakhau, Yuryi; Strickland, Kyle C; Krupenko, Sergey A

    2013-02-25

    Aldh1l1, also known as 10-formyltetrahydrofolate dehydrogenase (FDH), contains the carboxy-terminal domain (Ct-FDH), which is a structural and functional homolog of aldehyde dehydrogenases (ALDHs). This domain is capable of catalyzing the NADP(+)-dependent oxidation of short chain aldehydes to their corresponding acids, and similar to most ALDHs it has two conserved catalytic residues, Cys707 and Glu673. Previously, we demonstrated that in the Ct-FDH mechanism these residues define the conformation of the bound coenzyme and the affinity of its interaction with the protein. Specifically, the replacement of Cys707 with an alanine resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme. We suggested that this was due to the loss of a covalent bond between the cysteine and the C4N atom of nicotinamide ring of NADP(+) formed during Ct-FDH catalysis. To obtain further insight into the functional significance of the covalent bond between Cys707 and the coenzyme, and the overall role of the two catalytic residues in the coenzyme binding and positioning, we have now solved crystal structures of Ct-FDH in the complex with thio-NADP(+) and the complexes of the C707S mutant with NADP(+) and NADPH. This study has allowed us to trap the coenzyme in the contracted conformation, which provided a snapshot of the conformational processing of the coenzyme during the transition from oxidized to reduced form. Overall, the results of this study further support the previously proposed mechanism by which Cys707 helps to differentiate between the oxidized and reduced coenzyme during ALDH catalysis.

  7. Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production

    PubMed Central

    Zarei, Adel; Trobacher, Christopher P.; Shelp, Barry J.

    2016-01-01

    Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. Terminal catabolism of putrescine in Arabidopsis thaliana involves amine oxidase and the production of 4-aminobutanal, which is a substrate for NAD+-dependent aminoaldehyde dehydrogenase (AMADH). Here, two AMADH homologs were chosen (AtALDH10A8 and AtALDH10A9) as candidates for encoding 4-aminobutanal dehydrogenase activity for GABA synthesis. The two genes were cloned and soluble recombinant proteins were produced in Escherichia coli. The pH optima for activity and catalytic efficiency of recombinant AtALDH10A8 with 3-aminopropanal as substrate was 10.5 and 8.5, respectively, whereas the optima for AtALDH10A9 were approximately 9.5. Maximal activity and catalytic efficiency were obtained with NAD+ and 3-aminopropanal, followed by 4-aminobutanal; negligible activity was obtained with betaine aldehyde. NAD+ reduction was accompanied by the production of GABA and β-alanine, respectively, with 4-aminobutanal and 3-aminopropanal as substrates. Transient co-expression systems using Arabidopsis cell suspension protoplasts or onion epidermal cells and several organelle markers revealed that AtALDH10A9 was peroxisomal, but AtALDH10A8 was cytosolic, although the N-terminal 140 amino acid sequence of AtALDH10A8 localized to the plastid. Root growth of single loss-of-function mutants was more sensitive to salinity than wild-type plants, and this was accompanied by reduced GABA accumulation. PMID:27725774

  8. The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase (PDH)-E1α Phosphorylation

    PubMed Central

    Cerniglia, George J.; Dey, Souvik; Gallagher-Colombo, Shannon M.; Daurio, Natalie A; Tuttle, Stephen; Busch, Theresa M.; Lin, Alexander; Sun, Ramon; Esipova, Tatiana V.; Vinogradov, Sergei A.; Denko, Nicholas; Koumenis, Constantinos; Maity, Amit

    2015-01-01

    Inhibition of the PI3K/Akt pathway decreases hypoxia within SQ20B human head and neck cancer xenografts. We set out to understand the molecular mechanism underlying this observation. We measured oxygen consumption using both a Clark electrode and an extracellular flux analyzer. We made these measurements after various pharmacologic and genetic manipulations. Pharmacologic inhibition of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in vitro in SQ20B and other cell lines by 30–40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, which inhibits activity of this critical gatekeeper of mitochondrial respiration. Expressing wild type PTEN in a doxycycline-inducible manner in a cell line with mutant PTEN led to an increase in PDH-E1α phosphorylation and a decrease in OCR. Pre-treatment of SQ20B cells with dichloroacetate (DCA), which inhibits PDH-E1α phosphorylation by inhibiting dehydrogenase kinases (PDKs), reversed the decrease in OCR in response to PI3K/Akt/mTOR inhibition. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway. PMID:25995437

  9. Neutron diffraction investigations of L- and D-alanine at different temperatures: The search for structural evidence for parity violation

    SciTech Connect

    Wilson, Chick C.; Ghosh, Minakshi; Johnson, Louise N.; Wang, Wenging

    2005-09-01

    Single crystal neutron diffraction has been used in an investigation of the structures of the amino acids L- and D-alanine. The aim of the study was to look for proposed phase transitions around T{sub c} {approx} 270 K. Measurements of both structures at 295 K and 60 K - the neutron structure of D-alanine being determined for the first time - show no significant structural basis for this phase transition in alanine. Further, confirmatory, investigation of the structure of D-alanine at temperatures of 240, 250, 260 and 300 K also showed no significant changes in bond lengths or angles. We can thus offer no structural support to other physical measurements, which are indicative of the observable effect of parity violation of the electroweak force in these phase transitions.

  10. Alanine with the Precipitate of Tomato Juice Administered to Rats Enhances the Reduction in Blood Ethanol Levels

    PubMed Central

    Oshima, Shunji; Shiiya, Sachie; Tokumaru, Yoshimi; Kanda, Tomomasa

    2015-01-01

    Delay in gastric emptying (GE) lowers the blood ethanol concentration (BEC) after alcohol administration. We previously demonstrated that water-insoluble fractions, mainly comprising dietary fiber derived from many types of botanical foods, possessed the ability to absorb ethanol-containing aqueous solutions. Furthermore, there was a significant correlation between the absorption of ethanol and lowering of BEC because of delay in GE. Here we identified dietary nutrients that synergize with the water-insoluble fraction of tomatoes to lower BEC in rats. Consequently, unlike tomato juice without alanine, tomato juice with 5.0% alanine decreased BEC depending on the delay in GE and mediated the ethanol-induced decrease in the spontaneous motor activity (an indicator of drunkenness). Our findings indicate that the synergism between tomato juice and alanine to reduce the absorption of ethanol was attributable to the effect of alanine on precipitates such as the water-insoluble fraction of tomatoes. PMID:26713162

  11. Painting proteins blue: β-(1-azulenyl)-L-alanine as a probe for studying protein-protein interactions.

    PubMed

    Moroz, Yurii S; Binder, Wolfgang; Nygren, Patrik; Caputo, Gregory A; Korendovych, Ivan V

    2013-01-18

    We demonstrated that β-(1-azulenyl)-L-alanine, a fluorescent pseudoisosteric analog of tryptophan, exhibits weak environmental dependence and thus allows for using weak intrinsic quenchers, such as methionines, to monitor protein-protein interactions while not perturbing them.

  12. Alanine with the Precipitate of Tomato Juice Administered to Rats Enhances the Reduction in Blood Ethanol Levels.

    PubMed

    Oshima, Shunji; Shiiya, Sachie; Tokumaru, Yoshimi; Kanda, Tomomasa

    2015-01-01

    Delay in gastric emptying (GE) lowers the blood ethanol concentration (BEC) after alcohol administration. We previously demonstrated that water-insoluble fractions, mainly comprising dietary fiber derived from many types of botanical foods, possessed the ability to absorb ethanol-containing aqueous solutions. Furthermore, there was a significant correlation between the absorption of ethanol and lowering of BEC because of delay in GE. Here we identified dietary nutrients that synergize with the water-insoluble fraction of tomatoes to lower BEC in rats. Consequently, unlike tomato juice without alanine, tomato juice with 5.0% alanine decreased BEC depending on the delay in GE and mediated the ethanol-induced decrease in the spontaneous motor activity (an indicator of drunkenness). Our findings indicate that the synergism between tomato juice and alanine to reduce the absorption of ethanol was attributable to the effect of alanine on precipitates such as the water-insoluble fraction of tomatoes. PMID:26713162

  13. Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein-protein interactions by the MDM2 ligand nutlin-3.

    PubMed

    Way, Luke; Faktor, Jakub; Dvorakova, Petra; Nicholson, Judith; Vojtesek, Borek; Graham, Duncan; Ball, Kathryn L; Hupp, Ted

    2016-09-01

    Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53-independent MDM2-drug responsive-binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin-3 responsive MDM2-binding proteins that are perturbed independent of cell density using SWATH-MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin-3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin-3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin-3 treated cells. Mitotracker confirmed that Nutlin-3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin-3 treatment. Proximity ligation identified rearrangements of cellular protein-protein complexes in situ. In response to Nutlin-3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein-protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein-protein complexes in drug-treated cells. PMID:27273042

  14. Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

    PubMed Central

    Way, Luke; Faktor, Jakub; Dvorakova, Petra; Nicholson, Judith; Vojtesek, Borek; Graham, Duncan; Ball, Kathryn L.

    2016-01-01

    Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53‐independent MDM2‐drug responsive‐binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin‐3 responsive MDM2‐binding proteins that are perturbed independent of cell density using SWATH‐MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin‐3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin‐3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin‐3 treated cells. Mitotracker confirmed that Nutlin‐3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin‐3 treatment. Proximity ligation identified rearrangements of cellular protein–protein complexes in situ. In response to Nutlin‐3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein–protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein–protein complexes in drug‐treated cells. PMID:27273042

  15. Dehydrogenase activity of forest soils depends on the assay used

    NASA Astrophysics Data System (ADS)

    Januszek, Kazimierz; Długa, Joanna; Socha, Jarosław

    2015-01-01

    Dehydrogenases are exclusively intracellular enzymes, which play an important role in the initial stages of oxidation of soil organic matter. One of the most frequently used methods to estimate dehydrogenase activity in soil is based on the use of triphenyltetrazolium chloride as an artificial electron acceptor. The purpose of this study was to compare the activity of dehydrogenases of forest soils with varied physicochemical properties using different triphenyltetrazolium chloride assays. The determination was carried out using the original procedure by Casida et al., a modification of the procedure which involves the use of Ca(OH)2 instead of CaCO3, the Thalmann method, and the assay by Casida et al. without addition of buffer or any salt. Soil dehydrogenase activity depended on the assay used. Dehydrogenase determined by the Casida et al. method without addition of buffer or any salt correlated with the pH values of soils. The autoclaved strongly acidic samples of control soils showed high concentrations of triphenylformazan, probably due to chemical reduction of triphenyltetrazolium chloride. There is, therefore, a need for a sterilization method other than autoclaving, ie a process that results in significant changes in soil properties, thus helping to increase the chemical reduction of triphenyltetrazolium chloride.

  16. Characterization and purification of carbon monoxide dehydrogenase from Methanosarcina barkeri.

    PubMed Central

    Krzycki, J A; Zeikus, J G

    1984-01-01

    Carbon monoxide-dependent production of H2, CO2, and CH4 was detected in crude cell extracts of acetate-grown Methanosarcina barkeri. This metabolic transformation was associated with an active methyl viologen-linked CO dehydrogenase activity (5 to 10 U/mg of protein). Carbon monoxide dehydrogenase activity was inhibited 85% by 10 microM KCN and was rapidly inactivated by O2. The enzyme was nearly homogeneous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%). The native purified enzyme displayed a molecular weight of 232,000 and a two-subunit composition of 92,000 and 18,000 daltons. The enzyme was shown to contain nickel by isolation of radioactive CO dehydrogenase from cells grown in 63Ni. Analysis of enzyme kinetic properties revealed an apparent Km of 5 mM for CO and a Vmax of 1,300 U/mg of protein. The spectral properties of the enzyme were similar to those published for CO dehydrogenase from acetogenic anaerobes. The physiological functions of the enzyme are discussed. Images PMID:6425262

  17. Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme activity.

    PubMed

    Jadhav, Swati B; Bankar, Sandip B; Granström, Tom; Ojamo, Heikki; Singhal, Rekha S; Survase, Shrikant A

    2015-09-01

    Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.

  18. 6-Arylpyrido[2,3-d]pyrimidines as Novel ATP-Competitive Inhibitors of Bacterial D-Alanine:D-Alanine Ligase

    PubMed Central

    Škedelj, Veronika; Arsovska, Emilija; Tomašić, Tihomir; Kroflič, Ana; Hodnik, Vesna; Hrast, Martina; Bešter-Rogač, Marija; Anderluh, Gregor; Gobec, Stanislav; Bostock, Julieanne; Chopra, Ian; O'Neill, Alex J.; Randall, Christopher; Zega, Anamarija

    2012-01-01

    Background ATP-dependent D-alanine:D-alanine ligase (Ddl) is a part of biochemical machinery involved in peptidoglycan biosynthesis, as it catalyzes the formation of the terminal D-ala-D-ala dipeptide of the peptidoglycan precursor UDPMurNAc-pentapeptide. Inhibition of Ddl prevents bacterial growth, which makes this enzyme an attractive and viable target in the urgent search of novel effective antimicrobial drugs. To address the problem of a relentless increase in resistance to known antimicrobial agents we focused our attention to discovery of novel ATP-competitive inhibitors of Ddl. Methodology/Principal Findings Encouraged by recent successful attempts to find selective ATP-competitive inhibitors of bacterial enzymes we designed, synthesized and evaluated a library of 6-arylpyrido[2,3-d]pyrimidine-based compounds as inhibitors of Escherichia coli DdlB. Inhibitor binding to the target enzyme was subsequently confirmed by surface plasmon resonance and studied with isothermal titration calorimetry. Since kinetic analysis indicated that 6-arylpyrido[2,3-d]pyrimidines compete with the enzyme substrate ATP, inhibitor binding to the ATP-binding site was additionally studied with docking. Some of these inhibitors were found to possess antibacterial activity against membrane-compromised and efflux pump-deficient strains of E. coli. Conclusions/Significance We discovered new ATP-competitive inhibitors of DdlB, which may serve as a starting point for development of more potent inhibitors of DdlB that could include both, an ATP-competitive and D-Ala competitive moiety. PMID:22876277

  19. Feasibility on using composite gel-alanine dosimetry on the validation of a multiple brain metastasis radiosurgery VMAT technique

    NASA Astrophysics Data System (ADS)

    Pavoni, J. F.; Neves-Junior, W. F. P.; Silveira, M. A.; Ramos, P. A. M. M.; Haddad, C. M. K.; Baffa, O.

    2015-01-01

    This work presents an end-to-end test using a composite Gel-Alanine phantom, in order to validate 3-dimensionally the dose distribution delivered by a single isocenter VMAT technique on the simultaneous treatment of multiple brain metastases. The results obtained with the gel and alanine dosimeters are consistent with the expected by the treatment planning system, showing the potential of this multidosimetric approach and validating dosimetrically the multiple brain metastases treatment using VMAT.

  20. Barrier-Free Intermolecular Proton Transfer Induced by Excess Electron Attachment to the Complex of Alanine with Uracil

    SciTech Connect

    Dabkowska, Iwona; Rak, Janusz; Gutowski, Maciej S.; Nilles, J.M.; Stokes, Sarah; Bowen, Kit H.

    2004-04-01

    The photoelectron spectrum of the uracil-alanine anionic complex (UA)- has been recorded with 2.540 eV photons. This spectrum reveals a broad feature with a maximum between 1.6-2.1 eV. The vertical electron detachment energy is too large to be attributed to an (UA)- anionic complex in which an intact uracil anion is solvated by alanine, or vice versa. The neutral and anionic complexes of uracil and alanine were studied at the B3LYP and second order Moeller-Plesset level of theory with 6-31++G** basis sets. The neutral complexes form cyclic hydrogen bonds and the three most stable neutral complexes are bound by 0.72, 0.61 and 0.57 eV. The electron hole in complexes of uracil with alaninie is localized on uracil, but the formation of a complex with alanine strongly modulates the vertical ionization energy of uracil. The theoretical results indicate that the excess electron in (UA)- occupies a p* orbital localized on uracil. The excess electron attachment to the complex can induce a barrier-free proton transfer (BFPT) from the carboxylic group of alanine to the O8 atom of uracil. As a result, the four most stable structures of the uracil-alanine anionic complex can be characterized as the neutral radical of hydrogenated uracil solvated by the anion of deprotonated alanine. Our current results for the anionic complex of uracil with alanine are similar to our previous results for the anion of uracil with glycine [Eur. Phys. J. D 20, 431 (2002)], and together they indicate that the BFPT process is not very sensitive to the nature of the amino acid's hydrophobic residual group. The BFPT to the O8 atom of uracil may be relevant to the damage suffered by nucleic acid bases due to exposure to low energy electrons.

  1. The effect of beta-alanine supplementation on isokinetic force and cycling performance in highly trained cyclists.

    PubMed

    Howe, Samuel T; Bellinger, Phillip M; Driller, Matthew W; Shing, Cecilia M; Fell, James W

    2013-12-01

    Beta-alanine may benefit short-duration, high-intensity exercise performance. The aim of this randomized double-blind placebo-controlled study was to examine the effects of beta-alanine supplementation on aspects of muscular performance in highly trained cyclists. Sixteen highly trained cyclists (mean ± SD; age = 24 ± 7 yr; mass = 70 ± 7 kg; VO2max = 67 ± 4 ml · kg(-1) · min(-1)) supplemented with either beta-alanine (n = 8, 65 mg · kg - 1BM) or a placebo (n = 8; dextrose monohydrate) over 4 weeks. Pre- and postsupplementation cyclists performed a 4-minute maximal cycling test to measure average power and 30 reciprocal maximal isokinetic knee contractions at a fixed angular velocity of 180° · sec(-1) to measure average power/repetition, total work done (TWD), and fatigue index (%). Blood pH, lactate (La-) and bicarbonate (HCO3-) concentrations were measured pre- and postisokinetic testing at baseline and following the supplementation period. Beta-alanine supplementation was 44% likely to increase average power output during the 4-minute cycling time trial when compared with the placebo, although this was not statistically significant (p = .25). Isokinetic average power/repetition was significantly increased post beta-alanine supplementation compared with placebo (beta-alanine: 6.8 ± 9.9 W, placebo: -4.3 ± 9.5 W, p = .04, 85% likely benefit), while fatigue index was significantly reduced (p = .03, 95% likely benefit). TWD was 89% likely to be improved following beta-alanine supplementation; however, this was not statistically significant (p = .09). There were no significant differences in blood pH, lactate, and HCO3- between groups (p > .05). Four weeks of beta-alanine supplementation resulted in worthwhile changes in time-trial performance and short-duration muscular force production in highly trained cyclists.

  2. Effects of high-salinity seawater acclimation on the levels of D-alanine in the muscle and hepatopancreas of kuruma prawn, Marsupenaeus japonicus.

    PubMed

    Yoshikawa, Naoko; Yokoyama, Masahumi

    2015-12-10

    Changes in D- and L-alanine contents were determined in the muscle and hepatopancreas of kuruma prawn Marsupenaeus japonicus, during acclimation from seawater containing 100% salinity to artificial seawater containing 150% salinity. In the hepatopancreas, contents of both amino acids increased by approximately threefold. The activity of alanine racemase, which catalyzes the interconversion of D- and L-alanine, also increased in the high-salinity seawater. In addition, the expression of the gene encoding alanine racemase increased in the hepatopancreas with an increase in the alanine racemase activity. These data indicate that the biosynthesis of D- and L-alanine is controlled by the gene expression level of alanine racemase, and D-alanine in the hepatopancreas functions as a major osmolyte for isosmotic regulation. In contrast, the content of D-alanine and alanine racemase activity did not change in the muscle during hyper-osmotic acclimation. Therefore, we suggest that D-alanine, which exists in the several tissues of M. japonicus, is considered to be utilized in some different physiological phenomena in different tissues.

  3. Evidence-based evaluation of potential benefits and safety of beta-alanine supplementation for military personnel.

    PubMed

    Ko, Richard; Low Dog, Tieraona; Gorecki, Dennis K J; Cantilena, Louis R; Costello, Rebecca B; Evans, William J; Hardy, Mary L; Jordan, Scott A; Maughan, Ronald J; Rankin, Janet W; Smith-Ryan, Abbie E; Valerio, Luis G; Jones, Donnamaria; Deuster, Patricia; Giancaspro, Gabriel I; Sarma, Nandakumara D

    2014-03-01

    This Department of Defense-sponsored evidence-based review evaluates the safety and putative outcomes of enhancement of athletic performance or improved recovery from exhaustion in studies involving beta-alanine alone or in combination with other ingredients. Beta-alanine intervention studies and review articles were collected from 13 databases, and safety information was collected from adverse event reporting portals. Due to the lack of systematic studies involving military populations, all the available literature was assessed with a subgroup analysis of studies on athletes to determine if beta-alanine would be suitable for the military. Available literature provided only limited evidence concerning the benefits of beta-alanine use, and a majority of the studies were not designed to address safety. Overall, the strength of evidence in terms of the potential for risk of bias in the quality of the available literature, consistency, directness, and precision did not support the use of beta-alanine by military personnel. The strength of evidence for a causal relation between beta-alanine and paresthesia was moderate.

  4. Effects of beta-alanine supplementation on the onset of neuromuscular fatigue and ventilatory threshold in women.

    PubMed

    Stout, J R; Cramer, J T; Zoeller, R F; Torok, D; Costa, P; Hoffman, J R; Harris, R C; O'Kroy, J

    2007-01-01

    This study examined the effects of 28 days of beta-alanine supplementation on the physical working capacity at fatigue threshold (PWCFT), ventilatory threshold (VT), maximal oxygen consumption (VO2-MAX), and time-to-exhaustion (TTE) in women. Twenty-two women (age+/-SD 27.4+/-6.1 yrs) participated and were randomly assigned to either the beta-alanine (CarnoSyn) or Placebo (PL) group. Before (pre) and after (post) the supplementation period, participants performed a continuous, incremental cycle ergometry test to exhaustion to determine the PWCFT, VT, VO2-MAX, and TTE. There was a 13.9, 12.6 and 2.5% increase (p<0.05) in VT, PWCFT, and TTE, respectively, for the beta-alanine group, with no changes in the PL (p>0.05). There were no changes for VO2-MAX (p>0.05) in either group. Results of this study indicate that beta-alanine supplementation delays the onset of neuromuscular fatigue (PWCFT) and the ventilatory threshold (VT) at submaximal workloads, and increase in TTE during maximal cycle ergometry performance. However, beta-alanine supplementation did not affect maximal aerobic power (VO2-MAX). In conclusion, beta-alanine supplementation appears to improve submaximal cycle ergometry performance and TTE in young women, perhaps as a result of an increased buffering capacity due to elevated muscle carnosine concentrations.

  5. Properties of lactate dehydrogenase in a psychrophilic marine bacterium.

    PubMed Central

    Mitchell, P; Yen, H C; Mathemeier, P F

    1985-01-01

    Lactate dehydrogenase (EC 1.1.1.27) from Vibrio marinus MP-1 was purified 15-fold and ammonium activated. The optimum pH for pyruvate reduction was 7.4. Maximum lactate dehydrogenase activity occurred at 10 to 15 degrees C, and none occurred at 40 degrees C. The crude-extract enzyme was stable between 15 and 20 degrees C and lost 50% of its activity after 60 min at 45 degrees C. The partially purified enzyme was stable between 8 and 15 degrees C and lost 50% of its activity after 60 min at 30 degrees C. The thermal stability of lactate dehydrogenase was increased by mercaptoethanol, with 50% remaining activity at 42 degrees C. Images PMID:4004236

  6. [Features of glutamate dehydrogenase in fetal and adult rumen tissue].

    PubMed

    Kalachniuk, H I; Fomenko, I S; Kalachniuk, L H; Kavai, Sh; Marounek, M; Savka, O H

    2001-01-01

    Glutamate dehydrogenase (GDH) from rumen mucosa of cow fetus, liver and two forms from mucosa (bacterial and tissue) of the adult animal were partly purified and characterized. The activity of the bacterial glutamate dehydrogenase was shown to depend on qualities of a biomass of microbes, adhered on surface of rumen mucosa. All enzymes from tissues (GDHTRF, TRC, TLC), revealed the hypersensibility to increase in the concentration medium of Zn2+, guanosine triphosphate (GTP), acting here in a role of negative modulators, and also adenosine monophosphate (AMP) and leucine, which acted as activators. However, in the same concentrations these effectors do not influence the activity of the bacterial glutamate dehydrogenase. And if all tissues enzymes are highly specific to coenzyme NADH, the bacterial ones almost in 3 times is more active at NADPH use. PMID:11642036

  7. 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

  8. 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.

  9. The activity of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in the sera of patients with brain cancer.

    PubMed

    Jelski, Wojciech; Laniewska-Dunaj, Magdalena; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2014-12-01

    Human brain tissue contains various alcohol dehydrogenase (ADH) isoenzymes and possess also aldehyde dehydrogenase (ALDH) activity. In our last experiments we have shown that ADH and ALDH are present also in the brain tumour cells. Moreover the activities of total ADH and class I isoenzymes were significantly higher in cancer tissue than healthy cells. It can suggests that these changes may be reflected by enzyme activity in the serum of patients with brain cancer. Serum samples were taken for routine biochemical investigation from 62 patients suffering from brain cancer (36 glioblastoma, 26 meningioma). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, the fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. A statistically significant increase of class I alcohol dehydrogenase isoenzymes was found in the sera of patients with brain cancer. The median activity of this class isoenzyme in the patients group increased about 24 % in the comparison to the control level. The total alcohol dehydrogenase activity was also significantly higher (26 %) among patients with brain tumour than healthy ones. The activities of other tested ADH isoenzymes and total ALDH were unchanged. The increase of the activity of total ADH and class I alcohol dehydrogenase isoenzyme in the sera of patients with brain cancer seems to be caused by the release of this isoenzyme from tumour's cells.

  10. VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate dehydrogenase kinase, disrupts Warburg metabolism and induces context-dependent cytostasis in cancer cells.

    PubMed

    Moore, Jonathan D; Staniszewska, Anna; Shaw, Terence; D'Alessandro, Jalanie; Davis, Ben; Surgenor, Alan; Baker, Lisa; Matassova, Natalia; Murray, James; Macias, Alba; Brough, Paul; Wood, Mike; Mahon, Patrick C

    2014-12-30

    Pyruvate dehydrogenase kinase (PDK) is a pivotal enzyme in cellular energy metabolism that has previously been implicated in cancer through both RNAi based studies and clinical correlations with poor prognosis in several cancer types. Here, we report the discovery of a novel and selective ATP competitive pan-isoform inhibitor of PDK, VER-246608. Consistent with a PDK mediated MOA, VER-246608 increased pyruvate dehydrogenase complex (PDC) activity, oxygen consumption and attenuated glycolytic activity. However, these effects were only observed under D-glucose-depleted conditions and required almost complete ablation of PDC E1α subunit phosphorylation. VER-246608 was weakly anti-proliferative to cancer cells in standard culture media; however, depletion of either serum or combined D-glucose/L-glutamine resulted in enhanced cellular potency. Furthermore, this condition-selective cytostatic effect correlated with reduced intracellular pyruvate levels and an attenuated compensatory response involving deamination of L-alanine. In addition, VER-246608 was found to potentiate the activity of doxorubicin. In contrast, the lipoamide site inhibitor, Nov3r, demonstrated sub-maximal inhibition of PDK activity and no evidence of cellular activity. These studies suggest that PDK inhibition may be effective under the nutrient-depleted conditions found in the tumour microenvironment and that combination treatments should be explored to reveal the full potential of this therapeutic strategy.

  11. Structural insights into the efficient CO2-reducing activity of an NAD-dependent formate dehydrogenase from Thiobacillus sp. KNK65MA.

    PubMed

    Choe, Hyunjun; Ha, Jung Min; Joo, Jeong Chan; Kim, Hyunook; Yoon, Hye-Jin; Kim, Seonghoon; Son, Sang Hyeon; Gengan, Robert M; Jeon, Seung Taeg; Chang, Rakwoo; Jung, Kwang Deog; Kim, Yong Hwan; Lee, Hyung Ho

    2015-02-01

    CO2 fixation is thought to be one of the key factors in mitigating global warming. Of the various methods for removing CO2, the NAD-dependent formate dehydrogenase from Candida boidinii (CbFDH) has been widely used in various biological CO2-reduction systems; however, practical applications of CbFDH have often been impeded owing to its low CO2-reducing activity. It has recently been demonstrated that the NAD-dependent formate dehydrogenase from Thiobacillus sp. KNK65MA (TsFDH) has a higher CO2-reducing activity compared with CbFDH. The crystal structure of TsFDH revealed that the biological unit in the asymmetric unit has two conformations, i.e. open (NAD(+)-unbound) and closed (NAD(+)-bound) forms. Three major differences are observed in the crystal structures of TsFDH and CbFDH. Firstly, hole 2 in TsFDH is blocked by helix α20, whereas it is not blocked in CbFDH. Secondly, the sizes of holes 1 and 2 are larger in TsFDH than in CbFDH. Thirdly, Lys287 in TsFDH, which is crucial for the capture of formate and its subsequent delivery to the active site, is an alanine in CbFDH. A computational simulation suggested that the higher CO2-reducing activity of TsFDH is owing to its lower free-energy barrier to CO2 reduction than in CbFDH. PMID:25664741

  12. Structural insights into the efficient CO2-reducing activity of an NAD-dependent formate dehydrogenase from Thiobacillus sp. KNK65MA.

    PubMed

    Choe, Hyunjun; Ha, Jung Min; Joo, Jeong Chan; Kim, Hyunook; Yoon, Hye-Jin; Kim, Seonghoon; Son, Sang Hyeon; Gengan, Robert M; Jeon, Seung Taeg; Chang, Rakwoo; Jung, Kwang Deog; Kim, Yong Hwan; Lee, Hyung Ho

    2015-02-01

    CO2 fixation is thought to be one of the key factors in mitigating global warming. Of the various methods for removing CO2, the NAD-dependent formate dehydrogenase from Candida boidinii (CbFDH) has been widely used in various biological CO2-reduction systems; however, practical applications of CbFDH have often been impeded owing to its low CO2-reducing activity. It has recently been demonstrated that the NAD-dependent formate dehydrogenase from Thiobacillus sp. KNK65MA (TsFDH) has a higher CO2-reducing activity compared with CbFDH. The crystal structure of TsFDH revealed that the biological unit in the asymmetric unit has two conformations, i.e. open (NAD(+)-unbound) and closed (NAD(+)-bound) forms. Three major differences are observed in the crystal structures of TsFDH and CbFDH. Firstly, hole 2 in TsFDH is blocked by helix α20, whereas it is not blocked in CbFDH. Secondly, the sizes of holes 1 and 2 are larger in TsFDH than in CbFDH. Thirdly, Lys287 in TsFDH, which is crucial for the capture of formate and its subsequent delivery to the active site, is an alanine in CbFDH. A computational simulation suggested that the higher CO2-reducing activity of TsFDH is owing to its lower free-energy barrier to CO2 reduction than in CbFDH.

  13. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

    SciTech Connect

    Bulfer, Stacie L.; Hendershot, Jenna M.; Trievel, Raymond C.

    2013-09-18

    Lysine biosynthesis in fungi, euglena, and certain archaebacteria occurs through the {alpha}-aminoadipate pathway. Enzymes in the first steps of this pathway have been proposed as potential targets for the development of antifungal therapies, as they are absent in animals but are conserved in several pathogenic fungi species, including Candida, Cryptococcus, and Aspergillus. One potential antifungal target in the {alpha}-aminoadipate pathway is the third enzyme in the pathway, homoisocitrate dehydrogenase (HICDH), which catalyzes the divalent metal-dependent conversion of homoisocitrate to 2-oxoadipate (2-OA) using nicotinamide adenine dinucleotide (NAD{sup +}) as a cofactor. HICDH belogns to a family of {beta}-hydroxyacid oxidative decarboxylases that includes malate dehydrogenase, tartrate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase (ICDH), and 3-isopropylmalte dehydrogenase (IPMDH). ICDH and IPMDH are well-characterized enzymes that catalyze the decarboxylation of isocitrate to yield 2-oxoglutarate (2-OG) in the citric acid cycle and the conversion of 3-isopropylmalate to 2-oxoisovalerate in the leucine biosynthetic pathway, respectively. Recent structural and biochemical studies of HICDH reveal that this enzyme shares sequence, structural, and mechanistic homology with ICDH and IPMDH. To date, the only published structures of HICDH are from the archaebacteria Thermus thermophilus (TtHICDH). Fungal HICDHs diverge from TtHICDH in several aspects, including their thermal stability, oligomerization state, and substrate specificity, thus warranting further characterization. To gain insights into these differences, they determined crystal structures of a fungal Schizosaccharomyces pombe HICDH (SpHICDH) as an apoenzyme and as a binary complex with additive tripeptide glycyl-glycyl-glycine (GGG) to 1.55 {angstrom} and 1.85 {angstrom} resolution, respectively. Finally, a comparison of the SpHICDH and TtHICDH structures reveal differences in

  14. [Human semen lactate dehydrogenase isoenzymes in fertility studies (author's transl)].

    PubMed

    Gonzalez Buitrago, J M; García Díez, L C; de Castro, S

    1981-01-01

    The lactate dehydrogenase isoenzyme pattern has been obtained in the semen of 87 males undergoing fertility studies. The proportion of LDH-X, the isoenzyme specific to the spermatozoa, is reduced in proportion to the reduction of the sperm density and motility. LDH-X is the most abundant isoenzyme in the semen of normospermic subjects. As to the other isoenzymes, the most abundant ones are the LDH-2 and the LDH-3. The results obtained lead us to conclude that the measurement of the lactate dehydrogenase isoenzymes may be useful in studies of fertility as an indicative parameter of the quality of the semen.

  15. Malate dehydrogenase isozymes in the longnose dace, Rhinichthys cataractae.

    PubMed

    Starzyk, R M; Merritt, R B

    1980-08-01

    The interspecies homology of dace supernatant (A2,AB,B2) and mitochondrial (C2) malate dehydrogenase isozymes has been established through cell fractionation and tissue distribution studies. Isolated supernatant malate dehydrogenase (s-MDH) isozymes show significant differences in Michaelis constants for oxaloacetate and in pH optima. Shifts in s-MDH isozyme pH optima with temperature may result in immediate compensation for increase in ectotherm body pH with decrease in temperature, but duplicate s-MDH isozymes are probably maintained through selection for tissue specific regulation of metabolism.

  16. Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

    PubMed Central

    Kolk, A H; van Kuyk, L; Boettcher, B

    1978-01-01

    Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein. Images Fig. 2. Fig. 3. PMID:213050

  17. Purification and characterization of 3-isopropylmalate dehydrogenase from Thiobacillus thiooxidans.

    PubMed

    Kawaguchi, H; Inagaki, K; Matsunami, H; Nakayama, Y; Tano, T; Tanaka, H

    2000-01-01

    3-Isopropylmalate dehydrogenase was purified to homogeneity from the acidophilic autotroph Thiobacillus thiooxidans. The native enzyme was a dimer of molecular weight 40,000. The apparent K(m) values for 3-isopropylmalate and NAD+ were estimated to be 0.13 mM and 8.7 mM, respectively. The optimum pH for activity was 9.0 and the optimum temperature was 65 degrees C. The properties of the enzyme were similar to those of the Thiobacillus ferrooxidans enzyme, expect for substrate specificity. T. thiooxidans 3-isopropylmalate dehydrogenase could not utilize malate as a substrate.

  18. Reversible inactivation of CO dehydrogenase with thiol compounds

    SciTech Connect

    Kreß, Oliver; Gnida, Manuel; Pelzmann, Astrid M.; Marx, Christian; Meyer-Klaucke, Wolfram; Meyer, Ortwin

    2014-05-09

    Highlights: • Rather large thiols (e.g. coenzyme A) can reach the active site of CO dehydrogenase. • CO- and H{sub 2}-oxidizing activity of CO dehydrogenase is inhibited by thiols. • Inhibition by thiols was reversed by CO or upon lowering the thiol concentration. • Thiols coordinate the Cu ion in the [CuSMo(=O)OH] active site as a third ligand. - Abstract: Carbon monoxide dehydrogenase (CO dehydrogenase) from Oligotropha carboxidovorans is a structurally characterized member of the molybdenum hydroxylase enzyme family. It catalyzes the oxidation of CO (CO + H{sub 2}O → CO{sub 2} + 2e{sup −} + 2H{sup +}) which proceeds at a unique [CuSMo(=O)OH] metal cluster. Because of changing activities of CO dehydrogenase, particularly in subcellular fractions, we speculated whether the enzyme would be subject to regulation by thiols (RSH). Here we establish inhibition of CO dehydrogenase by thiols and report the corresponding K{sub i}-values (mM): L-cysteine (5.2), D-cysteine (9.7), N-acetyl-L-cysteine (8.2), D,L-homocysteine (25.8), L-cysteine–glycine (2.0), dithiothreitol (4.1), coenzyme A (8.3), and 2-mercaptoethanol (9.3). Inhibition of the enzyme was reversed by CO or upon lowering the thiol concentration. Electron paramagnetic resonance spectroscopy (EPR) and X-ray absorption spectroscopy (XAS) of thiol-inhibited CO dehydrogenase revealed a bimetallic site in which the RSH coordinates to the Cu-ion as a third ligand ([Mo{sup VI}(=O)OH{sub (2)}SCu{sup I}(SR)S-Cys]) leaving the redox state of the Cu(I) and the Mo(VI) unchanged. Collectively, our findings establish a regulation of CO dehydrogenase activity by thiols in vitro. They also corroborate the hypothesis that CO interacts with the Cu-ion first. The result that thiol compounds much larger than CO can freely travel through the substrate channel leading to the bimetallic cluster challenges previous concepts involving chaperone function and is of importance for an understanding how the sulfuration step in

  19. Pyruvate dehydrogenase complex from germinating castor bean endosperm.

    PubMed

    Rapp, B J; Randall, D D

    1980-02-01

    Subcellular organelles from castor bean (Ricinus communis) endosperm were isolated on discontinuous sucrose gradients from germinating seeds which were 1 to 7 days postimbibition. Marker enzyme activities of the organelles were measured (fumarase, catalase, and triose phosphate isomerase) and the homogeneity of the organelle fractions was examined by electron microscopy. Pyruvate dehydrogenase complex activity was measured only in the mitochondrial fraction and attempts to activate or release the enzyme from the proplastid were not successful. A pathway is proposed for the most efficient use of endosperm carbon for de novo fatty acid biosynthesis that does not require the presence of the pyruvate dehydrogenase complex in the proplastid to provide acetyl-coenzymeA.

  20. GMXPBSA 2.0: A GROMACS tool to perform MM/PBSA and computational alanine scanning

    NASA Astrophysics Data System (ADS)

    Paissoni, C.; Spiliotopoulos, D.; Musco, G.; Spitaleri, A.

    2014-11-01

    GMXPBSA 2.0 is a user-friendly suite of Bash/Perl scripts for streamlining MM/PBSA calculations on structural ensembles derived from GROMACS trajectories, to automatically calculate binding free energies for protein-protein or ligand-protein complexes. GMXPBSA 2.0 is flexible and can easily be customized to specific needs. Additionally, it performs computational alanine scanning (CAS) to study the effects of ligand and/or receptor alanine mutations on the free energy of binding. Calculations require only for protein-protein or protein-ligand MD simulations. GMXPBSA 2.0 performs different comparative analysis, including a posteriori generation of alanine mutants of the wild-type complex, calculation of the binding free energy values of the mutant complexes and comparison of the results with the wild-type system. Moreover, it compares the binding free energy of different complexes trajectories, allowing the study the effects of non-alanine mutations, post-translational modifications or unnatural amino acids on the binding free energy of the system under investigation. Finally, it can calculate and rank relative affinity to the same receptor utilizing MD simulations of proteins in complex with different ligands. In order to dissect the different MM/PBSA energy contributions, including molecular mechanic (MM), electrostatic contribution to solvation (PB) and nonpolar contribution to solvation (SA), the tool combines two freely available programs: the MD simulations software GROMACS and the Poisson-Boltzmann equation solver APBS. All the calculations can be performed in single or distributed automatic fashion on a cluster facility in order to increase the calculation by dividing frames across the available processors. The program is freely available under the GPL license. Catalogue identifier: AETQ_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AETQ_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, N. Ireland Licensing

  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. Free Energy Landscapes of Alanine Oligopeptides in Rigid-Body and Hybrid Water Models.

    PubMed

    Nayar, Divya; Chakravarty, Charusita

    2015-08-27

    Replica exchange molecular dynamics is used to study the effect of different rigid-body (mTIP3P, TIP4P, SPC/E) and hybrid (H1.56, H3.00) water models on the conformational free energy landscape of the alanine oligopeptides (acAnme and acA5nme), in conjunction with the CHARMM22 force field. The free energy landscape is mapped out as a function of the Ramachandran angles. In addition, various secondary structure metrics, solvation shell properties, and the number of peptide-solvent hydrogen bonds are monitored. Alanine dipeptide is found to have similar free energy landscapes in different solvent models, an insensitivity which may be due to the absence of possibilities for forming i-(i + 4) or i-(i + 3) intrapeptide hydrogen bonds. The pentapeptide, acA5nme, where there are three intrapeptide backbone hydrogen bonds, shows a conformational free energy landscape with a much greater degree of sensitivity to the choice of solvent model, though the three rigid-body water models differ only quantitatively. The pentapeptide prefers nonhelical, non-native PPII and β-sheet populations as the solvent is changed from SPC/E to the less tetrahedral liquid (H1.56) to an LJ-like liquid (H3.00). The pentapeptide conformational order metrics indicate a preference for open, solvent-exposed, non-native structures in hybrid solvent models at all temperatures of study. The possible correlations between the properties of solvent models and secondary structure preferences of alanine oligopeptides are discussed, and the competition between intrapeptide, peptide-solvent, and solvent-solvent hydrogen bonding is shown to be crucial in the relative free energies of different conformers.

  3. Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function

    PubMed Central

    Lee, Shirley Y.; Pullen, Lester; Virgil, Daniel J.; Castañeda, Carlos A.; Abeykoon, Dulith; Bolon, Daniel N. A.; Fushman, David

    2014-01-01

    Mutations at solvent inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. The two null mutants (I30A and L43A) were both less stable to temperature-induced unfolding in vitro than wild-type, but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to wild-type. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high molecular weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high molecular weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation. PMID:24361330

  4. Accumulation of D- vs. L-isomers of alanine and leucine in rat prostatic adenocarcinoma

    SciTech Connect

    Conti, P.S.; Schmall, B.; Bigler, R.E.; Zanzonico, P.B.; Kleinert, E.; Whitmore, W.F. Jr.

    1985-05-01

    It has been reported that tumor tissue may accumulate some D-amino acids preferentially over the L-isomers. In order to investigate the potential use of carbon-11 labeled amino acid isomers for in vivo tumor studies with positron emission tomography in patients, the tissue distributions of alanine and leucine, substrates for the A-type and L-type amino acid transport systems, respectively, were studied in Copenhagen rates bearing the Dunning R3327G prostatic adenocarcinoma. The authors have previously reported differences in the accumulation of A-type vs. L-type amino acids in rat prostatic adenocarcinoma and normal tissues. All compounds were labeled with C-14 in the carboxyl position with specific activities of 30.0-56.6 mCi/mmol. Higher levels of C-14 activity (Relative Concentration (RC)=dpm found per gm tissue + dpm inject per gm animal mass) were observed in tumor tissue using D-alanine (0.71) compared to L- (0.21) or DL-alanine (0.27) at 45 min post-injection. While tumor/prostate and tumor/liver ratios were above 2 for all three substrates, tumor/blood and tumor/muscle were above one for only the D-isomer. Comparisons made with D-, L-, and DL-leucine also demonstrated a higher level of RC in tumor tissue with the D-isomer (0.84) vs. the L-(0.66) and DL-leucine (0.63). In this case, however, tumor/blood, tumor/prostate, and tumor/muscle ratios were above one for all three substrates, while tumor/liver ratios were below one. These results support the observation of a preferential accumulation of D-amino acids in tumor tissue over the natural L-isomers. Observed differences in the accumulation of the isomers in normal tissues are discussed.

  5. Purification and kinetic characteristics of strombine dehydrogenase from the foot muscle of the hard clam (Meretrix lusoria).

    PubMed

    Lee, An-Chin; Lee, Kuen-Tsung; Pan, Li-Ying

    2011-01-01

    Strombine dehydrogenase (SDH, EC 1.5.1.22) from the foot of the hard clam Meretrix lusoria was purified over 470-fold to apparent homogeneity. It has a monomeric structure with a relative molecular mass of 46,000. Two isoenzymes were identified with isoelectric points of 6.83 and 6.88. SDH is heat labile, and has pH and temperature optima of 7.4-7.6 and 45-46°C, respectively. l-Alanine, glycine, and pyruvate are the preferred substrates. l-Serine is the third preferred amino acid. Iminodiacetate with the lowest K(i) of SDH at both pH 6.5 and 7.5 was the strongest inhibitor among succinate, acetate, iminodiacetate, oxaloacetate, and l-/d-lactate. The inhibitory activities of succinate at pH 6.5, and iminodiacetate and oxaloacetate at pH 7.5 on the SDH were higher. These inhibitors are either competitive or mixed-competitive inhibitors. Half of the enzymatic activity of SDH was inhibited by 0.2mM Fe(3+) and 0.6mM Zn(2+).

  6. Interactions across the interface contribute the stability of homodimeric 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase.

    PubMed

    Hwang, Chi-Ching; Hsu, Chao-Nan; Huang, Tzu-Jung; Chiou, Shean-Jaw; Hong, Yi-Ren

    2009-10-01

    The dimerization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. Substitution of alanine, lysine and serine for D249 decreased catalytic efficiency 30, 1400 and 1.4-fold, and lowered the melting temperature 6.9, 5.4 and 7.6 degrees C, respectively. The mutated enzymes have the dimeric species but the equilibrium between monomer and dimer for these mutants varies from each other, implying that these residues might contribute differently to the dimer stability. Thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appeared as a two-state transition and three-state transition, respectively. In addition, mutation on D249 breaks the salt bridges and causes different effects on the loss of enzymatic activity for D249A, D249K and D249S mutants in the urea-induced unfolding profiles. Hence, D249 at the dimeric interface in 3alpha-HSD/CR is essential for conformational stability, oligomeric integrity and enzymatic activity. PMID:19683506

  7. Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

    PubMed Central

    Packman, L C; Perham, R N

    1987-01-01

    The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented. Images Fig. 1. Fig. 2. PMID:3297046

  8. 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.

  9. GMXPBSA 2.1: A GROMACS tool to perform MM/PBSA and computational alanine scanning

    NASA Astrophysics Data System (ADS)

    Paissoni, C.; Spiliotopoulos, D.; Musco, G.; Spitaleri, A.

    2015-01-01

    GMXPBSA 2.1 is a user-friendly suite of Bash/Perl scripts for streamlining MM/PBSA calculations on structural ensembles derived from GROMACS trajectories, to automatically calculate binding free energies for protein-protein or ligand-protein complexes [R.T. Bradshaw et al., Protein Eng. Des. Sel. 24 (2011) 197-207]. GMXPBSA 2.1 is flexible and can easily be customized to specific needs and it is an improvement of the previous GMXPBSA 2.0 [C. Paissoni et al., Comput. Phys. Commun. (2014), 185, 2920-2929]. Additionally, it performs computational alanine scanning (CAS) to study the effects of ligand and/or receptor alanine mutations on the free energy of binding. Calculations require only for protein-protein or protein-ligand MD simulations. GMXPBSA 2.1 performs different comparative analyses, including a posteriori generation of alanine mutants of the wild-type complex, calculation of the binding free energy values of the mutant complexes and comparison of the results with the wild-type system. Moreover, it compares the binding free energy of different complex trajectories, allowing the study of the effects of non-alanine mutations, post-translational modifications or unnatural amino acids on the binding free energy of the system under investigation. Finally, it can calculate and rank relative affinity to the same receptor utilizing MD simulations of proteins in complex with different ligands. In order to dissect the different MM/PBSA energy contributions, including molecular mechanic (MM), electrostatic contribution to solvation (PB) and nonpolar contribution to solvation (SA), the tool combines two freely available programs: the MD simulations software GROMACS [S. Pronk et al., Bioinformatics 29 (2013) 845-854] and the Poisson-Boltzmann equation solver APBS [N.A. Baker et al., Proc. Natl. Acad. Sci. U.S.A 98 (2001) 10037-10041]. All the calculations can be performed in single or distributed automatic fashion on a cluster facility in order to increase the

  10. FT-Raman and FTIR spectroscopic studies of N-octadecanoyl-L-alanine amphiphiles.

    PubMed

    Du, Xuezhong; Liang, Yingqiu

    2004-01-01

    FTIR spectroscopy is used to compare the difference in molecular structure between Langmuir-Blodgett (LB) films (transferred at the surface pressure 40 mN/m with the vertical method and 0 mN/m with the horizontal method) and bulk sample of N-Octadecanoyl-L-alanine amphiphiles. The bulk sample possesses a very similar microstructure (intermolecular hydrogen-bonding interaction and triclinic chain packing) to the well-ordered LB films. Much information on molecular structure of the bulk sample is obtained using FT-Raman spectroscopy, and several weak Raman scattering peaks are assigned.

  11. ESR kinetics study of the decay of low-temperature radicals in glycine and. beta. -alanine

    SciTech Connect

    Smith, C.J.; Poole, C.P. Jr.; Farach, H.A.

    1981-01-15

    Monocrystals of glycine and ..beta..-alanine have been observed by electron spin resonance (ESR) after x-irradiation near 77 /sup 0/K. The rate constants of the decay of one signal in each have been determined over the ranges 127 /sup 0/--148 /sup 0/K and 98 /sup 0/--188 /sup 0/K, respectively. Both obey first-order kinetics with activation energies of approx.7.0 and approx.2.6 kcal/mole, respectively, and pre-exponential factors of 6.4 x 10/sup 8/ and 15 Hz, respectively.

  12. Evaluation of alanine as a reference dosimeter for therapy level dose comparisons in megavoltage electron beams

    NASA Astrophysics Data System (ADS)

    McEwen, Malcolm; Sharpe, Peter; Vörös, Sándor

    2015-04-01

    When comparing absorbed dose standards from different laboratories (e.g. National Measurement Institutes, NMIs, for Key or Supplementary comparisons) it is rarely possible to carry out a direct comparison of primary standard instruments, and therefore some form of transfer detector is required. Historically, air-filled, unsealed ionization chambers have been used because of the long history of using these instruments, very good stability over many years, and ease of transport. However, the use of ion chambers for therapy-level comparisons is not without its problems. Findings from recent investigations suggest that ion chambers are prone to non-random variations, they are not completely robust to standard courier practices, and failure at any step in a comparison can render all measurements potentially useless. An alternative approach is to identify a transfer system that is insensitive to some of these concerns—effectively a dosimeter that is inexpensive, simple to use, robust, but with sufficient precision and of a size relevant to the disseminated quantity in question. The alanine dosimetry system has been successfully used in a number of situations as an audit dosimeter and therefore the purpose of this investigation was to determine whether alanine could also be used as the transfer detector for dosimetric comparisons, which require a lower value for the measurement uncertainty. A measurement protocol was developed for comparing primary standards of absorbed dose to water in high-energy electron beams using alanine pellets irradiated in a water-equivalent plastic phantom. A trial comparison has been carried out between three NMIs and has indicated that alanine is a suitable alternative to ion chambers, with the system used achieving a precision of 0.1%. Although the focus of the evaluation was on the performance of the dosimeter, the comparison results are encouraging, showing agreement at the level of the combined uncertainties (~0.6%). Based on this

  13. Toxicity of the cyanobacterial neurotoxin beta-N-methylamino-L-alanine to three aquatic animal species.

    PubMed

    Purdie, Esme L; Metcalf, James S; Kashmiri, Shereen; Codd, Geoffrey A

    2009-01-01

    Beta-N-methylamino-L-alanine (BMAA), a neurotoxin and candidate contributory cause of neurodegenerative diseases including amyotrophic lateral sclerosis, is produced by aquatic and terrestrial cyanobacteria. We have determined BMAA toxicity to three aquatic animal species: zebra fish (Danio rerio), brine shrimp (Artemia salina) and the protozoan Nassula sorex. Responses included: clonus convulsions and abnormal spinal axis formation (D. rerio), loss of phototaxis (A. salina) and mortalities (all species). These systems offer potential to further understand BMAA toxicity and the bioaccumulation and fates of BMAA in aquatic food chains leading to potential human exposure.

  14. Verification of the pure alanine in PMMA tube dosimeter applicability for dosimetry of radiotherapy photon beams: a feasibility study.

    PubMed

    Al-Karmi, Anan M; Ayaz, Ali Asghar H; Al-Enezi, Mamdouh S; Abdel-Rahman, Wamied; Dwaikat, Nidal

    2015-09-01

    Alanine dosimeters in the form of pure alanine powder in PMMA plastic tubes were investigated for dosimetry in a clinical application. Electron paramagnetic resonance (EPR) spectroscopy was used to measure absorbed radiation doses by detection of signals from radicals generated in irradiated alanine. The measurements were performed for low-dose ranges typical for single-fraction doses often used in external photon beam radiotherapy. First, the dosimeters were irradiated in a solid water phantom to establish calibration curves in the dose range from 0.3 to 3 Gy for 6 and 18 MV X-ray beams from a clinical linear accelerator. Next, the dosimeters were placed at various locations in an anthropomorphic pelvic phantom to measure the dose delivery of a conventional four-field box technique treatment plan to the pelvis. Finally, the doses measured with alanine dosimeters were compared against the doses calculated with a commercial treatment planning system (TPS). The results showed that the alanine dosimeters have a highly sensitive dose response with good linearity and no energy dependence in the dose range and photon beams used in this work. Also, a fairly good agreement was found between the in-phantom dose measurements with alanine dosimeters and the TPS dose calculations. The mean value of the ratios of measured to calculated dose values was found to be near unity. The measured points in the in-field region passed dose-difference acceptance criterion of 3% and those in the penumbral region passed distance-to-agreement acceptance criterion of 3 mm. These findings suggest that the pure alanine powder in PMMA tube dosimeter is a suitable option for dosimetry of radiotherapy photon beams.

  15. Verification of the pure alanine in PMMA tube dosimeter applicability for dosimetry of radiotherapy photon beams: a feasibility study.

    PubMed

    Al-Karmi, Anan M; Ayaz, Ali Asghar H; Al-Enezi, Mamdouh S; Abdel-Rahman, Wamied; Dwaikat, Nidal

    2015-09-01

    Alanine dosimeters in the form of pure alanine powder in PMMA plastic tubes were investigated for dosimetry in a clinical application. Electron paramagnetic resonance (EPR) spectroscopy was used to measure absorbed radiation doses by detection of signals from radicals generated in irradiated alanine. The measurements were performed for low-dose ranges typical for single-fraction doses often used in external photon beam radiotherapy. First, the dosimeters were irradiated in a solid water phantom to establish calibration curves in the dose range from 0.3 to 3 Gy for 6 and 18 MV X-ray beams from a clinical linear accelerator. Next, the dosimeters were placed at various locations in an anthropomorphic pelvic phantom to measure the dose delivery of a conventional four-field box technique treatment plan to the pelvis. Finally, the doses measured with alanine dosimeters were compared against the doses calculated with a commercial treatment planning system (TPS). The results showed that the alanine dosimeters have a highly sensitive dose response with good linearity and no energy dependence in the dose range and photon beams used in this work. Also, a fairly good agreement was found between the in-phantom dose measurements with alanine dosimeters and the TPS dose calculations. The mean value of the ratios of measured to calculated dose values was found to be near unity. The measured points in the in-field region passed dose-difference acceptance criterion of 3% and those in the penumbral region passed distance-to-agreement acceptance criterion of 3 mm. These findings suggest that the pure alanine powder in PMMA tube dosimeter is a suitable option for dosimetry of radiotherapy photon beams. PMID:26138456

  16. Biosynthesis of d-Alanyl-Lipoteichoic Acid: Characterization of Ester-Linked d-Alanine in the In Vitro-Synthesized Product

    PubMed Central

    Childs, Warren C.; Neuhaus, Francis C.

    1980-01-01

    d-Alanyl-lipoteichoic acid (d-alanyl-LTA) contains d-alanine ester residues which control the ability of this polyer to chelate Mg2+. In Lactobacillus casei a two-step in vitro reaction sequence catalyzed by the d-alanine-activating enzyme and d-alanine:membrane acceptor ligase incorporates d-alanine into membrane acceptor. In this paper we provide additional evidence that the in vitro system catalyzes the covalent incorporation of d-[14C]alanine into membrane acceptor which is the poly([3H]glycerol phosphate) moiety of d-alanyl-LTA. This conclusion was supported by the observation that the d-[14C]alanine and [3H]glycerol labels of the partially purified product were co-precipitated by antiserum containing globulins specific for poly(glycerol phosphate). The isolation of d-[14C]alanyl-[3H]glycerol from d-[14C]alanine·[3H]glycerol-labeled d-alanyl-LTA synthesized in the in vitro system indicated that the d-alanine was linked to the poly(glycerol phosphate) chain of the LTA. A comparison of the reactivities of the d-alanine residues of d-alanyl-glycerol and d-alanyl-LTA supported the conclusion that the incorporated residue of d-alanine was attached by an ester linkage. Thus, the data indicated that the in vitro system catalyzes the incorporation of d-alanine covalently linked by ester linkages to the glycerol moieties of the poly(glycerol phosphate) chains of d-alanyl-LTA. New procedures are presented for the partial purification of d-alanyl-LTA with a high yield of ester-linked d-alanine and for the sequential degradation of the poly(glycerol phosphate) moiety substituted with d-alanine of d-alanyl-LTA with phosphodiesterase II/phosphatase from Aspergillus niger. PMID:6772629

  17. Amino acid sequence homology among the 2-hydroxy acid dehydrogenases: mitochondrial and cytoplasmic malate dehydrogenases form a homologous system with lactate dehydrogenase.

    PubMed Central

    Birktoft, J J; Fernley, R T; Bradshaw, R A; Banaszak, L J

    1982-01-01

    The amino acid sequence of porcine heart mitochondrial malate dehydrogenase (mMDH; L-malate: NAD+ oxidoreductase, EC 1.1.1.37) has been compared with the sequences of six different lactate dehydrogenases (LDH; L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) and with the "x-ray" sequence of cytoplasmic malate dehydrogenase (sMDH). The main points are that (i) all three enzymes are homologous; (ii) invariant residues in the catalytic center of these enzymes include a histidine and an internally located aspartate that function as a proton relay system; (iii) numerous residues important to coenzyme binding are conserved, including several glycines and charged residues; and (iv) amino acid side chains present in the subunit interface common to the MDHs and LDHs appear to be better conserved than those in the protein interior. It is concluded that LDH, sMDH, and mMDH are derived from a common ancestral gene and probably have similar catalytic mechanisms. PMID:6959107

  18. NADP+-Preferring D-Lactate Dehydrogenase from Sporolactobacillus inulinus.

    PubMed

    Zhu, Lingfeng; Xu, Xiaoling; Wang, Limin; Dong, Hui; Yu, Bo; Ma, Yanhe

    2015-09-01

    Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD(+) as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn(174) was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.

  19. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase

    SciTech Connect

    Mat-Jan, F.; Alam, K.Y.; Clark, D.P. )

    1989-01-01

    Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.

  20. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lactic dehydrogenase immunological test system. 866.5560 Section 866.5560 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the...

  1. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lactic dehydrogenase immunological test system. 866.5560 Section 866.5560 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the...

  2. A novel small-molecule inhibitor of 3-phosphoglycerate dehydrogenase.

    PubMed

    Mullarky, Edouard; Lairson, Luke L; Cantley, Lewis C; Lyssiotis, Costas A

    2016-07-01

    Serine metabolism is likely to play a critical role in cancer cell growth. A recent study reports the identification of a novel small-molecule inhibitor of serine synthesis that targets 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme of the serine synthesis pathway, and selectively abrogates the proliferation of PHGDH overexpressing breast cancer cells. PMID:27652319

  3. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false 6-Phosphogluconate dehydrogenase test system. 862.1565 Section 862.1565 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1565...

  4. Genetics Home Reference: 3-hydroxyacyl-CoA dehydrogenase deficiency

    MedlinePlus

    ... step that metabolizes groups of fats called medium-chain fatty acids and short-chain fatty acids. Mutations in the HADH gene lead ... a shortage of 3-hydroxyacyl-CoA dehydrogenase. Medium-chain and short-chain fatty acids cannot be metabolized ...

  5. Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene

    PubMed Central

    Sadeghi, H. Mir Mohammad; Ahmadi, R.; Aghaabdollahian, S.; Mofid, M.R.; Ghaemi, Y.; Abedi, D.

    2011-01-01

    Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. coli XL1-blue competent cells. Following plasmid preparation, the cloned gene was digested out and ligated into the expression vector pET-22b(+). Electrophoresis of PCR product showed a 789 bp band. Recombinant plasmid (rpTZ57R) was then constructed. This plasmid was double digested with XhoI and EcoRI resulting in 800 bp and 2900 bp bands. The obtained insert was ligated into pET-22b(+) vector and its orientation was confirmed with XhoI and BamHI restriction enzymes. In conclusion, in the present study the recombinant expression vector containing xylitol dehydrogenase gene has been constructed and can be used for the production of this enzyme in high quantities. PMID:22110522

  6. Effects of aerobic training on pyruvate dehydrogenase and pyruvate dehydrogenase kinase in human skeletal muscle.

    PubMed

    LeBlanc, Paul J; Peters, Sandra J; Tunstall, Rebecca J; Cameron-Smith, David; Heigenhauser, George J F

    2004-06-01

    This study examined the effects of short- and long-term aerobic training on the stable up-regulation of pyruvate dehydrogenase (PDH) and PDH kinase (PDK) in human skeletal muscle. We hypothesized that 8 weeks, but not 1 week, of aerobic training would increase total PDH (PDHt) and PDK activities compared to pretraining, and this would be detectable at the level of gene transcription (mRNA) and/or gene translation (protein). Resting muscle biopsies were taken before and after 1 and 8 weeks of aerobic cycle exercise training. PDHt and PDK activities, and their respective protein and mRNA expression, did not differ after 1 week of aerobic training. PDHt activity increased 31% after 8 weeks and this may be partially due to a 1.3-fold increase in PDH-E(1)alpha protein expression. PDK activity approximately doubled after 8 weeks of aerobic training and this was attributed to a 1.3-fold increase in PDK2 isoform protein expression. Similar to 1 week, no changes were observed at the mRNA level after 8 weeks of training. These findings suggest that aerobically trained human skeletal muscle has an increased maximal capacity to utilize carbohydrates, evident by increased PDHt, but increased metabolic control sensitivity to pyruvate through increased contribution of PDK2 to total PDK activity. PMID:15020699

  7. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  8. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  9. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  10. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  11. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  12. Alanine substitutions of noncysteine residues in the cysteine-stabilized αβ motif

    PubMed Central

    Yang, Ying-Fang; Cheng, Kuo-Chang; Tsai, Ping-Hsing; Liu, Chung-Cheng; Lee, Tian-Ren; Ping-Chiang Lyu

    2009-01-01

    The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized αβ motif (CSαβ) consists of an α-helix and an antiparallel triple-stranded β-sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering. PMID:19533758

  13. Mechano-responsive gelation of water by a short alanine-derivative.

    PubMed

    Reddy M, Amarendar; Srivastava, Aasheesh

    2014-07-21

    We report the design of a structurally concise alanine derivative (Ala-hyd) that has a rotationally flexible aromatic N-protecting group for alanine and a hydrazide functionality at its carboxylic end. Ala-hyd requires mechanical agitation (physically stirring, vortexing or sonicating) to form supramolecular hydrogels at medium concentrations (0.4-0.8 wt%). At higher concentrations (>0.8 wt%), it spontaneously gelates water on undisturbed cooling of the hot solution, while at lower concentrations (<0.4 wt%), only turbid suspensions were formed upon agitation. In the <0.8 wt% regime, hydrogelation by Ala-hyd is modulated by its concentration as well as by the extent of applied mechanical agitation. Turbidimetry and fluorescence spectroscopy indicate enhanced self-assembly of Ala-hyd upon agitation, and FTIR studies point towards stronger hydrogen bonds in the resulting assemblies. Since Ala-hyd requires mechanical agitation to undergo self-assembly, its aqueous sols exhibited mild shear-thickening behaviour in buffered as well as salt-free conditions. During shearing, the formation of an entangled mesh of long, helical nanofibers coincided with the maximum in the bulk shear viscosity. pH-dependent rheological investigations indicate that protonation of the amine unit (pKa = 8.9) of hydrazide diminishes the self-assembly propensity of this compound. The self-assembly of Ala-hyd can thus be modulated through mechanical as well as chemical cues.

  14. Dissociation of alkaliated alanine in the gas phase: the role of the metal cation.

    PubMed

    Abirami, Seduraman; Wong, Catherine Chiu Lan; Tsang, Chun Wai; Ma, Ngai Ling

    2005-09-01

    The dissociation of prototypical metal-cationized amino acid complexes, namely, alkaliated alanine ([Ala+M]+, M+ = Li+, Na+, K+), was studied by energy-resolved tandem mass spectrometry with an ion-trap mass analyzer and by density functional theory. Dissociation leads to formation of fragment ions arising from the loss of small neutrals, such as H2O, CO, NH3, (CO+NH3), and the formation of Na+/K+. The order of appearance threshold voltages for different dissociation pathways determined experimentally is consistent with the order of critical energies (energy barriers) obtained theoretically, and this provides the necessary confidence in both experimental and theoretical results. Although not explicitly involved in the reaction, the alkali metal cation plays novel and important roles in the dissociation of alkaliated alanine. The metal cation not only catalyzes the dissociation (via the formation of loosely bound ion-molecule complexes and by stabilizing the more polar intermediates and transition structures), but also affects the dissociation mechanisms, as the cation can alter the shape of the potential energy surfaces. This compression/expansion of the potential energy surface as a function of the alkali metal cation is discussed in detail, and how this affects the competitive loss of H2O versus CO/(CO+NH3) from [Ala+M]+ is illustrated. The present study provides new insights into the origin of the competition between various dissociation channels of alkaliated amino acid complexes.

  15. Tuning electronic transport via hepta-alanine peptides junction by tryptophan doping.

    PubMed

    Guo, Cunlan; Yu, Xi; Refaely-Abramson, Sivan; Sepunaru, Lior; Bendikov, Tatyana; Pecht, Israel; Kronik, Leeor; Vilan, Ayelet; Sheves, Mordechai; Cahen, David

    2016-09-27

    Charge migration for electron transfer via the polypeptide matrix of proteins is a key process in biological energy conversion and signaling systems. It is sensitive to the sequence of amino acids composing the protein and, therefore, offers a tool for chemical control of charge transport across biomaterial-based devices. We designed a series of linear oligoalanine peptides with a single tryptophan substitution that acts as a "dopant," introducing an energy level closer to the electrodes' Fermi level than that of the alanine homopeptide. We investigated the solid-state electron transport (ETp) across a self-assembled monolayer of these peptides between gold contacts. The single tryptophan "doping" markedly increased the conductance of the peptide chain, especially when its location in the sequence is close to the electrodes. Combining inelastic tunneling spectroscopy, UV photoelectron spectroscopy, electronic structure calculations by advanced density-functional theory, and dc current-voltage analysis, the role of tryptophan in ETp is rationalized by charge tunneling across a heterogeneous energy barrier, via electronic states of alanine and tryptophan, and by relatively efficient direct coupling of tryptophan to a Au electrode. These results reveal a controlled way of modulating the electrical properties of molecular junctions by tailor-made "building block" peptides.

  16. A single glycine-alanine exchange directs ligand specificity of the elephant progestin receptor.

    PubMed

    Wierer, Michael; Schrey, Anna K; Kühne, Ronald; Ulbrich, Susanne E; Meyer, Heinrich H D

    2012-01-01

    The primary gestagen of elephants is 5α-dihydroprogesterone (DHP), which is unlike all other mammals studied until now. The level of DHP in elephants equals that of progesterone in other mammals, and elephants are able to bind DHP with similar affinity to progesterone indicating a unique ligand-binding specificity of the elephant progestin receptor (PR). Using site-directed mutagenesis in combination with in vitro binding studies we here report that this change in specificity is due to a single glycine to alanine exchange at position 722 (G722A) of PR, which specifically increases DHP affinity while not affecting binding of progesterone. By conducting molecular dynamics simulations comparing human and elephant PR ligand-binding domains (LBD), we observed that the alanine methyl group at position 722 is able to push the DHP A-ring into a position similar to progesterone. In the human PR, the DHP A-ring position is twisted towards helix 3 of PR thereby disturbing the hydrogen bond pattern around the C3-keto group, resulting in a lower binding affinity. Furthermore, we observed that the elephant PR ligand-binding pocket is more rigid than the human analogue, which probably explains the higher affinity towards both progesterone and DHP. Interestingly, the G722A substitution is not elephant-specific, rather it is also present in five independent lineages of mammalian evolution, suggesting a special role of the substitution for the development of distinct mammalian gestagen systems. PMID:23209719

  17. Selective control of Cu(II) complex stability in histidine peptides by β-alanine.

    PubMed

    Nagaj, Justyna; Stokowa-Sołtys, Kamila; Zawisza, Izabela; Jeżowska-Bojczuk, Małgorzata; Bonna, Arkadiusz; Bal, Wojciech

    2013-02-01

    The cooperativity of formation of 5-membered and 6-membered chelate rings is the driving force for specificity and selectivity in Cu(II) peptidic complexes. α-Amino acids enable the formation of 5-membered rings, while a 6-membered ring is provided by the coordination of the His side chain imidazole. Introduction of β-alanine is another way of creating a 6-membered ring in the Cu(II) complex. The potentiometric and spectroscopic (UV-vis and CD) study of Cu(II) complexation by a series of four peptides, AAH-am, ABH-am, BAH-am, and BBH-am (where B stands for β-alanine, and -am for C-terminal amide) revealed a very strong effect of the sizes of individual rings, with the order of complex stability AAH-am (5,5,6)>BAH-am (6,5,6)>ABH-am (5,6,6)≫BBH-am (6,6,6). The stabilities of ABH-am and BAH-am complexes are intermediate between those of strong His-3 peptides but these complexes are still able to saturate the coordination sphere of the Cu(II) ion at neutral pH. This fact opens up new possibilities in engineering specific peptide-based chelates.

  18. Electron attachment to amino acid clusters in helium nanodroplets: Glycine, alanine, and serine

    NASA Astrophysics Data System (ADS)

    Ferreira da Silva, F.; Denifl, S.; Märk, T. D.; Ellis, A. M.; Scheier, P.

    2010-06-01

    The first detailed study of electron attachment to amino acid clusters is reported. The amino acids chosen for investigation were glycine, alanine, and serine. Clusters of these amino acids were formed inside helium nanodroplets, which provide a convenient low temperature (0.37 K) environment for growing noncovalent clusters. When subjected to low energy (2 eV) electron impact the chemistry for glycine and alanine clusters was found to be similar. In both cases, parent cluster anions were the major products, which contrasts with the corresponding monomers in the gas phase, where the dehydrogenated products ([AAn-H]-, where AA=amino acid monomer) dominate. Serine clusters are different, with the major product being the parent anion minus an OH group, an outcome presumably conferred by the facile loss of an OH group from the β carbon of serine. In addition to the bare parent anions and various fragment anions, helium atoms are also observed attached to both the parent anion clusters and the dehydrogenated parent anion clusters. Finally, we present the first anion yield spectra of amino acid clusters from doped helium nanodroplets as a function of incident electron energy.

  19. Characterization and biocompatibility of organogels based on L-alanine for parenteral drug delivery implants.

    PubMed

    Motulsky, Aude; Lafleur, Michel; Couffin-Hoarau, Anne-Claude; Hoarau, Didier; Boury, Frank; Benoit, Jean-Pierre; Leroux, Jean-Christophe

    2005-11-01

    The development of simple and efficient drug delivery systems for the sustained release of peptides/proteins and low molecular weight hydrophilic molecules is an ongoing challenge. The purpose of this work was to prepare and characterize novel biodegradable in situ-forming implants obtained via the self-assembly of L-alanine derivatives in pharmaceutical oils. Six different amphiphilic organogelators based on L-alanine were synthesized. These derivatives could successfully gel various vegetable and synthetic oils approved for parenteral administration. Gelation was thermoreversible, and phase transition temperatures depended on gelator structure, concentration and solvent. Hydrogen bonds and van der Waals interactions were shown to be the main forces implicated in network formation. Selected formulations were then injected subcutaneously in rats for preliminary assessment of biocompatibility. Histopathological analysis of the surrounding tissues revealed mild, chronic inflammation and an overall good biocompatibility profile of the implants over the 8 wk evaluation period. This study demonstrates that in situ-forming organogels represent a potentially promising platform for sustained drug delivery.

  20. Tuning electronic transport via hepta-alanine peptides junction by tryptophan doping.

    PubMed

    Guo, Cunlan; Yu, Xi; Refaely-Abramson, Sivan; Sepunaru, Lior; Bendikov, Tatyana; Pecht, Israel; Kronik, Leeor; Vilan, Ayelet; Sheves, Mordechai; Cahen, David

    2016-09-27

    Charge migration for electron transfer via the polypeptide matrix of proteins is a key process in biological energy conversion and signaling systems. It is sensitive to the sequence of amino acids composing the protein and, therefore, offers a tool for chemical control of charge transport across biomaterial-based devices. We designed a series of linear oligoalanine peptides with a single tryptophan substitution that acts as a "dopant," introducing an energy level closer to the electrodes' Fermi level than that of the alanine homopeptide. We investigated the solid-state electron transport (ETp) across a self-assembled monolayer of these peptides between gold contacts. The single tryptophan "doping" markedly increased the conductance of the peptide chain, especially when its location in the sequence is close to the electrodes. Combining inelastic tunneling spectroscopy, UV photoelectron spectroscopy, electronic structure calculations by advanced density-functional theory, and dc current-voltage analysis, the role of tryptophan in ETp is rationalized by charge tunneling across a heterogeneous energy barrier, via electronic states of alanine and tryptophan, and by relatively efficient direct coupling of tryptophan to a Au electrode. These results reveal a controlled way of modulating the electrical properties of molecular junctions by tailor-made "building block" peptides. PMID:27621456

  1. Glutamate dehydrogenases: the why and how of coenzyme specificity.

    PubMed

    Engel, Paul C

    2014-01-01

    NAD(+) and NADP(+), chemically similar and with almost identical standard oxidation-reduction potentials, nevertheless have distinct roles, NAD(+) serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD(+)-dependent for glutamate oxidation, NADP(+)-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD(+) reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD(+) but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP(+) reduction by NADH, maintaining the coenzyme pools at different oxidation-reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD(+)-dependent, NADP(+)-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD(+) or for NADP(+) has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2'- and 3'-hydroxyls, dictating NAD(+) specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD(+) only, NADP(+) only, or in higher animals both.

  2. Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts

    PubMed Central

    Kanoff, J.M.; Shankardas, J.; Dimitrijevich, S.

    2009-01-01

    Purpose To identify differentially expressed genes in keratoconus (KC) corneal fibroblasts. Methods Stromal keratocytes (having a fibroblast morphology) from KC keratoplasty specimens and eye bank donor corneas were isolated and expanded using a serum containing medium. RNA was isolated from three KC fibroblast cultures and five eye bank donor cornea fibroblast cultures. The targets from the cultured fibroblasts were hybridized to the Affymetrix U133 Plus 2.0 microarrays. Western blot analyses of cell lysates were performed to examine protein levels of interest in the two groups. Protein levels of select differentially expressed genes were further examined by immunohistochemistry. Keratocyte staining of archived KC keratoplasty specimens were graded using a 0 to 3+ scale and compared to five archived whole globes having normal corneas as well as to 10 Fuchs’ dystrophy keratoplasty specimens. Results Microarray analysis revealed up to a 212 fold reduction in the mRNA levels of alcohol dehydrogenase (class 1) beta polypeptide (ADH1B) in KC fibroblasts (p=0.04). Decreased alcohol dehydrogenase in KC fibroblasts was confirmed by western blot analysis of early passage primary keratocyte cell lysates. Immunohistochemistry using a monoclonal mouse immunoglobulin G (IgG) against human liver alcohol dehydrogenase revealed a dramatic difference in protein staining in the keratocytes of the KC group compared to the normal cornea group. Immunohistochemistry also showed decreased immunostaining against alcohol dehydrogenase in the KC stromal sections compared to those obtained from Fuchs’ endothelial corneal dystrophy samples. Conclusions Decreased alcohol dehydrogenase in KC corneal fibroblasts represents a strong marker and possible mediator of keratoconus. PMID:19365573

  3. Spatial variability of the dehydrogenase activity in forest soils

    NASA Astrophysics Data System (ADS)

    Błońska, Ewa; Lasota, Jarosław

    2014-05-01

    The aim of this study was to assess the spatial variability of the dehydrogenase activity (DH) in forest soils using geostatistics. We have studied variability soil dehydrogenase and their relationship with variability of some physic-chemical properties. Two study areas (A and B) were set up in southern Poland in the Zlotoryja Forest District. Study areas were covered by different types of vegetation (A- broadleaf forest with beech, ash and sycamore), B- coniferous forest with Norway spruce). The soils were classified as Dystric Cambisols (WRB 2006). The samples for laboratory testing were collected from 49 places on each areas. 15 cm of surface horizon of soil were taken (with previously removed litter). Dehydrogenase activity was marked with Lenhard's method according to the Casida procedure. Soil pH, nitrogen (N) and soil organic carbon (C) content (by LECO CNS 2000 carbon analyzer) was marked. C/N ratio was calculated. Particle size composition was determined using laser diffraction. Statistical analysis were performed using STATISTICA 10 software. Geostatistical analysis and mapping were done by application of GS 9+ (Gamma Design) and Surfer 11 (Golden Software). The activity of DH ranged between 5,02 and 71,20 mg TPP• kg-1 •24 h-1 on the A area and between 0,94 and 16,47 mg TPP• kg-1 •24 h-1. Differences in spatial variability of the analised features were noted. The variability of dehydrogenase activity on the A study area was described by an exponential model, whereas on the B study area the spatial correlation has not been noted. The relationship of dehydrogenase activity with the remaining parameters of soil was noted only in the case of A study area. The variability of organic carbon content on the A and B study areas were described by an exponential model. The variability of nitrogen content on both areas were described by an spherical model.

  4. A versatile proline/alanine transporter in the unicellular pathogen Leishmania donovani regulates amino acid homoeostasis and osmotic stress responses.

    PubMed

    Inbar, Ehud; Schlisselberg, Doreen; Suter Grotemeyer, Marianne; Rentsch, Doris; Zilberstein, Dan

    2013-01-15

    Unlike all other organisms, parasitic protozoa of the family Trypanosomatidae maintain a large cellular pool of proline that, together with the alanine pool, serve as alternative carbon sources as well as reservoirs of organic osmolytes. These reflect adaptation to their insect vectors whose haemolymphs are exceptionally rich in the two amino acids. In the present study we identify and characterize a new neutral amino acid transporter, LdAAP24, that translocates proline and alanine across the Leishmania donovani plasma membrane. This transporter fulfils multiple functions: it is the sole supplier for the intracellular pool of proline and contributes to the alanine pool; it is essential for cell volume regulation after osmotic stress; and it regulates the transport and homoeostasis of glutamate and arginine, none of which are its substrates. Notably, we provide evidence that proline and alanine exhibit different roles in the parasitic response to hypotonic shock; alanine affects swelling, whereas proline influences the rate of volume recovery. On the basis of our data we suggest that LdAAP24 plays a key role in parasite adaptation to its varying environments in host and vector, a phenomenon essential for successful parasitism.

  5. Rapid Crystallization of L-Alanine on Engineered Surfaces using Metal-Assisted and Microwave-Accelerated Evaporative Crystallization.

    PubMed

    Alabanza, Anginelle M; Pozharski, Edwin; Aslan, Kadir

    2012-01-01

    This study demonstrates the application of metal-assisted and microwave-accelerated evaporative crystallization (MA-MAEC) technique to rapid crystallization of L-alanine on surface engineered silver nanostructures. In this regard, silver island films (SIFs) were modified with hexamethylenediamine (HMA), 1-undecanethiol (UDET), and 11-mercaptoundecanoic acid (MUDA), which introduced -NH(2), -CH(3) and -COOH functional groups to SIFs, respectively. L-Alanine was crystallized on these engineered surfaces and blank SIFs at room temperature and using MA-MAEC technique. Significant improvements in crystal size, shape, and quality were observed on HMA-, MUDA- and UDET-modified SIFs at room temperature (crystallization time = 144, 40 and 147 min, respectively) as compared to those crystals grown on blank SIFs. Using the MA-MAEC technique, the crystallization time of L-alanine on engineered surfaces were reduced to 17 sec for microwave power level 10 (i.e., duty cycle 100%) and 7 min for microwave power level 1 (duty cycle 10%). Raman spectroscopy and powder x-ray diffraction (XRD) measurements showed that L-Alanine crystals grown on engineered surfaces using MA-MAEC technique had identical characteristic peaks of L-alanine crystals grown using traditional evaporative crystallization. PMID:22267957

  6. β-alanine Supplementation Fails to Increase Peak Aerobic Power or Ventilatory Threshold in Aerobically Trained Males.

    PubMed

    Greer, Beau Kjerulf; Katalinas, Matthew E; Shaholli, Danielle M; Gallo, Paul M

    2016-01-01

    The purpose of the present study was to determine the effect of 30 days of β-alanine supplementation on peak aerobic power and ventilatory threshold (VT) in aerobically fit males. Fourteen males (28.8 ± 9.8 yrs) were assigned to either a β-alanine (SUPP) or placebo (PLAC) group; groups were matched for VT as it was the primary outcome measure. β-alanine supplementation consisted of 3 g/day for 7 days, and 6 g/day for the remaining 23 days. Before and after the supplementation period, subjects performed a continuous, graded cycle ergometry test to determine VO2 peak and VT. Metabolic data were analyzed using a 2 × 2 ANOVA with repeated measures. Thirty days of β-alanine supplementation (SUPP) did not increase VO2 peak (4.05 ± 0.6 vs. 4.14 ± 0.6 L/min) as compared to the placebo (PLAC) group (3.88 ± 0.2 vs. 3.97 ± 0.2 L/min) (p > .05). VT did not significantly improve in either the SUPP (3.21 ± 0.5 vs. 3.33 ± 0.5 L/min) or PLAC (3.19 ± 0.1 vs. 3.20 ± 0.1 L/min) group (p > .05). In conclusion, 30 days of β-alanine supplementation had no effect on VO2 peak or VT in aerobically trained athletes.

  7. Monopeptide versus Monopeptoid: Insights on Structure and Hydration of Aqueous Alanine and Sarcosine via X-ray Absorption Spectroscopy

    SciTech Connect

    Uejio, Janel S.; Schwartz, Craig P.; Duffin, Andrew M.; England, Alice; Prendergast, David; Saykally, Richard J.

    2009-11-19

    Despite the obvious significance, the aqueous interactions of peptides remain incompletely understood. Their synthetic analogues called peptoids (poly-N-substituted glycines), have recently emerged as a promising biomimetic material, particularly due to their robust secondary structure and resistance to denaturation. We describe comparative near-edge x-ray absorption fine structure (NEXAFS) spectroscopy studies of aqueous sarcosine, the simplest peptoid, and alanine, its peptide isomer, interpreted by density functional theory calculations. The sarcosine nitrogen K-edge spectrum is blue-shifted with respect to that of alanine, in agreement with our calculations; we conclude that this shift results primarily from the methyl group substitution on the nitrogen of sarcosine. Our calculations indicate that the nitrogen K-edge spectrum of alanine differs significantly between dehydrated and hydrated scenarios, while that of the sarcosine zwitterion is less affected by hydration. In contrast, the computed sarcosine spectrum is greatly impacted by conformational variations, while the alanine spectrum is not. This relates to a predicted solvent dependence for alanine, as compared to sarcosine. Additionally, we show the theoretical nitrogen K-edge spectra to be sensitive to the degree of hydration, indicating that experimental X-ray spectroscopy may be able to distinguish between bulk and partial hydration, such as found in confined environments near proteins and in reverse micelles.

  8. 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.

  9. Crystal structure of quinone-dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates.

    PubMed

    Rozeboom, Henriëtte J; Yu, Shukun; Mikkelsen, Rene; Nikolaev, Igor; Mulder, Harm J; Dijkstra, Bauke W

    2015-12-01

    The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed β-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.

  10. Short Chain Dehydrogenase/Reductase Rdhe2 Is a Novel Retinol Dehydrogenase Essential for Frog Embryonic Development*

    PubMed Central

    Belyaeva, Olga V.; Lee, Seung-Ah; Adams, Mark K.; Chang, Chenbei; Kedishvili, Natalia Y.

    2012-01-01

    The enzymes responsible for the rate-limiting step in retinoic acid biosynthesis, the oxidation of retinol to retinaldehyde, during embryogenesis and in adulthood have not been fully defined. Here, we report that a novel member of the short chain dehydrogenase/reductase superfamily, frog sdr16c5, acts as a highly active retinol dehydrogenase (rdhe2) that promotes retinoic acid biosynthesis when expressed in mammalian cells. In vivo assays of rdhe2 function show that overexpression of rdhe2 in frog embryos leads to posteriorization and induction of defects resembling those caused by retinoic acid toxicity. Conversely, antisense morpholino-mediated knockdown of endogenous rdhe2 results in phenotypes consistent with retinoic acid deficiency, such as defects in anterior neural tube closure, microcephaly with small eye formation, disruption of somitogenesis, and curved body axis with bent tail. Higher doses of morpholino induce embryonic lethality. Analyses of retinoic acid levels using either endogenous retinoic acid-sensitive gene hoxd4 or retinoic acid reporter cell line both show that the levels of retinoic acid are significantly decreased in rdhe2 morphants. Taken together, these results provide strong evidence that Xenopus rdhe2 functions as a retinol dehydrogenase essential for frog embryonic development in vivo. Importantly, the retinol oxidizing activity of frog rdhe2 is conserved in its mouse homologs, suggesting that rdhe2-related enzymes may represent the previously unrecognized physiologically relevant retinol dehydrogenases that contribute to retinoic acid biosynthesis in higher vertebrates. PMID:22291023

  11. Crystal growth, structure and characterizations of a new semiorganic nonlinear optical material-{beta}-Alanine zinc chloride

    SciTech Connect

    Anbuchezhiyan, M.; Ponnusamy, S.; Muthamizhchelvan, C.; Sivakumar, K.

    2010-08-15

    The title compound, {beta}-alanine zinc chloride-a new semiorganic nonlinear optical crystal was grown by slow evaporation technique. Single crystals of {beta}-alanine zinc chloride have been subjected to X-ray diffraction analysis to determine the crystal structure. The powder X-ray diffractogram of the crystal has also been recorded. The amount of carbon, nitrogen and hydrogen in the crystals was also estimated. Fourier Transform Infrared and Raman spectral measurements have been carried out on the grown crystals in order to identify the functional groups. The presence of hydrogen and carbon in the {beta}-alanine zinc chloride was confirmed by using proton and carbon nuclear magnetic resonance spectral analyses. The percentage of zinc in the crystal was determined by atomic absorption spectroscopy. Optical behavior such as ultraviolet-vis-near infrared transmittance spectrum and second harmonic generation has been investigated. The mechanical strength and thermal behavior of the grown crystal have been analyzed.

  12. Ultraviolet radiation induces stress in etiolated Landoltia punctata, as evidenced by the presence of alanine, a universal stress signal: a ¹⁵N NMR study.

    PubMed

    Monselise, E B-I; Levkovitz, A; Kost, D

    2015-01-01

    Analysis with (15) N NMR revealed that alanine, a universal cellular stress signal, accumulates in etiolated duckweed plants exposed to 15-min pulsed UV light, but not in the absence of UV irradiation. The addition of 10 mm vitamin C, a radical scavenger, reduced alanine levels to zero, indicating the involvement of free radicals. Free D-alanine was detected in (15) N NMR analysis of the chiral amino acid content, using D-tartaric acid as solvent. The accumulation of D-alanine under stress conditions presents a new perspective on the biochemical processes taking place in prokaryote and eukaryote cells.

  13. Regulation of human dihydrodiol dehydrogenase by Michael acceptor xenobiotics.

    PubMed

    Ciaccio, P J; Jaiswal, A K; Tew, K D

    1994-06-01

    A human oxidoreductase (H-37) that is overexpressed in ethacrynic acid-resistant HT29 colon cells (Ciaccio, P. J., Stuart, J.E., and Tew, K.D. (1993) Mol. Pharmacol. 43, 845-853) has been identified as a dihydrodiol dehydrogenase. Translated protein from a dihydrodiol dehydrogenase cDNA isolated from a library prepared from ethacrynic acid-resistant HT29 cell poly(A+) RNA was recognized by anti-H-37 IgG and was identical in molecular weight with H-37. The isolated cDNA was identical in both nucleotide and amino acid sequences with the recently cloned liver dihydrodiol dehydrogenase (Stolz, A., Hammond, L., Lou, H., Takikawa, H., Ronk, M., and Shively, J.E. (1993) J. Biol. Chem. 268, 10448-10457). Using this cDNA as probe, we have examined its induction by Michael acceptors. The steady state dihydrodiol dehydrogenase mRNA level in the ethacrynic acid-resistant line was increased 30-fold relative to that of wild-type cells. Twenty-four hour treatment of wild-type cells with ethacrynic acid or dimethyl maleate increased mRNA 10-fold and 5-fold, respectively. These changes are accompanied by both increased protein expression and increased NADP-dependent 1-acenaphthenol oxidative activity in cell cytosol. In gel shift assays, compared to wild type controls, increased binding of NAD(P)H quinone oxidoreductase human antioxidant response element (hARE) DNA to redox labile protein complexes present in treated and resistant cell nuclear extract was observed. Ethacrynic acid induced CAT activity 2-fold in Hepa1 cells stably transfected with NAD(P)H quinone oxidoreductase hARE-tk-CAT chimeric gene construct. Thus, dihydrodiol dehydrogenase protein is inducible by de novo synthesis from mRNA by structurally related monofunctional inducer Michael acceptors. Altered in vitro binding of nuclear protein to the hARE is indirect evidence for the involvement of an element similar to hARE in the regulation of dihydrodiol dehydrogenase by these agents. PMID:7515059

  14. A structure-based design approach for the identification of novel inhibitors: application to an alanine racemase.

    PubMed

    Mustata, Gabriela Iurcu; Briggs, James M

    2002-12-01

    We report a new structure-based strategy for the identification of novel inhibitors. This approach has been applied to Bacillus stearothermophilus alanine racemase (AlaR), an enzyme implicated in the biosynthesis of the bacterial cell wall. The enzyme catalyzes the racemization of L- and D-alanine using pyridoxal 5'-phosphate (PLP) as a cofactor. The restriction of AlaR to bacteria and some fungi and the absolute requirement for D-alanine in peptidoglycan biosynthesis make alanine racemase a suitable target for drug design. Unfortunately, known inhibitors of alanine racemase are not specific and inhibit the activity of other PLP-dependent enzymes, leading to neurological and other side effects. This article describes the development of a receptor-based pharmacophore model for AllaR, taking into account receptor flexibility (i.e. a 'dynamic' pharmacophore model). In order to accomplish this, molecular dynamics (MD) simulations were performed on the full AlaR dimer from Bacillus stearothermophilus (PDB entry, 1 sft) with a D-alanine molecule in one active site and the non-covalent inhibitor, propionate, in the second active site of this homodimer. The basic strategy followed in this study was to utilize conformations of the protein obtained during MD simulations to generate a dynamic pharmacophore model using the property mapping capability of the LigBuilder program. Compounds from the Available Chemicals Directory that fit the pharmacophore model were identified and have been submitted for experimental testing. The approach described here can be used as a valuable tool for the design of novel inhibitors of other biomolecular targets.

  15. Site-directed mutagenesis provides insight into racemization and transamination of alanine catalyzed by Treponema denticola cystalysin.

    PubMed

    Cellini, Barbara; Bertoldi, Mariarita; Paiardini, Alessandro; D'Aguanno, Simona; Voltattorni, Carla Borri

    2004-08-27

    In addition to alpha, beta-elimination of L-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination. Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at Calpha/C4' of the external aldimine. In this investigation, two site-directed mutants, K238A and Y123F, have been characterized. The Lys --> Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward L-alanine. However, the K238A mutant is able to catalyze the overall transamination of D-alanine, and only D-alanine is the product of the reverse transamination. For Y123F the k(cat)/K(m) is reduced 3.5-fold for alpha, beta-elimination, whereas it is reduced 300-400-fold for racemization. Y123F has approximately 18% of wild type transaminase activity with L-alanine and an extremely low transaminase activity with D-alanine. Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124. All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor. Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face. PMID:15210695

  16. A mutant of Escherichia coli defective in penicillin-binding protein 5 and lacking D-alanine carboxypeptidase IA.

    PubMed Central

    Nishimura, Y; Suzuki, H; Hirota, Y; Park, J T

    1980-01-01

    A mutant of Escherichia coli defective in penicillin-binding protein 5 activity was isolated. The mutation (pfv) was shown to be located at 14.0 min on the E. coli chromosome map. Loss of penicillin-binding protein 5 in the pfv mutant was associated with the loss of D-alanine carboxypeptidase IA activity and increased sensitivity to beta-lactam antibiotics. We conclude that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity and that the enzyme activity, in vivo, protects E. coli cells from killing by low inhibitory concentrations of beta-lactam antibiotics. PMID:6995448

  17. High-temperature Raman study of L-alanine, L-threonine and taurine crystals related to thermal decomposition

    NASA Astrophysics Data System (ADS)

    Cavaignac, A. L. O.; Lima, R. J. C.; Façanha Filho, P. F.; Moreno, A. J. D.; Freire, P. T. C.

    2016-03-01

    In this work high-temperature Raman spectra are used to compare temperature dependence of the lattice mode wavenumber of L-alanine, L-threonine and taurine crystals. Anharmonic effects observed are associated with intermolecular N-H· · ·O hydrogen bond that plays an important role in thermal decomposition process of these materials. Short and strong hydrogen bonds in L-alanine crystal were associated with anharmonic effects in lattice modes leading to low thermal stability compared to taurine crystals. Connection between thermal decomposition process and anharmonic effects is furnished for the first time.

  18. The role of metal ions in chemical evolution - Polymerization of alanine and glycine in a cation-exchanged clay environment

    NASA Technical Reports Server (NTRS)

    Lawless, J. G.; Levi, N.

    1979-01-01

    The effect of the exchangeable cation on the condensation of glycine and alanine was investigated using a series of homoionic bentonites. A cycling procedure of drying, warming and wetting was employed. Peptide bond formation was observed, and the effectiveness of metal ions to catalyze the condensation was Cu(2+) greater than Ni(2) approximately equals Zn(2+) greater than Na(+). Glycine showed 6% of the monomer incorporated into oligomers with the largest detected being the pentamer. Alanine showed less peptide bond formation (a maximum of 2%) and only the dimer was observed.

  19. Growth, Structural And Optical Studies On Bis L-alanine Lithium Chloride (BLALC) Single Crystal

    NASA Astrophysics Data System (ADS)

    Rose, A. S. J. Lucia; Selvarajan, P.; Perumal, S.

    2011-10-01

    Bis L-alanine Lithium Chloride (BLALC) single crystals were grown successfully by solution method with slow evaporation technique at room temperature. Crystals of size 15 x 9 x 4 mm3 have been obtained in 28 days. The grown crystals were colourless and transparent. Single crystal X-ray diffraction (XRD) study showed that BLALC belongs to orthorhombic system with a non-centro-symmetric space group P212121. The crystallinity of BLALC crystal was confirmed by the powder X-ray diffraction study and diffraction peaks were indexed. The functional groups of the grown crystals have been identified by FTIR studies. UV-visible transmittance spectrum was recorded to study the optical transparency of BLALC crystal. The nonlinear optical (NLO) property of the grown crystal was confirmed by Kurtz-Perry powder technique.

  20. The influence of various cations on the catalytic properties of clays. [polymerization of alanine adenylate

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1978-01-01

    The polymerization of alanine adenylate in the presence of the sodium form of various clays was studied, and hectorite was found to cause more polymerization than nontronite and montmorillonite (in that order) although the differences were not great. The effect on polymerization of presaturating montmorillonite with different cations was determined. Hectorite, with increased basicity of the interspatial planes, allows polymerization of lysine, which montmorillonite does not. The general trend is that, for the same amino acid, higher degrees of polymerization are obtained when the cation in the octahedral lattice of the clay is divalent rather than trivalent. With the exchangeable cations the order is reversed, for a reason that is explained. The main role of clays in the polymerization mechanism of amino acids is concentration and neutralization of charges.

  1. Spectrofluorimetric determination of ofloxacin in milk with N-(9-fluorenylmethyloxycarbonyl)-L-alanine.

    PubMed

    Tong, Zhao; Bianfei, Yu; Wanjin, Tang; Haixia, Zhang

    2015-09-01

    It was found that N-(9-fluorenylmethyloxycarbonyl)-L-alanine (F-Ala) could interact with ofloxacin (OFL) and enhance the fluorescence of OFL, which could be used to set up a new fluorescence spectrophotometry method to detect OFL without isolation procedure. The experimental conditions were discussed and when MeOH was used as solvent, 0.001 μg mL(-1) of OFL could be detected. For making the method could be used easily in milk samples, MeOH/H2O (v/v, 4:1, pH 4.1) was used as the optimal solvent condition, the limit of quantification of OFL could be reached 0.01 μg mL(-1) in milk samples with recovery more than 88% at least.

  2. Thiophenyl-substituted triazolyl-thione L-alanine: asymmetric synthesis, aggregation and biological properties.

    PubMed

    Saghyan, Ashot S; Simonyan, Hayarpi M; Petrosyan, Satenik G; Geolchanyan, Arpine V; Roviello, Giovanni N; Musumeci, Domenica; Roviello, Valentina

    2014-10-01

    In this work, we report the asymmetric synthesis and characterization of an artificial amino acid based on triazolyl-thione L-alanine, which was modified with a thiophenyl-substituted moiety, as well as in vitro studies of its nucleic acid-binding ability. We found, by dynamic light scattering studies, that the synthetic amino acid was able to form supramolecular aggregates having a hydrodynamic diameter higher than 200 nm. Furthermore, we demonstrated, by UV and CD experiments, that the heteroaromatic amino acid, whose enzymatic stability was demonstrated by HPLC analysis also after 24 h of incubation in human serum, was able to bind a RNA complex, which is a feature of biomedical interest in view of innovative antiviral strategies based on modulation of RNA-RNA molecular recognition.

  3. Discovery of an L-alanine ester prodrug of the Hsp90 inhibitor, MPC-3100.

    PubMed

    Kim, Se-Ho; Tangallapally, Rajendra; Kim, In Chul; Trovato, Richard; Parker, Daniel; Patton, J Scott; Reeves, Leslie; Bradford, Chad; Wettstein, Daniel; Baichwal, Vijay; Papac, Damon; Bajji, Ashok; Carlson, Robert; Yager, Kraig M

    2015-11-15

    Various types of Hsp90 inhibitors have been and continue to undergo clinical investigation. One development candidate is the purine-based, synthetic Hsp90 inhibitor 1 (MPC-3100), which successfully completed a phase I clinical study. However, further clinical development of 1 was hindered by poor solubility and consequent formulation issues and promoted development of a more water soluble prodrug. Towards this end, numerous pro-moieties were explored in vitro and in vivo. These studies resulted in identification of L-alanine ester mesylate, 2i (MPC-0767), which exhibited improved aqueous solubility, adequate chemical stability, and rapid bioconversion without the need for solubilizing excipients. Based on improved physical characteristics and favorable PK and PD profiles, 2i mesylate was selected for further development. A convergent, scalable, chromatography-free synthesis for 2i mesylate was developed to support further clinical evaluation.

  4. Alanine-Scanning Mutational Analysis of Durancin GL Reveals Residues Important for Its Antimicrobial Activity.

    PubMed

    Ju, Xingrong; Chen, Xinquan; Du, Lihui; Wu, Xueyou; Liu, Fang; Yuan, Jian

    2015-07-22

    Durancin GL is a novel class IIa bacteriocin with 43 residues produced by Enterococcus durans 41D. This bacteriocin demonstrates narrow inhibition spectrum and potent antimicrobial activity against several Listeria monocytogenes strains, including nisin-resistant L. monocytogenes NR30. A systematic alanine-scanning mutational analysis with site-directed mutagenesis was performed to analyze durancin GL residues important for antimicrobial activity and specificity. Results showed that three mutations lost their antimicrobial activity, ten mutations demonstrated a decreased effect on the activity, and seven mutations exhibited relatively high activity. With regard to inhibitory spectrum, four mutants demonstrated a narrower antimicrobial spectrum than wild-type durancin GL. Another four mutants displayed a broader target cell spectrum and increased potency relative to wild-type durancin GL. These findings broaden our understanding of durancin GL residues important for its antimicrobial activity and contribute to future rational design of variants with increased potency.

  5. Pyridyl-alanine as a Hydrophilic, Aromatic Element in Peptide Structural Optimization.

    PubMed

    Mroz, Piotr A; Perez-Tilve, Diego; Liu, Fa; Gelfanov, Vasily; DiMarchi, Richard D; Mayer, John P

    2016-09-01

    Glucagon (Gcg) 1 serves a seminal physiological role in buffering against hypoglycemia, but its poor biophysical properties severely complicate its medicinal use. We report a series of novel glucagon analogues of enhanced aqueous solubility and stability at neutral pH, anchored by Gcg[Aib16]. Incorporation of 3- and 4-pyridyl-alanine (3-Pal and 4-Pal) enhanced aqueous solubility of glucagon while maintaining biological properties. Relative to native hormone, analogue 9 (Gcg[3-Pal6,10,13, Aib16]) demonstrated superior biophysical character, better suitability for medicinal purposes, and comparable pharmacology against insulin-induced hypoglycemia in rats and pigs. Our data indicate that Pal is a versatile surrogate to natural aromatic amino acids and can be employed as an alternative or supplement with isoelectric adjustment to refine the biophysical character of peptide drug candidates. PMID:27509198

  6. Alanine-Scanning Mutational Analysis of Durancin GL Reveals Residues Important for Its Antimicrobial Activity.

    PubMed

    Ju, Xingrong; Chen, Xinquan; Du, Lihui; Wu, Xueyou; Liu, Fang; Yuan, Jian

    2015-07-22

    Durancin GL is a novel class IIa bacteriocin with 43 residues produced by Enterococcus durans 41D. This bacteriocin demonstrates narrow inhibition spectrum and potent antimicrobial activity against several Listeria monocytogenes strains, including nisin-resistant L. monocytogenes NR30. A systematic alanine-scanning mutational analysis with site-directed mutagenesis was performed to analyze durancin GL residues important for antimicrobial activity and specificity. Results showed that three mutations lost their antimicrobial activity, ten mutations demonstrated a decreased effect on the activity, and seven mutations exhibited relatively high activity. With regard to inhibitory spectrum, four mutants demonstrated a narrower antimicrobial spectrum than wild-type durancin GL. Another four mutants displayed a broader target cell spectrum and increased potency relative to wild-type durancin GL. These findings broaden our understanding of durancin GL residues important for its antimicrobial activity and contribute to future rational design of variants with increased potency. PMID:26168032

  7. Experimental and DFT computational studies of L-alanine cadmium chloride crystals

    NASA Astrophysics Data System (ADS)

    Ignatius, I. Cicili; Dheivamalar, S.; Kirubavathi, K.; Selvaraju, K.

    2016-05-01

    In this work, we report the combined experimental and theoretical study on molecular structure and vibrational spectra of nonlinear optical crystal L-alanine cadmium chloride (LACC). The single X-ray diffraction studies have revealed that the compound crystallizes in monoclinic system C2 space group with cell parameters a = 16.270, b = 7.358, c = 7.887 and Z = 4. FTIR and Raman spectra of the nonlinear optical materials LACC have been recorded and analyzed. The optimized geometric bond length and bond angles are obtained with the help of density functional theory (DFT) (B3LYP) calculation. The optimized geometric bond lengths and bond angles obtained by using DFT show good agreement with the experimental data. Using the natural bond orbital analysis the electronic effect and hydrogen bonding were confirmed. The HOMO-LUMO energy gap and the first order hyperpolarizability were calculated and it supports the nonlinear optical activity of LACC crystal.

  8. Familial amyloid polyneuropathy: alanine-for-threonine substitution in the transthyretin (prealbumin) molecule.

    PubMed

    Koeppen, A H; Wallace, M R; Benson, M D; Altland, K

    1990-11-01

    A previously reported family with amyloid polyneuropathy (FAP) was reinvestigated to determine the type of mutation in the transthyretin (prealbumin) molecule. Transthyretin was isolated from amyloid-laden myocardium and serum, and tryptic peptides were resolved by high-performance liquid chromatography. Amino acid sequencing of an anomalous peptide revealed an alanine-for-threonine substitution corresponding to position No. 60 of the transthyretin monomer. Detection of the FAP gene in asymptomatic carriers was accomplished by hybrid isoelectric focusing of transthyretin in the presence of dithiothreitol and high concentrations of urea, and by Southern blotting of Pvull-digested leukocyte deoxyribonucleic acid. This type of FAP was found to be identical to the previously described Appalachian amyloid. Patients with FAP and their asymptomatic gene-carrying offspring had significantly reduced levels of serum transthyretin and retinol-binding protein.

  9. 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.

  10. Controlled radical polymerization of an acrylamide containing L-alanine moiety via ATRP.

    PubMed

    Rafiee, Zahra

    2016-02-01

    Homopolymerization of an optically active acrylamide having an amino acid moiety in the side chain, N-acryloyl-L-alanine (AAla) was carried out via atom transfer radical polymerization (ATRP) at room temperature using 2-hydroxyethyl-2'-methyl-2'-bromopropionate (HMB) or sodium-4-(bromomethyl)benzoate (SBB) as initiator in pure water, methanol/water mixture and pure methanol solvents. The polymerization reaction resulted in the optically active biocompatible amino acid-based homopolymer in good yield with narrow molecular weight distribution. The number average molecular weight increased with conversion and polydispersity was low. The structure and molecular weight of synthesized polymer were characterized by (1)H NMR, FT-IR spectroscopic techniques and size-exclusion chromatography.

  11. Synthesis of thiocarbamide derivatives of. beta. -alanine and aminalon and study of their antiradiation action

    SciTech Connect

    Golubev, A.A.; Shlykov, Yu.V.; Mandrugin, A.A.; Semenenko, M.N.; Fedoseev, V.M.; Dontsova, G.V.; Rakhmanina, O.N.; Konstantinova, M.M.

    1987-01-01

    The authors have synthesized the carboxyl derivatives of S-(2-Amino-ethyl)isothiourea (AET) and S-(3-Aminopropyl)isothiourea (3-APT), the dihydrobromides of S-(1-carboxy-2-aminoethyl)- and S-(1-carboxy-3-aminopropyl)isothioureas, and studied their toxicity and antiradiation effectiveness. Their work is based on data for the high radiation-protective activity of the corresponding aminoalkylisothioureas and on the fact that the introduction of the carboxylic group into the molecule influences the pharmacological properties of a compound. The starting compound for the preparation of S(1-carboxy-2-aminoethyl)isothiourea was beta-alanine. Mice were used in the investigations and in determining the toxicity of the compounds, the amounts of LD/sub 16/, LD/sub 50/, and LD/sub 84/ are given. In studying the anti-radiation activity, the mice were subjected to the action of /sup 60/Co-gamma rays.

  12. Spectrofluorimetric determination of ofloxacin in milk with N-(9-fluorenylmethyloxycarbonyl)-L-alanine

    NASA Astrophysics Data System (ADS)

    Tong, Zhao; Bianfei, Yu; Wanjin, Tang; Haixia, Zhang

    2015-09-01

    It was found that N-(9-fluorenylmethyloxycarbonyl)-L-alanine (F-Ala) could interact with ofloxacin (OFL) and enhance the fluorescence of OFL, which could be used to set up a new fluorescence spectrophotometry method to detect OFL without isolation procedure. The experimental conditions were discussed and when MeOH was used as solvent, 0.001 μg mL-1 of OFL could be detected. For making the method could be used easily in milk samples, MeOH/H2O (v/v, 4:1, pH 4.1) was used as the optimal solvent condition, the limit of quantification of OFL could be reached 0.01 μg mL-1 in milk samples with recovery more than 88% at least.

  13. Reanalysis of the effects of phenylalanine, alanine, and aspartame on food intake in human subjects.

    PubMed

    Rogers, P J; Blundell, J E

    1994-08-01

    In 1987 Ryan-Harshman et al. reported finding no effects on food intake after administering high doses (up to 10.08 g) of phenylalanine and aspartame in capsules to human volunteers. However, this is contrary to the results of other studies, and trends in their tabulated data suggest that certain effects may have been overlooked. This is confirmed by a reanalysis of the raw data (available from a Ph.D. thesis: Ryan-Harshman, 1987) that can be interpreted as showing a dose-related suppression of food intake by phenylalanine. Furthermore, the data are consistent with an anorexic action of aspartame and perhaps also of alanine (which was designated as the placebo treatment by Ryan-Harshman et al.). These, together with other findings, suggest that the appetite effects of amino acids and small peptides should be investigated further. In addition to its theoretical importance, such work may have potential for therapeutic applications.

  14. 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

  15. Anomalous behaviour of yeast isocitrate dehydrogenase during isoelectric focusing

    PubMed Central

    Illingworth, John A.

    1972-01-01

    Isoelectric focusing of yeast isocitrate dehydrogenase apparently reveals a number of `isoenzymes'. These have isoelectric points near pH5.5 in crude material, but during purification the mean isoelectric point progressively rises to pH7.0 and the band pattern changes. The shift in isoelectric point during purification is apparently genuine, since it is also manifested in the electrophoretic and chromatographic properties of the enzyme. The multiple forms, however, are an artifact, generated by exposure of the enzyme to Ampholine, since their activities vary with the protein/Ampholine ratio and they cannot be observed in any system from which Ampholine is excluded. There are no detectable isoenzymes of yeast isocitrate dehydrogenase. PMID:4571177

  16. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

    NASA Astrophysics Data System (ADS)

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

    2015-07-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

  17. Theoretical analysis of the glutamate dehydrogenase kinetics under physiological conditions.

    PubMed

    Popova, S V; Reich, J G

    1983-01-01

    A kinetic model of the glutamate dehydrogenase reaction has been formulated for the reversible reaction including all seven reactants (substrates and cofactors NAD(H) and NADP(H)). The model parameters have been evaluated from published initial-rate data. Analysis of the model at cofactor concentration near to that in the intact mitochondrion has shown that the competition for active sites between cofactors and substrates simultaneously present in mitochondria diminishes the steady-state rate of the reaction by a factor of 10 to 100 as compared to the maximal reaction rate. The model predicts near-equilibrium of the reaction substrates with NAD+/NADH cofactor pair and off-equilibrium with NADP+/NADPH. Substrate cycling with futile transfer of hydrogen from NADP+-system to NAD+-system has been found to account under in vivo conditions for no more than 2% of the maximal glutamate dehydrogenase activity in the mitochondria.

  18. Alanine screening mutagenesis establishes the critical inactivating damage of irradiated E. coli lactose repressor.

    PubMed

    Goffinont, Stephane; Villette, Sandrine; Spotheim-Maurizot, Melanie

    2012-06-01

    The function of the E. coli lactose operon requires the binding of lactose repressor to operator DNA. We have previously shown that γ rradiation destabilizes the repressor-operator complex because the repressor loses its DNA-binding ability. It was suggested that the observed oxidation of the four tyrosines (Y7, Y12, Y17, Y47) and the concomitant structural changes of the irradiated DNA-binding domains (headpieces) could be responsible for the inactivation. To pinpoint the tyrosine whose oxidation has the strongest effect, four headpieces containing the product of tyrosine oxidation, 3,4-dihydroxyphenylalanine (DOPA), were simulated by molecular dynamics. We have observed that replacing Y47 by DOPA triggers the largest change of structure and stability of the headpiece and have concluded that Y47 oxidation is the greatest contributor to the decrease of repressor binding to DNA. To experimentally verify this conclusion, we applied the alanine screening mutagenesis approach. Tetrameric mutated repressors bearing an alanine instead of each one of the tyrosines were prepared and their binding to operator DNA was checked. Their binding ability is quite similar to that of the wild-type repressor, except for the Y47A mutant whose binding is strongly reduced. Circular dichroism determinations revealed small reductions of the proportion of α helices and of the melting temperature for Y7A, Y12A and Y17A headpieces, but much larger ones were revealed for Y47A headpiece. These results established the critical role of Y47 oxidation in modifying the structure and stability of the headpiece, and in reduction of the binding ability of the whole lactose repressor. PMID:22551504

  19. Alanine and proline content modulate global sensitivity to discrete perturbations in disordered proteins.

    PubMed

    Perez, Romel B; Tischer, Alexander; Auton, Matthew; Whitten, Steven T

    2014-12-01

    Molecular transduction of biological signals is understood primarily in terms of the cooperative structural transitions of protein macromolecules, providing a mechanism through which discrete local structure perturbations affect global macromolecular properties. The recognition that proteins lacking tertiary stability, commonly referred to as intrinsically disordered proteins (IDPs), mediate key signaling pathways suggests that protein structures without cooperative intramolecular interactions may also have the ability to couple local and global structure changes. Presented here are results from experiments that measured and tested the ability of disordered proteins to couple local changes in structure to global changes in structure. Using the intrinsically disordered N-terminal region of the p53 protein as an experimental model, a set of proline (PRO) and alanine (ALA) to glycine (GLY) substitution variants were designed to modulate backbone conformational propensities without introducing non-native intramolecular interactions. The hydrodynamic radius (R(h)) was used to monitor changes in global structure. Circular dichroism spectroscopy showed that the GLY substitutions decreased polyproline II (PP(II)) propensities relative to the wild type, as expected, and fluorescence methods indicated that substitution-induced changes in R(h) were not associated with folding. The experiments showed that changes in local PP(II) structure cause changes in R(h) that are variable and that depend on the intrinsic chain propensities of PRO and ALA residues, demonstrating a mechanism for coupling local and global structure changes. Molecular simulations that model our results were used to extend the analysis to other proteins and illustrate the generality of the observed PRO and alanine effects on the structures of IDPs.

  20. Effect of lysine to alanine mutations on the phosphate activation and BPTES inhibition of glutaminase.

    PubMed

    McDonald, Charles J; Acheff, Eric; Kennedy, Ryan; Taylor, Lynn; Curthoys, Norman P

    2015-09-01

    The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme. These two segments also contain the binding sites for bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide (BPTES), a highly specific and potent uncompetitive inhibitor of this glutaminase. BPTES is also the lead compound for development of novel cancer chemotherapeutic agents. To provide a preliminary assessment of the potential effects of N-acetylation, the corresponding lysine to alanine mutations were constructed in the hGACΔ1 plasmid. The wild type and mutated proteins were purified by Ni(+)-affinity chromatography and their phosphate activation and BPTES inhibition profiles were analyzed. Two of the alanine substitutions in the loop segment (K311A and K328A) and the one in the dimer:dimer interface (K396A) form enzymes that require greater concentrations of phosphate to produce half-maximal activation and exhibit greater sensitivity to BPTES inhibition. By contrast, the K320A mutation results in a glutaminase that exhibits near maximal activity in the absence of phosphate and is not inhibited by BPTES. Thus, lysine N-acetylation may contribute to the acute regulation of glutaminase activity in various tissues and alter the efficacy of BPTES-type inhibitors.

  1. Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning.

    PubMed

    Peigneur, Steve; Yamaguchi, Yoko; Kawano, Chihiro; Nose, Takeru; Nirthanan, Selvanayagam; Gopalakrishnakone, Ponnampalam; Tytgat, Jan; Sato, Kazuki

    2016-05-31

    Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency. PMID:27159046

  2. β-Alanine supplementation slightly enhances repeated plyometric performance after high-intensity training in humans.

    PubMed

    Carpentier, Alain; Olbrechts, Naïma; Vieillevoye, Stéphanie; Poortmans, Jacques R

    2015-07-01

    β-Alanine (BA) supplementation has become an ergogenic aid amongst competitive athletes to augment intramuscular carnosine content, leading to higher buffer capacity and exercise performance. We investigated 27 regularly trained young males and females who were randomly allocated either to placebo (PL) or BA ingestion for 8 weeks. Every single day, BA or PL (4.0-5.6 g day(-1)) supplements were ingested by participants and associated with a strong plyometric high-intensity training (two sessions per week during the 8 weeks). Before and after training, maximal jump heights were recorded during squat jump (SJ) and countermovement jump (CMJ) and an index of fatigue was recorded as a mean height of 45 consecutive CMJ. Blood lactate was measured at rest, after completing the fatigue test and every 5 min thereafter up to 30 min recovery. After plyometric training, SJ and CMJ were increased, respectively, by 8.8 and 6.4 % in PL group and 9.9 and 11.0 % in BA group (p < 0.01, no difference between groups). Blood lactate reached a maximal value of 9.4 ± 1.6 mmol l(-1) in PL group, and 10.3 ± 1.3 mmol l(-1) in BA group, with a slight better performance in the fatigue test (+8.6 %, p ≤ 0.01) for BA group as compared to PL group. To conclude, 2-month β-alanine supplementation resulted in a slight improvement of explosive force after 45 maximal consecutive jumps in young athletes. However, the practical adequacy of supplementation remains questionable in an active and healthy population.

  3. Chiral selectivity of amino acid adsorption on chiral surfaces—The case of alanine on Pt

    SciTech Connect

    Franke, J.-H.; Kosov, D. S.

    2015-02-07

    We study the binding pattern of the amino acid alanine on the naturally chiral Pt surfaces Pt(531), Pt(321), and Pt(643). These surfaces are all vicinal to the (111) direction but have different local environments of their kink sites and are thus a model for realistic roughened Pt surfaces. Alanine has only a single methyl group attached to its chiral center, which makes the number of possible binding conformations computationally tractable. Additionally, only the amine and carboxyl group are expected to interact strongly with the Pt substrate. On Pt(531), we study the molecule in its pristine as well as its deprotonated form and find that the deprotonated one is more stable by 0.47 eV. Therefore, we study the molecule in its deprotonated form on Pt(321) and Pt(643). As expected, the oxygen and nitrogen atoms of the deprotonated molecule provide a local binding “tripod” and the most stable adsorption configurations optimize the interaction of this “tripod” with undercoordinated surface atoms. However, the interaction of the methyl group plays an important role: it induces significant chiral selectivity of about 60 meV on all surfaces. Hereby, the L-enantiomer adsorbs preferentially to the Pt(321){sup S} and Pt(643){sup S} surfaces, while the D-enantiomer is more stable on Pt(531){sup S}. The binding energies increase with increasing surface density of kink sites, i.e., they are largest for Pt(531){sup S} and smallest for Pt(643){sup S}.

  4. Exercise-induced oxidative stress: the effects of β-alanine supplementation in women.

    PubMed

    Smith, A E; Stout, J R; Kendall, K L; Fukuda, D H; Cramer, J T

    2012-07-01

    The purpose of this study was to evaluate the effects of β-alanine supplementation on markers of oxidative stress. Twenty-four women (age: 21.7±2.1 years; VO2max: 2.6±0.3 l min(-1)) were randomly assigned, in a double-blind fashion, to a β-alanine (BA, 2×800 mg tablets, 3× daily; CarnoSyn®; n=13) or placebo (PL, 2×800 mg maltodextrin tablets, 3× daily; n=11) group. A graded oxygen consumption test (VO2max) was performed to evaluate VO2max, time to exhaustion, ventilatory threshold and establish peak velocity (PV). A 40-min treadmill run was used to induce oxidative stress. Total antioxidant capacity, superoxide dismutase, 8-isoprostane (8ISO) and reduced glutathione were measured. Heart rate and ratings of perceived exertion were recorded during the 40 min run. Separate three- [4×2×2; acute (base vs. IP vs. 2 vs. 4 h)×chronic (pre- vs. post-)×treatment (BA vs. PL)] and two- [2×2; time (pre-supplement vs. post-supplement)×treatment (BA vs. PL)] way ANOVAs were used for analyses. There was a significant increase in VO2max (p=0.009), independent of treatment, with no significant changes in TTE (p=0.074) or VT (p=0.344). Ratings of perceived exertion values were significantly improved from pre- to post-supplementation for the BA group only at 40 min (p=0.02). The ANOVA model demonstrated no significant treatment effects on oxidative stress. The chronic effects of BA supplementation demonstrated little antioxidant potential, in women, and little influence on aerobic performance assessments.

  5. Ribitol dehydrogenase from Klebsiella aerogenes. Purification and subunit structure

    PubMed Central

    Taylor, Susan S.; Rigby, Peter W. J.; Hartley, Brian S.

    1974-01-01

    Ribitol dehydrogenase has been purified to homogeneity from several strains of Klebsiella aerogenes. One strain yields 3–6g of pure enzyme from 1kg of cells. The enzyme is a tetramer of four subunits, mol.wt. 27000. Preliminary studies of the activity of the enzyme are reported. Peptide `maps' together with the amino acid composition indicate that the subunits are identical. ImagesPLATE 2PLATE 1 PMID:4618776

  6. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria.

    PubMed

    Watanabe, Seiya; Sueda, Rui; Fukumori, Fumiyasu; Watanabe, Yasuo

    2015-01-01

    Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by "subunit-exchange". To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase. PMID:26382958

  7. Hydroxysteroid dehydrogenases (HSDs) in bacteria: a bioinformatic perspective.

    PubMed

    Kisiela, Michael; Skarka, Adam; Ebert, Bettina; Maser, Edmund

    2012-03-01

    Steroidal compounds including cholesterol, bile acids and steroid hormones play a central role in various physiological processes such as cell signaling, growth, reproduction, and energy homeostasis. Hydroxysteroid dehydrogenases (HSDs), which belong to the superfamily of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR), are important enzymes involved in the steroid hormone metabolism. HSDs function as an enzymatic switch that controls the access of receptor-active steroids to nuclear hormone receptors and thereby mediate a fine-tuning of the steroid response. The aim of this study was the identification of classified functional HSDs and the bioinformatic annotation of these proteins in all complete sequenced bacterial genomes followed by a phylogenetic analysis. For the bioinformatic annotation we constructed specific hidden Markov models in an iterative approach to provide a reliable identification for the specific catalytic groups of HSDs. Here, we show a detailed phylogenetic analysis of 3α-, 7α-, 12α-HSDs and two further functional related enzymes (3-ketosteroid-Δ(1)-dehydrogenase, 3-ketosteroid-Δ(4)(5α)-dehydrogenase) from the superfamily of SDRs. For some bacteria that have been previously reported to posses a specific HSD activity, we could annotate the corresponding HSD protein. The dominating phyla that were identified to express HSDs were that of Actinobacteria, Proteobacteria, and Firmicutes. Moreover, some evolutionarily more ancient microorganisms (e.g., Cyanobacteria and Euryachaeota) were found as well. A large number of HSD-expressing bacteria constitute the normal human gastro-intestinal flora. Another group of bacteria were originally isolated from natural habitats like seawater, soil, marine and permafrost sediments. These bacteria include polycyclic aromatic hydrocarbons-degrading species such as Pseudomonas, Burkholderia and Rhodococcus. In conclusion, HSDs are found in a wide variety of microorganisms including

  8. R-lipoic acid inhibits mammalian pyruvate dehydrogenase kinase.

    PubMed

    Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

    2004-10-01

    The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects. PMID:15512796

  9. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria

    PubMed Central

    Watanabe, Seiya; Sueda, Rui; Fukumori, Fumiyasu; Watanabe, Yasuo

    2015-01-01

    Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase. PMID:26382958

  10. GMXPBSA 2.0: A GROMACS tool to perform MM/PBSA and computational alanine scanning

    NASA Astrophysics Data System (ADS)

    Paissoni, C.; Spiliotopoulos, D.; Musco, G.; Spitaleri, A.

    2014-11-01

    GMXPBSA 2.0 is a user-friendly suite of Bash/Perl scripts for streamlining MM/PBSA calculations on structural ensembles derived from GROMACS trajectories, to automatically calculate binding free energies for protein-protein or ligand-protein complexes. GMXPBSA 2.0 is flexible and can easily be customized to specific needs. Additionally, it performs computational alanine scanning (CAS) to study the effects of ligand and/or receptor alanine mutations on the free energy of binding. Calculations require only for protein-protein or protein-ligand MD simulations. GMXPBSA 2.0 performs different comparative analysis, including a posteriori generation of alanine mutants of the wild-type complex, calculation of the binding free energy values of the mutant complexes and comparison of the results with the wild-type system. Moreover, it compares the binding free energy of different complexes trajectories, allowing the study the effects of non-alanine mutations, post-translational modifications or unnatural amino acids on the binding free energy of the system under investigation. Finally, it can calculate and rank relative affinity to the same receptor utilizing MD simulations of proteins in complex with different ligands. In order to dissect the different MM/PBSA energy contributions, including molecular mechanic (MM), electrostatic contribution to solvation (PB) and nonpolar contribution to solvation (SA), the tool combines two freely available programs: the MD simulations software GROMACS and the Poisson-Boltzmann equation solver APBS. All the calculations can be performed in single or distributed automatic fashion on a cluster facility in order to increase the calculation by dividing frames across the available processors. The program is freely available under the GPL license. Catalogue identifier: AETQ_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AETQ_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, N. Ireland Licensing

  11. Determination of β-Cyano-L-alanine, γ-Glutamyl-β-cyano-L-alanine, and Common Free Amino Acids in Vicia sativa (Fabaceae) Seeds by Reversed-Phase High-Performance Liquid Chromatography

    PubMed Central

    Megías, Cristina; Cortés-Giraldo, Isabel; Girón-Calle, Julio; Vioque, Javier; Alaiz, Manuel

    2014-01-01

    A method for determination of β-cyano-L-alanine, γ-glutamyl-β-cyano-L-alanine and other free amino acids in Vicia sativa is presented. Seed extracts were derivatized by reaction with diethyl ethoxymethylenemalonate and analyzed by reverse-phase high-performance liquid chromatography. Calibration curves showed very good linearity of the response. The limit of detection and quantification was 0.15 and 0.50 μM, respectively. The method has high intra- (RSD = 0.28–0.31%) and interrepeatability (RSD = 2.76–3.08%) and remarkable accuracy with a 99% recovery in spiked samples. The method is very easy to carry out and allows for ready analysis of large number of samples using very basic HPLC equipment because the derivatized samples are very stable and have very good chromatographic properties. The method has been applied to the determination of γ-glutamyl-β-cyano-L-alanine, β-cyano-L-alanine, and common free amino acids in eight wild populations of V. sativa from southwestern Spain. PMID:25587488

  12. 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

  13. Delineation of an in vivo inhibitor for Aspergillus glutamate dehydrogenase.

    PubMed

    Choudhury, Rajarshi; Noor, Shahid; Varadarajalu, Lakshmi Prabha; Punekar, Narayan S

    2008-01-01

    NADP-glutamate dehydrogenase (NADP-GDH) along with glutamine synthetase plays a pivotal role in ammonium assimilation. Specific inhibitors were valuable in defining the importance of glutamine synthetase in nitrogen metabolism. Selective in vivo inhibition of NADP-GDH has so far been an elusive desideratum. Isophthalate, a potent in vitro inhibitor of Aspergillus niger NADP-GDH [Noor S, Punekar NS. Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon-nitrogen interface. Microbiology 2005;151:1409-19], was evaluated for its efficacy in vivo. Dimethyl ester of isophthalate (DMIP), but not isophthalate, inhibited A. niger growth on agar as well as in liquid culture. This was ascribed to the inability of isophthalate to enter fungal mycelia. Subsequent to DMIP addition however, intracellular isophthalate could be demonstrated. Apart from NAD-GDH, no other enzyme including NAD-glutamate synthase was inhibited by isophthalate. A cross-over at NADP-GDH step of metabolism was observed as a direct consequence of isophthalate (formed in vivo from DMIP) inhibiting this enzyme. Addition of ammonium to DMIP-treated A. niger mycelia resulted in intensive vacuolation, retraction of cytoplasm and autolysis. Taken together, these results implicate glutamate dehydrogenase and NADP-GDH in particular, as a key target of in vivo isophthalate inhibition during ammonium assimilation. PMID:22578865

  14. Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

    SciTech Connect

    White, Tommi A.; Tanner, John J.

    2005-08-01

    Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ{sup 1}-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  15. Making biochemistry count: life among the amino acid dehydrogenases.

    PubMed

    Engel, Paul C

    2011-04-01

    The guiding principle of the IAS Medal Lecture and of the research it covered was that searching mathematical analysis, depending on good measurements, must underpin sound biochemical conclusions. This was illustrated through various experiences with the amino acid dehydrogenases. Topics covered in the present article include: (i) the place of kinetic measurement in assessing the metabolic role of GDH (glutamate dehydrogenase); (ii) the discovery of complex regulatory behaviour in mammalian GDH, involving negative co-operativity in coenzyme binding; (iii) an X-ray structure solution for a bacterial GDH providing insight into catalysis; (iv) almost total positive co-operativity in glutamate binding to clostridial GDH; (v) unexpected outcomes with mutations at the catalytic aspartate site in GDH; (vi) reactive cysteine as a counting tool in the construction of hybrid oligomers to probe the basis of allosteric interaction; (vii) tryptophan-to-phenylalanine mutations in analysis of allosteric conformational change; (viii) site-directed mutagenesis to alter substrate specificity in GDH and PheDH (phenylalanine dehydrogenase); and (ix) varying strengths of binding of the 'wrong' enantiomer in engineered mutant enzymes and implications for resolution of racemates.

  16. Characterization of a cellobiose dehydrogenase from Humicola insolens.

    PubMed Central

    Schou, C; Christensen, M H; Schülein, M

    1998-01-01

    The major cellobiose dehydrogenase (oxidase) (CBDH) secreted by the soft-rot thermophilic fungus Humicola insolens during growth on cellulose has been isolated and purified. It was shown to be a haemoflavoprotein with a molecular weight of 92 kDa and a pI of 4.0, capable of oxidizing the anomeric carbon of cellobiose, soluble cellooligosaccharides, lactose, xylobiose and maltose. Possible electron acceptors are 2,6-dichlorophenol-indophenol (DCPIP), Methylene Blue, 3,5-di-t-butyl-1,2-benzoquinone, potassium ferricyanide, cytochrome c and molecular oxygen. The oxidation of the prosthetic groups by oxygen was monitored at 449 nm for the flavin group and at 562 nm for the haem group. The curves were very similar to those of the cellobiose dehydrogenase from Phanerochaete chrysosporium, suggesting a similar mechanism. The pH-optima for the oxidation varied remarkably depending on the electron acceptor. For the organic electron acceptors, the pH-optima ranged from pH 4 for Methylene Blue to pH 7 for DCPIP and the benzoquinone. In the case of the FeIII-containing electron acceptors, the enzyme displayed alkaline pH-optima, in contrast to the properties of cellobiose dehydrogenases from Phanerochaete chrysosporium and Myceliophthora (Sporotrichum) thermophila. The enzyme has optimal activity at 65 degrees C. PMID:9461557

  17. Functional Analysis of a Mosquito Short Chain Dehydrogenase Cluster

    PubMed Central

    Mayoral, Jaime G.; Leonard, Kate T.; Defelipe, Lucas A.; Turjansksi, Adrian G.; Nouzova, Marcela; Noriegal, Fernando G.

    2013-01-01

    The short chain dehydrogenases (SDR) constitute one the oldest and largest families of enzymes with over 46,000 members in sequence databases. About 25% of all known dehydrogenases belong to the SDR family. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. This family is present in archaea, bacteria, and eukaryota, emphasizing their versatility and fundamental importance for metabolic processes. We identified a cluster of eight SDRs in the mosquito Aedes aegypti (AaSDRs). Members of the cluster differ in tissue specificity and developmental expression. Heterologous expression produced recombinant proteins that had diverse substrate specificities, but distinct from the conventional insect alcohol (ethanol) dehydrogenases. They are all NADP+-dependent and they have S-enantioselectivity and preference for secondary alcohols with 8–15 carbons. Homology modeling was used to build the structure of AaSDR1 and two additional cluster members. The computational study helped explain the selectivity towards the (10S)-isomers as well as the reduced activity of AaSDR4 and AaSDR9 for longer isoprenoid substrates. Similar clusters of SDRs are present in other species of insects, suggesting similar selection mechanisms causing duplication and diversification of this family of enzymes. PMID:23238893

  18. Histidine 51 facilitates proton transfer in alcohol dehydrogenase

    SciTech Connect

    Gould, R.M.; Plapp, B.V.

    1987-05-01

    Operating through a proton relay system, His-51 has been proposed to serve as a base during ethanol oxidation by alcohol dehydrogenase. This residue is highly conserved in alcohol dehydrogenases. They have used mutamer directed mutagenesis to change this residue to Gln-51. Diethyl pyrocarbonate treatment decreases the activity of the wild type enzyme 60-fold, whereas the Gln-51 enzyme is inactivated by only 5-fold. The rate of inactivation is also much slower with the mutant enzyme. They conclude that His-51 is the reactive residue in yeast alcohol dehydrogenase. The mutation also alters the Km for acetaldehyde and the pH dependence of several kinetic constants. At pH 7.0 the Km for acetaldehyde is 18-fold higher in the Gln-51 enzyme, whereas Vmax for acetaldehyde reduction is the same as with the wild type enzyme. For ethanol oxidation the pH dependence of the log of Vmax and V/K shows a linear dependence with a slope of 0.5 and no discernible pK. They propose a mechanism that can explain these data. For the Gln-51 enzyme, after the ternary complex has formed in an Ordered Bi mechanism, a random component for proton release and hydride transfer occurs. With histidine at position 51, serving as a base, a more rapid proton release from the enzyme-NAD-ethanol complex precedes product formation.

  19. An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

    PubMed

    Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

    2015-05-01

    An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity.

  20. X-linked glucose-6-phosphate dehydrogenase (G6PD) and autosomal 6-phosphogluconate dehydrogenase (6PGD) polymorphisms in baboons

    SciTech Connect

    VandeBerg, J.L.; Aivaliotis, M.J.; Samollow, P.B. )

    1992-12-01

    Electrophoretic polymorphisms of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were examined in captive colonies of five subspecies of baboons (Papio hamadryas). Phenotype frequencies and family data verified the X-linked inheritance of the G6PD polymorphism. Insufficient family data were available to confirm autosomal inheritance of the 6PGD polymorphism, but the electrophoretic patterns of variant types (putative heterozygotes) suggested the codominant expression of alleles at an autosomal locus. Implications of the G6PD polymorphism are discussed with regard to its utility as a marker system for research on X-chromosome inactivation during baboon development and for studies of clonal cell proliferation and/or cell selection during the development of atherosclerotic lesions in the baboon model. 61 refs., 1 fig., 4 tabs.