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Sample records for alcohol dehydrogenase activities

  1. Human gastric alcohol dehydrogenase activity: effect of age, sex, and alcoholism.

    PubMed Central

    Seitz, H K; Egerer, G; Simanowski, U A; Waldherr, R; Eckey, R; Agarwal, D P; Goedde, H W; von Wartburg, J P

    1993-01-01

    As various isoenzymes of gastric alcohol dehydrogenase exist and as the effect of sex and age on these enzymes is unknown, this study measured the activity of gastric alcohol dehydrogenase at high and low ethanol concentrations in endoscopic biopsy specimens from a total of 290 patients of various ages and from 10 patients with chronic alcoholism. Gastric alcohol dehydrogenase was also detected by immunohistological tests in biopsy specimens from 40 patients by the use of a polyclonal rabbit antibody against class I alcohol dehydrogenase. A significant correlation was found between the immunohistological reaction assessed by the intensity of the colour reaction in the biopsy specimen and the activity of alcohol dehydrogenase measured at 580 mM ethanol. While alcohol dehydrogenase activity measured at 16 mM ethanol was not significantly affected by age and sex, both factors influenced alcohol dehydrogenase activity measured at 580 mM ethanol. Young women below 50 years of age had significantly lower alcohol dehydrogenase activities in the gastric corpus and antrum when compared with age matched controls (SEM) (6.4 (0.7) v 8.8 (0.6) nmol/min/mg protein; p < 0.001 and 6.0 (1.3) v 9.5 (1.3) nmol/min/mg protein; p < 0.001). Over 50 years of age this sex difference was no longer detectable, as high Km gastric alcohol dehydrogenase activity decreases with age only in men and not in women. In addition, extremely low alcohol dehydrogenase activities have been found in gastric biopsy specimens from young male alcoholics (2.2 (0.5) nmol/min/mg protein), which returned to normal after two to three weeks of abstinence. The activity of alcohol dehydrogenase in the human stomach measured at 580 mM ethanol is decreased in young women, in elderly men, and in the subject with alcoholism. This decrease in alcohol dehydrogenase activity may contribute to the reduced first pass metabolism of ethanol associated with raised ethanol blood concentrations seen in these people. Images Figure

  2. [Effect Of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase].

    PubMed

    Dubrovsky, A V; Musina, E V; Kim, A L; Tikhonenko, S A

    2016-01-01

    Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 M and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect. PMID:27266256

  3. High blood alcohol levels in women. The role of decreased gastric alcohol dehydrogenase activity and first-pass metabolism.

    PubMed

    Frezza, M; di Padova, C; Pozzato, G; Terpin, M; Baraona, E; Lieber, C S

    1990-01-11

    After consuming comparable amounts of ethanol, women have higher blood ethanol concentrations than men, even with allowance for differences in size, and are more susceptible to alcoholic liver disease. Recently, we documented significant "first-pass metabolism" of ethanol due to its oxidation by gastric tissue. We report a study of the possible contribution of this metabolism to the sex-related difference in blood alcohol concentrations in 20 men and 23 women. Six in each group were alcoholics. The first-pass metabolism was determined on the basis of the difference in areas under the curves of blood alcohol concentrations after intravenous and oral administration of ethanol (0.3 g per kilogram of body weight). Alcohol dehydrogenase activity was also measured in endoscopic gastric biopsies. In nonalcoholic subjects, the first-pass metabolism and gastric alcohol dehydrogenase activity of the women were 23 and 59 percent, respectively, of those in the men, and there was a significant correlation (rs = 0.659) between first-pass metabolism and gastric mucosal alcohol dehydrogenase activity. In the alcoholic men, the first-pass metabolism and gastric alcohol dehydrogenase activity were about half those in the nonalcoholic men; in the alcoholic women, the gastric mucosal alcohol dehydrogenase activity was even lower than in the alcoholic men, and first-pass metabolism was virtually abolished. We conclude that the increased bioavailability of ethanol resulting from decreased gastric oxidation of ethanol may contribute to the enhanced vulnerability of women to acute and chronic complications of alcoholism. PMID:2248624

  4. Effect of amines as activators on the alcohol-oxidizing activity of pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenase.

    PubMed

    Takeda, Kouta; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Nakamura, Nobuhumi; Ohno, Hiroyuki

    2014-01-01

    Pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenases (PQQ-ADH) require ammonia or primary amines as activators in in vitro assays with artificial electron acceptors. We found that PQQ-ADH from Pseudomonas putida KT2440 (PpADH) was activated by various primary amines, di-methylamine, and tri-methylamine. The alcohol oxidation activity of PpADH was strongly enhanced and the affinity for substrates was also improved by pentylamine as an activator. PMID:25229857

  5. 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

  6. Alcohol dehydrogenase activity in Lactococcus chungangensis: application in cream cheese to moderate alcohol uptake.

    PubMed

    Konkit, Maytiya; Choi, Woo Jin; Kim, Wonyong

    2015-09-01

    Many human gastrointestinal facultative anaerobic and aerobic bacteria possess alcohol dehydrogenase (ADH) activity and are therefore capable of oxidizing ethanol to acetaldehyde. However, the ADH activity of Lactococcus spp., except Lactococcus lactis ssp. lactis, has not been widely determined, though they play an important role as the starter for most cheesemaking technologies. Cheese is a functional food recognized as an aid to digestion. In the current study, the ADH activity of Lactococcus chungangensis CAU 28(T) and 11 reference strains from the genus Lactococcus was determined. Only 5 strains, 3 of dairy origin, L. lactis ssp. lactis KCTC 3769(T), L. lactis ssp. cremoris KCCM 40699(T), and Lactococcus raffinolactis DSM 20443(T), and 2 of nondairy origin, Lactococcus fujiensis NJ317(T) and Lactococcus chungangensis CAU 28(T) KCTC 13185(T), showed ADH activity and possessed the ADH gene. All these strains were capable of making cheese, but the highest level of ADH activity was found in L. chungangensis, with 45.9nmol/min per gram in tryptic soy broth and 65.8nmol/min per gram in cream cheese. The extent that consumption of cheese, following imbibing alcohol, reduced alcohol uptake was observed by following the level of alcohol in the serum of mice. The results show a potential novel benefit of cheese as a dairy functional food. PMID:26142864

  7. Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation.

    PubMed Central

    Engeland, K; Höög, J O; Holmquist, B; Estonius, M; Jörnvall, H; Vallee, B L

    1993-01-01

    The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. The activities of the recombinant native and mutant enzymes toward ethanol are essentially identical, but mutagenesis greatly decreases the kcat/Km values for glutathione-dependent formaldehyde oxidation. The catalytic efficiency for the Asp variant is < 0.1% that of the unmutated enzyme, due to both a higher Km and a lower kcat value. As with the native enzyme, neither mutant can oxidize methanol, be saturated by ethanol, or be inhibited by 4-methylpyrazole; i.e., they retain these class III characteristics. In contrast, however, their activation by fatty acids, another characteristic unique to class III alcohol dehydrogenase, is markedly attenuated. The Ala mutant is activated only slightly, but the Asp mutant is not activated at all. The results strongly indicate that Arg-115 in class III alcohol dehydrogenase is a component of the binding site for activating fatty acids and is critical for the binding of S-hydroxymethylglutathione in glutathione-dependent formaldehyde dehydrogenase activity. PMID:8460164

  8. 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

  9. Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase.

    PubMed

    Li, Ming; Nie, Yao; Mu, Xiao Qing; Zhang, Rongzhen; Xu, Yan

    2016-07-01

    Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry. PMID:26178068

  10. A bifunctional enzyme from Rhodococcus erythropolis exhibiting secondary alcohol dehydrogenase-catalase activities.

    PubMed

    Martinez-Rojas, Enriqueta; Kurt, Tutku; Schmidt, Udo; Meyer, Vera; Garbe, Leif-Alexander

    2014-11-01

    Alcohol dehydrogenases have long been recognized as potential biocatalyst for production of chiral fine and bulk chemicals. They are relevant for industry in enantiospecific production of chiral compounds. In this study, we identified and purified a nicotinamide adenine dinucleotide (NAD)-dependent secondary alcohol dehydrogenase (SdcA) from Rhodococcus erythropolis oxidizing γ-lactols into γ-lactones. SdcA showed broad substrate specificity on γ-lactols; secondary aliphatic alcohols with 8 and 10 carbon atoms were also substrates and oxidized with (2S)-stereospecificity. The enzyme exhibited moderate stability with a half-life of 5 h at 40 °C and 20 days at 4 °C. Mass spectrometric identification revealed high sequence coverage of SdcA amino acid sequence to a highly conserved catalase from R. erythropolis. The corresponding encoding gene was isolated from genomic DNA and subsequently overexpressed in Escherichia coli BL21 DE3 cells. In addition, the recombinant SdcA was purified and characterized in order to confirm that the secondary alcohol dehydrogenase and catalase activity correspond to the same enzyme. PMID:24846734

  11. Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast.

    PubMed

    Schifferdecker, Anna Judith; Siurkus, Juozas; Andersen, Mikael Rørdam; Joerck-Ramberg, Dorte; Ling, Zhihao; Zhou, Nerve; Blevins, James E; Sibirny, Andriy A; Piškur, Jure; Ishchuk, Olena P

    2016-04-01

    Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions, respectively. The overexpression of ADH3 in D. bruxellensis also reduced the inhibition of fermentation by anaerobiosis, the "Custer effect". Thus, the fermentative capacity of D. bruxellensis could be further improved by metabolic engineering. PMID:26743658

  12. From Alcohol Dehydrogenase to a “One-way” Carbonyl Reductase by Active-site Redesign

    PubMed Central

    Klimacek, Mario; Nidetzky, Bernd

    2010-01-01

    Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol (Kint) was altered dramatically (104- to 105-fold) from being balanced in the wild-type enzyme (Kint ≈ 3) to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. The change in Kint reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn → Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD+-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn→Asp substitutions behaved as a slow “fructose reductase” at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8–10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively “one-way” reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis. PMID:20639204

  13. Cinnamyl Alcohol Dehydrogenase: Identification of New Sites of Promoter Activity in Transgenic Poplar.

    PubMed Central

    Hawkins, S.; Samaj, J.; Lauvergeat, V.; Boudet, A.; Grima-Pettenati, J.

    1997-01-01

    Stem sections from poplar that were stably transformed with a eucalypt cinnamyl alcohol dehydrogenase promoter-[beta]-glucuronidase construct were prepared by using either a technique routinely used in herbaceous species or a technique designed to take into account the particular anatomy of woody plants. Although both preparation techniques confirmed the pattern of expression previously observed (C. Feuillet, V. Lauvergeat, C. Deswarte, G. Pilate, A. Boudet and J. Grima-Pettenati [1995] Plant Mol Biol 27: 651-657), the latter technique also allowed the detection of other sites of promoter activity not revealed by the first technique. In situ hybridization confirmed the expression pattern obtained with the second sample preparation technique. PMID:12223610

  14. Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily

    PubMed Central

    Baker, Patrick J.; Britton, K. Linda; Fisher, Martin; Esclapez, Julia; Pire, Carmen; Bonete, Maria Jose; Ferrer, Juan; Rice, David W.

    2009-01-01

    Despite being the subject of intensive investigations, many aspects of the mechanism of the zinc-dependent medium chain alcohol dehydrogenase (MDR) superfamily remain contentious. We have determined the high-resolution structures of a series of binary and ternary complexes of glucose dehydrogenase, an MDR enzyme from Haloferax mediterranei. In stark contrast to the textbook MDR mechanism in which the zinc ion is proposed to remain stationary and attached to a common set of protein ligands, analysis of these structures reveals that in each complex, there are dramatic differences in the nature of the zinc ligation. These changes arise as a direct consequence of linked movements of the zinc ion, a zinc-bound bound water molecule, and the substrate during progression through the reaction. These results provide evidence for the molecular basis of proton traffic during catalysis, a structural explanation for pentacoordinate zinc ion intermediates, a unifying view for the observed patterns of metal ligation in the MDR family, and highlight the importance of dynamic fluctuations at the metal center in changing the electrostatic potential in the active site, thereby influencing the proton traffic and hydride transfer events. PMID:19131516

  15. Optimization of enzyme assisted extraction of Fructus Mori polysaccharides and its activities on antioxidant and alcohol dehydrogenase.

    PubMed

    Deng, Qingfang; Zhou, Xin; Chen, Huaguo

    2014-10-13

    In the present study, enzyme assisted extraction of Fructus Mori polysaccharides (FMPS) from F. mori using four kinds of enzymes and three compound enzymes were examined. Research found that glucose oxidase offered a better performance in enhancement of the extraction yields of FMPS, antioxidant and activate alcohol dehydrogenase activities. The glucose oxidase assisted extraction process was further optimized by using response surface method (RSM) to obtain maximum yield of crude FMPS. The results showed that optimized extraction conditions were ratio of enzyme amount 0.40%, enzyme treated time 38 min, treated temperature 58 °C and liquid-solid radio 11.0. Under these conditions, the mean experimental value of extraction yield (16.16 ± 0.14%) corresponded well with the predicted values and increased 160% than none enzyme treated ones. Pharmacological verification tests showed that F. mori crude polysaccharides had good antioxidant and activate alcohol dehydrogenase activities in vitro. PMID:25037415

  16. Fundamental molecular differences between alcohol dehydrogenase classes.

    PubMed Central

    Danielsson, O; Atrian, S; Luque, T; Hjelmqvist, L; Gonzàlez-Duarte, R; Jörnvall, H

    1994-01-01

    Two types of alcohol dehydrogenase in separate protein families are the "medium-chain" zinc enzymes (including the classical liver and yeast forms) and the "short-chain" enzymes (including the insect form). Although the medium-chain family has been characterized in prokaryotes and many eukaryotes (fungi, plants, cephalopods, and vertebrates), insects have seemed to possess only the short-chain enzyme. We have now also characterized a medium-chain alcohol dehydrogenase in Drosophila. The enzyme is identical to insect octanol dehydrogenase. It is a typical class III alcohol dehydrogenase, similar to the corresponding human form (70% residue identity), with mostly the same residues involved in substrate and coenzyme interactions. Changes that do occur are conservative, but Phe-51 is of functional interest in relation to decreased coenzyme binding and increased overall activity. Extra residues versus the human enzyme near position 250 affect the coenzyme-binding domain. Enzymatic properties are similar--i.e., very low activity toward ethanol (Km beyond measurement) and high selectivity for formaldehyde/glutathione (S-hydroxymethylglutathione; kcat/Km = 160,000 min-1.mM-1). Between the present class III and the ethanol-active class I enzymes, however, patterns of variability differ greatly, highlighting fundamentally separate molecular properties of these two alcohol dehydrogenases, with class III resembling enzymes in general and class I showing high variation. The gene coding for the Drosophila class III enzyme produces an mRNA of about 1.36 kb that is present at all developmental stages of the fly, compatible with the constitutive nature of the vertebrate enzyme. Taken together, the results bridge a previously apparent gap in the distribution of medium-chain alcohol dehydrogenases and establish a strictly conserved class III enzyme, consistent with an important role for this enzyme in cellular metabolism. Images PMID:8197167

  17. Biophysical and mutagenic analysis of Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase activity and specificity.

    PubMed Central

    Burdette, D S; Secundo, F; Phillips, R S; Dong, J; Scott, R A; Zeikus, J G

    1997-01-01

    The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (secondary ADH) was overexpressed in Escherichia coli at more than 10% of total protein. The recombinant enzyme was purified in high yield (67%) by heat-treatment at 85 degrees C and (NH4)2SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D150Eand D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)1(N,O)3 co-ordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type secondary ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively,suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indicating that an anionic side-chain moiety at this position preserves the bound Zn. All five mutant enzymes had activity, suggesting that the T. ethanolicus secondary ADH requires a properly co-ordinated catalytic Zn atom. The His-59 and Asp-150 mutations also altered secondary ADH affinity for propan-2-ol over a 140-fold range, whereas the overall change in affinity for ethanol spanned a range of only 7-fold, supporting the importance of the metal in secondary ADH substrate binding. The lack of significant changes in cofactor affinity as a result of these catalytic Zn ligand mutations suggested that secondary ADH substrate-and cofactor-binding sites are structurally distinct. Altering Gly198 to Asp reduced the enzyme specific activity 2.7-fold, increased the Km(app) for NADP+ 225-fold, and decreased the Km(app) for NAD+ 3-fold, supporting the prediction that the enzyme binds nicotinamide cofactor in a Rossmann fold. Our data indicate therefore that, unlike the liver primary ADH

  18. Carbon Dioxide Effects on Ethanol Production, Pyruvate Decarboxylase, and Alcohol Dehydrogenase Activities in Anaerobic Sweet Potato Roots 1

    PubMed Central

    Chang, Ling A.; Hammett, Larry K.; Pharr, David M.

    1983-01-01

    The effect of varied anaerobic atmospheres on the metabolism of sweet potato (Ipomoea batatas [L.] Lam.) roots was studied. The internal gas atmospheres of storage roots changed rapidly when the roots were submerged under water. O2 and N2 gases disappeared quickly and were replaced by CO2. There were no appreciable differences in gas composition among the four cultivars that were studied. Under different anaerobic conditions, ethanol concentration in the roots was highest in a CO2 environment, followed by submergence and a N2 environment in all the cultivars except one. A positive relationship was found between ethanol production and pyruvate decarboxylase activity from both 100% CO2-treated and 100% N2-treated roots. CO2 atmospheres also resulted in higher pyruvate decarboxylase activity than did N2 atmospheres. Concentrations of CO2 were higher within anaerobic roots than those in the ambient anaerobic atmosphere. The level of pyruvate decarboxylase and ethanol in anaerobic roots was proportional to the ambient CO2 concentration. The measurable activity of pyruvate decarboxylase that was present in the roots was about 100 times less than that of alcohol dehydrogenase. Considering these observations, it is suggested that the rate-limiting enzyme for ethanol biosynthesis in sweet potato storage roots under anoxia is likely to be pyruvate decarboxylase rather than alcohol dehydrogenase. PMID:16662798

  19. Sjögren-Larsson syndrome. Deficient activity of the fatty aldehyde dehydrogenase component of fatty alcohol:NAD+ oxidoreductase in cultured fibroblasts.

    PubMed Central

    Rizzo, W B; Craft, D A

    1991-01-01

    Sjögren-Larsson syndrome (SLS) is an inherited disorder associated with impaired fatty alcohol oxidation due to deficient activity of fatty alcohol:NAD+ oxidoreductase (FAO). FAO is a complex enzyme which consists of two separate proteins that sequentially catalyze the oxidation of fatty alcohol to fatty aldehyde and fatty acid. To determine which enzymatic component of FAO was deficient in SLS, we assayed fatty aldehyde dehydrogenase (FALDH) and fatty alcohol dehydrogenase in cultured fibroblasts from seven unrelated SLS patients. All SLS cells were selectively deficient in the FALDH component of FAO, and had normal activity of fatty alcohol dehydrogenase. The extent of FALDH deficiency in SLS cells depended on the aliphatic aldehyde used as substrate, ranging from 62% of mean normal activity using propionaldehyde as substrate to 8% of mean normal activity with octadecanal. FALDH activity in obligate SLS heterozygotes was partially decreased to 49 +/- 7% of mean normal activity using octadecanal as substrate. Differential centrifugation studies in fibroblasts indicated that this FALDH enzyme was largely particulate; soluble FALDH activity was normal in SLS cells. Intact SLS fibroblasts oxidized octadecanol to fatty acid at less than 10% of the normal rate, but oxidized free octadecanal normally, suggesting that the FALDH affected in SLS is chiefly involved in the oxidation of fatty alcohol to fatty acid. These results show that the primary enzymatic defect in SLS is the FALDH component of the FAO complex, which leads to deficient oxidation of fatty aldehyde derived from fatty alcohol. PMID:1939650

  20. Alcohol dehydrogenases from olive (Olea europaea) fruit.

    PubMed

    Salas, J J; Sánchez, J

    1998-05-01

    Alcohol dehydrogenase activity was detected in extracts from the pericarp tissues of developing olive fruits using hexanal as the substrate. Total activity in the crude extract was 20-fold higher with NADPH than with NADH. Three discrete enzymes were resolved by means of a purification protocol involving ammonium sulfate fractionation followed by ion-exchange and affinity chromatography. One of the enzymes was NAD-dependent and displayed a high K(m) for hexanal (K(m) = 2.1 mM). Two NADP-dependent alcohol dehydrogenases were resolved, one showing a high K(m) for hexanal (K(m) = 1.9 mM) and the second with a lower K(m) for the same substrate (K(m) = 0.04 mM). The three enzymes have been partially purified and their kinetic parameters and specificities for various aldehydes determined. The involvement of these enzymes in the biogenesis of six carbon alcohols constituent of the aroma of olive oil is discussed. PMID:9621451

  1. Activity and electrophoretic profiles of liver aldehyde dehydrogenases from mice of inbred strains with different alcohol preference.

    PubMed

    Yamazaki, H; Nishiguchi, K; Miyamoto, R; Ogita, Z I; Nakanishi, S

    1983-01-01

    1. The activity of low Km-aldehyde dehydrogenase (ALDH) in the liver mitochondrial fraction (MT-fraction) from male C57BL/6J strain mice (alcohol preferring) was significantly higher than that from DBA/2 mice (alcohol avoiding). The F1 hybrids (C57BL/6J X DBA/2) did not exhibit the intermediate activity to these two strains. 2. Strain differences in liver mitochondrial ALDH isozymes were observed by isoelectric focusing. C57BL/6J strain had two isozymes at pH 7.1 while DBA/2 had no band at this pH. F1 hybrid mice had similar two bands with lower density to those of C57BL/6J at pH 7.1. There was no difference in zymograms of the soluble fraction between C57BL/6J and DBA/2 strains. 3. The present results suggest that the difference in alcohol preference of mice may depend on some restricted ALDH isozymes with different pl or electric mobility rather than the enzymatic activity in the liver MT-fraction. PMID:6822317

  2. Identification of a long-range protein network that modulates active site dynamics in extremophilic alcohol dehydrogenases.

    PubMed

    Nagel, Zachary D; Cun, Shujian; Klinman, Judith P

    2013-05-17

    A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 Å away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496-499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases. PMID:23525111

  3. 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

  4. 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

  5. Effects of the cofactor binding sites on the activities of secondary alcohol dehydrogenase (SADH).

    PubMed

    Wang, Tao; Chen, Xiangjun; Han, Jun; Ma, Sichun; Wang, Jianmei; Li, Xufeng; Zhang, Hui; Liu, Zhibin; Yang, Yi

    2016-07-01

    SADHs from Thermoanaerobacter ethanolicus are enzymes that, together with various cofactors, catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. To explore how cofactors bind to SADH, TeSADH was cloned in this study, and Ser(199) and Arg(200) were replaced by Tyr and Asp, respectively. Both sites were expected to be inside or adjacent to the cofactor-binding domain according to computational a prediction. Analysis of TeSADH activities revealed that the enzymatic efficiency (kcat/Km) of the S199Y mutant was noticeably enhanced using by NADH, NADPH as cofactors, and similar with that of wild-type using by NADP(+), NAD(+). Conversely, the activity of the R200D mutant significantly decreased with all cofactors. Furthermore, in yeast, the S199Y mutant substantially elevated the ethanol concentration compared with the wild type. Molecular dynamics simulation results indicated the H-bonding network between TeSADH and the cofactors was stronger for the S199Y mutant and the binding energy was simultaneously increased. Moreover, the fluorescence results indicated the S199Y mutant exhibited an increased preference for NAD(P)H, binding with NAD(P)H more compactly compared with wild type. PMID:27016086

  6. Quinoprotein alcohol dehydrogenase from ethanol-grown Pseudomonas aeruginosa.

    PubMed Central

    Groen, B; Frank, J; Duine, J A

    1984-01-01

    Cell-free extracts of Pseudomonas aeruginosa strains, grown on ethanol, showed dye-linked alcohol dehydrogenase activities. The enzyme responsible for this activity was purified to homogeneity. It appeared to contain two molecules of pyrroloquinoline quinone per enzyme molecule. In many respects, it resembled other quinoprotein alcohol dehydrogenases (EC 1.1.99.8), having a substrate specificity intermediate between that of methanol dehydrogenases and ethanol dehydrogenases in this group. On the other hand, it also showed dissimilarities: the enzyme was found to be a monomer (Mr 101 000), to need only one molecule of the suicide substrate cyclopropanol to become fully inactivated, and to have a different aromatic amino acid composition. PMID:6439190

  7. Spaceflight exposure effects on transcription, activity, and localization of alcohol dehydrogenase in the roots of Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Porterfield, D. M.; Matthews, S. W.; Daugherty, C. J.; Musgrave, M. E.

    1997-01-01

    Although considerable research and speculation have been directed toward understanding a plant's perception of gravity and the resulting gravitropic responses, little is known about the role of gravity-dependent physical processes in normal physiological function. These studies were conducted to determine whether the roots of plants exposed to spaceflight conditions may be experiencing hypoxia. Arabidopsis thaliana (L.) Heynh. plants were grown in agar medium during 6 or 11 d of spaceflight exposure on shuttle missions STS-54 (CHROMEX-03) and STS-68 (CHROMEX-05), respectively. The analysis included measurement of agar redox potential and root alcohol dehydrogenase (ADH) activity, localization, and expression. ADH activity increased by 89% as a result of spaceflight exposure for both CHROMEX-03 and -05 experiments, and ADH RNase protection assays revealed a 136% increase in ADH mRNA. The increase in ADH activity associated with the spaceflight roots was realized by a 28% decrease in oxygen availability in a ground-based study; however, no reduction in redox potential was observed in measurements of the spaceflight bulk agar. Spaceflight exposure appears to effect a hypoxic response in the roots of agar-grown plants that may be caused by changes in gravity-mediated fluid and/or gas behavior.

  8. Spaceflight exposure effects on transcription, activity, and localization of alcohol dehydrogenase in the roots of Arabidopsis thaliana.

    PubMed Central

    Porterfield, D M; Matthews, S W; Daugherty, C J; Musgrave, M E

    1997-01-01

    Although considerable research and speculation have been directed toward understanding a plant's perception of gravity and the resulting gravitropic responses, little is known about the role of gravity-dependent physical processes in normal physiological function. These studies were conducted to determine whether the roots of plants exposed to spaceflight conditions may be experiencing hypoxia. Arabidopsis thaliana (L.) Heynh. plants were grown in agar medium during 6 or 11 d of spaceflight exposure on shuttle missions STS-54 (CHROMEX-03) and STS-68 (CHROMEX-05), respectively. The analysis included measurement of agar redox potential and root alcohol dehydrogenase (ADH) activity, localization, and expression. ADH activity increased by 89% as a result of spaceflight exposure for both CHROMEX-03 and -05 experiments, and ADH RNase protection assays revealed a 136% increase in ADH mRNA. The increase in ADH activity associated with the spaceflight roots was realized by a 28% decrease in oxygen availability in a ground-based study; however, no reduction in redox potential was observed in measurements of the spaceflight bulk agar. Spaceflight exposure appears to effect a hypoxic response in the roots of agar-grown plants that may be caused by changes in gravity-mediated fluid and/or gas behavior. PMID:9085569

  9. Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae) fruit-fly species and their hybrids

    PubMed Central

    2009-01-01

    The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits. PMID:21637665

  10. Drosophila alcohol dehydrogenase: developmental studies on cryptic variant lines.

    PubMed

    Miglani, G S; Ampy, F R

    1981-10-01

    Thirty-five cryptic variant lines were used to examine the mechanisms involved in genetic modulation of alcohol metabolism in Drosophila. Late third-instar larval, preemergence pupal, and adult stages cultured at 18 and 28 C were examined. Spectrophotometric analyses for native alcohol dehydrogenase (ADH) activity and residual ADH activity after treatment with guanidine hydrochloride and heat were performed. Differential response of cryptic variants to treatment with the denaturants during development suggested that this variation may have an adaptive significance. PMID:6800354

  11. Hydroperoxidic inhibitor of horse liver alcohol dehydrogenase activity, tightly bound to the enzyme-NAD+ complex, characteristically degrades the coenzyme.

    PubMed

    Skurský, L; Rezác, M; Khan, A N; Zídek, L; Rocek, J

    1992-01-01

    The strong inhibition of horse liver alcohol dehydrogenase (HLAD) by p-methylbenzyl hydroperoxide (XyHP) is only transient, XyHP behaves also as a pseudo-substrate of the enzyme and in the presence of NAD+, is degraded by HLAD to (as yet unidentified) non-inhibiting products while the NAD+ is converted to a derivative similar to the "NADX", originally observed in an analogous reaction of HLAD with hydrogen peroxide. The apparent KM for XyHP is approximately 10(4) times smaller than that for H2O2. The catalytic constant kcat for HLAD degradation of XyHP is two orders of magnitude less than that for ethanol dehydrogenation. XyHP inhibits both directions of the alcohol-aldehyde interconversion with equal potency. The first step of the inhibition mechanism is a tight binding of XyHP to the binary HLAD-NAD+ complex. PMID:1284958

  12. In vivo ethanol elimination in man, monkey and rat: A lack of relationship between the ethanol metabolism and the hepatic activities of alcohol and aldehyde dehydrogenases

    SciTech Connect

    Zorzano, A. ); Herrera, E. )

    1990-01-01

    The in vivo ethanol elimination in human subjects, monkeys and rats was investigated after an oral ethanol dosage. After 0.4 g. ethanol/kg of body weight, ethanol elimination was much slower in human subjects than in monkeys. In order to detect a rise in monkey plasma ethanol concentrations as early as observed in human subjects, ethanol had to be administered at a dose of 3 g/kg body weight. Ethanol metabolism in rats was also much faster than in human subjects. However, human liver showed higher alcohol dehydrogenase activity and higher low Km aldehyde dehydrogenase activity than rat liver. Thus, our data suggest a lack of relationship between hepatic ethanol-metabolizing activities and the in vivo ethanol elimination rate.

  13. Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis.

    PubMed Central

    Winberg, J O; Hovik, R; McKinley-McKee, J S; Juan, E; Gonzalez-Duarte, R

    1986-01-01

    Purified Drosophila lebanonensis alcohol dehydrogenase (Adh) revealed one enzymically active zone in starch gel electrophoresis at pH 8.5. This zone was located on the cathode side of the origin. Incubation of D. lebanonensis Adh with NAD+ and acetone altered the electrophoretic pattern to more anodal migrating zones. D. lebanonensis Adh has an Mr of 56,000, a subunit of Mr of 28 000 and is a dimer with two active sites per enzyme molecule. This agrees with a polypeptide chain of 247 residues. Metal analysis by plasma emission spectroscopy indicated that this insect alcohol dehydrogenase is not a metalloenzyme. In studies of the substrate specificity and stereospecificity, D. lebanonensis Adh was more active with secondary than with primary alcohols. Both alkyl groups in the secondary alcohols interacted hydrophobically with the alcohol binding region of the active site. The catalytic centre activity for propan-2-ol was 7.4 s-1 and the maximum velocity of most secondary alcohols was approximately the same and indicative of rate-limiting enzyme-coenzyme dissociation. For primary alcohols the maximum velocity varied and was much lower than for secondary alcohols. The catalytic centre activity for ethanol was 2.4 s-1. With [2H6]ethanol a primary kinetic 2H isotope effect of 2.8 indicated that the interconversion of the ternary complexes was rate-limiting. Pyrazole was an ethanol-competitive inhibitor of the enzyme. The difference spectra of the enzyme-NAD+-pyrazole complex gave an absorption peak at 305 nm with epsilon 305 14.5 X 10(3) M-1 X cm-1. Concentrations and amounts of active enzyme can thus be determined. A kinetic rate assay to determine the concentration of enzyme active sites is also presented. This has been developed from active site concentrations established by titration at 305 nm of the enzyme and pyrazole with NAD+. In contrast with the amino acid composition, which indicated that D. lebanonensis Adh and the D. melanogaster alleloenzymes were not

  14. Enzymic and structural studies on Drosophila alcohol dehydrogenase and other short-chain dehydrogenases/reductases.

    PubMed

    Smilda, T; Kamminga, A H; Reinders, P; Baron, W; van Hylckama Vlieg, J E; Beintema, J J

    2001-05-01

    Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several secondary alcohols were used to determine the kinetic parameters kcat and Km. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity for R(-) alcohols. At a high concentration of R(-) alcohols substrate activation occurs. The kcat and Km values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those at low substrate concentrations. Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic domains of these enzymes, but they do not have the structural features required for binding NAD+. Probably these lyases descend from an SDR, which has lost the capability to bind NAD+, but the enzyme reaction mechanisms may still be similar. PMID:11443349

  15. Activity of Yeast Alcohol Dehydrogenases on Benzyl Alcohols and Benzaldehydes. Characterization of ADH1 from Saccharomyces carlsbergensis and Transition State Analysis

    PubMed Central

    Pal, Suresh; Park, Doo-Hong; Plapp, Bryce V.

    2009-01-01

    The substrate specificities of yeast alcohol dehydrogenases I and II from Saccharomyces cerevisiae (SceADH1 and SceADH2) and Saccharomyces carlsbergensis (ScbADH1) were studied. For this work, the gene for the S. carlsbergensis ADH1 was cloned, sequenced and expressed. The amino acid sequence of ScbADH1 differs at four positions as compared to SceADH1, including substitutions of two glutamine residues with glutamic acid residues, and has the same sequence as the commercial yeast enzyme, which apparently is prepared from S. carlsbergensis. The electrophoretic mobilities of ScbADH1, SceADH2 and commercial ADH are similar. The kinetics and specificities of ScbADH1 and SceADH1 acting on branched, long-chain and benzyl alcohols are very similar, but the catalytic efficiency of SceADH2 is about 10 to 100-fold higher on these substrates. A three dimensional structure of SceADH1 shows that the substrate binding pocket has Met-270, whereas SceADH2 has Leu-270, which allows larger substrates to bind. The reduction of a series of p-substituted benzaldehydes catalyzed by SceADH2 is significantly enhanced by electron-withdrawing groups, whereas the oxidation of p-substituted aromatic alcohols may be only slightly affected by the substituents. The substituent effects on catalysis generally reflect the effects on the equilibrium constant for the reaction, where electron-withdrawing substituents favor alcohol. The results are consistent with a transition state that is electronically similar to the alcohol, supporting previous results obtained with commercial yeast ADH. PMID:19022233

  16. The alcohol dehydrogenase isoenzyme alcohol dehydrogenase IV as a candidate marker of Helicobacter pylori infection

    PubMed Central

    Laniewska-Dunaj, Magdalena; Strumnik, Anna; Szmitkowski, Maciej

    2014-01-01

    Introduction Helicobacter pylori infection is associated with decreased alcohol dehydrogenase (ADH) activity in the gastric mucosa. The decrease in gastric ADH activity depends on the severity of inflammation and mucosal injury. This damage can be a reason of the release of enzyme from gastric mucosa and leads to the increase of the ADH activity in the sera of patients with H. pylori infection. Material and methods Serum samples were taken from 140 patients with H. pylori infection. Total ADH activity was measured by photometric method with p-nitrosodimethylaniline as a substrate and ALDH activity by the fluorometric method with 6-methoxy-2-naphtaldehyde. For the measurement of the activity of class I and II isoenzymes we employed the fluorometric methods, with class-specific fluorogenic substrates. The activity of class III ADH was measured by the photometric method with n-octanol and class IV with m-nitrobenzaldehyde as a substrate. Results The activity of ADH IV in the serum of patients with H. pylori infection increased about 42% (7.86 mU/l) in the comparison to the control level (4.52 mU/l). Total activity of ADH was 1105 mU/l in patients group and 682 mU/l in control. The diagnostic sensitivity for ADH IV was 88%, specificity 90%, positive and negative predictive values were 91% and 84% respectively. Area under ROC curve for ADH IV was 0.84. Conclusions Helicobacter pylori infection of gastric mucosa is reflected in the serum by significant increase of class IV and total ADH activity. The results suggest a potential role for ADH IV as a marker of H. pylori infection. PMID:25395946

  17. 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.

  18. Stability of immobilized yeast alcohol dehydrogenase

    SciTech Connect

    Ooshima, H.; Genko, Y.; Harano, Y.

    1981-12-01

    The effects of substrate on stabilities of native (NA) and three kinds of immobilized yeast alcohol dehydrogenase (IMA), namely PGA (the carrier; porous glass), SEA (agarose gel) prepared covalently, and AMA (anion-exchange resin) prepared ionically, were studied. The following results were obtained. 1) The deactivations of NA and IMA free from the substrate or in the presence of ethanol obey the first-order kinetics, whereas, in the presence of butyraldehyde, their deactivation behaviors are explained on the basis of coexistence of two components of YADHs, namely the labile E1 and the comparatively stable E2, with different first-order deactivation constants. (2) A few attempts for stabilization of IMA were carried out from the viewpoint of the effects of crosslinkages among the subunits of YADH for PGA and the multibonding between the carrier and enzyme for SEA. The former is effective for the stabilization, whereas the latter is not. (Refs. 19).

  19. 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

  20. Tryptophan in Alcoholism Treatment II:  Inhibition of the Rat Liver Mitochondrial Low Km Aldehyde Dehydrogenase Activity, Elevation of Blood Acetaldehyde Concentration and Induction of Aversion to Alcohol by Combined Administration of Tryptophan and Benserazide

    PubMed Central

    Badawy, Abdulla A.-B.; Bano, Samina; Steptoe, Alex

    2011-01-01

    Aims: The aims were to provide proofs of mechanism and principle by establishing the ability of the amino acid L-tryptophan (Trp) combined with the kynureninase inhibitor benserazide (BSZ) to inhibit the liver mitochondrial low Km aldehyde dehydrogenase (ALDH) activity after administration and in vivo and to induce aversion to alcohol. Methods: Trp, BSZ or both were administered to male Wistar rats and ALDH activity was determined both in vitro in liver homogenates and in vivo (by measuring acetaldehyde accumulation in blood after ethanol administration). Alcohol consumption was studied in an aversion model in rats and in alcohol-preferring C57 mice. Results: Combined administration of Trp + BSZ, but neither compound alone, produced a strong inhibition of ALDH activity and an increase in blood acetaldehyde concentration after ethanol, and induced aversion to alcohol in rats and decreased preference in mice. Another kynureninase inhibitor, carbidopa, induced aversion to alcohol by itself, which was reversed by Trp co-administration. Conclusions: The present results establish a prior art for the use of a combination of Trp plus BSZ in the treatment of alcoholism by aversion, which merits rapid clinical development. PMID:21896551

  1. Application of nicotin amide-adenine dinucleotide analogs for clinical enzymology: alcohol dehydrogenase activity in liver injury.

    PubMed

    Fujisawa, K; Kimura, A; Minato, S; Tamaoki, H; Mizushima, H

    1976-06-01

    The activities of alcohol dehydrogease(ADH) in serum and in the subcellular fractions of rat liver were determined with n-amyl alcohol or ethanol as substrate and thionicotinamide-adenine dinucleotide as coenzyme. It was found that the enzyme's activity ratio on the amyl alcohol and ethanol(A/E value) of serum and on the particulate fractions of the liver were different, but the A/E value of the soluble fraction was similar to that of serum. The A/E value of the particulate fractions were higher than that of the soluble fraction. From the results of experimental liver damage in the rat, it seems that estimation of the A/E value of ADH activity in serum is a useful parameter for the diagnosis of active liver injury. Since the A/E values of patients' sera differed from those of the normal subjects, the estimation of the A/E value of serum may give diagnostic information on liver injury, especially in chronic liver injury. PMID:179739

  2. Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii.

    PubMed

    Alsafadi, Diya; Paradisi, Francesca

    2013-01-01

    The effect of various organic solvents on the catalytic activity, stability and substrate specificity of alchohol dehydrogenase from Haloferax volcanii (HvADH2) was evaluated. The HvADH2 showed remarkable stability and catalysed the reaction in aqueous-organic medium containing dimethyl sulfoxide (DMSO) and methanol (MeOH). Tetrahydrofuran and acetonitrile were also investigated and adversely affected the stability of the enzyme. High concentration of salt, essential to maintain the enzymatic activity and structural integrity of the halophilic enzyme under standard conditions may be partially replaced by DMSO and MeOH. The presence of organic solvents did not induce gross changes in substrate specificity. DMSO offered a protective effect for the stability of the enzyme at nonoptimal pHs such as 6 and 10. Salt and solvent effects on the HvADH2 conformation and folding were examined through fluorescence spectroscopy. The fluorescence findings were consistent with the activity and stability results and corroborated the denaturing properties of some solvents. The intrinsic tolerance of this enzyme to organic solvent makes it highly attractive to industry. PMID:23179592

  3. N-acylethanolamines as novel alcohol dehydrogenase 3 substrates.

    PubMed

    Ivkovic, Milena; Dempsey, Daniel R; Handa, Sumit; Hilton, Joshua H; Lowe, Edward W; Merkler, David J

    2011-02-15

    N-acylethanolamines (NAEs) are members of the fatty acid amide family. The NAEs have been proposed to serve as metabolic precursors to N-acylglycines (NAGs). The sequential oxidation of the NAEs by an alcohol dehydrogenase and an aldehyde dehydrogenase would yield the N-acylglycinals and/or the NAGs. Alcohol dehydrogenase 3 (ADH3) is one enzyme that might catalyze this reaction. To define a potential role for ADH3 in NAE catabolism, we synthesized a set of NAEs and evaluated these as ADH3 substrates. NAEs were oxidized by ADH3, yielding the N-acylglycinals as the product. The (V/K)(app) values for the NAEs included here were low relative to cinnamyl alcohol. Our data show that the NAEs can serve as alcohol dehydrogenase substrates. PMID:21144815

  4. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    PubMed

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. PMID:25280628

  5. Encapsulation of Alcohol Dehydrogenase in Mannitol by Spray Drying

    PubMed Central

    Shiga, Hirokazu; Joreau, Hiromi; Neoh, Tze Loon; Furuta, Takeshi; Yoshii, Hidefumi

    2014-01-01

    The retention of the enzyme activity of alcohol dehydrogenase (ADH) has been studied in various drying processes such as spray drying. The aim of this study is to encapsulate ADH in mannitol, either with or without additive in order to limit the thermal denaturation of the enzyme during the drying process. The retention of ADH activity was investigated at different drying temperatures. When mannitol was used, the encapsulated ADH was found inactive in all the dried powders. This is presumably due to the quick crystallization of mannitol during spray drying that resulted in the impairment of enzyme protection ability in comparison to its amorphous form. Maltodextin (dextrose equivalent = 11) was used to reduce the crystallization of mannitol. The addition of maltodextrin increased ADH activity and drastically changed the powder X-ray diffractogram of the spray-dried powders. PMID:24662364

  6. Contribution of liver alcohol dehydrogenase to metabolism of alcohols in rats.

    PubMed

    Plapp, Bryce V; Leidal, Kevin G; Murch, Bruce P; Green, David W

    2015-06-01

    The kinetics of oxidation of various alcohols by purified rat liver alcohol dehydrogenase (ADH) were compared with the kinetics of elimination of the alcohols in rats in order to investigate the roles of ADH and other factors that contribute to the rates of metabolism of alcohols. Primary alcohols (ethanol, 1-propanol, 1-butanol, 2-methyl-1-propanol, 3-methyl-1-butanol) and diols (1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol) were eliminated in rats with zero-order kinetics at doses of 5-20 mmol/kg. Ethanol was eliminated most rapidly, at 7.9 mmol/kgh. Secondary alcohols (2-propanol-d7, 2-propanol, 2-butanol, 3-pentanol, cyclopentanol, cyclohexanol) were eliminated with first order kinetics at doses of 5-10 mmol/kg, and the corresponding ketones were formed and slowly eliminated with zero or first order kinetics. The rates of elimination of various alcohols were inhibited on average 73% (55% for 2-propanol to 90% for ethanol) by 1 mmol/kg of 4-methylpyrazole, a good inhibitor of ADH, indicating a major role for ADH in the metabolism of the alcohols. The Michaelis kinetic constants from in vitro studies (pH 7.3, 37 °C) with isolated rat liver enzyme were used to calculate the expected relative rates of metabolism in rats. The rates of elimination generally increased with increased activity of ADH, but a maximum rate of 6±1 mmol/kg h was observed for the best substrates, suggesting that ADH activity is not solely rate-limiting. Because secondary alcohols only require one NAD(+) for the conversion to ketones whereas primary alcohols require two equivalents of NAD(+) for oxidation to the carboxylic acids, it appears that the rate of oxidation of NADH to NAD(+) is not a major limiting factor for metabolism of these alcohols, but the rate-limiting factors are yet to be identified. PMID:25641189

  7. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry.

    PubMed

    Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

    2015-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76-1.16] in abstainers with *2, 1.00 [0.80-1.26] in *1/*1 drinkers, 0.71 [0.54-0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28-6.13] in abstainers with *2, 1.89 [0.89-4.51] in *1/*1 drinkers, 2.35 [1.06-5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2 displayed a

  8. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry

    PubMed Central

    Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

    2015-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76–1.16] in abstainers with *2, 1.00 [0.80–1.26] in *1/*1 drinkers, 0.71 [0.54–0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28–6.13] in abstainers with *2, 1.89 [0.89–4.51] in *1/*1 drinkers, 2.35 [1.06–5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2

  9. Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum

    PubMed Central

    Vitale, Annalisa; Thorne, Natasha; Lovell, Scott; Battaile, Kevin P.; Hu, Xin; Shen, Min; D'Auria, Sabato; Auld, Douglas S.

    2013-01-01

    In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn2+) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn2+ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn2+ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes. PMID:23755111

  10. Regulation of human class I alcohol dehydrogenases by bile acids

    PubMed Central

    Langhi, Cédric; Pedraz-Cuesta, Elena; Haro, Diego; Marrero, Pedro F.; Rodríguez, Joan C.

    2013-01-01

    Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol and vitamin A (retinol) metabolism in the liver. Because previous studies have shown that human ADH1 enzymes may participate in bile acid metabolism, we investigated whether the bile acid-activated nuclear receptor farnesoid X receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064 increased ADH1 mRNA, protein, and activity. Moreover, overexpression of a constitutively active form of FXR induced ADH1A and ADH1B expression, whereas silencing of FXR abolished the effects of FXR agonists on ADH1 expression and activity. Transient transfection studies and electrophoretic mobility shift assays revealed functional FXR response elements in the ADH1A and ADH1B proximal promoters, thus indicating that both genes are direct targets of FXR. These findings provide the first evidence for direct connection of bile acid signaling and alcohol metabolism. PMID:23772048

  11. Characterization of alcohol dehydrogenase 1 and 3 from Neurospora crassa FGSC2489.

    PubMed

    Park, Yong-Cheol; San, Ka-Yiu; Bennett, George N

    2007-08-01

    Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD(+)-binding motif and showed 54-75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 +/- 9 mU/mg using ethanol and NAD(+) as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3-C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD(+) preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes. PMID:17516063

  12. Cinnamyl alcohol dehydrogenases in the mesocarp of ripening fruit of Prunus persica genotypes with different flesh characteristics: changes in activity and protein and transcript levels.

    PubMed

    Gabotti, Damiano; Negrini, Noemi; Morgutti, Silvia; Nocito, Fabio F; Cocucci, Maurizio

    2015-07-01

    Development of fruit flesh texture quality traits may involve the metabolism of phenolic compounds. This study presents molecular and biochemical results on the possible role played by cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) during ripening [S3, S4 I (pre-climacteric) and S4 III (climacteric) stages] of peach [Prunus persica (L.) Batsch] fruit with different flesh firmness [non-melting flesh (NMF) 'Oro A'/melting flesh (MF) 'Springcrest' and 'Sanguinella'] and color (blood-flesh Sanguinella). A total of 24 putative full-length PRUPE_CAD genes were identified (in silico analysis) in the peach genome. The most abundant CAD isoforms, encoded by genes located on scaffolds 8 and 6, were probed by specifically developed anti-PRUPE_CAD sc8 and by anti-FaCAD (PRUPE_CAD sc6) polyclonal antibodies, respectively. PRUPE_CAD sc8 proteins (SDS-PAGE and native-PAGE/western blot) appeared responsible for the CAD activity (in vitro/in-gel assays) that increased with ripening (parallel to PRUPE_ACO1 transcripts accumulation and ethylene evolution) only in the mesocarp of Oro A and blood-flesh Sanguinella. Accumulation of PRUPE_CAD sc8 transcripts (semi-quantitative RT-PCR) occurred in all three cultivars, but in Oro A and Springcrest it was not always accompanied by that of the related proteins, suggesting possible post-transcriptional regulation. Flesh firmness, as well as levels of lignin, total phenolics and, where present (Sanguinella), anthocyanins, declined with ripening, suggesting that, at least in the studied peach cultivars, CAD activity is related to neither lignification nor differences in flesh firmness (NMF/MF). Further studies are necessary to clarify whether the high levels of CAD activity/expression in Sanguinella play a role in determining the characteristics of this blood-flesh fruit. PMID:25534876

  13. Alcohol and aldehyde dehydrogenase polymorphisms in Chinese and Indian populations.

    PubMed

    Tan, Ene-Choo; Lim, Leslie; Leong, Jern-Yi; Lim, Jing-Yan; Lee, Arthur; Yang, Jun; Tan, Chay-Hoon; Winslow, Munidasa

    2010-01-01

    The association between two functional polymorphisms in alcohol dehydrogenase (ADH2/ADH1B) and aldehyde dehydrogenase (ALDH2) genes and alcohol dependence was examined in 182 Chinese and Indian patients undergoing treatment for alcohol dependence and 184 screened control subjects from Singapore. All subjects were screened by the Alcohol Use Disorders Identification Test (AUDIT). Patients were also administered the Severity of Alcohol Dependence Questionnaire (SADQ). Polymorphisms were genotyped by allele-specific polymerase chain reaction and selected genotypes confirmed by DNA sequencing or restriction fragment length polymorphism. Our results showed that frequencies of ADH1B*2 and ALDH2*2 were higher in controls compared to alcohol-dependent subjects for both Chinese and Indians. Frequencies of these two alleles were also higher in the 104 Chinese controls compared to the 80 Indian controls. None of the eight Chinese who were homozygous for both protective alleles was alcohol dependent. The higher frequencies of the protective alleles could explain the lower rate of alcohol dependence in Chinese. PMID:20025435

  14. Crystal structure of cod liver class I alcohol dehydrogenase: substrate pocket and structurally variable segments.

    PubMed Central

    Ramaswamy, S.; el Ahmad, M.; Danielsson, O.; Jörnvall, H.; Eklund, H.

    1996-01-01

    The structural framework of cod liver alcohol dehydrogenase is similar to that of horse and human alcohol dehydrogenases. In contrast, the substrate pocket differs significantly, and main differences are located in three loops. Nevertheless, the substrate pocket is hydrophobic like that of the mammalian class I enzymes and has a similar topography in spite of many main-chain and side-chain differences. The structural framework of alcohol dehydrogenase is also present in a number of related enzymes like glucose dehydrogenase and quinone oxidoreductase. These enzymes have completely different substrate specificity, but also for these enzymes, the corresponding loops of the substrate pocket have significantly different structures. The domains of the two subunits in the crystals of the cod enzyme further differ by a rotation of the catalytic domains by about 6 degrees. In one subunit, they close around the coenzyme similarly as in coenzyme complexes of the horse enzyme, but form a more open cleft in the other subunit, similar to the situation in coenzyme-free structures of the horse enzyme. The proton relay system differs from the mammalian class I alcohol dehydrogenases. His 51, which has been implicated in mammalian enzymes to be important for proton transfer from the buried active site to the surface is not present in the cod enzyme. A tyrosine in the corresponding position is turned into the substrate pocket and a water molecule occupies the same position in space as the His side chain, forming a shorter proton relay system. PMID:8845755

  15. A model system for QTL analysis: Effects of alcohol dehydrogenase genotype on alcohol pharmacokinetics

    SciTech Connect

    Martin, N.G.; Nightingale, B.; Whitfield, J.B.

    1994-09-01

    There is much interest in the detection of quantitative trait loci (QTL) - major genes which affect quantitative phenotypes. The relationship of polymorphism at known alcohol metabolizing enzyme loci to alcohol pharmacokinetics is a good model system. The three class I alcohol dehydrogenase genes are clustered on chromosome 4 and protein electrophoresis has revealed polymorphisms at the ADH2 and ADH3 loci. While different activities of the isozymes have been demonstrated in vitro, little work has been done in trying to relate ADH polymorphism to variation in ethanol metabolism in vivo. We previously measured ethanol metabolism and psychomotor reactivity in 206 twin pairs and demonstrated that most of the repeatable variation was genetic. We have now recontacted the twins to obtain DNA samples and used PCR with allele specific primers to type the ADH2 and ADH3 polymorphisms in 337 individual twins. FISHER has been used to estimate fixed effects of typed polymorphisms simultaneously with remaining linked and unlinked genetic variance. The ADH2*1-2 genotypes metabolize ethanol faster and attain a lower peak blood alcohol concentration than the more common ADH2*1-1 genotypes, although less than 3% of the variance is accounted for. There is no effect of ADH3 genotype. However, sib-pair linkage analysis suggests that there is a linked polymorphism which has a much greater effect on alcohol metabolism that those typed here.

  16. Structural aspects of the dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila.

    PubMed Central

    Bamforth, C W; Quayle, J R

    1979-01-01

    1. A dye-linked alcohol dehydrogenase was purified 60-fold from extracts of Rhodopseudomonas acidophila 10050 grown aerobically on ethanol. 2. The properties of this enzyme were identical with those of the alcohol dehydrogenase synthesized by this organism during growth on methanol anaerobically in the light, and they are judged to be the same enzyme. 3. The enzyme gave a single protein band, coincident with alcohol dehydrogenase activity, during electrophoresis on polyacrylamide gel. 4. The amino acid composition, ioselectric point, u.v. and visible absorption spectra of the enzyme were determined and compared with those of other similar enzymes. 5. The presence of 0.7--1.0 g-atom of non-haem, acidlabile iron/mol of enzyme was shown by atomic absorption spectrophotometry and colorimetric assay. The iron could not be dissociated from the enzyme by dialysis against chelating agents. 6. E.p.r. spectroscopy of the enzyme did not indicate any redox function for the iron during alcohol dehydrogenation, but showed a signal at g = 2.00 consistent with the presence of a protein-bound organic free radical. 8. Antisera were raised against alcohol (methanol) dehydrogenases purified from Rhodopseudomonas acidophila, Paracoccus denitrificans and Methylophilus methylotrophus. 9. The antiserum to the Rhodopseudomonas acidophila enzyme cross-reacted with neither of the two other antisera, nor with crude extracts of methanol-grown Hyphomicrobium X and Pseudomonas AM1, thus emphasizing its singular biochemical properties. PMID:229820

  17. Contribution of Liver Alcohol Dehydrogenase to Metabolism of Alcohols in Rats

    PubMed Central

    Plapp, Bryce V.; Leidal, Kevin G.; Murch, Bruce P.; Green, David W.

    2015-01-01

    The kinetics of oxidation of various alcohols by purified rat liver alcohol dehydrogenase (ADH) were compared with the kinetics of elimination of the alcohols in rats in order to investigate the roles of ADH and other factors that contribute to the rates of metabolism of alcohols. Primary alcohols (ethanol, 1-propanol, 1-butanol, 2-methyl-1-propanol, 3-methyl-1-butanol) and diols (1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol) were eliminated in rats with zero-order kinetics at doses of 5–20 mmole/kg. Ethanol was eliminated most rapidly, at 7.9 mmole/kg•h. Secondary alcohols (2-propanol-d7, 2-propanol, 2-butanol, 3-pentanol, cyclopentanol, cyclohexanol) were eliminated with first order kinetics at doses of 5–10 mmole/kg, and the corresponding ketones were formed and slowly eliminated with zero or first order kinetics. The rates of elimination of various alcohols were inhibited on average 73% (55% for 2-propanol to 90% for ethanol) by 1 mmole/kg of 4-methylpyrazole, a good inhibitor of ADH, indicating a major role for ADH in the metabolism of the alcohols. The Michaelis kinetic constants from in vitro studies (pH 7.3, 37 °C) with isolated rat liver enzyme were used to calculate the expected relative rates of metabolism in rats. The rates of elimination generally increased with increased activity of ADH, but a maximum rate of 6 ± 1 mmole/kg•h was observed for the best substrates, suggesting that ADH activity is not solely rate-limiting. Because secondary alcohols only require one NAD+ for the conversion to ketones whereas primary alcohols require two equivalents of NAD+ for oxidation to the carboxylic acids, it appears that the rate of oxidation of NADH to NAD+ is not a major limiting factor for metabolism of these alcohols, but the rate-limiting factors are yet to be identified. PMID:25641189

  18. Single motoneuron succinate dehydrogenase activity.

    PubMed

    Chalmers, G R; Edgerton, V R

    1989-07-01

    We have developed a quantitative histochemical assay for measurement of succinate dehydrogenase (SDH) activity in single motoneurons. A computer image processing system was used to quantify the histochemical enzyme reaction product and to follow the time course of the reaction. The optimal concentration for each of the ingredients of the incubation medium for the SDH reaction was determined and the importance of using histochemical "blanks" in the determination of enzymatic activity was demonstrated. The enzymatic activity was linear with respect to reaction time and tissue thickness. The procedure described meets the criteria generally considered essential for establishment of a quantitative histochemical assay. The assay was then used to examine the SDH activity of cat and rat motoneurons. It was found that motoneurons with a small soma size had a wide range of SDH activity, whereas those with a large soma size were restricted to low SDH activity. PMID:2732457

  19. Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase

    PubMed Central

    Napora-Wijata, Kamila; Strohmeier, Gernot A.; Sonavane, Manoj N.; Avi, Manuela; Robins, Karen; Winkler, Margit

    2013-01-01

    Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases. PMID:24970175

  20. Geometric specificity of alcohol dehydrogenases and its potential for separation of trans and cis isomers of unsaturated aldehydes.

    PubMed Central

    Klibanov, A M; Giannousis, P P

    1982-01-01

    The geometric specificity of three different alcohol dehydrogenases (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) (from yeast, from horse liver, and from Leuconostoc mesenteroides) in the reduction of trans- and cis-cinnamaldehydes has been investigated. All three enzymes display a remarkable trans specificity: they react with the trans isomer 7 to 647 times faster than with its cis counterpart. Experiments with the enzymatic reduction of 3-phenylpropionaldehyde, a saturated analog of cinnamaldehyde, have revealed that whereas trans-cinnamaldehyde possesses the "right" configuration for the active centers of the alcohol dehydrogenases, the cis isomer apparently does not fit the active centers well. All three alcohol dehydrogenases studied also exhibit a marked trans specificity in the reaction with alpha-methylcinnamaldehyde. The geometric specificity of alcohol dehydrogenases can be used for the production of otherwise hard to synthesize cis isomers of unsaturated aldehydes from their readily available trans counterparts: trans-cinnamaldehyde was irradiated with ultraviolet light (which converted it to a mixture of trans and cis isomers) then treated with NADH and yeast alcohol dehydrogenase (which selectively reduces only trans aldehyde into the alcohol), and finally the mixture of cis-cinnamaldehyde and trans-cinnamyl alcohol was separated easily by preparative column chromatography. PMID:7048306

  1. Inhibition of gastric alcohol dehydrogenase activity by histamine H2-receptor antagonists has no influence on the pharmacokinetics of ethanol after a moderate dose.

    PubMed Central

    Mallat, A; Roudot-Thoraval, F; Bergmann, J F; Trout, H; Simonneau, G; Dutreuil, C; Blanc, L E; Dhumeaux, D; Delchier, J C

    1994-01-01

    Ethanol undergoes gastric first pass metabolism by alcohol dehydrogenase (ADH). We have shown that cimetidine and famotidine both cause competitive inhibition of human gastric ADH in vitro. However, in a randomized 4-way cross-over study in 12 healthy subjects a 7-day course of treatment with cimetidine (800 mg day-1), ranitidine (300 mg day-1) or famotidine (40 mg day-1), did not modify the pharmacokinetics of ethanol given as a post-prandial 0.3 g kg-1 dose. We conclude that gastric mucosal concentrations of histamine H2-receptor blockers achieved after oral dosing are probably too low to cause significant inhibition of gastric ADH in vivo. PMID:7910473

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

    PubMed Central

    Pavlova, Sylvia I.; Jin, Ling; Gasparovich, Stephen R.

    2013-01-01

    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. PMID:23637459

  3. Action of shear on enzymes: studies with alcohol dehydrogenase.

    PubMed

    Thomas, C R; Nienow, A W; Dunnill, P

    1979-12-01

    Yeast alcohol dehydrogenase (ADH) solutions (approximately 1 mg/ml, pH 7) were sheared in a coaxial cylindrical viscometer. This was fitted with a lid sealing the contents from the atmosphere and preventing evaporation. At 30 degrees C after a total of 5 hr intermittent shearing at 683 sec-1 no losses of activity were observed. No losses were found after 5 hr continuous shearing and in a no-shear control. At 40 degrees C and 683 sec-1 there were only small activity losses in 5 hr. Shearing at 3440 sec-1 no measurable losses of activity were found with a 1.03 mg/ml solution in 5 hr at 30 degrees C, a 1.03 mg/ml solution in 8 hr at 5 degrees C, and with a 3.89 mg/ml solution in 3 hr at 5 degrees C. In all these cases, however, a white precipitate formed that was not observed in zero shear control experiments. The sheared 3.89 mg/ml solution was clarified by centrifugation. It was shown that there were no ADH aggregates in the supernatant and that the precipitate was less than 2% of the original protein. At 30 degrees C under adverse pH conditions (pH 8.8) there was no significant difference in activity losses of an approximately 1 mg/ml solution sheared at 65 and 744 sec-1. An approximately 0.5 mg/ml ADH solution, pH 7, was agitated in a small reactor with no free air-liquid interface. Peak shear rates near the impeller were estimated to be about 9000 sec-1. Only a small decrease in specific activity was observed until over 15 hr total running at 5 degrees C. PMID:42450

  4. Baboon alcohol dehydrogenase isozymes: phenotypic changes in liver following chronic consumption of alcohol.

    PubMed

    Holmes, R S; VandeBerg, J L

    1987-01-01

    According to the nomenclature of Vallee and Bazzone [1983] for mammalian alcohol dehydrogenase (ADH) isozymes, baboon ADHs comprise three major classes of activity, which were distinguished according to the following properties: Class I ADHs. These isozymes exhibited low-Km characteristics with ethanol as substrate, high isoelectric points (8.5-9.3), and sensitivity to 5 mM 4-methyl pyrazole inhibition, and were the major liver (ADH-2) and kidney (ADH-1) isozymes in the baboon. Class II ADHs. These isozymes showed high-Km values for ethanol, neutral isoelectric points (7.7 for the liver ADH-4 [pi-ADH] and 7.2 for the major stomach ADH [ADH-3], respectively), and were insensitive to inhibition with 5 mM 4-methyl pyrazole. Class III ADH. This enzyme was characterized by its inactivity with ethanol as substrate (up to 0.5 M), insensitivity to 4-methyl pyrazole inhibition, preference for medium-chain-length alcohols as substrate (trans-2-hexen-1-ol was routinely used in this study), and an isoelectric point (6.5) similar to that of the human liver chi-ADH (pI 6.4). Major activity variation of the liver pi-ADH (ADH-4) isozyme was observed among the 114 liver samples examined, with 34 percent exhibiting a null (or low-activity) phenotype. An electrophoretic variant phenotype for the major class II stomach isozyme (ADH-3) was also found in the population studied. The baboon was used as a model for studying alcohol-induced changes in liver ADH phenotype following chronic alcohol consumption. Prepuberal male baboons were pair-fed nutritionally adequate liquid diets containing ethanol (50 percent of calories) or isocaloric carbohydrates, and liver ADH isozyme patterns from biopsy samples were monitored for 20 weeks. Dramatic decreases in class II liver ADH activity (ADH-4, or pi-ADH) were observed within 4 weeks after the start of alcohol feeding, and a shift in liver class I isozymes was found during the later stages of alcohol consumption. These changes during chronic

  5. In vivo relationship between monoamine oxidase type B and alcohol dehydrogenase: effects of ethanol and phenylethylamine

    SciTech Connect

    Aliyu, S.U.; Upahi, L.

    1988-01-01

    The role of acute ethanol and phenylethylamine on the brain and platelet monoamine oxidase activities, hepatic cytosolic alcohol dehydrogenase, redox state and motor behavior were studied in male rats. Ethanol on its own decreased the redox couple ratio, as well as, alcohol dehydrogenase activity in the liver while at the same time it increased brain and platelet monoamine oxidase activity due to lower Km with no change in Vmax. The elevation in both brain and platelet MAO activity was associated with ethanol-induced hypomotility in the rats. Co-administration of phenylethylamine and ethanol to the animals, caused antagonism of the ethanol-induced effects described above. The effects of phenylethylamine alone, on the above mentioned biochemical and behavioral indices, are more complex. Phenylethylamine on its own, like ethanol, caused reduction of the cytosolic redox, ratio and elevation of monoamine oxidase activity in the brain and platelets. However, in contrast to ethanol, this monoamine produced hypermotility and activation of the hepatic cytosolic alcohol dehydrogenase activity in the animals.

  6. Alcohol dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecanse and hexadecanol metabolism

    SciTech Connect

    Singer, M.E.; Finnerty, W.R.

    1985-12-01

    Multiple alcohol dehydrogenases (ADH) were demonstrated in Acinetobacter sp. strain HO1-N. ADH-A and ADH-B were distinguished on the basis of electrophoretic mobility, pyridine nucleotide cofactor requirement, and substrate specificity. ADH-A is a soluble, NAD-linked, inducible ethanol dehydrogenase (EDH). An ethanol-negative mutant (Eth1) was isolated which contained 6.5% of wild-type EDH activity and was deficient in ADH-A. Eth1 exhibited normal growth on hexadecane and hexadecanol. A second ethanol-negative mutant (Eth3) was acetaldehyde dehydrogenase (ALDH) deficient, having 12.5% of wild-type ALDH activity. Eth3 had threefold-higher EDH activity than the wild-type strain. ALDH is a soluble, NAD-linked, ethanol-inducible enzyme. Eth3 exhibited normal growth on hexadecane, hexadecanol, and fatty aldehyde. ADH-B is soluble, constitutive, NADP-linked ADH which was active with medium-chain-length alcohols. Hexadecanol dehydrogenase (HDH), a soluble and membrane-bound, NAD-linked ADH, was induced 5- to 11-fold by growth on hexadecane or hexadecanol. HDH was distinct from ADH-A and ADH-B. NAD-linked HDH appears to possess a functional role in hexadecane and hexadecanol dissimilation.

  7. Direct Observation of Correlated Interdomain Motion in Alcohol Dehydrogenase

    SciTech Connect

    Biehl, Ralf; Monkenbusch, Michael; Richter, Dieter; Hoffmann, Bernd; Merkel, Rudolf; Falus, Peter; Preost, Sylvain

    2008-09-26

    Interdomain motions in proteins are essential to enable or promote biochemical function. Neutron spin-echo spectroscopy is used to directly observe the domain dynamics of the protein alcohol dehydrogenase. The collective motion of domains as revealed by their coherent form factor relates to the cleft opening dynamics between the binding and the catalytic domains enabling binding and release of the functional important cofactor. The cleft opening mode hardens as a result of an overall stiffening of the domain complex due to the binding of the cofactor.

  8. Isozyme multiplicity with anomalous dimer patterns in a class III alcohol dehydrogenase. Effects on the activity and quaternary structure of residue exchanges at "nonfunctional" sites in a native protein.

    PubMed

    Danielsson, O; Shafqat, J; Estonius, M; el-Ahmad, M; Jörnvall, H

    1996-11-19

    The isozymes of class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase from cod were characterized. They exhibited three unexpected properties of general interest. First, these dimeric isozymes, derived from two types of subunit (h and l, for high- and low-activity forms), were recovered from liver preparations in only the homodimeric ll and heterodimeric hl combinations. Dissociation and reassociation of the isolated hl form in vitro also resulted in lower yields of the hh than the ll homodimer, although class III subunits are usually freely associable over wide borders of divergence (human and Drosophila). The h and l primary structures show that both chain types are characteristic of class III enzymes, without large amino acid replacements at positions of known subunit interactions. Hence, the hh dimer partial restriction indicates nontraditional alterations at h-subunit interfaces. The structure provides a possible explanation, in the form of h-chain modifications that may influence the anchoring of a loop at positions of two potentially deamidative beta-aspartyl shifts at distant Asn-Gly structures. Second the ll and hl forms differ in enzymatic properties, having 5-fold different K(m) values for NAD+ at pH 8, different K(m) values for S-(hydroxymethyl)glutathione (10 versus 150 microM), and different specific activities (4.5 versus 41 units/mg), with ll resembling and hl deviating from human and other class III alcohol dehydrogenases. However, functional residues lining substrate and coenzyme pockets in the known conformations of homologous forms are largely identical in the two isozymes [only minor conservative exchanges of Val/Leu116, Val/Leu203, Ile/Val224, and Ile/Val269 (numbering system of the human class I enzyme)], again indicating effects from distantly positioned h-chain replacements. Third, the two isozymes differ a surprising amount in amino acid sequence (18%, the same as the piscine/ human difference), reflecting a

  9. The aromatic alcohol dehydrogenases in Pseudomonas putida N.C.I.B. 9869 grown on 3,5-xylenol and p-cresol.

    PubMed Central

    Keat, M J; Hopper, D J

    1978-01-01

    Whole cells of Pseudomonas putida N.C.I.B 9869, when grown on either 3,5-xylenol or p-cresol, oxidized both m- and p-hydroxybenzyl alcohols. Two distinct NAD+-dependent m-hydroxybenzyl alcohol dehydrogenases were purified from cells grown on 3,5-xylenol. Each is active with a range of aromatic alcohols, including both m- and p-hydroxybenzyl alcohol, but differ in their relative rates with the various substrates. An NAD+-dependent alcohol dehydrogenase was also partially purified from p-cresol grown cells. This too was active with m- and p-hydroxybenzyl alcohol and other aromatic alcohols, but was not identical with either of the other two dehydrogenases. All three enzymes were unstable, but were stabilized by dithiothreitol and all were inhibited with p-chloromercuribenzoate. All were specific for NAD+ and each was shown to catalyse conversion of alcohol into aldehyde. PMID:743216

  10. Amphibian alcohol dehydrogenase, the major frog liver enzyme. Relationships to other forms and assessment of an early gene duplication separating vertebrate class I and class III alcohol dehydrogenases

    SciTech Connect

    Cederlund, E.; Joernvall, H. ); Peralba, J.M.; Pares, X. )

    1991-03-19

    Submammalian alcohol dehydrogenase structures can be used to evaluate the origins and functions of different types of the mammalian enzyme. Two avian forms were recently reported, and the authors now define the major amphibian alcohol dehydrogenase. The enzyme from the liver of the Green frog Rana perezi was purified, carboxymethylated, and submitted to amino acid sequence determination by peptide analysis of six different digest. The protein has a 375-residue subunit and is a class I alcohol dehydrogenase, bridging the gap toward the original separation of the classes that are observable in the human alcohol dehydrogenase system. In relation to the human class I enzyme, the amphibian protein has residue identities exactly halfway (68%) between those for the corresponding avian enzyme (74%) and the human class III enzyme (62%), suggesting an origin of the alcohol dehnydrogenase classes very early in or close to the evolution of the vertebrate line. This conclusion suggests that these enzyme classes are more universal among animals than previously realized and constitutes the first real assessment of the origin of the duplications leading to the alcohol dehydrogenase classes. In conclusion, the amphibian enzyme allows a rough positioning of the divergence of the alcohol dehydrogenase classes, shows that the class I type is widesprread in vertebrates, and functionally conforms with greater variations at the substrate-binding than the coenzyme-binding site.

  11. Human liver alcohol dehydrogenase. 1. The primary structure of the beta 1 beta 1 isoenzyme.

    PubMed

    Hempel, J; Bühler, R; Kaiser, R; Holmquist, B; de Zalenski, C; von Wartburg, J P; Vallee, B; Jörnvall, H

    1984-12-17

    Determination of the amino acid sequence of the beta 1 subunit from the class I (pyrazole-sensitive) human liver alcohol dehydrogenase isoenzyme beta 1 beta 1 revealed a 373-residue structure differing at 48 positions (including a gap) from that of the subunit of the well studied horse liver alcohol dehydrogenase EE isoenzyme. The structure deduced is compatible with known differences in composition, ultraviolet absorbance, electrophoretic mobility and catalytic properties between the horse and human enzymes. All zinc-liganding residues of the horse E subunit are strictly conserved in the human beta 1 subunit, despite an earlier report of a mutation involving Cys-46. This residue therefore remains conserved in all known alcohol dehydrogenase structures. However, the total cysteine content of the beta 1 structure is raised from 14 in the subunit of the horse enzyme to 15 by a Tyr----Cys exchange. Most exchanges are on the surface of the molecule and of a well conserved nature. Substitutions close to the catalytic centre are of interest to explain the altered substrate specificity and different catalytic activity of the beta 1 homodimer. Functionally, a Ser----Thr exchange at position 48 appears to be of special importance, since Thr-48 in beta 1 instead of Ser-48 in the horse enzyme can restrict available space. Four other substitutions also line the active-site pocket, and appear to constitute partly compensated exchanges. PMID:6391920

  12. Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family.

    PubMed

    Duester, G; Farrés, J; Felder, M R; Holmes, R S; Höög, J O; Parés, X; Plapp, B V; Yin, S J; Jörnvall, H

    1999-08-01

    The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies. PMID:10424757

  13. Analysis of rat cytosolic 9-cis-retinol dehydrogenase activity and enzymatic characterization of rat ADHII.

    PubMed

    Popescu, G; Napoli, J L

    2000-01-01

    We report the characterization of two enzymes that catalyze NAD(+)-dependent 9-cis-retinol dehydrogenase activity in rat liver cystol. Alcohol dehydrogenase class I (ADHI) contributes > 80% of the NA D+-dependent 9-cis-retinol dehydrogenase activity recovered, whereas alcohol dehydrogenase class II (ADHII), not identified previously at the protein level, nor characterized enzymatically in rat, accounts for approximately 2% of the activity. Rat ADHII exhibits properties different from those described for human ADHII. Moreover, rat ADHII-catalyzed rates of ethanol dehydrogenation are markedly lower than octanol or retinoid dehydrogenation rates. Neither ethanol nor 4-methylpyrazole inhibits the 9-cis-retinol dehydrogenase activity of rat ADHII. We propose that ADHII represents the previously observed additional retinoid oxidation activity of rat liver cytosol which occurred in the presence of either ethanol or 4-methylpyrazole. We also show that human and rat ADHII differ considerably in enzymatic properties. PMID:10606766

  14. 11β-hydroxysteroid dehydrogenase inhibition as a new potential therapeutic target for alcohol abuse

    PubMed Central

    Sanna, P P; Kawamura, T; Chen, J; Koob, G F; Roberts, A J; Vendruscolo, L F; Repunte-Canonigo, V

    2016-01-01

    The identification of new and more effective treatments for alcohol abuse remains a priority. Alcohol intake activates glucocorticoids, which have a key role in alcohol's reinforcing properties. Glucocorticoid effects are modulated in part by the activity of 11β-hydroxysteroid dehydrogenases (11β-HSD) acting as pre-receptors. Here, we tested the effects on alcohol intake of the 11β-HSD inhibitor carbenoxolone (CBX, 18β-glycyrrhetinic acid 3β-O-hemisuccinate), which has been extensively used in the clinic for the treatment of gastritis and peptic ulcer and is active on both 11β-HSD1 and 11β-HSD2 isoforms. We observed that CBX reduces both baseline and excessive drinking in rats and mice. The CBX diastereomer 18α-glycyrrhetinic acid 3β-O-hemisuccinate (αCBX), which we found to be selective for 11β-HSD2, was also effective in reducing alcohol drinking in mice. Thus, 11β-HSD inhibitors may be a promising new class of candidate alcohol abuse medications, and existing 11β-HSD inhibitor drugs may be potentially re-purposed for alcohol abuse treatment. PMID:26978742

  15. Alcohol dehydrogenase polymorphism in barrel cactus populations of Drosophila mojavensis.

    PubMed

    Cleland, S; Hocutt, G D; Breitmeyer, C M; Markow, T A; Pfeiler, E

    1996-07-01

    Starch gel electrophoresis revealed that the alcohol dehydrogenase (ADH-2) locus was polymorphic in two populations (from Agua Caliente, California and the Grand Canyon, Arizona) of cactophilic Drosophila mojavensis that utilize barrel cactus (Ferocactus acanthodes) as a host plant. Electromorphs representing products of a slow (S) and a fast (F) allele were found in adult flies. The frequency of the slow allele was 0.448 in flies from Agua Caliente and 0.659 in flies from the Grand Canyon. These frequencies were intermediate to those of the low (Baja California peninsula, Mexico) and high (Sonora, Mexico and southern Arizona) frequency Adh-2S populations of D. mojavensis that utilize different species of host cacti. PMID:8765684

  16. Tea triterpenoidal saponins from the roots of Camellia sinensis have inhibitory effects against alcohol dehydrogenase.

    PubMed

    Varughese, Titto; Manir, Md Maniruzzaman; Rahaman, Mozahidur; Kim, Jeong Kee; Lee, Byeong-Gon; Moon, Surk-Sik

    2011-12-01

    Ten new polyhydroxyolean-12-ene pentacyclic triterpenoidal saponins, named rogchaponins 1-10, were isolated from the methanolic extract of the roots of Camellia sinensis by a series of chromatographic methods (silica gel flash column and C18 MPLC followed by C18 HPLC). Their structures were established by 1D and 2D-NMR techniques along with IR and HR-TOF-MS. Rogchaponins R4 ( 4) and R5 (5) showed inhibitory activities against yeast alcohol dehydrogenase (ADH) with IC (50) values of 16.1 ± 3.2 and 15.4 ± 3.3 µM, respectively. A 4-methylpyrazole positive control exhibited an IC (50) of 2750 ± 50 µM. However, the saponins showed no inhibitory activity against yeast aldehyde dehydrogenase (ALDH). PMID:21786220

  17. Ethanol-Induced Alcohol Dehydrogenase E (AdhE) Potentiates Pneumolysin in Streptococcus pneumoniae

    PubMed Central

    Luong, Truc Thanh; Kim, Eun-Hye; Bak, Jong Phil; Nguyen, Cuong Thach; Choi, Sangdun; Briles, David E.; Pyo, Suhkneung

    2014-01-01

    Alcohol impairs the host immune system, rendering the host more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae, including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here, we showed that the S. pneumoniae type 2 D39 strain is ethanol tolerant and that alcohol upregulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae, as well as host cell myeloperoxidase activity, proinflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (ΔadhE strain) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE level under the ethanol condition conferred an increase in Ply and H2O2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than the ΔadhE strain under the ethanol stress condition, and ethanol-fed mice (alcoholic mice) were more susceptible to infection with the D39 wild-type bacteria than with the ΔadhE strain. Taken together, these data indicate that AdhE increases Ply under the ethanol stress condition, thus potentiating pneumococcal virulence. PMID:25312953

  18. Use of the anti-Prelog stereospecific alcohol dehydrogenase from Leifsonia and Pseudomonas for producing chiral alcohols.

    PubMed

    Itoh, Nobuya

    2014-05-01

    The asymmetric reduction of ketones is one of the most promising processes for producing chiral alcohols. However, dehydrogenases or reductases that can catalyze the reduction of ketones to give anti-Prelog chiral alcohols have been limited to some NADP(+)/NADPH-dependent enzymes. Recently, we reported a novel NAD(+)/NADH-dependent alcohol dehydrogenase (ADH) from Leifsonia sp. and Pseudomonas ADH homologs from soil metagenomes. Moreover, we have established an efficient hydrogen-transfer bioreduction process with 2-propanol as a hydrogen donor using Leifsonia ADH. This review focuses on the recent development of novel ADHs for producing industrially useful anti-Prelog chiral alcohols from various ketones. PMID:24615386

  19. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis

    PubMed Central

    Kim, Sung-Jin; Kim, Mi-Ran; Bedgar, Diana L.; Moinuddin, Syed G. A.; Cardenas, Claudia L.; Davin, Laurence B.; Kang, ChulHee; Lewis, Norman G.

    2004-01-01

    Of 17 genes annotated in the Arabidopsis genome database as cinnamyl alcohol dehydrogenase (CAD) homologues, an in silico analysis revealed that 8 genes were misannotated. Of the remaining nine, six were catalytically competent for NADPH-dependent reduction of p-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes, whereas three displayed very low activity and only at very high substrate concentrations. Of the nine putative CADs, two (AtCAD5 and AtCAD4) had the highest activity and homology (≈83% similarity) relative to bona fide CADs from other species. AtCAD5 used all five substrates effectively, whereas AtCAD4 (of lower overall catalytic capacity) poorly used sinapyl aldehyde; the corresponding 270-fold decrease in kenz resulted from higher Km and lower kcat values, respectively. No CAD homologue displayed a specific requirement for sinapyl aldehyde, which was in direct contrast with unfounded claims for a so-called sinapyl alcohol dehydrogenase in angiosperms. AtCAD2, 3, as well as AtCAD7 and 8 (highest homology to sinapyl alcohol dehydrogenase) were catalytically less active overall by at least an order of magnitude, due to increased Km and lower kcat values. Accordingly, alternative and/or bifunctional metabolic roles of these proteins in plant defense cannot be ruled out. Comprehensive analyses of lignified tissues of various Arabidopsis knockout mutants (for AtCAD5, 6, and 9) at different stages of growth/development indicated the presence of functionally redundant CAD metabolic networks. Moreover, disruption of AtCAD5 expression had only a small effect on either overall lignin amounts deposited, or on syringyl-guaiacyl compositions, despite being the most catalytically active form in vitro. PMID:14745009

  20. Alcohol dehydrogenases and an alcohol oxidase involved in the assimilation of exogenous fatty alcohols in Yarrowia lipolytica.

    PubMed

    Iwama, Ryo; Kobayashi, Satoshi; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi

    2015-05-01

    The yeast Yarrowia lipolytica can assimilate hydrophobic substrates, including n-alkanes and fatty alcohols. Here, eight alcohol dehydrogenase genes, ADH1-ADH7 and FADH, and a fatty alcohol oxidase gene, FAO1, were analyzed to determine their roles in the metabolism of hydrophobic substrates. A mutant deleted for all of these genes (ALCY02 strain) showed severely defective growth on fatty alcohols, and enhanced sensitivity to fatty alcohols in glucose-containing media. The ALCY02 strain grew normally on n-tetradecane or n-hexadecane, but exhibited slightly defective growth on n-decane or n-dodecane. It accumulated more 1-dodecanol and less dodecanoic acid than the wild-type strain when n-dodecane was fed. Expression of ADH1, ADH3 or FAO1, but not that of other ADH genes or FADH, in the ALCY02 strain restored its growth on fatty alcohols. In addition, a triple deletion mutant of ADH1, ADH3 and FAO1 showed similarly defective growth on fatty alcohols and on n-dodecane to the ALCY02 strain. Microscopic observation suggests that Adh1p and Adh3p are localized in the cytosol and Fao1p is in the peroxisome. These results suggest that Adh1p, Adh3p and Fao1p are responsible for the oxidation of exogenous fatty alcohols but play less prominent roles in the oxidation of fatty alcohols derived from n-alkanes. PMID:25805841

  1. Temperature-Jump Fluorescence Provides Evidence for Fully Reversible Microsecond Dynamics in a Thermophilic Alcohol Dehydrogenase

    PubMed Central

    2015-01-01

    Protein dynamics on the microsecond (μs) time scale were investigated by temperature-jump fluorescence spectroscopy as a function of temperature in two variants of a thermophilic alcohol dehydrogenase: W87F and W87F:H43A. Both mutants exhibit a fast, temperature-independent μs decrease in fluorescence followed by a slower full recovery of the initial fluorescence. The results, which rule out an ionizing histidine as the origin of the fluorescence quenching, are discussed in the context of a Trp49-containing dimer interface that acts as a conduit for thermally activated structural change within the protein interior. PMID:26223665

  2. Redesigning alcohol dehydrogenases/reductases for more efficient biosynthesis of enantiopure isomers.

    PubMed

    Zhang, Rongzhen; Xu, Yan; Xiao, Rong

    2015-12-01

    Alcohol dehydrogenases/reductases predominantly catalyze the asymmetric biosynthesis of optically pure stereoisomers because of their unique chiral constitutions. The enantioselectivities of alcohol dehydrogenases/reductases are substrate- and cofactor-dependent, and therefore they usually catalyze specific reactions with high enantioselectivity under physiological conditions; this may not be suitable for asymmetric biosynthesis with non-natural substrates or non-natural cofactors, and under nonphysiological conditions. It is therefore necessary to modify alcohol dehydrogenases/reductases using various redesigning tools such as directed evolution and rational design, and their combinations, as well as engineering enzyme modules for more efficient production of "non-natural" products. In this article, progress in these aspects of alcohol dehydrogenase/reductase design is reviewed, and future challenges are discussed. PMID:26320091

  3. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis

    DOEpatents

    Ingram, Lonnie O.; Conway, Tyrrell

    1992-01-01

    The alcohol dehydrogenase II gene from Zymomonas mobilis has been cloned and sequenced. This gene can be expressed at high levels in other organisms to produce acetaldehyde or to convert acetaldehyde to ethanol.

  4. Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

    PubMed

    Winberg, J O; McKinley-McKee, J S

    1998-02-01

    Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain

  5. Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

    PubMed Central

    Winberg, J O; McKinley-McKee, J S

    1998-01-01

    Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain

  6. Purification and Characterization of Cinnamyl Alcohol Dehydrogenase Isoforms from the Periderm of Eucalyptus gunnii Hook.

    PubMed Central

    Hawkins, S. W.; Boudet, A. M.

    1994-01-01

    Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) isoforms were purified from the periderm (containing both suberized and lignified cell layers) of Eucalyptus gunnii Hook stems. Two isoforms (CAD 1P and CAD 2P) were initially characterized, and the major form, CAD 2P, was resolved into three further isoforms by ion-exchange chromatography. Crude extracts contained two aliphatic alcohol dehydrogenases (ADH) and one aromatic ADH, which was later resolved into two further isoforms. Aliphatic ADHs did not use hydroxycinnamyl alcohols as substrates, whereas both aromatic ADH isoforms used coniferyl and sinapyl alcohol as substrates but with a much lower specific activity when compared with benzyl alcohol. The minor form, CAD 1P, was a monomer with a molecular weight of 34,000 that did not co-elute with either aromatic or aliphatic ADH activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis demonstrated that this protein was very similar to another CAD isoform purified from Eucalyptus xylem tissue. CAD 2P had a native molecular weight of approximately 84,000 and was a dimer consisting of two heterogenous subunits (with molecular weights of 42,000 and 44,000). These subunits were differentially combined to give the heterodimer and two homodimers. SDS-PAGE, western blots, and nondenaturing PAGE indicated that the CAD 2P heterodimer was very similar to the main CAD isoform previously purified in our laboratory from differentiating xylem tissue of E. gunnii (D. Goffner, I. Joffroy, J. Grima-Pettenati, C. Halpin, M.E. Knight, W. Schuch, A.M. Boudet [1992] Planta 188: 48-53). Kinetic data indicated that the different CAD 2P isoforms may be implicated in the preferential production of different monolignols used in the synthesis of lignin and/or suberin. PMID:12232063

  7. Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens).

    PubMed

    Iwamoto, Ryoko; Kubota, Humie; Hosoki, Tomoko; Ikehara, Kenji; Tanaka, Mieko

    2002-02-15

    A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex. PMID:11831851

  8. Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain.

    PubMed

    Bühler, R; Hempel, J; Kaiser, R; de Zalenski, C; von Wartburg, J P; Jörnvall, H

    1984-12-17

    The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE. PMID:6391921

  9. Pancreatic injury in hepatic alcohol dehydrogenase-deficient deer mice after subchronic exposure to ethanol

    SciTech Connect

    Kaphalia, Bhupendra S.; Bhopale, Kamlesh K.; Kondraganti, Shakuntala; Wu Hai; Boor, Paul J.; Ansari, G.A. Shakeel

    2010-08-01

    Pancreatitis caused by activation of digestive zymogens in the exocrine pancreas is a serious chronic health problem in alcoholic patients. However, mechanism of alcoholic pancreatitis remains obscure due to lack of a suitable animal model. Earlier, we reported pancreatic injury and substantial increases in endogenous formation of fatty acid ethyl esters (FAEEs) in the pancreas of hepatic alcohol dehydrogenase (ADH)-deficient (ADH{sup -}) deer mice fed 4% ethanol. To understand the mechanism of alcoholic pancreatitis, we evaluated dose-dependent metabolism of ethanol and related pancreatic injury in ADH{sup -} and hepatic ADH-normal (ADH{sup +}) deer mice fed 1%, 2% or 3.5% ethanol via Lieber-DeCarli liquid diet daily for 2 months. Blood alcohol concentration (BAC) was remarkably increased and the concentration was {approx} 1.5-fold greater in ADH{sup -} vs. ADH{sup +} deer mice fed 3.5% ethanol. At the end of the experiment, remarkable increases in pancreatic FAEEs and significant pancreatic injury indicated by the presence of prominent perinuclear space, pyknotic nuclei, apoptotic bodies and dilation of glandular ER were found only in ADH{sup -} deer mice fed 3.5% ethanol. This pancreatic injury was further supported by increased plasma lipase and pancreatic cathepsin B (a lysosomal hydrolase capable of activating trypsinogen), trypsinogen activation peptide (by-product of trypsinogen activation process) and glucose-regulated protein 78 (endoplasmic reticulum stress marker). These findings suggest that ADH-deficiency and high alcohol levels in the body are the key factors in ethanol-induced pancreatic injury. Therefore, determining how this early stage of pancreatic injury advances to inflammation stage could be important for understanding the mechanism(s) of alcoholic pancreatitis.

  10. Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol.

    PubMed

    Plapp, Bryce V; Charlier, Henry A; Ramaswamy, S

    2016-02-01

    Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 Å where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 Å to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxylate of Glu-67, while the oxygen of trifluoroethanol is 3.5 Å from the zinc. Subunit B may represent an intermediate in the mechanism after coenzyme and alcohol bind and before the conformation changes to the closed form and the alcohol oxygen binds to the zinc and displaces Glu-67. PMID:26743849

  11. Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

    PubMed

    Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

    2011-06-01

    Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability. PMID:21517848

  12. Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum, but aldehyde dehydrogenase is dispensable.

    PubMed

    Segers, G; Bradshaw, N; Archer, D; Blissett, K; Oliver, R P

    2001-03-01

    Cladosporiumfulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxidase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta. PMID:11277434

  13. Alcohol Dehydrogenase-1B (rs1229984) and Aldehyde Dehydrogenase-2 (rs671) Genotypes Are Strong Determinants of the Serum Triglyceride and Cholesterol Levels of Japanese Alcoholic Men

    PubMed Central

    Yokoyama, Akira; Yokoyama, Tetsuji; Matsui, Toshifumi; Mizukami, Takeshi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

    2015-01-01

    Background Elevated serum triglyceride (TG) and high-density-lipoprotein cholesterol (HDL-C) levels are common in drinkers. The fast-metabolizing alcohol dehydrogenase-1B encoded by the ADH1B*2 allele (vs. ADH1B*1/*1 genotype) and inactive aldehyde dehydrogenase-2 encoded by the ALDH2*2 allele (vs. ALDH2*1/*1 genotype) modify ethanol metabolism and are prevalent (≈90% and ≈40%, respectively) in East Asians. We attempted to evaluate the associations between the ADH1B and ALDH2 genotypes and lipid levels in alcoholics. Methods The population consisted of 1806 Japanese alcoholic men (≥40 years) who had undergone ADH1B and ALDH2 genotyping and whose serum TG, total cholesterol, and HDL-C levels in the fasting state had been measured within 3 days after admission. Results High serum levels of TG (≥150 mg/dl), HDL-C (>80 mg/dl), and low-density-lipoprotein cholesterol (LDL-C calculated by the Friedewald formula ≥140 mg/dl) were observed in 24.3%, 16.8%, and 15.6%, respectively, of the subjects. Diabetes, cirrhosis, smoking, and body mass index (BMI) affected the serum lipid levels. Multivariate analysis revealed that the presence of the ADH1B*2 allele and the active ALDH2*1/*1 genotype increased the odds ratio (OR; 95% confidence interval) for a high TG level (2.22 [1.67–2.94] and 1.39 [0.99–1.96], respectively), and decreased the OR for a high HDL-C level (0.37 [0.28–0.49] and 0.51 [0.37–0.69], respectively). The presence of the ADH1B*2 allele decreased the OR for a high LDL-C level (0.60 [0.45–0.80]). The ADH1B*2 plus ALDH2*1/*1 combination yielded the highest ORs for high TG levels and lowest OR for a high HDL-C level. The genotype effects were more prominent in relation to the higher levels of TG (≥220 mg/dl) and HDL-C (≥100 mg/dl). Conclusions The fast-metabolizing ADH1B and active ALDH2, and especially a combination of the two were strongly associated with higher serum TG levels and lower serum HDL-C levels of alcoholics. The fast

  14. S-Nitrosoglutathione is a substrate for rat alcohol dehydrogenase class III isoenzyme.

    PubMed

    Jensen, D E; Belka, G K; Du Bois, G C

    1998-04-15

    An enzyme isolated from rat liver cytosol (native molecular mass 78. 3 kDa; polypeptide molecular mass 42.5 kDa) is capable of catalysing the NADH/NADPH-dependent degradation of S-nitrosoglutathione (GSNO). The activity utilizes 1 mol of coenzyme per mol of GSNO processed. The isolated enzyme has, as well, several characteristics that are unique to alcohol dehydrogenase (ADH) class III isoenzyme: it is capable of catalysing the NAD+-dependent oxidations of octanol (insensitive to inhibition by 4-methylpyrazole), methylcrotyl alcohol (stimulated by added pentanoate) and 12-hydroxydodecanoic acid, and also the NADH/NADPH-dependent reduction of octanal. Methanol and ethanol oxidation activity is minimal. The enzyme has formaldehyde dehydrogenase activity in that it is capable of catalysing the NAD+/NADP+-dependent oxidation of S-hydroxymethylglutathione. Treatment with the arginine-specific reagent phenylglyoxal prevents the pentanoate stimulation of methylcrotyl alcohol oxidation and markedly diminishes the enzymic activity towards octanol, 12-hydroxydodecanoic acid and S-hydroxymethylglutathione; the capacity to catalyse GSNO degradation is also checked. Additionally, limited peptide sequencing indicates 100% correspondence with known ADH class III isoenzyme sequences. Kinetic studies demonstrate that GSNO is an exceptionally active substrate for this enzyme. S-Nitroso-N-acetylpenicillamine and S-nitrosated human serum albumin are not substrates; the activity towards S-nitrosated glutathione mono- and di-ethyl esters is minimal. Product analysis suggests that glutathione sulphinamide is the major stable product of enzymic GSNO processing, with minor yields of GSSG and NH3; GSH, hydroxylamine, nitrite, nitrate and nitric oxide accumulations are minimal. Inclusion of GSH in the reaction mix decreases the yield of the supposed glutathione sulphinamide in favor of GSSG and hydroxylamine. PMID:9531510

  15. Characterization of a Zinc-Containing Alcohol Dehydrogenase with Stereoselectivity from the Hyperthermophilic Archaeon Thermococcus guaymasensis▿

    PubMed Central

    Ying, Xiangxian; Ma, Kesen

    2011-01-01

    An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ± 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and l-threonine dehydrogenases with binding motifs of catalytic zinc and NADP+. Metal analyses revealed that this NADP+-dependent enzyme contained 0.9 ± 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic (R/S)-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent Km values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP+, respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation. PMID:21515780

  16. New inhibitors of alcohol dehydrogenase: studies in vivo and in vitro in the rat.

    PubMed

    Delmas, C; de Saint Blanquat, G; Freudenreich, C; Biellmann, J F

    1983-01-01

    Two compounds bearing an amide group, p-butoxyphenol acetamide (BPA) and N-(p-butoxybenzyl)formamide (BBF) were studied as inhibitors of alcohol dehydrogenase (ADH) and their action compared with that of 4-methyl-pyrazole (4-MP), a known inhibitor of this enzyme. In vitro studies on pure horse liver ADH showed that BPA and BBF were noncompetitive inhibitors with respect to ethanol and that their Ki values were 22 and 0.14 micrometer, respectively. The apparent Ki values of BPA and BBF for rat liver ADH were found to be 90 and 2.3 micrometers, respectively (noncompetitive inhibition). Several in vivo experiments were carried out in the rat. Administration intraperitoneally of the substance (460 mumol/kg) 1 hr before intraperitoneal injection of alcohol (2 g/kg body weight) led to a significant decrease in ethanol catabolism. Injection of the substances at 460 mumol/kg brought about a decrease in rat liver ADH activity, but the activity of mitochondrial aldehyde dehydrogenase was only decreased in animals treated with BBF. PMID:6353976

  17. Alcohol dehydrogenases from Scheffersomyces stipitis involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion.

    PubMed

    Ma, Menggen; Wang, Xu; Zhang, Xiaoping; Zhao, Xianxian

    2013-09-01

    Aldehyde inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) are generated from biomass pretreatment. Scheffersomyces stipitis is able to reduce furfural and HMF to less toxic furanmethanol and furan-2,5-dimethanol; however, the enzymes involved in the reductive reaction still remain unknown. In this study, transcription responses of two known and five putative alcohol dehydrogenase genes from S. stipitis were analyzed under furfural and HMF stress conditions. All the seven alcohol dehydrogenase genes were also cloned and overexpressed for their activity analyses. Our results indicate that transcriptions of SsADH4 and SsADH6 were highly induced under furfural and HMF stress conditions, and the proteins encoded by them exhibited NADH- and/or NADPH-dependent activities for furfural and HMF reduction, respectively. For furfural reduction, NADH-dependent activity was also observed in SsAdh1p and NAD(P)H-dependent activities were also observed in SsAdh5p and SsAdh7p. For HMF reduction, NADPH-dependent activities were also observed in SsAdh5p and SsAdh7p. SsAdh4p displayed the highest NADPH-dependent specific activity and catalytic efficiency for reduction of both furfural and HMF among the seven alcohol dehydrogenases. Enzyme activities of all SsADH proteins were more stable under acidic condition. For most SsADH proteins, the optimum temperature for enzyme activities was 30 °C and more than 50 % enzyme activities remained at 60 °C. Reduction activities of formaldehyde, acetaldehyde, isovaleraldehyde, benzaldehyde, and phenylacetaldehyde were also observed in some SsADH proteins. Our results indicate that multiple alcohol dehydrogenases in S. stipitis are involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion. PMID:23912116

  18. Origin and evolution of medium chain alcohol dehydrogenases.

    PubMed

    Jörnvall, Hans; Hedlund, Joel; Bergman, Tomas; Kallberg, Yvonne; Cederlund, Ella; Persson, Bengt

    2013-02-25

    Different lines of alcohol dehydrogenases (ADHs) have separate superfamily origins, already recognized but now extended and re-evaluated by re-screening of the latest databank update. The short-chain form (SDR) is still the superfamily with most abundant occurrence, most multiple divergence, most prokaryotic emphasis, and most non-complicated architecture. This pattern is compatible with an early appearance at the time of the emergence of prokaryotic cellular life. The medium-chain form (MDR) is also old but second in terms of all the parameters above, and therefore compatible with a second emergence. However, this step appears seemingly earlier than previously considered, and may indicate sub-stages of early emergences at the increased resolution available from the now greater number of data entries. The Zn-MDR origin constitutes a third stage, possibly compatible with the transition to oxidative conditions on earth. Within all these three lines, repeated enzymogeneses gave the present divergence. MDR-ADH origin(s), at a fourth stage, may also be further resolved in multiple or extended modes, but the classical liver MDR-ADH of the liver type can still be traced to a gene duplication ~550 MYA (million years ago), at the early vertebrate radiation, compatible with the post-eon-shift, "Cambrian explosion". Classes and isozymes correspond to subsequent and recent duplicatory events, respectively. They illustrate a peculiar pattern with functional and emerging evolutionary distinctions between parent and emerging lines, suggesting a parallelism between duplicatory and mutational events, now also visible at separate sub-stages. Combined, all forms show distinctive patterns at different levels and illustrate correlations with global events. They further show that simple molecular observations on patterns, multiplicities and occurrence give much information, suggesting common divergence rules not much disturbed by horizontal gene transfers after the initial origins. PMID

  19. Bifunctional aldehyde/alcohol dehydrogenase (ADHE) in chlorophyte algal mitochondria.

    PubMed

    Atteia, Ariane; van Lis, Robert; Mendoza-Hernández, Guillermo; Henze, Katrin; Martin, William; Riveros-Rosas, Hector; González-Halphen, Diego

    2003-09-01

    Protein profiles of mitochondria isolated from the heterotrophic chlorophyte Polytomella sp. grown on ethanol at pH 6.0 and pH 3.7 were analyzed by Blue Native and denaturing polyacrylamide gel electrophoresis. Steady-state levels of oxidative phosphorylation complexes were influenced by external pH. Levels of an abundant, soluble, mitochondrial protein of 85 kDa and its corresponding mRNA increased at pH 6.0 relative to pH 3.7. N-terminal and internal sequencing of the 85 kDa mitochondrial protein together with the corresponding cDNA identified it as a bifunctional aldehyde/alcohol dehydrogenase (ADHE) with strong similarity to homologues from eubacteria and amitochondriate protists. A mitochondrial targeting sequence of 27 amino acids precedes the N-terminus of the mature mitochondrial protein. A gene encoding an ADHE homologue was also identified in the genome of Chlamydomonas reinhardtii, a photosynthetic relative of Polytomella. ADHE reveals a complex picture of sequence similarity among homologues. The lack of ADHE from archaebacteria indicates a eubacterial origin for the eukaryotic enzyme. Among eukaryotes, ADHE has hitherto been characteristic of anaerobes since it is essential to cytosolic energy metabolism of amitochondriate protists such as Giardia intestinalis and Entamoeba histolytica. Its abundance and expression pattern suggest an important role for ADHE in mitochondrial metabolism of Polytomella under the conditions studied. The current data are compatible with the view that Polytomella ADHE could be involved either in ethanol production or assimilation, or both, depending upon environmental conditions. Presence of ADHE in an oxygen-respiring algal mitochondrion and co-expression at ambient oxygen levels with respiratory chain components is unexpected with respect to the view that eukaryotes acquired ADHE genes specifically as an adaptation to an anaerobic lifestyle. PMID:14756315

  20. Secondary alcohol dehydrogenase catalyzes the reduction of exogenous acetone to 2-propanol in Trichomonas vaginalis.

    PubMed

    Sutak, Robert; Hrdy, Ivan; Dolezal, Pavel; Cabala, Radomir; Sedinová, Miroslava; Lewin, Joern; Harant, Karel; Müller, Miklos; Tachezy, Jan

    2012-08-01

    Secondary alcohols such as 2-propanol are readily produced by various anaerobic bacteria that possess secondary alcohol dehydrogenase (S-ADH), although production of 2-propanol is rare in eukaryotes. Specific bacterial-type S-ADH has been identified in a few unicellular eukaryotes, but its function is not known and the production of secondary alcohols has not been studied. We purified and characterized S-ADH from the human pathogen Trichomonas vaginalis. The kinetic properties and thermostability of T. vaginalis S-ADH were comparable with bacterial orthologues. The substantial activity of S-ADH in the parasite's cytosol was surprising, because only low amounts of ethanol and trace amounts of secondary alcohols were detected as metabolic end products. However, S-ADH provided the parasite with a high capacity to scavenge and reduce external acetone to 2-propanol. To maintain redox balance, the demand for reducing power to metabolize external acetone was compensated for by decreased cytosolic reduction of pyruvate to lactate and by hydrogenosomal metabolism of pyruvate. We speculate that hydrogen might be utilized to maintain cytosolic reducing power. The high activity of Tv-S-ADH together with the ability of T. vaginalis to modulate the metabolic fluxes indicate efficacious metabolic responsiveness that could be advantageous for rapid adaptation of the parasite to changes in the host environment. PMID:22686835

  1. Physiological Studies of Methane- and Methanol-Oxidizing Bacteria: Comparison of a Primary Alcohol Dehydrogenase from Methylococcus capsulatus (Texas Strain) and Pseudomonas Species M27

    PubMed Central

    Patel, R. N.; Bose, H. R.; Mandy, W. J.; Hoare, D. S.

    1972-01-01

    A primary alcohol dehydrogenase has been purified from Methylococcus capsulatus (Texas strain). The purified enzyme catalyzes the oxidation of methanol and formaldehyde to formate; other primary alcohols are oxidized to their corresponding aldehydes. Ammonium ions are required for enzyme activity. The enzyme has a molecular weight of 120,000 daltons and consists of two 62,000 molecular-weight subunits which dissociate at acidic pH. The enzyme is similar to an alcohol dehydrogenase enzyme isolated from Pseudomonas sp. M27. Images PMID:5022170

  2. Unexpected properties of NADP-dependent secondary alcohol dehydrogenase (ADH-1) in Trichomonas vaginalis and other microaerophilic parasites.

    PubMed

    Leitsch, David; Williams, Catrin F; Lloyd, David; Duchêne, Michael

    2013-07-01

    Our previous observation that NADP-dependent secondary alcohol dehydrogenase (ADH-1) is down-regulated in metronidazole-resistant Trichomonas vaginalis isolates prompted us to further characterise the enzyme. In addition to its canonical enzyme activity as a secondary alcohol dehydrogenase, a pronounced, so far unknown, background NADPH-oxidising activity in absence of any added substrate was observed when the recombinant enzyme or T. vaginalis extract were used. This activity was strongly enhanced at low oxygen concentrations. Unexpectedly, all functions of ADH-1 were efficiently inhibited by coenzyme A which is a cofactor of a number of key enzymes in T. vaginalis metabolism, i.e. pyruvate:ferredoxin oxidoreductase (PFOR). These observations could be extended to Entamoeba histolytica and Tritrichomonas foetus, both of which have a homologue of ADH-1, but not to Giardia lamblia which lacks an NADP-dependent secondary alcohol dehydrogenase. Although we could not identify the substrate of the observed background activity, we propose that ADH-1 functions as a major sink for NADPH in microaerophilic parasites at low oxygen tension. PMID:23578856

  3. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

    SciTech Connect

    Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V; Parks, Jerry M; Smolin, Nikolai; Yang, Shihui; Land, Miriam L; Klingeman, Dawn Marie; Bhandiwad, Ashwini; Rodriguez, Jr., Miguel; Raman, Babu; Shao, Xiongjun; Mielenz, Jonathan R; Smith, Jeremy C; Keller, Martin; Lynd, Lee R

    2011-01-01

    Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

  4. Expression of Alcohol Dehydrogenase 3 in Tissue and Cultured Cells from Human Oral Mucosa

    PubMed Central

    Hedberg, Jesper J.; Höög, Jan-Olov; Nilsson, Jan A.; Xi, Zheng; Elfwing, Åsa; Grafström, Roland C.

    2000-01-01

    Because formaldehyde exposure has been shown to induce pathological changes in human oral mucosa, eg, micronuclei, the potential enzymatic defense by alcohol dehydrogenase 3 (ADH3)/glutathione-dependent formaldehyde dehydrogenase was characterized in oral tissue specimens and cell lines using RNA hybridization and immunological methods as well as enzyme activity measurements. ADH3 mRNA was expressed in basal and parabasal cell layers of oral epithelium, whereas the protein was detected throughout the cell layers. ADH3 mRNA and protein were further detected in homogenates of oral tissue and various oral cell cultures, including, normal, SV40T antigen-immortalized, and tumor keratinocyte lines. Inhibition of the growth of normal keratinocytes by maintenance at confluency significantly decreased the amount of ADH3 mRNA, a transcript with a determined half-life of 7 hours. In contrast, decay of ADH3 protein was not observed throughout a 4-day period in normal keratinocytes. In samples from both tissue and cells, the ADH3 protein content correlated to oxidizing activity for the ADH3-specific substrate S-hydroxymethylglutathione. The composite analyses associates ADH3 mRNA primarily to proliferative keratinocytes where it exhibits a comparatively short half-life. In contrast, the ADH3 protein is extremely stable, and consequently is retained during the keratinocyte life span in oral mucosa. Finally, substantial capacity for formaldehyde detoxification is shown from quantitative assessments of alcohol- and aldehyde-oxidizing activities including Km determinations, indicating that ADH3 is the major enzyme involved in formaldehyde oxidation in oral mucosa. PMID:11073833

  5. Isolation of an alcohol dehydrogenase cDNA from and characterization of its expression in chrysanthemum under waterlogging.

    PubMed

    Yin, Dongmei; Ni, Dian; Song, Lili; Zhang, Zhiguo

    2013-11-01

    A PCR strategy was used to isolate a full-length CgADH (alcohol dehydrogenase) cDNA from chrysanthemum. The gene putatively encodes a 378 residue polypeptides, which shares 95% homology with tomato alcohol dehydrogenase class III. Endogenous ethylene generated in waterlogged Chrysanthemum zawadskii was enhanced by exogenous ethylene but decreased by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action. In waterlogged roots, the transcription of the gene encoding alcohol dehydrogenase (ADH, EC 1.1.1.1) increased rapidly but transiently, peaking at 7.5 fold the non-waterlogged level after 2h of stress. Waterlogging elevated ADH activity after a prolonged episode of stress. The exogenous supply of 40μLL(-1) ethylene suppressed the production of ethanol, while that of 4μLL(-1) 1-MCP enhanced it. Ethylene appeared to suppress an acceleration of both CgADH expression and fermentation, and alleviates ethanolic fermentation probably through by as a signal to acceleration of waterlogging-induced aerenchyma formation. This supports the previously observed phenomenon that the expression level of ADH gene is regulated by the local level of physiologically active ethylene. The relevance of the CgADH gene in relation to chrysanthemum waterlogging was discussed as well. PMID:24094053

  6. Dehydrogenation of 3-phenoxybenzyl alcohol in isolated perfused rabbit skin, skin homogenate and purified dehydrogenases.

    PubMed

    Bast, G E; Kampffmeyer, H G

    1998-01-01

    The formation of 3-phenoxybenzoic acid from 3-phenoxybenzyl alcohol was determined in (a) rabbit ears, single-pass perfused with a protein-free buffer, pH 7.4; (b) the microsomal fraction and its supernatant from homogenized rabbit skin; and (c) purified alcohol dehydrogenase from horse liver and baker's yeast. The inhibition of product formation in (a) was about 60% by various 4-methylpyrazole concentrations, but metyrapone had no effect. Following ultracentrifugation, only the supernatant of homogenized skin showed product formation (apparent Vmay: 32 pmol/min per cm2 skin; apparent Km: 64 microM). 3-Phenoxybenzyl alcohol and ethanol dehydrogenation was similar by alcohol dehydrogenase from horse liver (apparent Km: 0.7 vs. 0.4 mM; apparent Vmax: 0.3 vs. 0.2 U/ microg protein). In baker's yeast, the apparent Km of 3-phenoxybenzoic acid formation was several times larger than that for ethanol dehydrogenation. The KI of 4-methylpyrazole for alcohol dehydrogenase from horse liver was 0.6 (3-phenoxybenzyl alcohol) vs. 0.04 microM (ethanol). The KI for ethanol in baker's yeast was 470 microM. In conclusion dehydrogenation is an important metabolic pathway in the skin for xenobiotics with an aliphatic alcohol at a side chain. PMID:9885409

  7. Direct Electrochemical Addressing of Immobilized Alcohol Dehydrogenase for the Heterogeneous Bioelectrocatalytic Reduction of Butyraldehyde to Butanol

    PubMed Central

    Schlager, S; Neugebauer, H; Haberbauer, M; Hinterberger, G; Sariciftci, N S

    2015-01-01

    Modified electrodes using immobilized alcohol dehydrogenase enzymes for the efficient electroreduction of butyraldehyde to butanol are presented as an important step for the utilization of CO2-reduction products. Alcohol dehydrogenase was immobilized, embedded in an alginate–silicate hybrid gel, on a carbon felt (CF) electrode. The application of this enzyme to the reduction of an aldehyde to an alcohol with the aid of the coenzyme nicotinamide adenine dinucleotide (NADH), in analogy to the final step in the natural reduction cascade of CO2 to alcohol, has been already reported. However, the use of such enzymatic reductions is limited because of the necessity of providing expensive NADH as a sacrificial electron and proton donor. Immobilization of such dehydrogenase enzymes on electrodes and direct pumping of electrons into the biocatalysts offers an easy and efficient way for the biochemical recycling of CO2 to valuable chemicals or alternative synthetic fuels. We report the direct electrochemical addressing of immobilized alcohol dehydrogenase for the reduction of butyraldehyde to butanol without consumption of NADH. The selective reduction of butyraldehyde to butanol occurs at room temperature, ambient pressure and neutral pH. Production of butanol was detected by using liquid-injection gas chromatography and was estimated to occur with Faradaic efficiencies of around 40 %. PMID:26113881

  8. Theoretical Calculations of the Catalytic Triad in Short-Chain Alcohol Dehydrogenases/Reductases

    PubMed Central

    Gani, Osman A. B. S. M.; Adekoya, Olayiwola A.; Giurato, Laura; Spyrakis, Francesca; Cozzini, Pietro; Guccione, Salvatore; Winberg, Jan-Olof; Sylte, Ingebrigt

    2008-01-01

    Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that might be possible when inhibitors bind to the enzyme cofactor complex, were constructed. The binding of the two alcohol competitive inhibitors were studied using automatic docking by the Internal Coordinate Mechanics program, molecular dynamic (MD) simulations with the AMBER program package, calculation of the free energy of ligand binding by the linear interaction energy method, and the hydropathic interactions force field. The calculations indicated that deprotonated Tyr acts as a strong base in the binary enzyme-NAD+ complex. Molecular dynamic simulations for 5 ns confirmed that deprotonated Tyr is essential for anchoring and orientating the inhibitors at the active site, which might be a general trend for the family of SDRs. The findings here have implications for the development of therapeutically important SDR inhibitors. PMID:17981907

  9. Biochemical characterization of a bifunctional acetaldehyde-alcohol dehydrogenase purified from a facultative anaerobic bacterium Citrobacter sp. S-77.

    PubMed

    Tsuji, Kohsei; Yoon, Ki-Seok; Ogo, Seiji

    2016-03-01

    Acetaldehyde-alcohol dehydrogenase (ADHE) is a bifunctional enzyme consisting of two domains of an N-terminal acetaldehyde dehydrogenase (ALDH) and a C-terminal alcohol dehydrogenase (ADH). The enzyme is known to be important in the cellular alcohol metabolism. However, the role of coenzyme A-acylating ADHE responsible for ethanol production from acetyl-CoA remains uncertain. Here, we present the purification and biochemical characterization of an ADHE from Citrobacter sp. S-77 (ADHES77). Interestingly, the ADHES77 was unable to be solubilized from membrane with detergents either 1% Triton X-100 or 1% Sulfobetaine 3-12. However, the enzyme was easily dissociated from membrane by high-salt buffers containing either 1.0 M NaCl or (NH4)2SO4 without detergents. The molecular weight of a native protein was estimated as approximately 400 kDa, consisting of four identical subunits of 96.3 kDa. Based on the specific activity and kinetic analysis, the ADHES77 tended to have catalytic reaction towards acetaldehyde elimination rather than acetaldehyde formation. Our experimental observation suggests that the ADHES77 may play a pivotal role in modulating intracellular acetaldehyde concentration. PMID:26216639

  10. Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignin is a significant recalcitrant in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired...

  11. Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Morgan, Chad; Moir, Neil

    1996-11-01

    A rapid microscale procedure has been developed for the isolation and purification of yeast alcohol dehydrogenase. Glass beads are used for cytolysis, PEG precipitation for partial purification and a cibacron blue affinity column for the final step. A 27.5 fold purification can be achieved in 2 - 3 hours.

  12. Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

    ERIC Educational Resources Information Center

    Nash, Barbara T.

    2007-01-01

    SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

  13. An enzyme-amplified microtiter plate assay for ethanol: Its application to the detection of peanut ethanol and alcohol dehydrogenase

    SciTech Connect

    Chung, S.Y.; Vercellotti, J.R.; Sanders, T.H.

    1995-12-01

    A calorimetric microliter plate assay for ethanol amplified by aldehyde dehydrogenase (ALDH) was developed. In the assay ethanol from a sample took part in a chain-reaction catalyzed by alcohol dehydrogenase (ADH) and amplified by ALDH in the presence of NAD{sup +}, diaphorase, and p-ibdonitrotetrazolium-violet (INT-violet)(a precursor of red product). The resultant reaction gave a red color, the intensity of which was proportional to the amount of ethanol present. Using the technique, the content of activity from peanuts of differing maturity and curing stages were determined respectively. Data showed that immature peanuts had a higher level of ethanol and a lower ADH activity than mature peanuts, and that the level of ethanol and ADH activity decreased with the curing time. This indicates that peanut maturity and curing have an effect on ethanol. Also, this implies that other peanut volatiles could be affected in the same way as ethanol, a major volatile in peanuts.

  14. Postischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity

    PubMed Central

    Richards, Erica M.; Rosenthal, Robert E.; Kristian, Tibor; Fiskum, Gary

    2008-01-01

    The pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme that catalyzes the oxidative decarboxylation of pyruvate and represents the sole bridge between anaerobic and aerobic cerebral energy metabolism. Previous studies demonstrating loss of PDHC enzyme activity and immunoreactivity during reperfusion after cerebral ischemia suggest that oxidative modifications are involved. This study tested the hypothesis that hyperoxic reperfusion exacerbates loss of PDHC enzyme activity, possibly due to tyrosine nitration or S-nitrosation. We used a clinically relevant canine ventricular fibrillation cardiac arrest model in which, after resuscitation and ventilation on either 100% O2 (hyperoxic) or 21–30% O2 (normoxic), animals were sacrificed at 2 h reperfusion and the brains removed for enzyme activity and immunoreactivity measurements. Animals resuscitated under hyperoxic conditions exhibited decreased PDHC activity and elevated 3-nitrotyrosine immunoreactivity in the hippocampus but not the cortex, compared to nonischemic controls. These measures were unchanged in normoxic animals. In vitro exposure of purified PDHC to peroxynitrite resulted in a dose-dependent loss of activity and increased nitrotyrosine immunoreactivity. These results support the hypothesis that oxidative stress contributes to loss of hippocampal PDHC activity during cerebral ischemia and reperfusion and suggest that PDHC is a target of peroxynitrite. PMID:16716897

  15. Optical isopropanol biosensor using NADH-dependent secondary alcohol dehydrogenase (S-ADH).

    PubMed

    Chien, Po-Jen; Ye, Ming; Suzuki, Takuma; Toma, Koji; Arakawa, Takahiro; Iwasaki, Yasuhiko; Mitsubayashi, Kohji

    2016-10-01

    Isopropanol (IPA) is an important solvent used in industrial activity often found in hospitals as antiseptic alcohol rub. Also, IPA may have the potential to be a biomarker of diabetic ketoacidosis. In this study, an optical biosensor using NADH-dependent secondary alcohol dehydrogenase (S-ADH) for IPA measurement was constructed and evaluated. An ultraviolet light emitting diode (UV-LED, λ=340nm) was employed as the excitation light to excite nicotinamide adenine dinucleotide (NADH). A photomultiplier tube (PMT) was connected to a two-way branch optical fiber for measuring the fluorescence emitted from the NADH. S-ADH was immobilized on the membrane to catalyze IPA to acetone and reduce NAD(+) to be NADH. This IPA biosensor shows highly sensitivity and selectivity, the calibration range is from 500 nmol L(-1) to 1mmolL(-1). The optimization of buffer pH, temperature, and the enzyme-immobilized method were also evaluated. The detection of IPA in nail related cosmetic using our IPA biosensor was also carried out. The results showed that large amounts of IPA were used in these kinds of cosmetics. This IPA biosensor comes with the advantages of rapid reaction, good reproducibility, and wide dynamic range, and is also expected to use for clinical IPA detections in serum or other medical and health related applications. PMID:27474326

  16. Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

    PubMed Central

    Henningsen, Brooks M.; Hon, Shuen; Covalla, Sean F.; Sonu, Carolina; Argyros, D. Aaron; Barrett, Trisha F.; Wiswall, Erin; Froehlich, Allan C.

    2015-01-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter−1 acetate during fermentation of 114 g liter−1 glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter−1, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter−1 and raised the ethanol yield to 7% above the wild-type level. PMID:26386051

  17. Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

    PubMed

    Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. PMID:26386051

  18. Probes of hydrogen tunneling with horse liver alcohol dehydrogenase at subzero temperatures.

    PubMed

    Tsai, S; Klinman, J P

    2001-02-20

    The temperature dependence of steady-state kinetics has been studied with horse liver alcohol dehydrogenase (HLADH) using protonated and deuterated benzyl alcohol as substrates in methanol/water mixtures between +3 and -50 degrees C. Additionally, the competitive isotope effects, k(H)/k(T) and k(D)/k(T), were measured. The studies indicate increasing kinetic complexity for wild-type HLADH at subzero temperatures. Consistent with earlier findings at 25 degrees C [Bahnson et al. (1993) Biochemistry 31, 5503], the F93W mutant shows much less kinetic complexity than the wild-type enzyme between 3 and -35 degrees C. An analysis of noncompetitive deuterium isotope effects and competitive tritium isotope effects leads to the conclusion that the reaction of F93W involves substantial hydrogen tunneling down to -35 degrees C. The effect of methanol on kinetic properties for the F93W mutant was analyzed, showing a dependence of competitive KIEs on the NAD(+) concentration. This indicates a more random bi--bi kinetic mechanism, in comparison to an ordered bi-bi kinetic mechanism in water. Although MeOH also affects the magnitude of the reaction rates and, to some extent, the observed KIEs, the ratio of ln k(H)/k(T) to ln k(D)/k(T) for primary isotope effects has not changed in methanol, and we conclude little or no change in kinetic complexity. Importantly, the degree of tunneling, as shown from the relationship between the secondary k(H)/k(T) and k(D)/k(T) values, is the same in water and MeOH/water mixtures, implicating similar trajectories for H transfer in both solvents. In a recent study of a thermophilic alcohol dehydrogenase [Kohen et al. (1999) Nature 399, 496], it was shown that decreases in temperatures below a transition temperature lead to decreased tunneling. This arises because of a change in protein dynamics below a break point in enzyme activity [Kohen et al. (2000) J. Am. Chem. Soc. 122, 10738-10739]. For the mesophilic HLADH described herein, an opposite

  19. [Possible ways of regulating detoxifying processes in the alcohol dehydrogenase reaction with pantothenic acid derivatives].

    PubMed

    Chernikevich, I P; Dorofeev, B F; Moĭseenok, A G

    1993-01-01

    Oxidation of derivatives and precursors of pantothenic acid was studied in alcohol dehydrogenase reactions. Despite the presence of free hydroxymethyl groups in a number of pantothenic acid derivatives only panthenol with Km = 8 x 10(-3) M was shown to serve as a substrate for alcohol dehydrogenase from horse liver tissue (EC 1.1.1.1) Pantethine, sodium phosphopantothenate, CoA and acetyl-CoA decreased the rate of ethanol oxidation, where pantethine and sodium phosphopantothenate were competitive inhibitors, while CoA and acetyl-CoA inhibited the enzyme noncompetitively Ki = 1.2 x 10(-2) M, 2.1 x 10(-2) M, 4.4 x 10(-4) M and 5.1 x 10(-4) M, respectively. Metabolic precursors, which were different from pantothenic acid in their structure, were not involved in the alcohol dehydrogenase reaction. Possible regulation of alcohol intoxication using derivatives and precursors of vitamin B3 is discussed. PMID:8511887

  20. Evaluation of alcohol dehydrogenase and aldehyde dehydrogenase enzymes as bi-enzymatic anodes in a membraneless ethanol microfluidic fuel cell

    NASA Astrophysics Data System (ADS)

    Galindo-de-la-Rosa, J.; Arjona, N.; Arriaga, L. G.; Ledesma-García, J.; Guerra-Balcázar, M.

    2015-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldH) enzymes were immobilized by covalent binding and used as the anode in a bi-enzymatic membraneless ethanol hybrid microfluidic fuel cell. The purpose of using both enzymes was to optimize the ethanol electro-oxidation reaction (EOR) by using ADH toward its direct oxidation and AldH for the oxidation of aldehydes as by-products of the EOR. For this reason, three enzymatic bioanode configurations were evaluated according with the location of enzymes: combined, vertical and horizontally separated. In the combined configuration, a current density of 16.3 mA cm-2, a voltage of 1.14 V and a power density of 7.02 mW cm-2 were obtained. When enzymes were separately placed in a horizontal and vertical position the ocp drops to 0.94 V and to 0.68 V, respectively. The current density also falls to values of 13.63 and 5.05 mA cm-2. The decrease of cell performance of bioanodes with separated enzymes compared with the combined bioanode was of 31.7% and 86.87% for the horizontal and the vertical array.

  1. Aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) polymorphisms exacerbate bladder cancer risk associated with alcohol drinking: gene-environment interaction.

    PubMed

    Masaoka, Hiroyuki; Ito, Hidemi; Soga, Norihito; Hosono, Satoyo; Oze, Isao; Watanabe, Miki; Tanaka, Hideo; Yokomizo, Akira; Hayashi, Norio; Eto, Masatoshi; Matsuo, Keitaro

    2016-06-01

    Although a range of chemical exposures (cigarette smoking and occupational exposure) are recognized risk factors for the development of bladder cancer (BCa), many epidemiological studies have demonstrated that alcohol drinking is not associated with BCa risk. Aldehyde dehydrogenase 2 (ALDH2; rs671, Glu504Lys) and alcohol dehydrogenase 1B (ADH1B; rs1229984, His47Arg) polymorphisms impact the accumulation of acetaldehyde, resulting in an increased risk of various cancers. To date, however, no studies evaluating the association between BCa risk and alcohol drinking have considered these polymorphisms. Here, we conducted a matched case-control study to investigate whether ALDH2 and ADH1B polymorphisms influence BCa risk associated with alcohol drinking. Cases were 74 BCa patients and controls were 740 first-visit outpatients without cancer at Aichi Cancer Center Hospital between January 2001 and December 2005. Odds ratio (OR), 95% confidence interval (CI) and gene-environment interaction were assessed by conditional logistic regression analysis with adjustment for potential confounders. Results showed that ALDH2 Glu/Lys was associated with a significantly increased risk of BCa compared with Glu/Glu (OR 2.03, 95% CI 1.14-3.62, P = 0.017). In contrast, ALDH2 Glu/Lys showed no increase in risk among the stratum of never drinkers compared with Glu/Glu, indicating a gene-environment interaction. ADH1B His/Arg had an OR of 1.98 (1.20-3.24, P = 0.007) compared with His/His. ADH1B Arg+ showed a similar OR and 95% CI. Individuals with ALDH2 Glu/Lys and ADH1B Arg+ had the highest risk of BCa compared with ALDH2 Glu/Glu and ADH1B His/His [OR 4.00 (1.81-8.87), P = 0.001]. PMID:26992901

  2. Regulation of human alcohol dehydrogenase gene ADH7: importance of an AP-1 site.

    PubMed

    Kotagiri, S; Edenberg, H J

    1998-07-01

    The structure and function of the human alcohol dehydrogenase 7 (ADH7) promoter were analyzed. A promoter fragment extending to bp -232 functioned well in H4IIE-C3, CV-1, and HeLa cells, whereas the region extending further upstream to bp -799 had no significant effect on activity. We identified cis-acting elements in the proximal 232 bp and examined their effect on promoter activity. Mutation of site A, where c-Jun bound, caused a drastic decrease in the promoter activity in H4IIE-C3 and CV-1 cells, suggesting that AP-1 plays an important role in the regulation of ADH7. Mutation of site B also caused a large drop in promoter activity in both cell lines; C/EBPalpha can bind to this site, but because the site affects activity approximately equally in CV-1 cells that lack C/EBPalpha and in H4IIE-C3 cells that contain low levels, other proteins are likely to play the major roles in vivo. Mutation of site C, where C/EBP bound and c-Jun bound weakly, had different effects in the two cell lines: in H4IIE-C3 cells, the site C mutation did not significantly increase promoter activity, whereas in CV-1 cells, which lack C/EBPalpha, it led to a doubling of activity. Surprisingly, cotransfection of the wild-type promoter with C/EBPa or C/EBPbeta led to a decrease in promoter activity, which might in part explain the lack of activity of ADH7 in adult liver. PMID:9703017

  3. Insight into the stereospecificity of short-chain thermus thermophilus alcohol dehydrogenase showing pro-S hydride transfer and prelog enantioselectivity.

    PubMed

    Pennacchio, Angela; Giordano, Assunta; Esposito, Luciana; Langella, Emma; Rossi, Mosè; Raia, Carlo A

    2010-04-01

    The stereochemistry of the hydride transfer in reactions catalyzed by NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus HB27 was determined by means of (1)H-NMR spectroscopy. The enzyme transfers the pro-S hydrogen of [4R-(2)H]NADH and exhibits Prelog specificity. Enzyme-substrate docking calculations provided structural details about the enantioselectivity of this thermophilic enzyme. These results give additional insights into the diverse active site architectures of the largely versatile short-chain dehydrogenase superfamily enzymes. A feasible protocol for the synthesis of [4R-(2)H]NADH with high yield was also set up by enzymatic oxidation of 2-propanol-d(8) catalyzed by Bacillus stearothermophilus alcohol dehydrogenase. PMID:19807673

  4. The first step in polyethylene glycol degradation by sphingomonads proceeds via a flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide.

    PubMed

    Sugimoto, M; Tanabe, M; Hataya, M; Enokibara, S; Duine, J A; Kawai, F

    2001-11-01

    Several Sphingomonas spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound. The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E. coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%). PMID:11673442

  5. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase.

    PubMed

    Li, Qunrui; Metthew Lam, L K; Xun, Luying

    2011-11-01

    Ethanol is a renewable biofuel, and it can be produced from lignocellulosic biomass. The biomass is usually converted to hydrolysates that consist of sugar and sugar derivatives, such as furfural. Yeast ferments sugar to ethanol, but furfural higher than 3 mM is inhibitory. It can take several days for yeast cells to reduce furfural to non-inhibitory furfuryl alcohol before producing ethanol. Bioreduction of furfural to furfuryl alcohol before fermentation may relieve yeast from furfural toxicity. We observed that Cupriavidus necator JMP134, a strict aerobe, rapidly reduced 17 mM furfural to less than 3 mM within 14 min with cell turbidity of 1.0 at 600 nm at 50°C. The rapid reduction consumed ethanol. The "furfural reductase" (FurX) was purified, and it oxidized ethanol to acetaldehyde and reduced furfural to furfuryl alcohol with NAD(+) as the cofactor. The protein was identified with mass spectrometry fingerprinting to be a hypothetical protein belonging to Zn-dependent alcohol dehydrogenase family. The furX-inactivation mutant of C. necator JMP134 lost the ability to rapidly reduce furfural, and Escherichia coli producing recombinant FurX gained the ability. Thus, an alcohol dehydrogenase enabled bacteria to rapidly reduce furfural with ethanol as the reducing power. PMID:21526390

  6. Red Xylem and Higher Lignin Extractability by Down-Regulating a Cinnamyl Alcohol Dehydrogenase in Poplar.

    PubMed Central

    Baucher, M.; Chabbert, B.; Pilate, G.; Van Doorsselaere, J.; Tollier, M. T.; Petit-Conil, M.; Cornu, D.; Monties, B.; Van Montagu, M.; Inze, D.; Jouanin, L.; Boerjan, W.

    1996-01-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Populus tremula X Populus alba) by both antisense and co-suppression strategies. Several antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCl staining was different in the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity in trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry. PMID:12226459

  7. Monoterpene alcohol metabolism: identification, purification, and characterization of two geraniol dehydrogenase isoenzymes from Polygonum minus leaves.

    PubMed

    Hassan, Maizom; Maarof, Nur Diyana; Ali, Zainon Mohd; Noor, Normah Mohd; Othman, Roohaida; Mori, Nobuhiro

    2012-01-01

    NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+). PMID:22878188

  8. Enhanced Stability and Reusability of Alcohol Dehydrogenase Covalently Immobilized on Magnetic Graphene Oxide Nanocomposites.

    PubMed

    Liu, Liangliang; Yu, Jingang; Chen, Xiaoqing

    2015-02-01

    Graphene oxide (GO) has a unique planar structure and contains many functional groups. As a functional material, it can be functionalized with biomolecules and nanomaterials for various applications. In this study, Magnetic GO (MGO) nanocomposites were synthesized according to covalent binding of amino Fe3O4 nanoparticles onto the GO surface and the as-made nanocomposites were successfully applied as supports for the immobilization of alcohol dehydrogenase (ADH). Compared with free ADH and Fe3O4 nanoparticles immobilized ADH (MNP-ADH), the MGO immobilized ADH (MGO-ADH) exhibited a wider pH stability range and a better thermal stability. Furthermore, the MGO-ADH exhibited better storage stability and reusability than MNP-ADH after recovered by magnetic separations. The MGO-ADH maintained 35.1% activity after 20 days storage and lost about 20.4% activity after ten times usage. The Michaelis constant (Km) of MGO-ADH was close to that of free ADH. The results showed the MGO nanocomposites were appropriate for the immobilization of enzyme. As a novel support, MGO nanocomposites effectively increased the stability of enzyme, allowed the reuse or continuous use of enzymes and therefore improved the potential use in practical. PMID:26353636

  9. Molecular control of the induction of alcohol dehydrogenase by ethanol in Drosophila melanogaster larvae

    SciTech Connect

    Kapoun, A.M.; Geer, B.W.; Heinstra, P.W.H. ); Corbin, V. ); McKechnie, S.W. )

    1990-04-01

    The activity of alcohol dehydrogenase, the initial enzyme in the major pathway for ethanol degradation, is induced in Drosophila melanogaster larvae by low concentrations of dietary ethanol. Two lines of evidence indicate that the metabolic products of the ADH pathway for ethanol degradation are not directly involved in the induction of Adh. First, the accumulation of the proximal transcript in Adh{sup n2} larvae was increased when the intracellular level of ethanol was elevated. In addition, the ADH activity, the proximal Adh mRNA, and the intracellular concentration of ethanol were elevated coordinately in wild-type larvae fed hexadeuterated-ethanol, which is metabolized more slowly than normal ethanol.l An examination of P element transformant lines with specific deletions in the 5{prime} regulatory DNA of the Adh gene showed that the DNA sequence between +604 and +634 of the start site of transcription from the distal promoter was essential for this induction. The DNA sequence between {minus}660 and about {minus}5,000 of the distal transcript start site was important for the down-regulation of the induction response.

  10. Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

    PubMed Central

    Zarnt, Grit; Schräder, Thomas; Andreesen, Jan R.

    2001-01-01

    The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparent kcat/Km and Ki values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a kcat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases. PMID:11222593

  11. Suitability of the hydrocarbon-hydroxylating molybdenum-enzyme ethylbenzene dehydrogenase for industrial chiral alcohol production.

    PubMed

    Tataruch, M; Heider, J; Bryjak, J; Nowak, P; Knack, D; Czerniak, A; Liesiene, J; Szaleniec, M

    2014-12-20

    The molybdenum/iron-sulfur/heme protein ethylbenzene dehydrogenase (EbDH) was successfully applied to catalyze enantiospecific hydroxylation of alkylaromatic and alkylheterocyclic compounds. The optimization of the synthetic procedure involves use of the enzyme in a crude purification state that saves significant preparation effort and is more stable than purified EbDH without exhibiting unwanted side reactions. Moreover, immobilization of the enzyme on a crystalline cellulose support and changes in reaction conditions were introduced in order to increase the amounts of product formed (anaerobic atmosphere, electrochemical electron acceptor recycling or utilization of ferricyanide as alternative electron acceptor in high concentrations). We report here on an extension of effective enzyme activity from 4h to more than 10 days and final product yields of up to 0.4-0.5g/l, which represent a decent starting point for further optimization. Therefore, we expect that the hydrocarbon-hydroxylation capabilities of EbDH may be developed into a new process of industrial production of chiral alcohols. PMID:24998764

  12. Genetic basis of the difference in alcohol dehydrogenase expression between Drosophila melanogaster and Drosophila simulans.

    PubMed Central

    Laurie, C C; Heath, E M; Jacobson, J W; Thomson, M S

    1990-01-01

    Drosophila melanogaster and its sibling species, Drosophila simulans, differ in expression of the enzyme alcohol dehydrogenase (ADH). Adult melanogaster flies that are homozygous for the Slow allozyme have approximately twice the level of ADH activity and crossreacting material as simulans adults. There is no corresponding difference in ADH mRNA, however, so this difference in ADH protein level is evidently due to a difference in the rate of translation of the two RNAs and/or to a difference in protein stability. Here we report an interspecific gene-transfer experiment, using P-element transformation, to determine whether this expression difference is due to genetic background differences between the species (trans-acting modifiers) or to cis-acting factors within the Adh gene. When the Adh genes from D. melanogaster and D. simulans are put into the same genetic background, there is no detectable difference in their level of expression. The level is relatively high in the melanogaster background and relatively low in the simulans background. Therefore, the interspecific difference in Adh expression is due entirely to trans-acting modifiers, in spite of the many sequence differences between the Adh genes of the two species, which include two amino acid substitutions. PMID:2124699

  13. Computational optimization of AG18051 inhibitor for amyloid-beta binding alcohol dehydrogenase enzyme

    NASA Astrophysics Data System (ADS)

    Marques, Alexandra T.; Antunes, Agostinho; Fernandes, Pedro A.; Ramos, Maria J.

    Amyloid-beta (Abeta) binding alcohol dehydrogenase (ABAD) is a multifunctional enzyme involved in maintaining the homeostasis. The enzyme can also mediate some diseases, including genetic diseases, Alzheimer's disease, and possibly some prostate cancers. Potent inhibitors of ABAD might facilitate a better clarification of the functions of the enzyme under normal and pathogenic conditions and might also be used for therapeutic intervention in disease conditions mediated by the enzyme. The AG18051 is the only presently available inhibitor of ABAD. It binds in the active-site cavity of the enzyme and reacts with the NAD+ cofactor to form a covalent adduct. In this work, we use computational methods to perform a rational optimization of the AG18051 inhibitor, through the introduction of chemical substitutions directed to improve the affinity of the inhibitor to the enzyme. The molecular mechanics-Poisson-Boltzmann surface area methodology was used to predict the relative free binding energy of the different modified inhibitor-NAD-enzyme complexes. We show that it is possible to increase significantly the affinity of the inhibitor to the enzyme with small modifications, without changing the overall structure and ADME (absorption, distribution, metabolism, and excretion) properties of the original inhibitor.

  14. Amino ketone formation and aminopropanol-dehydrogenase activity in rat-liver preparations

    PubMed Central

    Turner, J. M.; Willetts, A. J.

    1967-01-01

    1. Rat tissue homogenates convert dl-1-aminopropan-2-ol into aminoacetone. Liver homogenates have relatively high aminopropanol-dehydrogenase activity compared with kidney, heart, spleen and muscle preparations. 2. Maximum activity of liver homogenates is exhibited at pH9·8. The Km for aminopropanol is approx. 15mm, calculated for a single enantiomorph, and the maximum activity is approx. 9mμmoles of aminoacetone formed/mg. wet wt. of liver/hr.at 37°. Aminoacetone is also formed from l-threonine, but less rapidly. An unidentified amino ketone is formed from dl-4-amino-3-hydroxybutyrate, the Km for which is approx. 200mm at pH9·8. 3. Aminopropanol-dehydrogenase activity in homogenates is inhibited non-competitively by dl-3-hydroxybutyrate, the Ki being approx. 200mm. EDTA and other chelating agents are weakly inhibitory, and whereas potassium chloride activates slightly at low concentrations, inhibition occurs at 50–100mm. 4. It is concluded that aminopropanol-dehydrogenase is located in mitochondria, and in contrast with l-threonine dehydrogenase can be readily solubilized from mitochondrial preparations by ultrasonic treatment. 5. Soluble extracts of disintegrated mitochondria exhibit maximum aminopropanol-dehydrogenase activity at pH9·1 At this pH, Km values for the amino alcohol and NAD+ are approx. 200 and 1·3mm respectively. Under optimum conditions the maximum velocity is approx. 70mμmoles of aminoacetone formed/mg. of protein/hr. at 37°. Chelating agents and thiol reagents appear to have little effect on enzyme activity, but potassium chloride inhibits at all concentrations tested up to 80mm. dl-3-Hydroxybutyrate is only slightly inhibitory. 6. Dehydrogenase activities for l-threonine and dl-4-amino-3-hydroxybutyrate appear to be distinct from that for aminopropanol. 7. Intraperitoneal injection of aminopropanol into rats leads to excretion of aminoacetone in the urine. Aminoacetone excretion proportional to the amount of the amino alcohol

  15. Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

    NASA Astrophysics Data System (ADS)

    Hamid, Nur Athirah Abd; Ismail, Ismanizan

    2013-11-01

    Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

  16. CARDIAC OVEREXPRESSION OF ALCOHOL DEHYDROGENASE EXACERBATES CARDIAC CONTRACTILE DYSFUNCTION, LIPID PEROXIDATION, AND PROTEIN DAMAGE AFTER CHRONIC ETHANOL INGESTION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alcoholic cardiomyopathy is manifested as ventricular dysfunction although its specific toxic mechanism(s) remains obscure. This study was designed to examine the impact of enhanced acetaldehyde (ACA) exposure on cardiac function via cardiac-specific over-expression of alcohol dehydrogenase (ADH) fo...

  17. ETHANOL INDUCES AND INSULIN INHIBITS ALCOHOL DEHYDROGENASE CLASS 1 IN FGC-4 CELLS: BOTH APPEAR TO WORK THROUGH SREBP-1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously reported that chronic feeding of alcohol-containing diets (via intragastric infusion) to Sprague-Dawley rats induces hepatic alcohol dehydrogenase (ADH) Class 1 by interfering with signaling via the sterol regulatory element binding protein (SREBP-1). We have studied the effects ...

  18. Purification and characterization of an anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol.

    PubMed

    Meng, Fantao; Xu, Yan

    2010-04-01

    An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic methyl ketones (chain length > or =6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45 degrees C and at pH 8.0. PMID:20035369

  19. Three alcohol dehydrogenase genes and one acetyl-CoA synthetase gene are responsible for ethanol utilization in Yarrowia lipolytica.

    PubMed

    Gatter, Michael; Ottlik, Stephanie; Kövesi, Zsolt; Bauer, Benjamin; Matthäus, Falk; Barth, Gerold

    2016-10-01

    The non-conventional yeast Yarrowia lipolytica is able to utilize a wide range of different substrates like glucose, glycerol, ethanol, acetate, proteins and various hydrophobic molecules. Although most metabolic pathways for the utilization of these substrates have been clarified by now, it was not clear whether ethanol is oxidized by alcohol dehydrogenases or by an alternative oxidation system inside the cell. In order to detect the genes that are required for ethanol utilization in Y. lipolytica, eight alcohol dehydrogenase (ADH) genes and one alcohol oxidase gene (FAO1) have been identified and respective deletion strains were tested for their ability to metabolize ethanol. As a result of this, we found that the availability of ADH1, ADH2 or ADH3 is required for ethanol utilization in Y. lipolytica. A strain with deletions in all three genes is lacking the ability to utilize ethanol as sole carbon source. Although Adh2p showed by far the highest enzyme activity in an in vitro assay, the availability of any of the three genes was sufficient to enable a decent growth. In addition to ADH1, ADH2 and ADH3, an acetyl-CoA synthetase encoding gene (ACS1) was found to be essential for ethanol utilization. As Y. lipolytica is a non-fermenting yeast, it is neither able to grow under anaerobic conditions nor to produce ethanol. To investigate whether Y. lipolytica may produce ethanol, the key genes of alcoholic fermentation in S. cerevisiae, ScADH1 and ScPDC1, were overexpressed in an ADH and an ACS1 deletion strain. However, instead of producing ethanol, the respective strains regained the ability to use ethanol as single carbon source and were still not able to grow under anaerobic conditions. PMID:27486067

  20. Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase

    PubMed Central

    Köpke, Michael; Gerth, Monica L.; Maddock, Danielle J.; Mueller, Alexander P.; Liew, FungMin

    2014-01-01

    Acetogenic bacteria use CO and/or CO2 plus H2 as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogen Clostridium autoethanogenum is known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize the C. autoethanogenum enzymes for lactate and 2,3-butanediol biosynthesis. The putative C. autoethanogenum lactate dehydrogenase was active when expressed in Escherichia coli. The 2,3-butanediol pathway was reconstituted in E. coli by cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resulting E. coli strain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 μM h−1 optical density unit−1), which is comparable to the level produced by C. autoethanogenum during growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, that C. autoethanogenum can act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway from C. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential of C. autoethanogenum as a platform for sustainable chemical production. PMID:24657865

  1. Reconstruction of an acetogenic 2,3-butanediol pathway involving a novel NADPH-dependent primary-secondary alcohol dehydrogenase.

    PubMed

    Köpke, Michael; Gerth, Monica L; Maddock, Danielle J; Mueller, Alexander P; Liew, FungMin; Simpson, Séan D; Patrick, Wayne M

    2014-06-01

    Acetogenic bacteria use CO and/or CO2 plus H2 as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogen Clostridium autoethanogenum is known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize the C. autoethanogenum enzymes for lactate and 2,3-butanediol biosynthesis. The putative C. autoethanogenum lactate dehydrogenase was active when expressed in Escherichia coli. The 2,3-butanediol pathway was reconstituted in E. coli by cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resulting E. coli strain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 μM h(-1) optical density unit(-1)), which is comparable to the level produced by C. autoethanogenum during growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, that C. autoethanogenum can act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway from C. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential of C. autoethanogenum as a platform for sustainable chemical production. PMID:24657865

  2. Effect of Hofmeister anions and protein concentration on the activity and stability of some immobilized made-independent dehydrogenases

    SciTech Connect

    Carrea, G.; Bovara, R.; Pasta, P.; Cremonesi, P.

    1982-01-01

    The effect of several factors on the activity and stability of alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and 20-beta-hydroxysteroid dehydrogenase, both free and immobilized on CNBr-activated Sepharose 4B, was investigated. Enzymes were immobilized under different conditions including various degrees of matrix activation, variable amounts of protein, in the presence, or in the absence of, additives (coenzymes, dithiothreitol, salts). Activity recovery was in general satisfactorily high with 20-beta-hydroxysteroid dehydrogenase, low with glyceraldehyde-3-phosphate dehydrogenase, and markedly linked to the concentration of immobilized protein with alcohol dehydrogenase. In the latter case the advantageous stabilizing effect of high enzyme concentrations was notably diminished by the paralled decrease of the effectiveness factor. The effect of high concentrations of anions of the Hofmeister series was examined. It was found that 1M phosphate and 0.5M sulfate dramatically stabilize both free and immobilized enzymes against inactivation by temperature and urea. Km values of apolar substrates were considerably lowered by the two anions while Km values of polar substrates were not affected. In some cases Vmax values also were influenced by high concentrations of these anions. The present results appear of interest particularly in view of enzyme utilization for analytical as well as for preparative purposes. (Refs. 13).

  3. Syringyl Lignin Is Unaltered by Severe Sinapyl Alcohol Dehydrogenase Suppression in Tobacco[W

    PubMed Central

    Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N.; Marshall, David; Hancock, Robert D.; Lapierre, Catherine; Morreel, Kris; Boerjan, Wout; Halpin, Claire

    2011-01-01

    The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference–inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem. PMID:22158465

  4. Assessment of toxicity using dehydrogenases activity and mathematical modeling.

    PubMed

    Matyja, Konrad; Małachowska-Jutsz, Anna; Mazur, Anna K; Grabas, Kazimierz

    2016-07-01

    Dehydrogenase activity is frequently used to assess the general condition of microorganisms in soil and activated sludge. Many studies have investigated the inhibition of dehydrogenase activity by various compounds, including heavy metal ions. However, the time after which the measurements are carried out is often chosen arbitrarily. Thus, it can be difficult to estimate how the toxic effects of compounds vary during the reaction and when the maximum of the effect would be reached. Hence, the aim of this study was to create simple and useful mathematical model describing changes in dehydrogenase activity during exposure to substances that inactivate enzymes. Our model is based on the Lagergrens pseudo-first-order equation, the rate of chemical reactions, enzyme activity, and inactivation and was created to describe short-term changes in dehydrogenase activity. The main assumption of our model is that toxic substances cause irreversible inactivation of enzyme units. The model is able to predict the maximum direct toxic effect (MDTE) and the time to reach this maximum (TMDTE). In order to validate our model, we present two examples: inactivation of dehydrogenase in microorganisms in soil and activated sludge. The model was applied successfully for cadmium and copper ions. Our results indicate that the predicted MDTE and TMDTE are more appropriate than EC50 and IC50 for toxicity assessments, except for long exposure times. PMID:27021434

  5. Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

    PubMed Central

    Ying, Xiangxian; Wang, Yifang; Xiong, Bin; Wu, Tingting; Xie, Liping; Yu, Meilan

    2014-01-01

    A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg−1 for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg−1 using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP+, suggesting the nature of being an aldehyde reductase. PMID:24509923

  6. Characterization of an allylic/benzyl alcohol dehydrogenase from Yokenella sp. strain WZY002, an organism potentially useful for the synthesis of α,β-unsaturated alcohols from allylic aldehydes and ketones.

    PubMed

    Ying, Xiangxian; Wang, Yifang; Xiong, Bin; Wu, Tingting; Xie, Liping; Yu, Meilan; Wang, Zhao

    2014-04-01

    A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg(-1) for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg(-1) using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP(+), suggesting the nature of being an aldehyde reductase. PMID:24509923

  7. Classical Raman spectroscopic studies of NADH and NAD+ bound to liver alcohol dehydrogenase by difference techniques

    SciTech Connect

    Chen, D.; Yue, K.T.; Martin, C.; Rhee, K.W.; Sloan, D.; Callender, R.

    1987-07-28

    We report the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) and adenosine 5'-diphosphate ribose (ADPR) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by taking the classical Raman difference spectrum of the binary complex, LADH/NADH, with that of LADH. We have investigated how the bound NADH spectrum is affected when the ternary complexes with inhibitors are formed with dimethyl sulfoxide (Me2SO) or isobutyramide (IBA), i.e., LADH/NADH/Me2SO or LADH/NADH/IBA. Similarly, the difference spectra of LADH/NAD+/pyrazole or LADH/ADPR with LADH are calculated. The magnitude of these difference spectra is on the order of a few percent of the protein Raman spectrum. We report and discuss the experimental configuration and control procedures we use in reliably calculating such small difference signals. These sensitive difference techniques could be applied to a large number of problems where the classical Raman spectrum of a ''small'' molecule, like adenine, bound to the active site of a protein is of interest. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD+ spectra to normal coordinates located primarily on either the nicotinamide or the adenine moiety. By comparing the spectra of the bound coenzymes with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant molecular changes upon binding.

  8. NAD(P)-Dependent Aldehyde Dehydrogenases Induced during Growth of Ralstonia eutropha Strain Bo on Tetrahydrofurfuryl Alcohol

    PubMed Central

    Schräder, Thomas; Zarnt, Grit; Andreesen, Jan R.

    2001-01-01

    Different aldehyde dehydrogenases (AlDHs) were formed during growth of Ralstonia eutropha Bo on tetrahydrofurfuryl alcohol (THFA). One of these enzymes, AlDH 4, was purified and characterized as a homodimer containing no prosthetic groups, showing a strong substrate inhibition, and having an N-terminal sequence similar to those of various NAD(P)-dependent AlDHs. The conversion rate of THFA by the quinohemoprotein THFA dehydrogenase was increased by AlDH 4. PMID:11717302

  9. Genic Heterogeneity at Two Alcohol Dehydrogenase Loci in DROSOPHILA PSEUDOOBSCURA and DROSOPHILA PERSIMILIS

    PubMed Central

    Coyne, Jerry A.; Felton, Alexander A.

    1977-01-01

    A sequential electrophoretic survey of the second chromosome loci, alcohol dehydrogenase-6 (Adh-6) and octanol dehydrogenase ( Odh), was performed on 147 isochromosomal lines of Drosophila pseudoobscura and 60 lines of its sibling species, D. persimilis. Gels run with a variety of acrylamide concentrations and buffer pH's revealed the presence of 18 alleles of Adh-6 in the two species, where only eight had been previously detected by conventional electrophoretic methods. Only two alleles were added with our techniques to the previous total of nine in both species at the largely monomorphic Odh locus. Both enzymes show a predominance of one allele, with the other variants being fairly rare. There was no evidence of increased genetic divergence between the two species, but we found a striking increase in differentiation of Adh-6 alleles between the main body of D. pseudoobscura populations and the conspecific isolate from Bogotá, Colombia. These results are compared with our previous surveys of xanthine dehydrogenase in these species and discussed in reference to theories of genic polymorphism. PMID:17248763

  10. 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.

  11. Ranitidine as an alcohol dehydrogenase inhibitor in acute methanol toxicity in rats.

    PubMed

    El-Bakary, Amal A; El-Dakrory, Sahar A; Attalla, Sohayla M; Hasanein, Nawal A; Malek, Hala A

    2010-02-01

    Methanol poisoning is a hazardous intoxication characterized by visual impairment and formic acidemia. The therapy for methanol poisoning is alcohol dehydrogenase (ADH) inhibitors to prevent formate accumulation. Ranitidine has been considered to be an inhibitor of both gastric alcohol and hepatic aldehyde dehydrogenase enzymes. This study aimed at testing ranitidine as an antidote for methanol acute toxicity and comparing it with ethanol and 4-methyl pyrazole (4-MP). This study was conducted on 48 Sprague-Dawley rats, divided into 6 groups, with 8 rats in each group (one negative control group [C1], two positive control groups [C2, C3] and three test groups [1, 2 and 3]). C2, C3 and all test groups were exposed to nitrous oxide by inhalation, then, C3 group was given methanol (3 g/kg orally). The three test groups 1, 2 and 3 were given ethanol (0.5 g/kg orally), 4-MP (15 mg/kg intraperitoneally) and ranitidine (30 mg/kg intraperitoneally), respectively, 4 hours after giving methanol. Rats were sacrificed and heparinized, cardiac blood samples were collected for blood pH and bicarbonate. Non-heparinized blood samples were collected for formate levels by high performance liquid chromatography. Eye balls were enucleated for histological examination of the retina. Ranitidine corrected metabolic acidosis (p = .025), decreased formate levels (p = .014) and improved the histological findings in the retina induced by acute methanol toxicity. PMID:20026516

  12. Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

    PubMed Central

    2012-01-01

    Background The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied. Results The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations. Conclusion In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene

  13. 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

  14. Identification and characterization of a mycobacterial NAD⁺-dependent alcohol dehydrogenase with superior reduction of diacetyl to (S)-acetoin.

    PubMed

    Takeda, Minoru; Anamizu, Shiori; Motomatsu, Shigekazu; Chen, Xue; Thapa Chhetri, Rajan

    2014-01-01

    An enzyme capable of reducing acetoin in the presence of NADH was purified from Mycobacterium sp. B-009, a non-clinical bacterial strain of soil origin. The enzyme is a homotetramer and can be classified as a medium-chain alcohol dehydrogenase/reductase based on the molecular weight of the monomer. Identification of the structural gene revealed a limited distribution of homologous genes only among actinomycetes. In addition to its activity as a reductase specific for (S)-acetoin (EC 1.1.1.76), the enzyme showed both diacetyl reductase (EC 1.1.1.304) and NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1) activities. (S)-Acetoin and diacetyl reductases belong to a group of short-chain alcohol dehydrogenase/reductases but do not have superior abilities to dehydrogenate monoalcohols. Thus, the purified enzyme can be readily distinguished from other enzymes. We used the dual functionality of the enzyme to effectively reduce diacetyl to (S)-acetoin, coupled with the oxidation of 1-butanol. PMID:25082080

  15. Phylogeny and structure of the cinnamyl alcohol dehydrogenase gene family in Brachypodium distachyon.

    PubMed

    Bukh, Christian; Nord-Larsen, Pia Haugaard; Rasmussen, Søren K

    2012-10-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step of the monolignol biosynthesis, the conversion of cinnamyl aldehydes to alcohols, using NADPH as a cofactor. Seven members of the CAD gene family were identified in the genome of Brachypodium distachyon and five of these were isolated and cloned from genomic DNA. Semi-quantitative reverse-transcription PCR revealed differential expression of the cloned genes, with BdCAD5 being expressed in all tissues and highest in root and stem while BdCAD3 was only expressed in stem and spikes. A phylogenetic analysis of CAD-like proteins placed BdCAD5 on the same branch as bona fide CAD proteins from maize (ZmCAD2), rice (OsCAD2), sorghum (SbCAD2) and Arabidopsis (AtCAD4, 5). The predicted three-dimensional structures of both BdCAD3 and BdCAD5 resemble that of AtCAD5. However, the amino-acid residues in the substrate-binding domains of BdCAD3 and BdCAD5 are distributed symmetrically and BdCAD3 is similar to that of poplar sinapyl alcohol dehydrogenase (PotSAD). BdCAD3 and BdCAD5 expressed and purified from Escherichia coli both showed a temperature optimum of about 50 °C and molar weight of 49 kDa. The optimal pH for the reduction of coniferyl aldehyde were pH 5.2 and 6.2 and the pH for the oxidation of coniferyl alcohol were pH 8 and 9.5, for BdCAD3 and BdCAD5 respectively. Kinetic parameters for conversion of coniferyl aldehyde and coniferyl alcohol showed that BdCAD5 was clearly the most efficient enzyme of the two. These data suggest that BdCAD5 is the main CAD enzyme for lignin biosynthesis and that BdCAD3 has a different role in Brachypodium. All CAD enzymes are cytosolic except for BdCAD4, which has a putative chloroplast signal peptide adding to the diversity of CAD functions. PMID:23028019

  16. 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.

  17. Fructophilic characteristics of Fructobacillus spp. may be due to the absence of an alcohol/acetaldehyde dehydrogenase gene (adhE).

    PubMed

    Endo, Akihito; Tanaka, Naoto; Oikawa, Yo; Okada, Sanae; Dicks, Leon

    2014-04-01

    Fructophilic strains of Leuconostoc spp. have recently been reclassified to a new genus, i.e., Fructobacillus. Members of the genus are differentiated from Leuconostoc spp. by their preference for fructose on growth, requirement of an electron acceptor for glucose metabolism, and the inability to produce ethanol from the fermentation of glucose. In the present study, enzyme activities and genes involved in ethanol production were studied, since this is the key pathway for NAD(+)/NADH cycling in heterofermentative lactic acid bacteria. Fructobacillus spp. has a weak alcohol dehydrogenase activity and has no acetaldehyde dehydrogenase activity, whereas both enzymes are active in Leuconostoc mesenteroides. The bifunctional alcohol/acetaldehyde dehydrogenase gene, adhE, was described in Leuconostoc spp., but not in Fructobacillus spp. These results suggested that, due to the deficiency of the adhE gene, the normal pathway for ethanol production is absent in Fructobacillus spp. This leads to a shortage of NAD(+), and the requirement for an electron acceptor in glucose metabolism. Fructophilic characteristics, as observed for Fructobacillus spp., are thus due to the absence of the adhE gene, and a phenotype that most likely evolved as a result of regressive evolution. PMID:24352296

  18. Use of an ionic liquid in a two-phase system to improve an alcohol dehydrogenase catalysed reduction.

    PubMed

    Eckstein, Marrit; Villela Filho, Murillo; Liese, Andreas; Kragl, Udo

    2004-05-01

    Due to favourable partition coefficients the highly enantioselective reduction of 2-octanone, catalysed by an alcohol dehydrogenase from Lactobacillus brevis, is faster in a biphasic system containing buffer and the ionic liquid [BMIM][(CF(3)SO(2))(2)N] compared to the reduction in a biphasic system containing buffer and methyl tert-butyl ether. PMID:15116196

  19. DOWNREGULATION OF CINNAMYL-ALCOHOL DEHYDROGENASE IN SWITCHGRASS BY RNA SILENCING RESULTS IN ENHANCED GLUCOSE RELEASE AFTER CELLULASE TREATMENT

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cinnamyl alcohol dehydrogenase (CAD), catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switch...

  20. Atomic-Resolution Structures of Horse Liver Alcohol Dehydrogenase with NAD[superscript +] and Fluoroalcohols Define Strained Michaelis Complexes

    SciTech Connect

    Plapp, Bryce V.; Ramaswamy, S.

    2013-01-16

    Structures of horse liver alcohol dehydrogenase complexed with NAD{sup +} and unreactive substrate analogues, 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol, were determined at 100 K at 1.12 or 1.14 {angstrom} resolution, providing estimates of atomic positions with overall errors of 0.02 {angstrom}, the geometry of ligand binding, descriptions of alternative conformations of amino acid residues and waters, and evidence of a strained nicotinamide ring. The four independent subunits from the two homodimeric structures differ only slightly in the peptide backbone conformation. Alternative conformations for amino acid side chains were identified for 50 of the 748 residues in each complex, and Leu-57 and Leu-116 adopt different conformations to accommodate the different alcohols at the active site. Each fluoroalcohol occupies one position, and the fluorines of the alcohols are well-resolved. These structures closely resemble the expected Michaelis complexes with the pro-R hydrogens of the methylene carbons of the alcohols directed toward the re face of C4N of the nicotinamide rings with a C-C distance of 3.40 {angstrom}. The oxygens of the alcohols are ligated to the catalytic zinc at a distance expected for a zinc alkoxide (1.96 {angstrom}) and participate in a low-barrier hydrogen bond (2.52 {angstrom}) with the hydroxyl group of Ser-48 in a proton relay system. As determined by X-ray refinement with no restraints on bond distances and planarity, the nicotinamide rings in the two complexes are slightly puckered (quasi-boat conformation, with torsion angles of 5.9{sup o} for C4N and 4.8{sup o} for N1N relative to the plane of the other atoms) and have bond distances that are somewhat different compared to those found for NAD(P){sup +}. It appears that the nicotinamide ring is strained toward the transition state on the path to alcohol oxidation.

  1. Regulated Expression of Three Alcohol Dehydrogenase Genes in Barley Aleurone Layers 1

    PubMed Central

    Hanson, Andrew D.; Jacobsen, John V.; Zwar, John A.

    1984-01-01

    Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity. Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O2 deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O2 levels were reduced below 5%, and were most intense at 0% O2. In vivo35S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O2, synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O2, the synthesis of all three ADH peptides increased markedly. Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O2 deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O2, and about 100-fold after 1 day at 0% O2. We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O2, that regulation of mRNA level is likely to be a major controlling factor, and

  2. Manipulating cinnamyl alcohol dehydrogenase (CAD) expression in flax affects fibre composition and properties

    PubMed Central

    2014-01-01

    Background In recent decades cultivation of flax and its application have dramatically decreased. One of the reasons for this is unpredictable quality and properties of flax fibre, because they depend on environmental factors, retting duration and growing conditions. These factors have contribution to the fibre composition, which consists of cellulose, hemicelluloses, lignin and pectin. By far, it is largely established that in flax, lignin reduces an accessibility of enzymes either to pectin, hemicelluloses or cellulose (during retting or in biofuel synthesis and paper production). Therefore, in this study we evaluated composition and properties of flax fibre from plants with silenced CAD (cinnamyl alcohol dehydrogenase) gene, which is key in the lignin biosynthesis. There is evidence that CAD is a useful tool to improve lignin digestibility and/or to lower the lignin levels in plants. Results Two studied lines responded differentially to the introduced modification due to the efficiency of the CAD silencing. Phylogenetic analysis revealed that flax CAD belongs to the “bona-fide” CAD family. CAD down-regulation had an effect in the reduced lignin amount in the flax fibre cell wall and as FT-IR results suggests, disturbed lignin composition and structure. Moreover introduced modification activated a compensatory mechanism which was manifested in the accumulation of cellulose and/or pectin. These changes had putative correlation with observed improved fiber’s tensile strength. Moreover, CAD down-regulation did not disturb at all or has only slight effect on flax plants’ development in vivo, however, the resistance against flax major pathogen Fusarium oxysporum decreased slightly. The modification positively affected fibre possessing; it resulted in more uniform retting. Conclusion The major finding of our paper is that the modification targeted directly to block lignin synthesis caused not only reduced lignin level in fibre, but also affected amount and

  3. Azotobacter vinelandii Aldehyde Dehydrogenase Regulated by ς54: Role in Alcohol Catabolism and Encystment

    PubMed Central

    Gama-Castro, Socorro; Núñez, Cinthia; Segura, Daniel; Moreno, Soledad; Guzmán, Josefina; Espín, Guadalupe

    2001-01-01

    Encystment in Azotobacter vinelandii is induced by n-butanol or β-hydroxybutyrate (BHB). We identified a gene, encoding an aldehyde dehydrogenase, that was named aldA. An aldA mutation impaired bacterial growth on n-butanol, ethanol, or hexanol as the sole carbon source. Expression of aldA increased in cells shifted from sucrose to n-butanol and was shown to be dependent on the alternative ς54 factor. A mutation in rpoN encoding the ς54 factor also impaired growth on alcohols. Encystment on n-butanol, but not on BHB, was impaired in aldA or rpoN mutants, indicating that n-butanol is not an inducer of encystment by itself but must be catabolized in order to induce encystment. PMID:11591659

  4. Characterization of a transient intermediate formed in the liver alcohol dehydrogenase catalyzed reduction of 3-hydroxy-4-nitrobenzaldehyde

    SciTech Connect

    MacGibbon, A.K.H.; Koerber, S.C.; Pease, K.; Dunn, M.F.

    1987-06-02

    The compounds 3-hydroxy-4-nitrobenzaldehyde and 3-hydroxy-4-nitrobenzyl alcohol are introduced as new chromophoric substrates for probing the catalytic mechanism of horse liver alcohol dehydrogenase (LADH). Ionization of the phenolic hydroxyl group shifts the spectrum of the aldehyde from 360 to 433 nm (pK/sub a/ = 6.0), whereas the spectrum of the alcohol shifts from 350 to 417 nm (pK/sub a/ = 6.9). Rapid-scanning, stopped-flow (RSSF) studies at alkaline pH show that the LADH-catalyzed interconversion of these compounds occurs via the formation of an enzyme-bound intermediate with a blue-shifted spectrum. When reaction is limited to a single turnover of enzyme sites, the formation and decay of the intermediate when aldehyde reacts with enzyme-bound reduced nicotinamide adenine dinucleotide E(NADH) are characterized by two relaxations. Detailed stopped-flow kinetic studies were carried out to investigate the disappearance of aldehyde and NADH, the formation and decay of the intermediate, the displacement of Auramine O by substrate, and /sup 2/H kinetic isotope effects. It was found that (1) NADH oxidation takes place at the rate of the slower relaxation (2) when NADD is substituted for NADH, lambda/sub s/ is subject to a small primary isotope effect; and (3) the events that occur in lambda/sub s/ precede lambda/sub f/. These findings identify the intermediate as a ternary complex containing bound oxidized nicotinamide adenine dinucleotide (NAD/sup +/) and some form of 3-hydroxy-4-nitrobenzyl alcohol. The authors conclude that the LADH substrate site can be divided into two subsites: a highly polar, electropositive subsite in the vicinity of the active-site zinc and, just a few angstroms away, a rather nonpolar region.

  5. Two zebrafish alcohol dehydrogenases share common ancestry with mammalian class I, II, IV, and V alcohol dehydrogenase genes but have distinct functional characteristics.

    PubMed

    Reimers, Mark J; Hahn, Mark E; Tanguay, Robert L

    2004-09-10

    Ethanol is teratogenic to many vertebrates. We are utilizing zebrafish as a model system to determine whether there is an association between ethanol metabolism and ethanol-mediated developmental toxicity. Here we report the isolation and characterization of two cDNAs encoding zebrafish alcohol dehydrogenases (ADHs). Phylogenetic analysis of these zebrafish ADHs indicates that they share a common ancestor with mammalian class I, II, IV, and V ADHs. The genes encoding these zebrafish ADHs have been named Adh8a and Adh8b by the nomenclature committee. Both genes were genetically mapped to chromosome 13. The 1450-bp Adh8a is 82, 73, 72, and 72% similar at the amino acid level to the Baltic cod ADH8 (previously named ADH1), the human ADH1B2, the mouse ADH1, and the rat ADH1, respectively. Also, the 1484-bp Adh8b is 77, 68, 67, and 66% similar at the amino acid level to the Baltic cod ADH8, the human ADH1B2, the mouse ADH1, and the rat ADH1, respectively. ADH8A and ADH8B share 86% amino acid similarity. To characterize the functional properties of ADH8A and ADH8B, recombinant proteins were purified from SF-9 insect cells. Kinetic studies demonstrate that ADH8A metabolizes ethanol, with a V(max) of 13.4 nmol/min/mg protein, whereas ADH8B does not metabolize ethanol. The ADH8A K(m) for ethanol as a substrate is 0.7 mm. 4-Methyl pyrazole, a classical competitive inhibitor of class I ADH, failed to inhibit ADH8A. ADH8B has the capacity to efficiently biotransform longer chain primary alcohols (>/=5 carbons) and S-hydroxymethlyglutathione, whereas ADH8A does not efficiently metabolize these substrates. Finally, mRNA expression studies indicate that both ADH8A and ADH8B mRNA are expressed during early development and in the adult brain, fin, gill, heart, kidney, muscle, and liver. Together these results indicate that class I-like ADH is conserved in zebrafish, albeit with mixed functional properties. PMID:15231826

  6. Lysophosphatidylethanolamine Is a Substrate for the Short-Chain Alcohol Dehydrogenase SocA from Myxococcus xanthus▿ †

    PubMed Central

    Avadhani, Madhavi; Geyer, Roland; White, David C.; Shimkets, Lawrence J.

    2006-01-01

    Short-chain alcohol dehydrogenases (SCADHs) synthesize a variety of intercellular signals and other chemically diverse products. It is difficult to predict the substrate of a SCADH on the basis of amino acid sequence homology, as the substrates are not known for most SCADHs. In Myxococcus xanthus, the SCADH CsgA is responsible for C signaling during fruiting body development, although the mechanism is unclear. Overexpression of the SCADH SocA compensates for the lack of CsgA and restores development and C signaling in csgA mutants. The potential of SocA in generating the C signal enzymatically was explored by developing a dehydrogenase assay-based screen to purify the SocA substrate(s). A SocA substrate was extracted from M. xanthus cells with acidified ethyl acetate and sequentially purified by solid-phase extraction on silica gel and by reverse-phase high-performance liquid chromatography. The fraction with the highest SocA dehydrogenase activity contained the lysophospholipid 1-acyl 2-hydroxy-sn-glycerophosphoethanolamine (lyso-PE) as indicated by the fragment ions and a phosphatidylethanolamine-specific neutral loss scan following liquid chromatography coupled to mass spectrometry. The abundant lysophospholipid with the mass m/z 450 (molecular ion [M-H]−) had a monounsaturated acyl chain with 16 carbons. SocA oxidizes lyso-PE containing either saturated or unsaturated fatty acids but exhibits poor activity on l-α-glycerophosphorylethanolamine, suggesting that an acyl chain is important for activity. Of the five different head groups, only ethanolamine showed appreciable activity. The apparent Km and Vmax for lyso-PE 18:1 were 116 μM and 875 μmol min−1 mg−1, respectively. The catalytic efficiency (kcat/Km) was 1 × 108 M−1 s−1. The proposed product, 1-acyloxy-3-(2-aminoethylphosphatyl) acetone was unstable, and the fragmented products were unable to rescue csgA mutant development. The active fraction from thin-layer chromatography also contained an

  7. Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu

    PubMed Central

    Wee, Ching Ching; Roslan, Hairul Azman

    2012-01-01

    Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group.

  8. Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu.

    PubMed

    Wee, Ching Ching; Roslan, Hairul Azman

    2012-01-01

    Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group. PMID:27335670

  9. A human alcohol dehydrogenase gene (ADH6) encoding an additional class of isozyme.

    PubMed Central

    Yasunami, M; Chen, C S; Yoshida, A

    1991-01-01

    The human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) gene family consists of five known loci (ADH1-ADH5), which have been mapped close together on chromosome 4 (4q21-25). ADH isozymes encoded by these genes are grouped in three distinct classes in terms of their enzymological properties. A moderate structural similarity is observed between the members of different classes. We isolated an additional member of the ADH gene family by means of cross-hybridization with the ADH2 (class I) cDNA probe. cDNA clones corresponding to this gene were derived from PCR-amplified libraries as well. The coding sequence of a 368-amino-acid-long open reading frame was interrupted by introns into eight exons and spanned approximately 17 kilobases on the genome. The gene contains a glucocorticoid response element at the 5' region. The transcript was detected in the stomach and liver. The deduced amino acid sequence of the open reading frame showed about 60% positional identity with known human ADHs. This extent of homology is comparable to interclass similarity in the human ADH family. Thus, the newly identified gene, which is designated ADH6, governs the synthesis of an enzyme that belongs to another class of ADHs presumably with a distinct physiological role. Images PMID:1881901

  10. Low Km aldehyde dehydrogenase (ALDH2) polymorphism, alcohol-drinking behavior, and chromosome alterations in peripheral lymphocytes.

    PubMed Central

    Morimoto, K; Takeshita, T

    1996-01-01

    Excessive drinking of alcohol is now widely known to be one of the major lifestyle choices that ca effect health. Among the various effects of alcohol drinking, cytogenetic and other genotoxic effects are of major concern from the viewpoint of prevention of alcohol-related diseases. Alcohol is first metabolized to acetaldehyde, which directly causes various types of chromosomal DNA lesions and alcohol-related diseases, and is then further detoxified to the much less toxic metabolite acetate. About 50% of Oriental people are deficient in the aldehyde-dehydrogenase 2 isozyme (ALDH2) that can most efficiently detoxify acetaldehyde. We have performed a series of experiments to investigate how the genetic deficiency in ALDH2 affects the behavioral pattern for alcohol drinking and the sensitivity of peripheral lymphocytes to the induction of chromosome alterations by exposure to alcohol and alcohol-related chemicals. We found great effects of the ALDH2 genotypes on alcohol sensitivity and alcohol-drinking behavior. We also show that lymphocytes from habitual drinkers with the deficient ALDH2 enzyme had significantly higher frequencies of sister chromatid exchanges than those from ALDH2-proficient individuals. PMID:8781384

  11. Alcohol dehydrogenase 1C (ADH1C) gene polymorphism and alcoholic liver cirrhosis risk: a meta analysis

    PubMed Central

    He, Lei; Deng, Tao; Luo, He-Sheng

    2015-01-01

    The association between alcohol dehydrogenase 1C (ADH1C) gene polymorphism and alcoholic liver cirrhosis (ALC) has been analyzed in several studies, but results have been conflicting. In this study, a meta-analysis was performed to assess the associations between the ADH1C polymorphism and risk of ALC. Relevant studies were identified using PubMed, Web of Science, CNKI and Wanfang databases up to January 10, 2015. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association using the fixed or random effect model. A total of 16 case-control studies, including 1375 cases and 1802 controls, were included. Overall, no significant association between the ADH1C polymorphism and ALC risk was found (dominant model: OR=0.87, 95% CI: 0.62-1.23; recessive model: OR=1.30, 95% CI: 0.84-1.99; *1/*2 vs. *1/*1: OR=0.87, 95% CI: 0.63-1.21; *2/*2 vs. *1/*1: OR=1.10, 95% CI: 0.71-1.70). In the subgroup analysis by ethnicity, we observed a significant association in Asian descent (*1/*2 vs. *1/*1: OR=1.63, 95% CI: 1.07-2.49), while a decreased risk was found among Caucasians (dominant model: OR=0.81, 95% CI: 0.66-0.99; *1/*2 vs. *1/*1: OR=0.76, 95% CI: 0.61-0.95). This meta-analysis demonstrated that the ADH1C polymorphism might increase the risk of ALC in Asians, while it may be a protective factor for ALC among Caucasians. PMID:26379912

  12. Microbial production of methylketones: properties of purified yeast secondary alcohol dehydrogenase

    SciTech Connect

    Patel, R.N.; Hou, C.T.; Laskin, A.I.; Derelanko, P.

    1981-06-01

    Secondary alcohol dehydrogenase (SADH) was purified from extracts of a methanol-grown yeast, Pichia sp. The purified enzyme was homogeneous as judged by ultracentrifugation and by polyacrylamide gel electrophoresis. The purified SADH has a molecular weight of 98,000 as determined by gel filtration and 102,000 as determined by sedimentation equilibrium analysis. The sedimentation constant s/sub 20,w/ was 6.0. The subunit size of the SADH was 48,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it consists of two subunits. The purified SADH contained two atoms of zinc per mole of enzyme protein. SADH catalyzed the oxidation of secondary alcohols. Primary alcohols (C/sub 1/ to C/sub 8/ tested) were not oxidized. The purified SADH and extracts of various yeasts and bacteria also catalyzed the reduction of methylketones to the corresponding secondary alcohols in the presence of reduced NAD/sup +/ as an electron donor. Both reactions (oxidation of secondary alcohols in the presence of NAD/sup +/ and reduction of methylketones in the presence of reduced NAD/sup +/) catalyzed by the purified SADH were inhibited by metal-chelating agents, thio reagent, and by antisera prepared against the purified enzyme. The apparent K/sub m/ values for NAD/sup +/, reduced NAD/sup +/, reduced NAD/sup +/, 2-butanol, and 2-butanone are 0.05, 0.1, 0.4, and 1 mM, respectively. The purified enzyme preferentially oxidized (-)-2-butanol and (-)-2-octanol, the rate of oxidation of (+)-2-butanol and (+)-2-octanol was 36% and 13% of that of 100% with (-)-2-butanol and (-)-2-octanol, respectively. The K/sub m/ values for (-)-2-butanol and (+)-2-butanol were 3.0 and 0.75 mM, respectively. Antisera prepared against purified Pichia SADH cross-reacted with the SADH derived from bacteria. This suggests difference in immunological properties between yeast and bacterial SADH.

  13. Evaluation of NAD(P)-Dependent Dehydrogenase Activities in Neutrophilic Granulocytes by the Bioluminescent Method.

    PubMed

    Savchenko, A A

    2015-09-01

    Bioluminescent method for measurements of the neutrophilic NAD(P)-dependent dehydrogenases (lactate dehydrogenase, NAD-dependent malate dehydrogenase, NADP-dependent decarboxylating malate dehydrogenase, NAD-dependent isocitrate dehydrogenase, and glucose- 6-phosphate dehydrogenase) is developed. The sensitivity of the method allows minimization of the volume of biological material for measurements to 104 neutrophils per analysis. The method is tried in patients with diffuse purulent peritonitis. Low levels of NADPH synthesis enzymes and high levels of enzymes determining the substrate flow by the Krebs cycle found in these patients can lead to attenuation of functional activity of cells. PMID:26468025

  14. Stability engineering of the Geobacillus stearothermophilus alcohol dehydrogenase and application for the synthesis of a polyamide 12 precursor.

    PubMed

    Kirmair, Ludwig; Seiler, Daniel Leonard; Skerra, Arne

    2015-12-01

    The thermostable NAD(+)-dependent alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH) was exploited with regard to the biocatalytic synthesis of ω-oxo lauric acid methyl ester (OLAMe), a key intermediate for biobased polyamide 12 production, from the corresponding long-chain alcohol. Recombinant BsADH was produced in Escherichia coli as a homogeneous tetrameric enzyme and showed high activity towards the industrially relevant substrate ω-hydroxy lauric acid methyl ester (HLAMe) with K M = 86 μM and 44 U mg(-1). The equilibrium constant for HLAMe oxidation to the aldehyde (OLAMe) with NAD(+) was determined as 2.16 × 10(-3) from the kinetic parameters of the BsADH-catalyzed forward and reverse reactions. Since BsADH displayed limited stability under oxidizing conditions, the predominant oxidation-prone residue Cys257 was mutated to Leu based on sequence homology with related enzymes and computational simulation. This substitution resulted in an improved BsADH variant exhibiting prolonged stability and an elevated inactivation temperature. Semi-preparative biocatalysis at 60 °C using the stabilized enzyme, employing butyraldehyde for in situ cofactor regeneration with only catalytic amounts of NAD(+), yielded up to 23 % conversion of HLAMe to OLAMe after 30 min. In contrast to other oxidoreductases, no overoxidation to the dodecanoic diacid monomethyl ester was detected. Thus, the mutated BsADH offers a promising biocatalyst for the selective oxidation of fatty alcohols to yield intermediates for industrial polymer production. PMID:26329849

  15. Metabolic basis of ethylene glycol monobutyl ether (2-butoxyethanol) toxicity: role of alcohol and aldehyde dehydrogenases

    SciTech Connect

    Ghanayem, B.I.; Burka, L.T.; Matthews, H.B.

    1987-07-01

    2-Butoxyethanol (BE) is a massively produced glycol ether of which more than 230 million pounds was produced in the United States in 1983. It is extensively used in aerosols and cleaning agents intended for household use. This creates a high potential for human exposure during its manufacturing and use. A single exposure of rats to BE causes severe hemolytic anemia accompanied by secondary hemoglobinuria as well as liver and kidney damage. Butoxyacetic acid (BAA) was earlier identified as a urinary metabolite of BE. In addition, we have recently identified two additional urinary metabolites of BE, namely, BE-glucuronide and BE-sulfate conjugates. The current studies were undertaken to investigate the metabolic basis of BE-induced hematotoxicity in male F344 rats. Treatment of rats with pyrazole (alcohol dehydrogenase inhibitor) protected rats against BE-induced hematotoxicity and inhibited BE metabolism to BAA. Pyrazole inhibition of BE metabolism to BAA was accompanied by increased BE metabolism to BE-glucuronide and BE-sulfate as determined by quantitative high-performance liquid chromatography analysis of BE metabolites in urine. There was approximately a 10-fold decrease in the ratio of BAA to BE-glucuronide + BE-sulfate in the urine of rats treated with pyrazole + BE compared to rats treated with BE alone. Pretreatment of rats with cyanamide (aldehyde dehydrogenase inhibitor) also significantly protected rats against BE-induced hematotoxicity and modified BE metabolism in a manner similar to that caused by pyrazole. Administration of equimolar doses of BE, the metabolic intermediate butoxyacetaldehyde, or the ultimate metabolite BAA caused similar hematotoxic effects. Cyanamide also protected rats against butoxyacetaldehyde-induced hematotoxicity.

  16. Novel yeast cell dehydrogenase activity assay in situ.

    PubMed

    Berłowska, Joanna; Kregiel, Dorota; Klimek, Leszek; Orzeszyna, Bartosz; Ambroziak, Wojciech

    2006-01-01

    The aim of this research was to develop a suitable method of succinate dehydrogenase activity assay in situ for different industrial yeast strains. For this purpose different compounds: EDTA, Triton X-100, sodium deoxycholate, digitonin, nystatin and beta-mercaptoethanol were used. The permeabilization process was controlled microscopically by primuline staining. Enzyme assay was conducted in whole yeast cells with Na-succinate as substrate, phenazine methosulfate (PMS) as electron carrier and in the presence one of two different tetrazolium salts: tetrazolium blue chloride (BT) or cyanoditolyl tetrazolium chloride (CTC) reduced during the assay. In comparabile studies of yeast vitality the amount of intracellular ATP was determined according to luciferin/luciferase method. During the succinate dehydrogenase assay in intact yeast cells without permeabilization, BT formazans were partially visualized in the cells, but CTC formazans appeared to be totally extracellular or associated with the plasma membrane. Under these conditions there was no linear relationship between formazan color intensity signal and yeast cell density. From all chemical compounds tested, only digitonin was effective in membrane permeabilization without negative influence on cell morphology. Furthermore, with digitonin-treated cells a linear relationship between formazan color intensity signal and yeast cell number was noticed. Significant decreasing of succinate dehydrogenase activity and ATP content were observed during aging of the tested yeast strains. PMID:17419290

  17. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains

    PubMed Central

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  18. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains.

    PubMed

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  19. NAD-dependent aromatic alcohol dehydrogenase in wheats (Triticum L.) and goatgrasses (Aegilops L.): evolutionary genetics.

    PubMed

    Jaaska, V

    1984-04-01

    Evolutionary electrophoretic variation of a NAD-specific aromatic alcohol dehydrogenase, AADH-E, in wheat and goatgrass species is described and discussed in comparison with a NAD-specific alcohol dehydrogenase (ADH-A) and a NADP-dependent AADH-B studied previously. Cultivated tetraploid emmer wheats (T. turgidum s. l.) and hexaploid bread wheats (T. aestivum s. l.) are all fixed for a heterozygous triplet, E(0.58)/E(0.64). The slowest isoenzyme, E(0.58), is controlled by a homoeoallelic gene on the chromosome arm 6AL of T. aestivum cv. 'Chinese Spring' and is inherent in all diploid wheats, T. monococcum s. Str., T. boeoticum s. l. and T. urartu. The fastest isoenzyme, E(0.64), is presumably controlled by the B- and D-genome homoeoalleles of the bread wheat and is the commonest alloenzyme of diploid goat-grasses, including Ae. speltaides and Ae. tauschii. The tetraploid T. timopheevii s. str. has a particular heterozygous triplet E(0.56)/E(0.71), whereas the hexaploid T. zhukovskyi exhibited polymorphism with electromorphs characteristic of T. timopheevii and T. monococcum. Wild tetraploid wheats, T. dicoccoides and T. araraticum, showed partially homologous intraspecific variation of AADH-E with heterozygous triplets E(0.58)/E(0.64) (the commonest), E(0.58)/E(0.71), E(0.45)/E(0.58), E(0.48)/E(0.58) and E(0.56)/E(0.58) recorded. Polyploid goatgrasses of the D-genome group, excepting Ae. cylindrica, are fixed for the common triplet E(0.58)/E(0.64). Ae. cylindrica and polyploid goatgrasses of the C(u)-genome group, excepting Ae. kotschyi, are homozygous for E(0.64). Ae. kotschyi is exceptional, showing fixed heterozygosity for both AADH-E and ADH-A with unique triplets E(0.56)/E(0.64) and A(0.49)/A(0.56). PMID:24258843

  20. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed.

    PubMed

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  1. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

    PubMed Central

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R.; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  2. Thiodiglycol, the hydrolysis product of sulfur mustard: Analysis of in vitro biotransformation by mammalian alcohol dehydrogenases using nuclear magnetic resonance

    SciTech Connect

    Brimfield, A.A.; Hodgson, Ernest

    2006-06-15

    Thiodiglycol (2,2'-bis-hydroxyethylsulfide, TDG), the hydrolysis product of the chemical warfare agent sulfur mustard, has been implicated in the toxicity of sulfur mustard through the inhibition of protein phosphatases in mouse liver cytosol. The absence of any inhibitory activity when TDG was present in assays of pure enzymes, however, led us to investigate the possibility for metabolic activation of TDG to inhibitory compound(s) by cytosolic enzymes. We have successfully shown that mammalian alcohol dehydrogenases (ADH) rapidly oxidize TDG in vitro, but the classic spectrophotometric techniques for following this reaction provided no information on the identity of TDG intermediates and products. The use of proton NMR to monitor the oxidative reaction with structural confirmation by independent synthesis allowed us to establish the ultimate product, 2-hydroxyethylthioacetic acid, and to identify an intermediate equilibrium mixture consisting of 2-hydroxyethylthioacetaldehyde, 2-hydroxyethylthioacetaldehyde hydrate and the cyclic 1,4-oxathian-2-ol. The intermediate nature of this mixture was determined spectrophotometrically when it was shown to drive the production of NADH when added to ADH and NAD.

  3. Selection variability for Arg48His in alcohol dehydrogenase ADH1B among Asian populations.

    PubMed

    Evsyukov, Alexey; Ivanov, Denis

    2013-08-01

    The variant His at codon 48 of the alcohol dehydrogenase gene (ADH1B) results in more efficient ethanol metabolism than with the "typical" codon 48Arg. In this study we introduced selection properties of Arg48His genotypes of ADH1B and estimated fitness in four ethnic-geographical clusters in Asia. Population genetics models were employed that derive observed gene frequencies from fitness relationships among genotypes, to infer the selection pattern of polymorphisms in an indirect manner. The data were analyzed using the model of "complete stationary distribution" by Wright that takes into account random genetic drift, pressure of migrations, mutations, and selection as influential factors of gene frequency. We found that the different population groups showed some variation in the types of selection for Arg48His. Han Chinese from eastern and southeastern China and the Japanese and Korean populations showed stabilizing selection, while the groups from Central Asian and Indochina showed divergent selection. However, all the groups demonstrated a strong positive selection for Arg48His. PMID:25019189

  4. Alcohol and Aldehyde Dehydrogenases Contribute to Sex-Related Differences in Clearance of Zolpidem in Rats

    PubMed Central

    Peer, Cody J.; Strope, Jonathan D.; Beedie, Shaunna; Ley, Ariel M.; Holly, Alesia; Calis, Karim; Farkas, Ronald; Parepally, Jagan; Men, Angela; Fadiran, Emmanuel O.; Scott, Pamela; Jenkins, Marjorie; Theodore, William H.; Sissung, Tristan M.

    2016-01-01

    Objectives: The recommended zolpidem starting dose was lowered in females (5 mg vs. 10 mg) since side effects were more frequent and severe than those of males; the mechanism underlying sex differences in pharmacokinetics (PK) is unknown. We hypothesized that such differences were caused by known sex-related variability in alcohol dehydrogenase (ADH) expression. Methods: Male, female, and castrated male rats were administered 2.6 mg/kg zolpidem, ± disulfiram (ADH/ALDH pathway inhibitor) to compare PK changes induced by sex and gonadal hormones. PK analyses were conducted in rat plasma and rat brain. Key findings: Sex differences in PK were evident: females had a higher CMAX (112.4 vs. 68.1 ug/L) and AUC (537.8 vs. 231.8 h∗ug/L) than uncastrated males. Castration induced an earlier TMAX (0.25 vs. 1 h), greater CMAX (109.1 vs. 68.1 ug/L), and a corresponding AUC increase (339.7 vs. 231.8 h∗ug/L). Administration of disulfiram caused more drastic CMAX and TMAX changes in male vs. female rats that mirrored the effects of castration on first-pass metabolism, suggesting that the observed PK differences may be caused by ADH/ALDH expression. Brain concentrations paralleled plasma concentrations. Conclusion: These findings indicate that sex differences in zolpidem PK are influenced by variation in the expression of ADH/ALDH due to gonadal androgens. PMID:27574509

  5. Determination of Kinetic Isotope Effects in Yeast Alcohol Dehydrogenase Using Transition Path Sampling

    NASA Astrophysics Data System (ADS)

    Varga, Matthew; Schwartz, Steven

    2015-03-01

    The experimental determination of kinetic isotope effects in enzymatic systems can be a difficult, time-consuming, and expensive process. In this study, we use the Chandler-Bolhius method for the determination of reaction rates within transition path sampling (rTPS) to determine the primary kinetic isotope effect in yeast alcohol dehydrogenase (YADH). In this study, normal mode centroid molecular dynamics (CMD) was applied to the transferring hydride/deuteride in order to correctly incorporate quantum effects into the molecular simulations. Though previous studies have used rTPS to calculate reaction rate constants in various model and real systems, it has not been applied to a system as large as YADH. Due to the fact that particle transfer is not wholly indicative of the chemical step, this method cannot be used to determine reaction rate constants in YADH. However, it is possible to determine the transition rate constant of the particle transfer, and the kinetic isotope effect of that step. This method provides a set of tools to determine kinetic isotope effects with the atomistic detail of molecular simulations.

  6. Furfural reduction mechanism of a zinc-dependent alcohol dehydrogenase from Cupriavidus necator JMP134

    PubMed Central

    Kang, ChulHee; Hayes, Robert; Sanchez, Emiliano J.; Webb, Brian N.; Li, Qunrui; Hooper, Travis; Nissen, Mark S.; Xun, Luying

    2012-01-01

    Summary FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn2+ coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn2+ coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD+ dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD+ to NADH that is subsequently used for furfural reduction. PMID:22081946

  7. Study on immobilization of yeast alcohol dehydrogenase on nanocrystalline Ni-Co ferrites as magnetic support.

    PubMed

    Shakir, Mohammad; Nasir, Zeba; Khan, Mohd Shoeb; Lutfullah; Alam, Md Fazle; Younus, Hina; Al-Resayes, Saud Ibrahim

    2015-01-01

    The covalent binding of yeast alcohol dehydrogenase (YADH) enzyme complex in a series of magnetic crystalline Ni-Co nanoferrites, synthesized via sol-gel auto combustion technique was investigated. The structural analysis, morphology and magnetic properties of Ni-Co nanoferrites were determined by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), vibrating-sample magnetometer (VSM), high resolution transmission electron microscopy (HRTEM) and Fourier transform infrared spectroscopy (FTIR). The comparative analysis of the HRTEM micrographs of bare magnetic nanoferrite particles and particles immobilized with enzyme revealed an uniform distribution of the particles in both the cases without undergoing change in the size which was found to be in the range 20-30 nm. The binding of YADH to Ni-Co nanoferrites and the possible binding mechanism have been suggested by comparing the FTIR results. The binding properties of the immobilized YADH enzyme were also studied by kinetic parameters, optimum operational pH, temperature, thermal stability and reusability. The immobilized YADH exhibits enhanced thermal stability as compared to the free enzyme over a wide range of temperature and pH, and showed good durability after recovery by magnetic separation for repeated use. PMID:25450541

  8. Mechanisms of mutagenesis: Analysis through the use of alcohol dehydrogenase in Drosophila: Final report

    SciTech Connect

    Sofer, W.H.

    1986-12-01

    Our original objective was to understand the mechanism of mutagenesis of several important mutagens in higher organisms. Our approach was to try to deduce this mechanism by working backwards from its final effects. The strategy that we used in an effort to carry out our studies was to make mutations in the alcohol dehydrogenase gene of Drosophila melanogaster and sequence the modified genes. Most of our work was focused on an array of mutants that we had induced with formaldehyde, a potent mutagen in Drosophila, and with ethyl methane sulfonate. Over the course of the project period we cloned and sequenced the ADH gene from four formalde-induced mutants and from one EMS mutant. We showed that the four formaldehyde-induced mutants contained small deletions within the protein-coding region of their ADH genes ranging in size from between 6 and 34 bp. The one EMS-induced mutant was shown by DNA sequencing to bear an AT to GC sequence change at a tryptophan codon near the c-terminal coding portion of the gene. These results have significantly increased our understanding of the mechanism(s) of mutagenesis in higher organisms. 20 refs., 1 fig.

  9. High current density PQQ-dependent alcohol and aldehyde dehydrogenase bioanodes.

    PubMed

    Aquino Neto, Sidney; Hickey, David P; Milton, Ross D; De Andrade, Adalgisa R; Minteer, Shelley D

    2015-10-15

    In this paper, we explore the bioelectrooxidation of ethanol using pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenase (ADH and AldDH) enzymes for biofuel cell applications. The bioanode architectures were designed with both direct electron transfer (DET) and mediated electron transfer (MET) mechanisms employing high surface area materials such as multi-walled carbon nanotubes (MWCNTs) and MWCNT-decorated gold nanoparticles, along with different immobilization techniques. Three different polymeric matrices were tested (tetrabutyl ammonium bromide (TBAB)-modified Nafion; octyl-modified linear polyethyleneimine (C8-LPEI); and cellulose) in the DET studies. The modified Nafion membrane provided the best electrical communication between enzymes and the electrode surface, with catalytic currents as high as 16.8 ± 2.1 µA cm(-2). Then, a series of ferrocene redox polymers were evaluated for MET. The redox polymer 1,1'-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI) provided the best electrochemical response. Using this polymer, the electrochemical assays conducted in the presence of MWCNTs and MWCNTs-Au indicated a Jmax of 781 ± 59 µA cm(-2) and 925 ± 68 µA cm(-2), respectively. Overall, from the results obtained here, DET using the PQQ-dependent ADH and AldDH still lacks high current density, while the bioanodes that operate via MET employing ferrocene-modified LPEI redox polymers show efficient energy conversion capability in ethanol/air biofuel cells. PMID:25988787

  10. Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment

    NASA Technical Reports Server (NTRS)

    Chung, H. J.; Ferl, R. J.

    1999-01-01

    It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.

  11. The alcohol dehydrogenase gene is nested in the outspread locus of Drosophila melanogaster

    SciTech Connect

    McNabb, S.; Greig, S.; Davis, T.

    1996-06-01

    This report describes the structure and expression of the outspread (osp) gene of Drosophila melanogaster. Previous work showed that chromosomal breakpoints associated with mutations of the osp locus map to both sides of the alcohol dehydrogenase gene (Adh), suggesting that Adh and the adjacent gene Adh{sup r} are nested in osp. We extended a chromosomal walk and mapped additional osp mutations to define the maximum molecular limit of osp as 119 kb. We identified a 6-kb transcript that hybridizes to osp region DNA and is altered or absent in osp mutants. Accumulation of this RNA peaks during embryonic and pupal periods. The osp cDNAs comprise two distinct classes based on alternative splicing patterns. The 5{prime} end of the longest cDNA was extended by PCR amplification. When hybridized to the osp walk, the 5{prime} extension verifies that Adh and Adh{sup r} are nested in osp and shows that osp has a transcription unit of {ge}74 kb. In situ hybridization shows that osp is expressed both maternally and zygotically. In the ovary, osp is transcribed in nurse cells and localized in the oocyte. In embryos, expression is most abundant in the developing visceral and somatic musculature. 55 refs., 11 figs., 1 tab.

  12. Measuring Selection Coefficients Affecting the Alcohol Dehydrogenase Polymorphism in DROSOPHILA MELANOGASTER

    PubMed Central

    Wilson, S. R.; Oakeshott, J. G.; Gibson, J. B.; Anderson, P. R.

    1982-01-01

    This paper describes a perturbation experiment on the frequency of the F and S Alcohol dehydrogenase (Adh) alleles of D. melanogaster. Fifty-four isofemale lines set up from three wild populations and with initial F frequencies of either 0.25, 0.50 or 0.75 were maintained on standard laboratory food medium at 22°. At generations 4, 12 and 20 the lines were again scored for Adh gene frequencies. Maximum likelihood procedures were used to estimate selection coefficients for the Adh genotypes. An analysis of deviance was used to compare the coefficients against expectations under the hypotheses of neutrality and of constant values for the three base populations, and for the three initial gene frequency classes. Highly-significant departures from neutrality were observed; over all 54 lines, the set of relative fitnesses for S/S:F/S:F/F was estimated as 1.00:1.08:1.08. In addition, there were significant differences between lines in the outcome of selection which were not attributable to differences between base populations or initial F frequencies. These residual between-line differences, as well as some between-generation, within-line differences are discussed in terms of linkage disequilibria with background genes and electrophoretically cryptic variation at the Adh locus. PMID:6807750

  13. Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.

    PubMed

    Maddock, Danielle J; Patrick, Wayne M; Gerth, Monica L

    2015-08-01

    Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2'-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary-secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2'-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme-cofactor interactions. PMID:26034298

  14. Changes in soluble sugar, starch, and alcohol dehydrogenase in Arabidopsis thaliana exposed to N2 diluted atmospheres

    NASA Technical Reports Server (NTRS)

    Porterfield, D. M.; Crispi, M. L.; Musgrave, M. E.

    1997-01-01

    Proper exchange of atmospheric gases is important for normal root and shoot metabolism in plants. This study was conducted to determine how restricted air supply affects foliar carbohydrates, while using the marker enzyme alcohol dehydrogenase (ADH) to report on the oxygenation status of the rootzone. Fourteen-day-old Arabidopsis thaliana (L.) Heynh. plants grown singly in 7-ml tubes containing agarified nutrient medium were placed in coupled Magenta vessels and exposed for six days to either ambient air or one of six different air/nitrogen dilutions. Redox potential of the agar medium was measured immediately after harvesting and freezing leaf tissue, and then root systems were quickly extracted from the agar and frozen for subsequent analyses. Redox potential measurements indicated that this series of gas mixtures produced a transition from hypoxia to anoxia in the root zones. Root ADH activity increased at higher rates as the redox potential neared anoxic levels. In contrast, ADH mRNA expression quickly neared its maximum as the medium became hypoxic and showed little further increase as it became anoxic. Foliar carbohydrate levels increased 1.5- to 2-fold with decreased availability of metabolic gases, with starch increasing at higher concentrations of air than soluble carbohydrate. The results serve as a model for plant performance under microgravity conditions, where absence of convective air movement prevents replenishment of metabolic gases.

  15. Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity

    PubMed Central

    Maddock, Danielle J.; Patrick, Wayne M.; Gerth, Monica L.

    2015-01-01

    Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2′-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary–secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2′-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme–cofactor interactions. PMID:26034298

  16. Microbial metabolic activity in soil as measured by dehydrogenase determinations

    NASA Technical Reports Server (NTRS)

    Casida, L. E., Jr.

    1977-01-01

    The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 C incubation with either glucose or yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.

  17. Characterization of transposable element-associated mutations that alter yeast alcohol dehydrogenase II expression.

    PubMed Central

    Williamson, V M; Cox, D; Young, E T; Russell, D W; Smith, M

    1983-01-01

    Seven cis-dominant, constitutively expressed mutations of the normally glucose-repressible isozyme of alcohol dehydrogenase (ADHII) from the yeast Saccharomyces cerevisiae are caused by insertion of transposable elements from the Ty1 family in front of the ADHII structural gene (ADR2) (V. M. Williamson, E. T. Young, and M. Ciriacy, Cell 23:605-614, 1981). We cloned ADR2 with its associated Ty1 element from five S. cerevisiae strains carrying these mutations. Comparison of the Ty1 elements by heteroduplex studies and restriction enzyme analyses indicated that four were very similar; the fifth, although the same size as the others (about 5.6 kilobases), differed by the presence of two large substitutions of approximately 1 and 2 kilobases. The DNA sequences of the terminal direct repeats (deltas) were very homologous but not identical and were similar to previously reported Ty1 element direct repeats. We determined the 5'-flanking sequences of the ADR2 gene isolated from a wild-type strain and from five Ty1-associated mutations. The 5-base pair target sequence at the site of Ty1 insertion was present at both ends of each Ty1 element. The sites of insertion of the elements were all different and occurred from 125 to 210 base pairs in front of the coding region of ADR2. The 5' end of the major transcript as determined by S1 mapping was the same in wild-type cells and in Ty1-associated constitutive mutants and was approximately 54 base pairs upstream from the coding region. ADR2 transcripts were not detected when a solo delta sequence was present in the 5'-flanking region of this gene. Images PMID:6298605

  18. Some properties of an alcohol dehydrogenase partially purified from baker's yeast grown without added zinc.

    PubMed Central

    Dickenson, C J; Dickinson, F M

    1976-01-01

    Alcohol dehydrogenase was partially purified from yeast (Saccharomyces cerevisiae) grown in the presence of 20 muM-MnSO4 without added Zn2+ and from yeast grown in the presence of 1.8 muM-MnSO4. The enzyme from yeast grown with added Zn2+ has the same properties as the crystalline enzyme from commercial supplies of baker's yeast. The enzyme from yeast grown without added An2+ has quite different properties. It has a mol.wt. in the region of 72000 and an S 20 w of 5.8S. The values can be compared with a mol.wt. of 141000 and an S 20 w of 7.6S for the crystalline enzyme. ADP-ribose, a common impurity in commercial samples of NAD+, is a potent competitive inhibitor of the new enzyme (K1 = 0.5 muM), but is not so for the crystalline enzyme. The observed maximum rate of ethanol oxidation at pH 7.05 and 25 degrees C was decreased 12-fold by the presence of 0.06 mol of inhibitor/mol of NAD+ when using the enzyme from Zn2+-deficient yeast, but with crystalline enzyme the maximum rate was essentially unchanged by this concentration of inhibitor. The kinetic characteristics for the two enzymes with ethanol, butan-1-ol, acetaldehyde and butyraldehyde as substrates are markedly different. These kinetic differences are discussed in relation to the mechanism of catalysis for the enzyme from Zn2+-deficient yeast. PMID:179534

  19. The Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns

    PubMed Central

    Jin, Yazhong; Zhang, Chong; Liu, Wei; Tang, Yufan; Qi, Hongyan; Chen, Hao; Cao, Songxiao

    2016-01-01

    Alcohol dehydrogenases (ADH), encoded by multigene family in plants, play a critical role in plant growth, development, adaptation, fruit ripening and aroma production. Thirteen ADH genes were identified in melon genome, including 12 ADHs and one formaldehyde dehydrogenease (FDH), designated CmADH1-12 and CmFDH1, in which CmADH1 and CmADH2 have been isolated in Cantaloupe. ADH genes shared a lower identity with each other at the protein level and had different intron-exon structure at nucleotide level. No typical signal peptides were found in all CmADHs, and CmADH proteins might locate in the cytoplasm. The phylogenetic tree revealed that 13 ADH genes were divided into three groups respectively, namely long-, medium-, and short-chain ADH subfamily, and CmADH1,3-11, which belongs to the medium-chain ADH subfamily, fell into six medium-chain ADH subgroups. CmADH12 may belong to the long-chain ADH subfamily, while CmFDH1 may be a Class III ADH and serve as an ancestral ADH in melon. Expression profiling revealed that CmADH1, CmADH2, CmADH10 and CmFDH1 were moderately or strongly expressed in different vegetative tissues and fruit at medium and late developmental stages, while CmADH8 and CmADH12 were highly expressed in fruit after 20 days. CmADH3 showed preferential expression in young tissues. CmADH4 only had slight expression in root. Promoter analysis revealed several motifs of CmADH genes involved in the gene expression modulated by various hormones, and the response pattern of CmADH genes to ABA, IAA and ethylene were different. These CmADHs were divided into ethylene-sensitive and –insensitive groups, and the functions of CmADHs were discussed. PMID:27242871

  20. Acrylamide quenching of Trp phosphorescence in liver alcohol dehydrogenase: evidence of gated quencher penetration.

    PubMed

    Strambini, Giovanni B; Gonnelli, Margherita

    2009-08-11

    Notwithstanding the relevance of their biological function, slow motions in proteins, beyond the microsecond range, are still poorly understood and often elusive. We propose that acrylamide quenching of Trp phosphorescence of deeply buried residues, when extended over the entire accessible range of lifetime measurements (tau > 10 micros), may help to unveil low-frequency protein motions that allow penetration of solute into the protein interior. The work examines in some detail acrylamide quenching of Trp phosphorescence in a model protein (liver alcohol dehydrogenase) over an extended submillimolar to molar acrylamide concentration range. The results, which encompass a >10(4)-fold variation in the quenching rate, provide the first evidence of a downward-curving lifetime Stern-Volmer plot, indicative of a nonlinear dependence of the quenching rate on the quencher concentration. From an analysis of saturation effects in terms of a protein-gated acrylamide diffusion mechanism, we infer two main routes for acrylamide to penetrate the globular fold and come into the proximity of internal W314: a low-frequency gate [36 s(-1) (at 25 degrees C)] tentatively assigned to partial opening of the dimer interface and a higher-frequency one (11800 s(-1)) tentatively assigned to a channel blocked by the side chains of V276 and L307. These motions are sharply inhibited in the rigid protein complexes formed with the coenzyme NAD(+) and the coenzyme analogue adenine diphosphate ribose, as well as by the frictional drag of the solvent in viscous glycerol solutions, evidence that rules out an alternative quenching mechanism involving acrylamide binding to the protein. PMID:19594170

  1. Development of an Alcohol Dehydrogenase Biosensor for Ethanol Determination with Toluidine Blue O Covalently Attached to a Cellulose Acetate Modified Electrode

    PubMed Central

    Alpat, Şenol; Telefoncu, Azmi

    2010-01-01

    In this work, a novel voltammetric ethanol biosensor was constructed using alcohol dehydrogenase (ADH). Firstly, alcohol dehydrogenase was immobilized on the surface of a glassy carbon electrode modified by cellulose acetate (CA) bonded to toluidine blue O (TBO). Secondly, the surface was covered by a glutaraldehyde/bovine serum albumin (BSA) cross-linking procedure to provide a new voltammetric sensor for the ethanol determination. In order to fabricate the biosensor, a new electrode matrix containing insoluble Toluidine Blue O (TBO) was obtained from the process, and enzyme/coenzyme was combined on the biosensor surface. The influence of various experimental conditions was examined for the characterization of the optimum analytical performance. The developed biosensor exhibited sensitive and selective determination of ethanol and showed a linear response between 1 × 10−5 M and 4 × 10−4 M ethanol. A detection limit calculated as three times the signal-to-noise ratio was 5.0 × 10−6 M. At the end of the 20th day, the biosensor still retained 50% of its initial activity. PMID:22315566

  2. CHRONIC FEEDING ALCOHOL-CONTAINING DIETS VIA TOTAL ENTERAL NUTRITION INDUCES ALCOHOL DEHYDROGENASE (ADH) AND INSULIN RESISTANCE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Induction of Class 1 ADH occurs in rats fed alcohol chronically, and we have reported that C/EBPs and SREBP-1 are important signaling factors in this process. Chronic alcohol intake in humans can result in alcohol-induced diabetes. We have studied insulin signaling pathways in adult male Sprague-D...

  3. Elemental sulfur: toxicity in vivo and in vitro to bacterial luciferase, in vitro yeast alcohol dehydrogenase, and bovine liver catalase.

    PubMed

    Cetkauskaite, Anolda; Pessala, Piia; Södergren, Anders

    2004-08-01

    The aim of this research was to analyze the effects and the modes of action of elemental sulfur (S(0)) in bioluminescence and respiration of Vibrio fischeri cells and the enzymes crude luciferase, pure catalase, and alcohol dehydrogenase (ADH). Metallic copper removed sulfur and reduced the toxicity of acetone extracts of sediment samples analyzed in the bioluminescence test. The sulfur inhibition of cell bioluminescence was noncompetitive with decanal, the luciferase substrate; reversible, with maximum toxicity after 15 min (EC(50) = 11.8 microg/L); and almost totally recovered after 2 h. In vitro preincubation of crude luciferase extract with sulfur (0.28 ppm) weakly inhibited bioluminescence at 5 min, but at 30 min the inhibition reached 60%. Increasing the concentration of sulfur in the parts per million concentration range in vitro decreased bioluminescence, which was not constant, but depended on exposure time, and no dead-end/total inhibition was observed. The redox state of enzymes in the in vitro system significantly affected inhibition. Hydrogen peroxide restored fully and the reducing agent dithiothreitol, itself toxic, restored only partially luciferase activity in the presence of sulfur. Sulfur (5.5 ppm) slightly inhibited ADH and catalase, and dithiothreitol enhanced sulfur inhibition. High sulfur concentrations (2.2 ppm) inhibited the bioluminescence and enhanced the respiration rate of V. fischeri cells. Elemental sulfur data were interpreted to show that sulfur acted on at least a few V. fischeri cell sites: reversibly modifying luciferase at sites sensitive to/protected by oxidative and reducing agents and by affecting electron transport processes, resulting in enhanced oxygen consumption. Sulfur together with an enzyme reducing agent inhibited the oxidoreductive enzymes ADH and catalase, which have --SH groups, metal ion cofactors, or heme, respectively, in their active centers. PMID:15269910

  4. 17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

    SciTech Connect

    Mendoza-Hernandez, G.; Lopez-Solache, I.; Rendon, J.L.; Diaz-Sanchez, V.; Diaz-Zagoya, J.C.

    1988-04-15

    The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively.

  5. A Case of Hyperammonemia Associated with High Dihydropyrimidine Dehydrogenase Activity.

    PubMed

    Nagaharu, Keiki; Ikemura, Kenji; Yamashita, Yoshiki; Oda, Hiroyasu; Ishihara, Mikiya; Sugawara, Yumiko; Tamaru, Satoshi; Mizuno, Toshiro; Katayama, Naoyuki

    2016-01-01

    Over the past decades, 5-Fluorouracil (5-FU) has been widely used to treat several types of carcinoma, including esophageal squamous cell carcinoma. In addition to its common side effects, including diarrhea, mucositis, neutropenia, and anemia, 5-FU treatment has also been reported to cause hyperammonemia. However, the exact mechanism responsible for 5-FU-induced hyperammonemia remains unknown. We encountered an esophageal carcinoma patient who developed hyperammonemia when receiving 5-FU-containing chemotherapy but did not exhibit any of the other common adverse effects of 5-FU treatment. At the onset of hyperammonemia, laboratory tests revealed high dihydropyrimidine dehydrogenase (DPD) activity and rapid 5-FU clearance. Our findings suggested that 5-FU hypermetabolism may be one of the key mechanisms responsible for hyperammonemia during 5-FU treatment. PMID:27195162

  6. A Case of Hyperammonemia Associated with High Dihydropyrimidine Dehydrogenase Activity

    PubMed Central

    Nagaharu, Keiki; Ikemura, Kenji; Yamashita, Yoshiki; Oda, Hiroyasu; Ishihara, Mikiya; Sugawara, Yumiko; Tamaru, Satoshi; Mizuno, Toshiro; Katayama, Naoyuki

    2016-01-01

    Over the past decades, 5-Fluorouracil (5-FU) has been widely used to treat several types of carcinoma, including esophageal squamous cell carcinoma. In addition to its common side effects, including diarrhea, mucositis, neutropenia, and anemia, 5-FU treatment has also been reported to cause hyperammonemia. However, the exact mechanism responsible for 5-FU-induced hyperammonemia remains unknown. We encountered an esophageal carcinoma patient who developed hyperammonemia when receiving 5-FU-containing chemotherapy but did not exhibit any of the other common adverse effects of 5-FU treatment. At the onset of hyperammonemia, laboratory tests revealed high dihydropyrimidine dehydrogenase (DPD) activity and rapid 5-FU clearance. Our findings suggested that 5-FU hypermetabolism may be one of the key mechanisms responsible for hyperammonemia during 5-FU treatment. PMID:27195162

  7. Strong Protective Effect of The Aldehyde Dehydrogenase Gene (ALDH2) 504lys (*2) Allele Against Alcoholism And Alcohol-Induced Medical Diseases in Asians

    PubMed Central

    Li, Dawei; Zhao, Hongyu; Gelernter, Joel

    2013-01-01

    Alcohol is oxidized to acetaldehyde, which in turn is oxidized to acetate. The aldehyde dehydrogenase 2 gene (ALDH2) is the most important gene responsible for acetaldehyde metabolism. Individuals heterozygous or homozygous for the lys (A or *2) allele at the single nucleotide polymorphism (SNP) glu504lys (rs671) of ALDH2 have greatly reduced ability to metabolize acetaldehyde, which greatly decreases their risk for alcohol dependence (AD). Case-control studies have shown association between this SNP and alcohol dependence as well as alcohol-induced liver disease. However, some studies have produced insignificant results. Using cumulative data from the past 20 years predominately from Asian populations (from both English and Chinese publications), this meta-analysis sought to examine and update whether the aggregate data provide new evidence of statistical significance for the proposed association. Our results (9,678 cases and 7,331 controls from 53 studies) support a strong association of alcohol abuse and dependence, with allelic P value of 3×10−56 and OR of 0.23 (0.2, 0.28) under the random effects model. The dominant model (lys-lys + lys-glu vs. glu-glu) also showed strong association with P value of 1×10−44 and OR of 0.22 (0.18, 0.27). When stricter criteria and various sub-group analyses were applied, the association remained strong (for example, OR = 0.23 (0.18, 0.3) and P = 2×10−28 for the alcoholic patients with alcoholic liver disease, cirrhosis, or pancreatitis). These findings provide confirmation of the involvement of the human ALDH2 gene in the pathogenesis of AD as well as alcohol-induced medical illnesses in East-Asians. PMID:22102315

  8. Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1.

    PubMed

    Wu, Tung-Yun; Chen, Chang-Ting; Liu, Jessica Tse-Jin; Bogorad, Igor W; Damoiseaux, Robert; Liao, James C

    2016-06-01

    Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation. PMID:26846745

  9. Inhibition of human alcohol and aldehyde dehydrogenases by aspirin and salicylate: assessment of the effects on first-pass metabolism of ethanol.

    PubMed

    Lee, Shou-Lun; Lee, Yung-Pin; Wu, Min-Li; Chi, Yu-Chou; Liu, Chiu-Ming; Lai, Ching-Long; Yin, Shih-Jiun

    2015-05-01

    Previous studies have reported that aspirin significantly reduced the first-pass metabolism (FPM) of ethanol in humans thereby increasing adverse effects of alcohol. The underlying causes, however, remain poorly understood. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), principal enzymes responsible for metabolism of ethanol, are complex enzyme families that exhibit functional polymorphisms among ethnic groups and distinct tissue distributions. We investigated the inhibition profiles by aspirin and its major metabolite salicylate of ethanol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and acetaldehyde oxidation by ALDH1A1 and ALDH2, at pH 7.5 and 0.5 mM NAD(+). Competitive inhibition pattern was found to be a predominant type among the ADHs and ALDHs studied, although noncompetitive and uncompetitive inhibitions were also detected in a few cases. The inhibition constants of salicylate for the ADHs and ALDHs were considerably lower than that of aspirin with the exception of ADH1A that can be ascribed to a substitution of Ala-93 at the bottom of substrate pocket as revealed by molecular docking experiments. Kinetic inhibition equation-based simulations show at higher therapeutic levels of blood plasma salicylate (1.5 mM) that the decrease of activities at 2-10 mM ethanol for ADH1A/ADH2 and ADH1B2/ADH1B3 are predicted to be 75-86% and 31-52%, respectively, and that the activity decline for ALDH1A1 and ALDH2 at 10-50 μM acetaldehyde to be 62-73%. Our findings suggest that salicylate may substantially inhibit hepatic FPM of alcohol at both the ADH and ALDH steps when concurrent intaking aspirin. PMID:25772736

  10. The Cinnamyl Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns

    PubMed Central

    Jin, Yazhong; Zhang, Chong; Liu, Wei; Qi, Hongyan; Chen, Hao; Cao, Songxiao

    2014-01-01

    Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. However, little was known about CADs in melon. Five CAD-like genes were identified in the genome of melons, namely CmCAD1 to CmCAD5. The signal peptides analysis and CAD proteins prediction showed no typical signal peptides were found in all CmCADs and CmCAD proteins may locate in the cytoplasm. Multiple alignments implied that some motifs may be responsible for the high specificity of these CAD proteins, and may be one of the key residues in the catalytic mechanism. The phylogenetic tree revealed seven groups of CAD and melon CAD genes fell into four main groups. CmCAD1 and CmCAD2 belonged to the bona fide CAD group, in which these CAD genes, as representative from angiosperms, were involved in lignin synthesis. Other CmCADs were distributed in group II, V and VII, respectively. Semi-quantitative PCR and real time qPCR revealed differential expression of CmCADs, and CmCAD5 was expressed in different vegetative tissues except mature leaves, with the highest expression in flower, while CmCAD2 and CmCAD5 were strongly expressed in flesh during development. Promoter analysis revealed several motifs of CAD genes involved in the gene expression modulated by various hormones. Treatment of abscisic acid (ABA) elevated the expression of CmCADs in flesh, whereas the transcript levels of CmCAD1 and CmCAD5 were induced by auxin (IAA); Ethylene induced the expression of CmCADs, while 1-MCP repressed the effect, apart from CmCAD4. Taken together, these data suggested that CmCAD4 may be a pseudogene and that all other CmCADs may be involved in the lignin biosynthesis induced by both abiotic and biotic stresses and in tissue-specific developmental lignification through a CAD genes family network, and CmCAD2 may be the main CAD enzymes for lignification of melon flesh and CmCAD5 may also function in flower development. PMID:25019207

  11. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    PubMed Central

    Bemer, Meagan J.; Risler, Linda J.; Phillips, Brian R.; Wang, Joanne; Storer, Barry E.; Sandmaier, Brenda M.; Duan, Haichuan; Raccor, Brianne S.; Boeckh, Michael J.; McCune, Jeannine S.

    2014-01-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5’- monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNC) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T-cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation, but not with chronic GVHD, relapse, non-relapse mortality, or overall mortality. We conclude that quantitation of the recipient’s pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient’s sensitivity to MMF, but confirmatory studies are needed. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  12. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

    PubMed Central

    Jean, Elise; Laoudj-Chenivesse, Dalila; Notarnicola, Cécile; Rouger, Karl; Serratrice, Nicolas; Bonnieu, Anne; Gay, Stéphanie; Bacou, Francis; Duret, Cédric; Carnac, Gilles

    2011-01-01

    Abstract Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56+ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non-human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H2O2)-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. PMID:19840193

  13. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling

    PubMed Central

    Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  14. Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum

    PubMed Central

    Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

    2016-01-01

    Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production. PMID:27321949

  15. Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum.

    PubMed

    Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

    2016-01-01

    Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production. PMID:27321949

  16. Furosemide and 11beta-hydroxysteroid dehydrogenase activity, in man.

    PubMed

    Palermo, M; Armanini, D; Shackleton, C H L; Sorba, G; Cossu, M; Roitman, E; Scaroni, C; Delitala, G

    2002-09-01

    Mineralocorticoid receptors possess the same affinity for aldosterone and for cortisol and preferential binding of aldosterone is modulated by the 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) enzyme, which converts cortisol to its inactive metabolite cortisone. Several endogenous or exogenous compounds able to inhibit the enzyme have been described and, as a consequence, produce the syndrome of apparent mineralocorticoid excess (AME) characterized by hypertension, hypokalemia, volume repletion and suppression of the renin-angiotensin-aldosterone system. High doses of furosemide, a diuretic that works in the luminal surface of the thick ascending limb of Henle's loop, have been reported to inhibit 11 beta-OHSD activity to the same extent as licorice in vivo and in vitro, in rat. The aim of our study was to verify the effect of the drug on 11 beta-OHSD activity in man at the doses currently used in clinical practice. We tested the activity of 11 beta-OHSD following both acute and protracted administration of furosemide. In the acute study, the drug was administered at low (40 mg i.v. in bolo) and high doses (infusion of 10 mg/kg bw i.v for six hours); the protracted furosemide administration consisted in 50 mg/day for 20 days, by mouth. The ratios between the cortisol metabolites tetrahydrocortisol plus allo-tetrahydrocortisol to tetra-hydrocortisone and urinary free cortisol to urinary free cortisone were used to measure the activity of 11 beta-OHSD. Urinary cortisol, cortisone and their metabolites were tested by a gas-chromatographic/mass spectrometric method. Neither acute nor prolonged administration of furosemide did affect the activity of 11 beta-OHSD although the drug was able to modify plasma aldosterone and PRA secretion and to determine hypokalemia. Our results suggest that furosemide does not play a significant role in 11 beta-OHSD modulation in humans, at least at the dosage used in clinical practice. PMID:12373630

  17. 11 beta-Hydroxysteroid dehydrogenase activity in hypothalamic obesity.

    PubMed

    Tiosano, Dov; Eisentein, Israel; Militianu, Daniela; Chrousos, George P; Hochberg, Ze'ev

    2003-01-01

    After extensive suprasellar operations for hypothalamic tumor removal, some patients develop Cushing-like morbid obesity while they receive replacement doses of glucocorticoids. In this study, we examined the hypothesis that target tissue conversion of inactive 11-ketosteroids to active 11 beta-OH glucocorticoids might explain the obesity of some patients with hypothalamic lesions. Toward this aim, we studied 10 patients with hypothalamic obesity and secondary adrenal insufficiency and 6 control Addisonian patients while they were on glucocorticoid replacement therapy. Pituitary hormone deficiencies were replaced when medically indicated. Twenty-four-hour urine was collected after a single oral dose of 12 mg/m(2) hydrocortisone acetate. The ratios of free and conjugated cortisol (F) to cortisone (E) and their metabolites, [tetrahydrocortisol (THF)+5 alpha THF]/tetrahyrdocortisone (THE), dihydrocortisols/dihydrocortisones, cortols/cortolones, and (F+E)/(THF+THE+5 alpha THF), were considered to represent 11 beta-hydroxysteroid dehydrogenase (HSD) activity. The 11-OH/11-oxo ratios were significantly higher in the urine of patients with hypothalamic obesity. The 11-OH/11-oxo ratios, however, did not correlate with the degree of obesity, yet a significant correlation was found between conjugated F/E and the ratio of visceral fat to sc fat measured by computerized tomography at the umbilical level. The consequence of increased 11 beta-HSD1 activity and the shift of the interconversion toward cortisol may contribute to the effects of the latter in adipose tissue. We propose that deficiency of hypothalamic messengers after surgical injury induces a paracrine/autocrine effect of enhanced glucocorticoid activity due to up-regulated 11 beta-HSD1 activity. PMID:12519880

  18. Environmental Stresses of Field Growth Allow Cinnamyl Alcohol Dehydrogenase-Deficient Nicotiana attenuata Plants to Compensate for their Structural Deficiencies1[C][W][OA

    PubMed Central

    Kaur, Harleen; Shaker, Kamel; Heinzel, Nicolas; Ralph, John; Gális, Ivan; Baldwin, Ian T.

    2012-01-01

    The organized lignocellulosic assemblies of cell walls provide the structural integrity required for the large statures of terrestrial plants. Silencing two CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes in Nicotiana attenuata produced plants (ir-CAD) with thin, red-pigmented stems, low CAD and sinapyl alcohol dehydrogenase activity, low lignin contents, and rubbery, structurally unstable stems when grown in the glasshouse (GH). However, when planted into their native desert habitat, ir-CAD plants produced robust stems that survived wind storms as well as the wild-type plants. Despite efficient silencing of NaCAD transcripts and enzymatic activity, field-grown ir-CAD plants had delayed and restricted spread of red stem pigmentation, a color change reflecting blocked lignification by CAD silencing, and attained wild-type-comparable total lignin contents. The rubbery GH phenotype was largely restored when field-grown ir-CAD plants were protected from wind, herbivore attack, and ultraviolet B exposure and grown in restricted rooting volumes; conversely, it was lost when ir-CAD plants were experimentally exposed to wind, ultraviolet B, and grown in large pots in growth chambers. Transcript and liquid chromatography-electrospray ionization-time-of-flight analysis revealed that these environmental stresses enhanced the accumulation of various phenylpropanoids in stems of field-grown plants; gas chromatography-mass spectrometry and nuclear magnetic resonance analysis revealed that the lignin of field-grown ir-CAD plants had GH-grown comparable levels of sinapaldehyde and syringaldehyde cross-linked into their lignins. Additionally, field-grown ir-CAD plants had short, thick stems with normal xylem element traits, which collectively enabled field-grown ir-CAD plants to compensate for the structural deficiencies associated with CAD silencing. Environmental stresses play an essential role in regulating lignin biosynthesis in lignin-deficient plants. PMID:22645069

  19. Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction.

    PubMed Central

    Larroy, Carol; Fernández, M Rosario; González, Eva; Parés, Xavier; Biosca, Josep A

    2002-01-01

    YMR318C represents an open reading frame from Saccharomyces cerevisiae with unknown function. It possesses a conserved sequence motif, the zinc-containing alcohol dehydrogenase (ADH) signature, specific to the medium-chain zinc-containing ADHs. In the present study, the YMR318C gene product has been purified to homogeneity from overexpressing yeast cells, and found to be a homodimeric ADH, composed of 40 kDa subunits and with a pI of 5.0-5.4. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (linear and branched-chain) and aromatic primary alcohols and aldehydes. Aldehydes were processed with a 50-fold higher catalytic efficiency than that for the corresponding alcohols. The highest k(cat)/K(m) values were found with pentanal>veratraldehyde > hexanal > 3-methylbutanal >cinnamaldehyde. Taking into consideration the substrate specificity and sequence characteristics of the YMR318C gene product, we have proposed this gene to be called ADH6. The disruption of ADH6 was not lethal for the yeast under laboratory conditions. Although S. cerevisiae is considered a non lignin-degrading organism, the catalytic activity of ADHVI can direct veratraldehyde and anisaldehyde, arising from the oxidation of lignocellulose by fungal lignin peroxidases, to the lignin biodegradation pathway. ADHVI is the only S. cerevisiae enzyme able to significantly reduce veratraldehyde in vivo, and its overexpression allowed yeast to grow under toxic concentrations of this aldehyde. The enzyme may also be involved in the synthesis of fusel alcohols. To our knowledge this is the first NADPH-dependent medium-chain ADH to be characterized in S. cerevisiae. PMID:11742541

  20. Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis[C][W

    PubMed Central

    Pan, Haiyun; Zhou, Rui; Louie, Gordon V.; Mühlemann, Joëlle K.; Bomati, Erin K.; Bowman, Marianne E.; Dudareva, Natalia; Dixon, Richard A.; Noel, Joseph P.; Wang, Xiaoqiang

    2014-01-01

    The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. PMID:25217505

  1. Reliability of a flushing questionnaire and the ethanol patch test in screening for inactive aldehyde dehydrogenase-2 and alcohol-related cancer risk.

    PubMed

    Yokoyama, A; Muramatsu, T; Ohmori, T; Kumagai, Y; Higuchi, S; Ishii, H

    1997-12-01

    Molecular epidemiology of esophageal and upper aerodigestive tract cancers revealed that alcohol is more carcinogenic in persons with inactive aldehyde dehydrogenase-2 (ALDH2) than in those with active ALDH2. A simple questionnaire has been developed to screen for the facial flushing that occurs in persons with inactive ALDH2 when they drink even a single glass of beer. In this study, 266 of 284 consecutive male Japanese clinic patients (age > or = 50 years) completed the flushing questionnaire, and 239 underwent the ethanol patch test (a cutaneous model for the flushing response). Blinded genotyping showed inactive ALDH2 for 94.4% (102 of 108) of subjects who reported always flushing (early in their drinking history or currently) and for 47.7% (21 of 44) of those who reported sometimes flushing, whereas 95.6% (109 of 114) of subjects reporting that they never exhibited facial flushing had active ALDH2. When all three categories of flushing (current always, former always, and sometimes) were collapsed into one, the questionnaire's sensitivity and specificity for identifying inactive ALDH2 were 96.1 and 79.0%, respectively, compared with 72.4 and 71.4% for the ethanol patch test. The results suggest the utility of this simple flushing questionnaire in daily practice, as well as large-scale studies to assess cancer risks associated with drinking and ALDH2 and for activities aimed at preventing alcohol-related cancer. PMID:9419411

  2. Antimicrobial activity of alcohols from Musca domestica.

    PubMed

    Gołębiowski, Marek; Dawgul, Małgorzata; Kamysz, Wojciech; Boguś, Mieczysława I; Wieloch, Wioletta; Włóka, Emilia; Paszkiewicz, Monika; Przybysz, Elżbieta; Stepnowski, Piotr

    2012-10-01

    Information on the stimulatory and inhibitory effects of cuticular alcohols on growth and virulence of insecticidal fungi is unavailable. Therefore, we set out to describe the content of cuticular and internal alcohols in the body of housefly larvae, pupae, males and females. The total cuticular alcohols in larvae, males and females of Musca domestica were detected in comparable amounts (4.59, 3.95 and 4.03 μg g(-1) insect body, respectively), but occurred in smaller quantities in pupae (2.16 μg g(-1)). The major free alcohol in M. domestica larvae was C(12:0) (70.4%). Internal alcohols of M. domestica larvae were not found. Among cuticular pupae alcohols, C(12:0) (31.0%) was the most abundant. In the internal lipids of pupae, only five alcohols were identified in trace amounts. The most abundant alcohol in males was C(24:0) (57.5%). The percentage content of cuticular C(24:0) in males and females (57.5 and 36.5%, respectively) was significantly higher than that of cuticular lipids in larvae and pupae (0.9 and 5.6%, respectively). Only two alcohols were present in the internal lipids of males in trace amounts (C(18:0) and C(20:0)). The most abundant cuticular alcohols in females were C(24:0) (36.5%) and C(12:0) (26.8%); only two alcohols (C(18:0) and C(20:0)) were detected in comparable amounts in internal lipids (3.61±0.32 and 5.01±0.42 μg g(-1), respectively). For isolated alcohols, antimicrobial activity against 10 reference strains of bacteria and fungi was determined. Individual alcohols showed approximately equal activity against fungal strains. C(14:0) was effective against gram-positive bacteria, whereas gram-negative bacteria were resistant to all tested alcohols. Mixtures of alcohols found in cuticular lipids of larvae, pupae, males and females of M. domestica generally presented higher antimicrobial activity than individual alcohols. In contrast, crude extracts containing both cuticular and internal lipids showed no antifungal activity against the

  3. Microbial and xanthine dehydrogenase inhibitory activity of some flavones.

    PubMed

    Khobragade, C N; Bodade, Ragini G; Shinde, M S; Jaju, Deepa R; Bhosle, R B; Dawane, B S

    2008-06-01

    Xanthine dehydrogenase (XDH) is responsible for the pathological condition called Gout. In the present study different flavones synthesized from chalcone were evaluated in vitro for their inhibitory activity. Inhibitory activity of flavones on XDH was determined in terms of inhibition of uric acid synthesis from Xanthine. The enzymatic activity was found maximum at pH 7.5 and temperature 40 degrees C. The flavones 6-chloro-2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(1)) and 6-chloro-7methyl-2-[3-(4-chloro-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one(F(2)),were noncompetitive and competitive inhibitor with Ki values 1.1 and 0.22 respectively. The flavones (F(1)), (F(2)), 6-chloro-2-[3-(4-chloro-phenyl)-1phenyl-1-H-pyrazol-4-yl]-chromen-4-one(F(3)), 8-bromo-6-chloro-2-[3-(4-chloro-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(4)), 2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(5)) and 6-methyl-2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(6)) were also screened for their antimicrobial activity, measured in terms of zone of inhibition. A broad spectrum antifungal activity was obtained against Trichoderma viridae, Candida albicans, Microsporum cannis, Penicillium chrysogenum and Fusarium moniliformae. In case of Aspergillus niger and Aspergillus flavous only spore formation was affected, while antibacterial activity was observed against Staphylococcus aureus, Bacillus subtilis and Serratia marsecens only. The flavones were further analyzed for quantitative structural activity relationship study (QSAR) by using PASS, online software to determine their Pa value. Toxicity and drug relevant properties were revealed by PALLAS software in terms of their molecular weight. Log P values were also studied. The result showed both the F(1) and F(2) flavones as antigout and therefore supports the development of novel drugs for the treatment of gout. PMID:18569337

  4. Folate, alcohol, and aldehyde dehydrogenase 2 polymorphism and the risk of oral and pharyngeal cancer in Japanese.

    PubMed

    Matsuo, Keitaro; Rossi, Marta; Negri, Eva; Oze, Isao; Hosono, Satoyo; Ito, Hidemi; Watanabe, Miki; Yatabe, Yasushi; Hasegawa, Yasuhisa; Tanaka, Hideo; Tajima, Kazuo; La Vecchia, Carlo

    2012-03-01

    Folate consumption is inversely associated with the risk of oral and pharyngeal cancer (OPC) and potentially interacts with alcohol drinking in the risk of OPC. Aldehyde dehydrogenase 2 (ALDH2) gene polymorphism is known to interact with alcohol consumption. The aim of this study was to investigate potential interaction between folate, alcohol drinking, and ALDH2 polymorphism in the risk of OPC in a Japanese population. The study group comprised 409 head and neck cancer cases and 1227 age-matched and sex-matched noncancer controls; of these, 251 cases and 759 controls were evaluated for ALDH rs671 polymorphism. Associations were assessed by odds ratios and 95% confidence intervals in multiple logistic regression models. We observed an inverse association between folate consumption and OPC risk. The odds ratio for high folate intake was 0.53 (95% confidence interval: 0.36-0.77) relative to low intake (P trend=0.003). This association was consistent across strata of sex, age, smoking, and ALDH2 genotypes. Interaction between folate consumption, drinking, and ALDH2 genotype was remarkable (three-way interaction, P<0.001). We observed significant interaction among folate, drinking, and ALDH2 genotype in the Japanese population. PMID:21946912

  5. Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.

    PubMed

    Wang, Qingzhao; Ingram, Lonnie O; Shanmugam, K T

    2011-11-22

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates. PMID:22065761

  6. The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

    PubMed Central

    Zammit, V A; Newsholme, E A

    1976-01-01

    Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is

  7. Synthesis of cinnamyl alcohol from cinnamaldehyde with Bacillus stearothermophilus alcohol dehydrogenase as the isolated enzyme and in recombinant E. coli cells.

    PubMed

    Pennacchio, Angela; Rossi, Mosè; Raia, Carlo A

    2013-07-01

    The synthesis of the aroma chemical cinnamyl alcohol (CMO) by means of enzymatic reduction of cinnamaldehyde (CMA) was investigated using NADH-dependent alcohol dehydrogenase from Bacillus stearothermophilus both as an isolated enzyme, and in recombinant Escherichia coli whole cells. The influence of parameters such as reaction time and cofactor, substrate, co-substrate 2-propanol and biocatalyst concentrations on the bioreduction reaction was investigated and an efficient and sustainable one-phase system developed. The reduction of CMA (0.5 g/L, 3.8 mmol/L) by the isolated enzyme occurred in 3 h at 50 °C with 97% conversion, and yielded high purity CMO (≥98%) with a yield of 88% and a productivity of 50 g/genzyme. The reduction of 12.5 g/L (94 mmol/L) CMA by whole cells in 6 h, at 37 °C and no requirement of external cofactor occurred with 97% conversion, 82% yield of 98% pure alcohol and a productivity of 34 mg/gwet cell weight. The results demonstrate the microbial system as a practical and efficient method for larger-scale synthesis of CMO. PMID:23686507

  8. Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification.

    PubMed

    Daniellou, Richard; Zheng, Hongyan; Langill, David M; Sanders, David A R; Palmer, David R J

    2007-06-26

    The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant. PMID:17539607

  9. I86A/C295A mutant secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus has broadened substrate specificity for aryl ketones.

    PubMed

    Nealon, Christopher M; Welsh, Travis P; Kim, Chang Sup; Phillips, Robert S

    2016-09-15

    Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (SADH) reduces aliphatic ketones according to Prelog's Rule, with binding pockets for small and large substituents. It was shown previously that the I86A mutant SADH reduces acetophenone, which is not a substrate of wild-type SADH, to give the anti-Prelog R-product (Musa, M. M.; Lott, N.; Laivenieks, M.; Watanabe, L.; Vieille, C.; Phillips, R. S. ChemCatChem2009, 1, 89-93.). However, I86A SADH did not reduce aryl ketones with substituents larger than fluorine. We have now expanded the small pocket of the active site of I86A SADH by mutation of Cys-295 to alanine to allow reaction of substituted acetophenones. As predicted, the double mutant I86A/C295A SADH has broadened substrate specificity for meta-substituted, but not para-substituted, acetophenones. However, the increase of the substrate specificity of I86A/C295A SADH is accompanied by a decrease in the kcat/Km values of acetophenones, possibly due to the substrates fitting loosely inside the more open active site. Nevertheless, I86A/C295A SADH gives high conversions and very high enantiomeric excess of the anti-Prelog R-alcohols from the tested substrates. PMID:27495738

  10. MOLECULAR SYSTEMATICS OF THE GENUS NEOTOMA BASED ON DNA SEQUENCES FROM INTRON 2 OF THE ALCOHOL DEHYDROGENASE GENE

    PubMed Central

    Longhofer, Lisa K.; Bradley, Robert D.

    2009-01-01

    Phylogenetic relationships were evaluated among 13 species of Neotoma based on DNA sequences from intron 2 of the nuclear alcohol dehydrogenase gene 1 (Adh1-I2). Sequences were analyzed using parsimony, likelihood, and Bayesian methods. Three major clades (I–III) consistently were recovered and relationships among taxa within 2 of the clades remained unchanged between analyses; however, relationships within clade III were largely unresolved. Average genetic divergence values were 2.12% among species, 4% between subgenera (Teonoma and Neotoma), and 5.1% between genera (Hodomys and Neotoma). Adh1-I2 sequences were concatenated with mitochondrial cytochrome-b sequences generated from the same individuals. Examination of the combined data resulted in a phylogeny whose topology was similar to that based only on cytochrome-b sequences. PMID:19907669

  11. Aldehyde Dehydrogenase-2 (ALDH2) Ameliorates Chronic Alcohol Ingestion-Induced Hepatic Steatosis and Inflammation: Role of Autophagy

    PubMed Central

    Guo, Rui; Xu, Xihui; Babcock, Sara A.; Zhang, Yingmei; Ren, Jun

    2014-01-01

    Background & Aims Mitochondrial aldehyde dehydrogenase (ALDH2) plays a critical role in the detoxification of the ethanol metabolite acetaldehyde. This study was designed to examine the impact of global ALDH2 overexpression on alcohol-induced hepatic steatosis. Methods Wild-type friendly virus B (FVB) and ALDH2 transgenic mice were placed on a 4% alcohol or control diet for 12 weeks. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin and cholesterol, hepatic triglyceride, steatosis, fat metabolism-related proteins, pro-inflammatory cytokines, glutathione (GSH), oxidized glutathione (GSSG), autophagy and autophagy signaling were examined. The role of autophagy was evaluated in ADH1-transfected human hepatocellular liver carcinoma cells (VA-13) treated with or without autophagy inducer rapamycin and lysosomal inhibitors. Results Chronic alcohol intake led to elevated AST, ALT, bilirubin, AST/ALT ratio, cholesterol, hepatic triglycerides, hepatic fat deposition as evidenced by H&E and oil Red O staining, associated with disturbed fat metabolism-related proteins (fatty acid synthase, SCD1), upregulated interleukin-6, TNF-α, cyclooxygenase, oxidative stress, and loss of autophagy, the effects of which were attenuated or ablated by ALDH2 transgene. Moreover, ethanol (100 mM) and acetaldehyde (100, 500 μM) increased levels of IL-6 and IFN-γ, and suppressed autophagy in VA-13 cells, the effects of which were markedly alleviated by rapamycin. In addition, lysosomal inhibitors mimicked ethanol-induced p62 accumulation with little additive effect with ethanol. Ethanol significantly suppressed LC3 conversion in the presence of lysosomal inhibitors. Conclusions In summary, our results revealed that ALDH2 plays a beneficial role in ameliorating chronic alcohol intake-induced hepatic steatosis and inflammation through regulation of autophagy. PMID:25457208

  12. Degradation of Swainsonine by the NADP-Dependent Alcohol Dehydrogenase A1R6C3 in Arthrobacter sp. HW08

    PubMed Central

    Wang, Yan; Zhai, A’guan; Zhang, Yanqi; Qiu, Kai; Wang, Jianhua; Li, Qinfan

    2016-01-01

    Swainsonine is an indolizidine alkaloid that has been found in locoweeds and some fungi. Our previous study demonstrated that Arthrobacter sp. HW08 or its crude enzyme extract could degrade swainsonie efficiently. However, the mechanism of swainsonine degradation in bacteria remains unclear. In this study, we used label-free quantitative proteomics method based on liquid chromatography-electrospray ionization-tandem mass spectrometry to dissect the mechanism of swainsonine biodegradation by Arthrobacter sp. HW08. The results showed that 129 differentially expressed proteins were relevant to swainsonine degradation. These differentially expressed proteins were mostly related to the biological process of metabolism and the molecular function of catalytic activity. Among the 129 differentially expressed proteins, putative sugar phosphate isomerase/epimerase A1R5X7, Acetyl-CoA acetyltransferase A0JZ95, and nicotinamide adenine dinucleotide phosphate (NADP)-dependent alcohol dehydrogenase A1R6C3 were found to contribute to the swainsonine degradation. Notably, NADP-dependent alcohol dehyrodgenase A1R6C3 appeared to play a major role in degrading swainsonine, but not as much as Arthrobacter sp. HW08 did. Collectively, our findings here provide insights to understand the mechanism of swainsonine degradation in bacteria. PMID:27196926

  13. Elevated glutathione level does not protect against chronic alcohol mediated apoptosis in recombinant human hepatoma cell line VL-17A over-expressing alcohol metabolizing enzymes--alcohol dehydrogenase and Cytochrome P450 2E1.

    PubMed

    Chandrasekaran, Karthikeyan; Swaminathan, Kavitha; Kumar, S Mathan; Chatterjee, Suvro; Clemens, Dahn L; Dey, Aparajita

    2011-06-01

    Chronic consumption of alcohol leads to liver injury. Ethanol-inducible Cytochrome P450 2E1 (CYP2E1) plays a critical role in alcohol mediated oxidative stress due to its ability to metabolize ethanol. In the present study, using the recombinant human hepatoma cell line VL-17A that over-expresses the alcohol metabolizing enzymes-alcohol dehydrogenase (ADH) and CYP2E1; and control HepG2 cells, the mechanism and mode of cell death due to chronic ethanol exposure were studied. Untreated VL-17A cells exhibited apoptosis and oxidative stress when compared with untreated HepG2 cells. Chronic alcohol exposure, i.e., 100 mM ethanol treatment for 72 h caused a significant decrease in viability (47%) in VL-17A cells but not in HepG2 cells. Chronic ethanol mediated cell death in VL-17A cells was predominantly apoptotic, with increased oxidative stress as the underlying mechanism. Chronic ethanol exposure of VL-17A cells resulted in 1.1- to 2.5-fold increased levels of ADH and CYP2E1. Interestingly, the level of the antioxidant GSH was found to be 3-fold upregulated in VL-17A cells treated with ethanol, which may be a metabolic adaptation to the persistent and overwhelming oxidative stress. In conclusion, the increased GSH level may not be sufficient enough to protect VL-17A cells from chronic alcohol mediated oxidative stress and resultant apoptosis. PMID:21414402

  14. 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.

  15. Effects of Cavities at the Nicotinamide Binding Site of Liver Alcohol Dehydrogenase on Structure, Dynamics and Catalysis

    PubMed Central

    2015-01-01

    A role for protein dynamics in enzymatic catalysis of hydrogen transfer has received substantial scientific support, but the connections between protein structure and catalysis remain to be established. Valine residues 203 and 207 are at the binding site for the nicotinamide ring of the coenzyme in liver alcohol dehydrogenase and have been suggested to facilitate catalysis with “protein-promoting vibrations” (PPV). We find that the V207A substitution has small effects on steady-state kinetic constants and the rate of hydrogen transfer; the introduced cavity is empty and is tolerated with minimal effects on structure (determined at 1.2 Å for the complex with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol). Thus, no evidence is found to support a role for Val-207 in the dynamics of catalysis. The protein structures and ligand geometries (including donor–acceptor distances) in the V203A enzyme complexed with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or 2,2,2-trifluoroethanol (determined at 1.1 Å) are very similar to those for the wild-type enzyme, except that the introduced cavity accommodates a new water molecule that contacts the nicotinamide ring. The structures of the V203A enzyme complexes suggest, in contrast to previous studies, that the diminished tunneling and decreased rate of hydride transfer (16-fold, relative to that of the wild-type enzyme) are not due to differences in ground-state ligand geometries. The V203A substitution may alter the PPV and the reorganization energy for hydrogen transfer, but the protein scaffold and equilibrium thermal motions within the Michaelis complex may be more significant for enzyme catalysis. PMID:24437493

  16. Ethanol metabolism, oxidative stress, and endoplasmic reticulum stress responses in the lungs of hepatic alcohol dehydrogenase deficient deer mice after chronic ethanol feeding

    SciTech Connect

    Kaphalia, Lata; Boroumand, Nahal; Hyunsu, Ju; Kaphalia, Bhupendra S.; Calhoun, William J.

    2014-06-01

    Consumption and over-consumption of alcoholic beverages are well-recognized contributors to a variety of pulmonary disorders, even in the absence of intoxication. The mechanisms by which alcohol (ethanol) may produce disease include oxidative stress and prolonged endoplasmic reticulum (ER) stress. Many aspects of these processes remain incompletely understood due to a lack of a suitable animal model. Chronic alcohol over-consumption reduces hepatic alcohol dehydrogenase (ADH), the principal canonical metabolic pathway of ethanol oxidation. We therefore modeled this situation using hepatic ADH-deficient deer mice fed 3.5% ethanol daily for 3 months. Blood ethanol concentration was 180 mg% in ethanol fed mice, compared to < 1.0% in the controls. Acetaldehyde (oxidative metabolite of ethanol) was minimally, but significantly increased in ethanol-fed vs. pair-fed control mice. Total fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) were 47.6 μg/g in the lungs of ethanol-fed mice as compared to 1.5 μg/g in pair-fed controls. Histological and immunohistological evaluation showed perivascular and peribronchiolar lymphocytic infiltration, and significant oxidative injury, in the lungs of ethanol-fed mice compared to pair-fed controls. Several fold increases for cytochrome P450 2E1, caspase 8 and caspase 3 found in the lungs of ethanol-fed mice as compared to pair-fed controls suggest role of oxidative stress in ethanol-induced lung injury. ER stress and unfolded protein response signaling were also significantly increased in the lungs of ethanol-fed mice. Surprisingly, no significant activation of inositol-requiring enzyme-1α and spliced XBP1 was observed indicating a lack of activation of corrective mechanisms to reinstate ER homeostasis. The data suggest that oxidative stress and prolonged ER stress, coupled with formation and accumulation of cytotoxic FAEEs may contribute to the pathogenesis of alcoholic lung disease. - Highlights: • Chronic

  17. Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol dehydrogenases and the inhibition by ethanol and 4-methylpyrazole.

    PubMed

    Lee, Shou-Lun; Shih, Hsuan-Ting; Chi, Yu-Chou; Li, Yeung-Pin; Yin, Shih-Jiun

    2011-05-30

    Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1M sodium phosphate buffer, at pH 7.5 and 25°C, containing 0.5 mM NAD(+) and varied concentrations of substrate. K(M) values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12-57 mM, for methanol to be 2.0-3500 mM, for ethylene glycol to be 4.3-2600mM, and for isopropanol to be 0.73-3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for V(max) for the toxic alcohols were much less than that of the K(M) across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (K(is)) for the whole family being 0.062-960 μM and the intercept inhibition constants (K(ii)), 33-3000 μM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50mM are largely inhibited by 20 mM ethanol or by 50 μM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives. PMID:21167143

  18. Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations.

    PubMed

    Sillers, Ryan; Al-Hinai, Mohab Ali; Papoutsakis, Eleftherios T

    2009-01-01

    Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity. PMID:18726959

  19. Cloning and expression of a putative alcohol dehydrogenase gene of Entamoeba histolytica and its application to immunological examination.

    PubMed Central

    Kimura, A; Hara, Y; Kimoto, T; Okuno, Y; Minekawa, Y; Nakabayashi, T

    1996-01-01

    To clone and express the genes encoding major antigens of Entamoeba histolytica, we constructed a lambda gt11 cDNA library for E. histolytica HM1:IMSS and screened it with pooled sera from patients with amoebiasis. A 1,223-bp cDNA was cloned (clone 1223), and its nucleotide sequence was determined. The amino acid sequence predicted to be encoded by the open reading frame of clone 1223 consisted of 396 residues and showed 32.5 and 32.3% homology to the NADH-dependent butanol dehydrogenases I and II (bdhA and bdhB) of Clostridium acetobutylicum, respectively. In addition, 29 of the 34 consensus positions of bdhA and bdhB were also well conserved in clone 1223. The recombinant protein expressed from clone 1223 had an estimated molecular mass of 43.5 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenicity and specificity of the recombinant protein were evaluated by an enzyme-linked immunosorbent assay using sera obtained from two clinical groups of patients with amoebiasis and a group of healthy controls. The recombinant protein had potent and specific antigenicity. In all, 53 serum samples (88.3%) from 60 patients with amoebiasis were positive for immunoglobulin G antibody against the recombinant protein, with a mean optical density value of 0.42. In contrast, 53 of 54 healthy control serum samples were negative, with only 1 positive serum sample showing the lower optical density value. These results suggested that clone 1223 is promising in terms of providing a useful antigen for the accurate serodiagnosis of amoebiasis and that the gene encodes a putative alcohol dehydrogenase of E. histolytica. PMID:8705667

  20. Mutation of Tyr-218 to Phe in Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase: effects on bioelectronic interface performance.

    PubMed

    Hassler, Brian L; Dennis, Megan; Laivenieks, Maris; Zeikus, J Gregory; Worden, Robert M

    2007-10-01

    Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2 degrees ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, beta-nicotinamide adenine dinucleotide phosphate (NADP+), is more expensive and less stable than beta-nicotinamide adenine dinucleotide (NAD+). PCR-based, site-directed mutagenesis was performed on 2 degrees ADH in an attempt to adjust the cofactor specificity toward NAD+ by mutating Tyr218 to Phe (Y218F 2 degrees ADH). This mutation increased the Km(app) for NADP+ 200-fold while decreasing the Km(app) for NAD+ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD+, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2 degrees ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD+. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD+ was electrically recycled 6.7 x 10(4) times, suggesting that the Y218F 2 degrees ADH-modified bioelectronic interface is stable. PMID:18025592

  1. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    PubMed Central

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-01-01

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

  2. Simulated ischaemia-reperfusion conditions increase xanthine dehydrogenase and oxidase activities in rat brain slices.

    PubMed

    Battelli, M G; Buonamici, L; Virgili, M; Abbondanza, A; Contestabile, A

    1998-01-01

    Xanthine dehydrogenase and oxidase activities increased by 87% in rat brain slices after 30 min in vitro ischaemia. A further 41% increase was induced by 30 min simulated reperfusion of ischaemic slices. No conversion from the dehydrogenase to the oxidase activity was observed. The increment of enzyme activity was not due to neosynthesis of the enzyme, since it was not affected by the addition of cycloheximide during the ischaemic incubation. The increased oxygen-dependent form of the enzyme could aggravate the ischaemic brain injury by free radicals production, in particular after reperfusion. PMID:9460697

  3. The short-chain alcohol dehydrogenase ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde.

    PubMed

    González-Guzmán, Miguel; Apostolova, Nadezda; Bellés, José M; Barrero, José M; Piqueras, Pedro; Ponce, María R; Micol, José L; Serrano, Ramón; Rodríguez, Pedro L

    2002-08-01

    Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobreño (sañ). The sre and sañ mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1, sre1-2, sañ3-1, and sañ3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a K(m) value for xanthoxin of 19 micro M and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC-mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin-->abscisic aldehyde-->ABA transition as the last steps of the major ABA biosynthetic pathway. PMID:12172025

  4. Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

    PubMed Central

    Zheng, Tianyong; Olson, Daniel G.; Tian, Liang; Bomble, Yannick J.; Himmel, Michael E.; Lo, Jonathan; Hon, Shuen; Shaw, A. Joe; van Dijken, Johannes P.

    2015-01-01

    ABSTRACT Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticum produce ethanol with a yield of 90% of the theoretical maximum, engineered strains of C. thermocellum produce ethanol at lower yields (∼50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in their adhE genes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, the adhE genes from six strains of C. thermocellum and T. saccharolyticum were cloned and expressed in Escherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains of T. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain of C. thermocellum has acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced into C. thermocellum and T. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. IMPORTANCE This work describes the characterization of the AdhE enzyme from different strains of C. thermocellum and T. saccharolyticum. C. thermocellum and T. saccharolyticum are thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of

  5. Physiological Studies of Methane- and Methanol-Oxidizing Bacteria: Immunological Comparison of a Primary Alcohol Dehydrogenase from Methylococcus capsulatus and Pseudomonas sp. M27

    PubMed Central

    Patel, R. N.; Mandy, W. J.; Hoare, D. S.

    1973-01-01

    A primary alcohol dehydrogenase was purified from cell extracts of two apparently unrelated microorganisms, namely, Pseudomonas sp. M27 and Methylococcus capsulatus. Rabbit antiserum prepared against the purified enzyme from M. capsulatus revealed distinctive antigenic determinants by quantitative and gel precipitin reactions. Rabbit antiserum to M27 enzyme detected both distinctive and shared antigenic determinants. Certain methane- and methanol-oxidizing bacteria were grouped on the basis of serological cross-reacting enzyme specificities. Images PMID:4120569

  6. Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4

    SciTech Connect

    Short, Duncan M.; Lyon, Robert; Watson, David G.; Barski, Oleg A.; McGarvie, Gail; Ellis, Elizabeth M. . E-mail: Elizabeth.ellis@strath.ac.uk

    2006-01-15

    The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a V {sub max} of 2141 {+-} 500 nmol/min/mg and a K {sub m} of 11 {+-} 4 {mu}M. This enzyme was inhibited by pyrazole with a K {sub I} of 3.1 {+-} 0.57 {mu}M. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a V {sub max} of 115 nmol/min/mg and a K {sub m} of 15 {+-} 2 {mu}M and was not inhibited by pyrazole.

  7. A novel electrochemiluminescence ethanol biosensor based on tris(2,2'-bipyridine) ruthenium (II) and alcohol dehydrogenase immobilized in graphene/bovine serum albumin composite film.

    PubMed

    Gao, Wenhua; Chen, Yunsheng; Xi, Jing; Lin, Shaoyu; Chen, Yaowen; Lin, Yuejuan; Chen, Zhanguang

    2013-03-15

    We developed a novel electrochemiluminescence (ECL) ethanol biosensor based on Ru(bpy)(3)(2+) and alcohol dehydrogenase (ADH) immobilized by graphene/bovine serum albumin composite film. The graphene film was directly formed on a glassy carbon electrode surface via an in situ reduction of graphene oxide (GO) and Ru(bpy)(3)(2+) was immobilized during its formation. The graphene film acted as both a decorating agent for immobilization of Ru(bpy)(3)(2+) and a matrix to immobilize bovine serum albumin (BSA), meanwhile BSA not only acted as a reductant to reduce GO, but also provided a friendly environment for ADH immobilization. Furthermore, ADH was separated from Ru(bpy)(3)(2+) by the electron-conductive graphene/BSA composite film to retain its enzymatic activity. The experimental results indicated that the biosensor had excellent electrochemical activity, ECL response to ethanol and stability. Such a design of Ru(bpy)(3)(2+)-graphene/BSA film to modify electrode holds a great promise as a new biocompatible platform for the development of enzyme-based ECL biosensors. PMID:23122751

  8. SDHAF4 promotes mitochondrial succinate dehydrogenase activity and prevents neurodegeneration

    PubMed Central

    Van Vranken, Jonathan G.; Bricker, Daniel K.; Dephoure, Noah; Gygi, Steven P.; Cox, James E.; Thummel, Carl S.; Rutter, Jared

    2014-01-01

    SUMMARY Succinate dehydrogenase (SDH) occupies a central place in cellular energy production, linking the tricarboxylic cycle with the electron transport chain. As a result, a subset of cancers and neuromuscular disorders result from mutations affecting any of the four SDH structural subunits or either of two known SDH assembly factors. Herein we characterize a novel evolutionarily conserved SDH assembly factor designated Sdh8/SDHAF4, using yeast, Drosophila, and mammalian cells. Sdh8 interacts specifically with the catalytic Sdh1 subunit in the mitochondrial matrix, facilitating its association with Sdh2 and the subsequent assembly of the SDH holocomplex. These roles for Sdh8 are critical for preventing motility defects and neurodegeneration in Drosophila as well as the excess ROS generated by free Sdh1. These studies provide insights into the mechanisms by which SDH is assembled and raise the possibility that some forms of neuromuscular disease may be associated with mutations that affect this SDH assembly factor. PMID:24954416

  9. Estimates of Gene Flow in Drosophila Pseudoobscura Determined from Nucleotide Sequence Analysis of the Alcohol Dehydrogenase Region

    PubMed Central

    Schaeffer, S. W.; Miller, E. L.

    1992-01-01

    The genetic structure of Drosophila pseudoobscura populations was inferred from a nucleotide sequence analysis of a 3.4-kb segment of the alcohol dehydrogenase (Adh) region. A total of 99 isochromosomal strains collected from 13 populations in North and South America were used to determine if any population departed from a neutral model and to estimate levels of gene flow between populations. This study also included the nucleotide sequences from two sibling species, D. persimilis and D. miranda. We estimated the neutral mutation parameter, 4Nμ, in synonymous and noncoding sites for 17 subregions of Adh in each of nine populations with sample sizes greater than three. The nucleotide diversity data in the nine populations was tested for departures from an equilibrium neutral model with two statistical tests. The Tajima and the Hudson, Kreitman, Aguade tests showed that each population fails to reject a neutral model. Tests for genetic differentiation between populations fail to show any population substructure among the North American populations of D. pseudoobscura. The nucleotide diversity data is consistent with direct and indirect measures of gene flow that show extensive dispersal between populations of D. pseudoobscura. PMID:1427038

  10. Aldehyde Dehydrogenase 2 (ALDH2) Polymorphism and the Risk of Alcoholic Liver Cirrhosis among East Asians: A Meta-Analysis

    PubMed Central

    He, Lei; Luo, Hesheng

    2016-01-01

    Purpose The aldehyde dehydrogenase 2 (ALDH2) gene has been implicated in the development of alcoholic liver cirrhosis (ALC) in East Asians. However, the results are inconsistent. In this study, a meta-analysis was performed to assess the associations between the ALDH2 polymorphism and the risk of ALC. Materials and Methods Relevant studies were retrieved by searching PubMed, Web of Science, CNKI, Wanfang and Veipu databases up to January 10, 2015. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using either the fixed- or random effects model. Results A total of twelve case-control studies included 1003 cases and 2011 controls were included. Overall, the ALDH2 polymorphism was associated with a decreased risk of ALC (*1/*2 vs. *1/*1: OR=0.78, 95% CI: 0.61–0.99). However, in stratification analysis by country, we failed to detect any association among Chinese, Korean or Japanese populations. Conclusion The pooled evidence suggests that ALDH2 polymorphism may be an important protective factor for ALC in East Asians. PMID:27189280

  11. Slowed Diffusion and Excluded Volume Both Contribute to the Effects of Macromolecular Crowding on Alcohol Dehydrogenase Steady-State Kinetics.

    PubMed

    Schneider, Samuel H; Lockwood, Schuyler P; Hargreaves, Dominique I; Slade, David J; LoConte, Micaela A; Logan, Bridget E; McLaughlin, Erin E; Conroy, Michael J; Slade, Kristin M

    2015-09-29

    To understand the consequences of macromolecular crowding, studies have largely employed in vitro experiments with synthetic polymers assumed to be both pure and "inert". These polymers alter enzyme kinetics by excluding volume that would otherwise be available to the enzymes, substrates, and products. Presented here is evidence that other factors, in addition to excluded volume, must be considered in the interpretation of crowding studies with synthetic polymers. Dextran has a weaker effect on the Michaelis-Menten kinetic parameters of yeast alcohol dehydrogenase (YADH) than its small molecule counterpart, glucose. For glucose, the decreased Vmax values directly correlate with slower translational diffusion and the decreased Km values likely result from enhanced substrate binding due to YADH stabilization. Because dextran is unable to stabilize YADH to the same extent as glucose, this polymer's ability to decrease Km is potentially due to the nonideality of the solution, a crowding-induced conformational change, or both. Chronoamperometry reveals that glucose and dextran have surprisingly similar ferricyanide diffusion coefficients. Thus, the reduction in Vmax values for glucose is partially offset by an additional macromolecular crowding effect with dextran. Finally, this is the first report that supplier-dependent impurities in dextran affect the kinetic parameters of YADH. Taken together, our results reveal that caution should be used when interpreting results obtained with inert synthetic polymeric agents, as additional effects from the underlying monomer need to be considered. PMID:26333028

  12. The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa)

    PubMed Central

    Gómez-Manzo, Saúl; Escamilla, José E.; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M. H.; Sosa-Torres, Martha Elena

    2015-01-01

    Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2–C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde. PMID:25574602

  13. Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae

    SciTech Connect

    Conway, T. ); Ingram, L.O. )

    1989-07-01

    The gene that encodes 1,2-propanediol oxidoreductase (fucO) from Escherichia coli was sequenced. The reading frame specified a protein of 383 amino acids (including the N-terminal methionine), with an aggregate molecular weight of 40,642. The induction of fucO transcription, which occurred in the presence of fucose, was confirmed by Northern blot analysis. In E. coli, the primary fucO transcript was approximately 2.1 kilobases in length. The 5{prime} end of the transcript began more than 0.7 kilobase upstream of the fucO start codon within or beyond the fucA gene. Propanediol oxidoreductase exhibited 41.7% identity with the iron-containing alcohol dehydrogenase II from Zymomonas mobilis and 39.5% identity with ADH4 from Saccharomyces cerevisiae. These three proteins did not share homology with either short-chain or long-chain zinc-containing alcohol dehydrogenase enzymes. We propose that these three unusual alcohol dehydrogenases define a new family of enzymes.

  14. The oxidative fermentation of ethanol in Gluconacetobacter diazotrophicus is a two-step pathway catalyzed by a single enzyme: alcohol-aldehyde Dehydrogenase (ADHa).

    PubMed

    Gómez-Manzo, Saúl; Escamilla, José E; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M H; Sosa-Torres, Martha Elena

    2015-01-01

    Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde. PMID:25574602

  15. Relationship of lactate dehydrogenase activity to body measurements of Angus x Charolais cows and calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives were to examine 1) relationships between lactate dehydrogenase (LDH) activity and body measurements of grazing beef cows, and 2) the association between maternal LDH activity in late gestation and subsequent calf birth weight (BRW), hip height (HH) at weaning, and adjusted weaning weight ...

  16. Relationship of lactate dehydrogenase activity with body measeurements of Angus x Charolais cows and calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Angus x Charolais cows (n = 87) and their Angus-sired, spring-born calves (n = 86) were utilized to examine relationships between lactate dehydrogenase (LDH) activity and body measurements of beef cows; and the relationship between maternal LDH activity in late gestation and subsequent calf birth we...

  17. Active surfaces engineered by immobilizing protein-polymer nanoreactors for selectively detecting sugar alcohols.

    PubMed

    Zhang, Xiaoyan; Lomora, Mihai; Einfalt, Tomaz; Meier, Wolfgang; Klein, Noreen; Schneider, Dirk; Palivan, Cornelia G

    2016-05-01

    We introduce active surfaces generated by immobilizing protein-polymer nanoreactors on a solid support for sensitive sugar alcohols detection. First, such selective nanoreactors were engineered in solution by simultaneous encapsulation of specific enzymes in copolymer polymersomes, and insertion of membrane proteins for selective conduct of sugar alcohols. Despite the artificial surroundings, and the thickness of the copolymer membrane, functionality of reconstituted Escherichia coli glycerol facilitator (GlpF) was preserved, and allowed selective diffusion of sugar alcohols to the inner cavity of the polymersome, where encapsulated ribitol dehydrogenase (RDH) enzymes served as biosensing entities. Ribitol, selected as a model sugar alcohol, was detected quantitatively by the RDH-nanoreactors with GlpF-mediated permeability in a concentration range of 1.5-9 mM. To obtain "active surfaces" for detecting sugar alcohols, the nanoreactors optimized in solution were then immobilized on a solid support: aldehyde groups exposed at the compartment external surface reacted via an aldehyde-amino reaction with glass surfaces chemically modified with amino groups. The nanoreactors preserved their architecture and activity after immobilization on the glass surface, and represent active biosensing surfaces for selective detection of sugar alcohols, with high sensitivity. PMID:26950167

  18. The intrinsic topological information of the wild-type and of up-promoter mutations of the Saccharomyces cerevisiae alcohol dehydrogenase II regulatory region.

    PubMed

    Della Seta, F; Camilloni, G; Venditti, S; Di Mauro, E

    1988-11-01

    A 569-base pair fragment encompassing the upstream regulatory region, the RNA initiation sites, and the initial part of the coding region of the Saccharomyces cerevisiae alcohol dehydrogenase II gene has been analyzed for the presence of sites which undergo conformational modification under torsional stress. Fine mapping of P1 and S1 endonuclease-sensitive sites was obtained on single topoisomers produced by in vitro ligation. It was shown that the upstream activator sequence, the TATA sequence, a region directly upstream to the RNA initiation sites, and several positions in the first segment of the transcribed region change conformation as a function of the applied torsional stress in a precisely coordinate fashion. The superhelical density optima for this coordinate modifications have been determined. Analysis of the conformational changes of the promoter sequence in several naturally occurring (Young, E. T., Williamson, V. M., Taguchi, A., Smith, M., Sledziewski, L., Russel, D., Osterman, J., Denis, C., Cox, D., and Beier, D., (1982) in Genetic Engineering of Microorganisms for Chemicals (Hollander, A., De Moss, R. D., Kaplan, S., Konisky, J., Savage, D., and Wolle, R. S., eds) pp. 335-361, Plenum Publishing Corp., New York) up-promoter constitutive mutants was performed. This analysis has shown that the conformation of functionally relevant sites changes as a function of sequence mutations that have taken place elsewhere; this shows that the conformational behavior of the whole promoter region is linked and suggests transmission in cis of topological effects in RNA polymerase II promoters. PMID:3053683

  19. CINNAMYL ALCOHOL DEHYDROGENASE-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis.

    PubMed

    Sibout, Richard; Eudes, Aymerick; Mouille, Gregory; Pollet, Brigitte; Lapierre, Catherine; Jouanin, Lise; Séguin, Armand

    2005-07-01

    During lignin biosynthesis in angiosperms, coniferyl and sinapyl aldehydes are believed to be converted into their corresponding alcohols by cinnamyl alcohol dehydrogenase (CAD) and by sinapyl alcohol dehydrogenase (SAD), respectively. This work clearly shows that CAD-C and CAD-D act as the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis thaliana by supplying both coniferyl and sinapyl alcohols. An Arabidopsis CAD double mutant (cad-c cad-d) resulted in a phenotype with a limp floral stem at maturity as well as modifications in the pattern of lignin staining. Lignin content of the mutant stem was reduced by 40%, with a 94% reduction, relative to the wild type, in conventional beta-O-4-linked guaiacyl and syringyl units and incorportion of coniferyl and sinapyl aldehydes. Fourier transform infrared spectroscopy demonstrated that both xylem vessels and fibers were affected. GeneChip data and real-time PCR analysis revealed that transcription of CAD homologs and other genes mainly involved in cell wall integrity were also altered in the double mutant. In addition, molecular complementation of the double mutant by tissue-specific expression of CAD derived from various species suggests different abilities of these genes/proteins to produce syringyl-lignin moieties but does not indicate a requirement for any specific SAD gene. PMID:15937231

  20. A Nonsense Mutation in a Cinnamyl Alcohol Dehydrogenase Gene Is Responsible for the Sorghum brown midrib6 Phenotype1[W][OA

    PubMed Central

    Sattler, Scott E.; Saathoff, Aaron J.; Haas, Eric J.; Palmer, Nathan A.; Funnell-Harris, Deanna L.; Sarath, Gautam; Pedersen, Jeffrey F.

    2009-01-01

    brown midrib6 (bmr6) affects phenylpropanoid metabolism, resulting in reduced lignin concentrations and altered lignin composition in sorghum (Sorghum bicolor). Recently, bmr6 plants were shown to have limited cinnamyl alcohol dehydrogenase activity (CAD; EC 1.1.1.195), the enzyme that catalyzes the conversion of hydroxycinnamoyl aldehydes (monolignals) to monolignols. A candidate gene approach was taken to identify Bmr6. Two CAD genes (Sb02g024190 and Sb04g005950) were identified in the sorghum genome based on similarity to known CAD genes and through DNA sequencing a nonsense mutation was discovered in Sb04g005950 that results in a truncated protein lacking the NADPH-binding and C-terminal catalytic domains. Immunoblotting confirmed that the Bmr6 protein was absent in protein extracts from bmr6 plants. Phylogenetic analysis indicated that Bmr6 is a member of an evolutionarily conserved group of CAD proteins, which function in lignin biosynthesis. In addition, Bmr6 is distinct from the other CAD-like proteins in sorghum, including SbCAD4 (Sb02g024190). Although both Bmr6 and SbCAD4 are expressed in sorghum internodes, an examination of enzymatic activity of recombinant Bmr6 and SbCAD4 showed that Bmr6 had 1 to 2 orders of magnitude greater activity for monolignol substrates. Modeling of Bmr6 and SbCAD4 protein structures showed differences in the amino acid composition of the active site that could explain the difference in enzyme activity. These differences include His-57, which is unique to Bmr6 and other grass CADs. In summary, Bmr6 encodes the major CAD protein involved in lignin synthesis in sorghum, and the bmr6 mutant is a null allele. PMID:19363091

  1. Picosecond-resolved fluorescence studies of substrate and cofactor-binding domain mutants in a thermophilic alcohol dehydrogenase uncover an extended network of communication.

    PubMed

    Meadows, Corey W; Tsang, Jonathan E; Klinman, Judith P

    2014-10-22

    Time-resolved fluorescence dynamics are investigated in two mutants of a thermophilic alcohol dehydrogenase (ht-ADH): Y25A (at the dimer interface) and V260A (at the cofactor-binding domain). These residues, ca. 32 Å apart, are shown to exhibit opposing low-temperature effects on the hydride tunneling step. Using single-tryptophan constructs at the active site (Trp87) and a remote, surface-exposed site (Trp167), time-dependent Stokes shifts and collisional quenching data allow an analysis of intra-protein dynamical communication. A double mutant, Y25A:V260A, was also inserted into each single-Trp construct and analyzed accordingly. None of the mutations affect fluorescence lifetimes, Stokes shift relaxation rates, and quenching data for the surface-exposed Trp167 to an appreciable extent. By contrast, fluorescent probes of the active-site tryptophan 87 reveal distinctive forms of dynamical communication. Stokes shifts show that the distal Y25A increases active-site flexibility, V260A introduces a temperature-dependent equilibration process not previously reported by such measurements, and the double mutant (Y25A:V260A) eliminates the temperature-dependent transition sensed by the active-site tryptophan in the presence of V260A. Collisional quenching data at Trp87 further show a structural change in the active-site environment/solvation for V260A. In the aggregate, the temperature dependencies of the fluorescence data are distinct from the breaks in behavior previously reported for catalysis and hydrogen/deuterium exchange, attributed to time scales for the interconversion of protein conformational substates that are slower and more global than the local motions monitored within. An extended network of dynamical communication between the protein dimer surface and substrate- and cofactor-binding domains emerges from the flourescent data. PMID:25314615

  2. Re-design of Saccharomyces cerevisiae flavocytochrome b2: introduction of L-mandelate dehydrogenase activity.

    PubMed

    Sinclair, R; Reid, G A; Chapman, S K

    1998-07-01

    Flavocytochrome b2 from Saccharomyces cerevisiae is an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate. The double mutation of Ala-198-->Gly and Leu-230-->Ala results in an enzyme with a kcat value (25 degrees C) with L-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with L-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of L-mandelate at the active site of the enzyme. To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme. In addition, the double mutation of Ala-198-->Gly and Leu-230-->Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with L-mandelate than with l-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase. PMID:9639570

  3. Dehydrin, alcohol dehydrogenase, and central metabolite levels are associated with cold tolerance in diploid strawberry (Fragaria spp.).

    PubMed

    Davik, Jahn; Koehler, Gage; From, Britta; Torp, Torfinn; Rohloff, Jens; Eidem, Petter; Wilson, Robert C; Sønsteby, Anita; Randall, Stephen K; Alsheikh, Muath

    2013-01-01

    The use of artificial freezing tests, identification of biomarkers linked to or directly involved in the low-temperature tolerance processes, could prove useful in applied strawberry breeding. This study was conducted to identify genotypes of diploid strawberry that differ in their tolerance to low-temperature stress and to investigate whether a set of candidate proteins and metabolites correlate with the level of tolerance. 17 Fragaria vesca, 2 F. nilgerrensis, 2 F. nubicola, and 1 F. pentaphylla genotypes were evaluated for low-temperature tolerance. Estimates of temperatures where 50 % of the plants survived (LT₅₀) ranged from -4.7 to -12.0 °C between the genotypes. Among the F. vesca genotypes, the LT₅₀ varied from -7.7 °C to -12.0 °C. Among the most tolerant were three F. vesca ssp. bracteata genotypes (FDP821, NCGR424, and NCGR502), while a F. vesca ssp. californica genotype (FDP817) was the least tolerant (LT₅₀) -7.7 °C). Alcohol dehydrogenase (ADH), total dehydrin expression, and content of central metabolism constituents were assayed in select plants acclimated at 2 °C. The LT₅₀ estimates and the expression of ADH and total dehydrins were highly correlated (r(adh) = -0.87, r (dehyd) = -0.82). Compounds related to the citric acid cycle were quantified in the leaves during acclimation. While several sugars and acids were significantly correlated to the LT₅₀ estimates early in the acclimation period, only galactinol proved to be a good LT₅₀ predictor after 28 days of acclimation (r(galact) = 0.79). It is concluded that ADH, dehydrins, and galactinol show great potential to serve as biomarkers for cold tolerance in diploid strawberry. PMID:23014928

  4. Dissociation and rate constants of some human liver alcohol dehydrogenase isoenzymes.

    PubMed

    Pietruszko, R; de Zalenski, C; Theorell, H

    1976-01-01

    ADH from human liver forms binary complexes with NADH, associated with a blue shift of the peak of the fluorescence emission of NADH. The wavelength shift is the same for all isoenzymes but the accompanying intensification of the fluorescence is different. The fluorescence is further increased by the formation of the very tight ternary enzyme-NADH-isobutyramide complexes. These properties are similar to those for the horse liver ADH, as well as the molecular weight of E=40 000 per active site of the dimer molecule (EE). "Stopped-flow" determined velocity constants (ER in equilibrium E+R) were found to be in good agreement with ethanol activity constants previously determined by activity measurement, confirming the validity of the ordered ternary complex mechanism also for the human ADH. No single isoenzyme activity as high as that reported by Mourad and Woronick or Drum has been found. PMID:184631

  5. An enantioselective NADP(+)-dependent alcohol dehydrogenase responsible for cooxidative production of (3S)-5-hydroxy-3-methyl-pentanoic acid.

    PubMed

    Takeda, Minoru; Matsumura, Aline Tiemi; Kurosaki, Kaishi; Chhetri, Rajan Thapa; Motomatsu, Shigekazu; Suzuki, Ichiro; Sahabi, Danladi Mahuta

    2016-06-01

    A soil bacterium, Mycobacterium sp. B-009, is able to grow on racemic 1,2-propanediol (PD). The strain was revealed to oxidize 3-methyl-1,5-pentanediol (MPD) to 5-hydroxy-3-methyl-pentanoic acid (HMPA) during growth on PD. MPD was converted into an almost equimolar amount of the S-form of HMPA (S-HMPA) at 72%ee, suggesting the presence of an enantioselective MPD dehydrogenase (MPD-DH). As expected, an NADP(+)-dependent alcohol dehydrogenase, which catalyzes the initial step of MPD oxidation, was detected and purified from the cell-free extract. This enzyme was suggested to be a homodimeric medium-chain alcohol dehydrogenase/reductase (MDR). The catalytic and kinetic parameters indicated that MPD is the most suitable substrate for the enzyme. The enzyme was encoded by a 1047-bp gene (mpd1) and several mycobacterial strains were found to have putative MDR genes similar to mpd1. In a phylogenetic tree, MPD-DH formed an independent clade together with the putative MDR of Mycobacterium neoaurum, which produces opportunistic infections. PMID:26923741

  6. Activation and injury of Clostridium perfringens spores by alcohols.

    PubMed Central

    Craven, S E; Blankenship, L C

    1985-01-01

    The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation. PMID:2864897

  7. Picosecond-resolved fluorescent probes at functionally distinct tryptophans within a thermophilic alcohol dehydrogenase: relationship of temperature-dependent changes in fluorescence to catalysis.

    PubMed

    Meadows, Corey W; Ou, Ryan; Klinman, Judith P

    2014-06-12

    Two single-tryptophan variants were generated in a thermophilic alcohol dehydrogenase with the goal of correlating temperature-dependent changes in local fluorescence with the previously demonstrated catalytic break at ca. 30 °C (Kohen et al., Nature 1999, 399, 496). One tryptophan variant, W87in, resides at the active site within van der Waals contact of bound alcohol substrate; the other variant, W167in, is a remote-site surface reporter located >25 Å from the active site. Picosecond-resolved fluorescence measurements were used to analyze fluorescence lifetimes, time-dependent Stokes shifts, and the extent of collisional quenching at Trp87 and Trp167 as a function of temperature. A subnanosecond fluorescence decay rate constant has been detected for W87in that is ascribed to the proximity of the active site Zn(2+) and shows a break in behavior at 30 °C. For the remainder of the reported lifetime measurements, there is no detectable break between 10 and 50 °C, in contrast with previously reported hydrogen/deuterium exchange experiments that revealed a temperature-dependent break analogous to catalysis (Liang et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556). We conclude that the motions that lead to the rigidification of ht-ADH below 30 °C are likely to be dominated by global processes slower than the picosecond to nanosecond motions measured herein. In the case of collisional quenching of fluorescence by acrylamide, W87in and W167in behave in a similar manner that resembles free tryptophan in water. Stokes shift measurements, by contrast, show distinctive behaviors in which the active-site tryptophan relaxation is highly temperature-dependent, whereas the solvent-exposed tryptophan's dynamics are temperature-independent. These data are concluded to reflect a significantly constrained environment surrounding the active site Trp87 that both increases the magnitude of the Stokes shift and its temperature-dependence. The results are discussed in the context

  8. Picosecond-Resolved Fluorescent Probes at Functionally Distinct Tryptophans within a Thermophilic Alcohol Dehydrogenase: Relationship of Temperature-Dependent Changes in Fluorescence to Catalysis

    PubMed Central

    2015-01-01

    Two single-tryptophan variants were generated in a thermophilic alcohol dehydrogenase with the goal of correlating temperature-dependent changes in local fluorescence with the previously demonstrated catalytic break at ca. 30 °C (Kohen et al., Nature1999, 399, 496). One tryptophan variant, W87in, resides at the active site within van der Waals contact of bound alcohol substrate; the other variant, W167in, is a remote-site surface reporter located >25 Å from the active site. Picosecond-resolved fluorescence measurements were used to analyze fluorescence lifetimes, time-dependent Stokes shifts, and the extent of collisional quenching at Trp87 and Trp167 as a function of temperature. A subnanosecond fluorescence decay rate constant has been detected for W87in that is ascribed to the proximity of the active site Zn2+ and shows a break in behavior at 30 °C. For the remainder of the reported lifetime measurements, there is no detectable break between 10 and 50 °C, in contrast with previously reported hydrogen/deuterium exchange experiments that revealed a temperature-dependent break analogous to catalysis (Liang et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556). We conclude that the motions that lead to the rigidification of ht-ADH below 30 °C are likely to be dominated by global processes slower than the picosecond to nanosecond motions measured herein. In the case of collisional quenching of fluorescence by acrylamide, W87in and W167in behave in a similar manner that resembles free tryptophan in water. Stokes shift measurements, by contrast, show distinctive behaviors in which the active-site tryptophan relaxation is highly temperature-dependent, whereas the solvent-exposed tryptophan’s dynamics are temperature-independent. These data are concluded to reflect a significantly constrained environment surrounding the active site Trp87 that both increases the magnitude of the Stokes shift and its temperature-dependence. The results are discussed in the context

  9. The hormonal regulation of hepatic microsomal 11beta-hydroxysteroid dehydrogenase activity in the rat.

    PubMed

    Lax, E R; Ghraf, R; Schriefers, H

    1978-10-01

    Hepatic microsomal 11beta-hydroxysteroid dehydrogenase activity is higher in male than in female rat liver. Gonadectomy on day 25 of life only affects the activity in the adult male animal, causing a decrease towards the normal female level. Administration of testosterone to gonadectomized rats of either sex causes the induction of typical male activity levels. On the basis of these experiments, this enzyme activity may be classified as an drogen-dependent. However, 11beta-hydroxysteroid dehydrogenase differs from other known androgen-dependent activities in that administration of oestradiol to gonadectomized animals of either sex causes a further significant repression of the activity to levels close to the limits of detection. Hypophysectomy on day 50 of life does not affect the activity in 75 day-old male rats, but causes the appearance of typically male activity levels in females. These results indicate that the hypophysis exerts a repressive influence on hepatic 11beta-hydroxysteroid dehydrogenase in female rats. The facts that this activity is not influenced by androgen or oestrogen administration once the pituitary has been removed demonstrates the obligatory role of the hypophysis for sex hormone action. PMID:696183

  10. Separation of dehydrogenases on polyaminomethylstyrene.

    PubMed

    Schöpp, W; Meinert, S; Thyfronitou, J; Aurich, H

    1975-01-29

    The binding of dehydrogenases, especially alcohol dehydrogenase, and other proteins to several ion exchangers and hydrophobic polymers was investigated. Quantitative parameters for the stability of the polymer-protein complexes (obtained form double reciprocal plots) indicate a high but different affinity of many proteins for polyaminomethylstyrene. The chromatography of a mixture of five dehydrogenases and human serum albumin on polyaminomethylstyrene is described. PMID:237012

  11. Crystal structure of Pseudomonas fluorescens mannitol 2-dehydrogenase: evidence for a very divergent long-chain dehydrogenase family.

    PubMed

    Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

    2003-02-01

    Mannitol 2-dehydrogenase from Pseudomonas fluorescens (pfMDH) is a secondary alcohol dehydrogenase that catalyzes the reversible NAD(P)-dependent oxidation of D-mannitol to D-fructose, D-arabinitol to D-xylulose, and D-sorbitol to L-sorbose. It is a member of the mostly prokaryotic family of long-chain mannitol dehydrogenases that so far includes 66 members. Unlike other alcohol and polyol dehydrogenases that utilize metal cofactors or a conserved active-site tyrosine for catalysis, an invariant lysine is the general base. The crystal structure of pfMDH in a binary complex with NAD(H) and a ternary complex with NAD(H) and D-mannitol have been determined to 1.7 and 1.8 A resolution respectively. Comparison of secondary structure assignment to sequence alignments suggest the shortest members of this family, mannitol-1-phosphate 5-dehydrogenases, retain core elements but lack secondary structural components found on the surface of pfMDH. The elements predicted to be absent are distributed throughout the primary sequence, implying that a simple truncation or fusion did not occur. The closest structural neighbors are 6-phosphogluconate dehydrogenase, UDP-glucose dehydrogenase, N-(1-D-carboxyethyl)-L-norvaline dehydrogenase, and glycerol-3-phosphate dehydrogenase. Although sequence identity is only a barely recognizable 7-10%, conservation of secondary structural elements as well as homologous residues that are contributed to the active site indicates they may be related by divergent evolution. PMID:12604241

  12. Inhibition of alcohol dehydrogenase after 2-propanol exposure in different geographic races of Drosophila mojavensis: lack of evidence for selection at the Adh-2 locus.

    PubMed

    Pfeiler, Edward; Reed, Laura K; Markow, Therese A

    2005-03-15

    High frequencies of the fast allele of alcohol dehydrogenase-2 (Adh-2F) are found in populations of Drosophila mojavensis that inhabit the Baja California peninsula (race BII) whereas the slow allele (Adh-2S) predominates at most other localities within the species' geographic range. Race BII flies utilize necrotic tissue of pitaya agria cactus (Stenocereus gummosus) which contains high levels of 2-propanol, whereas flies from most other localities utilize different cactus hosts in which 2-propanol levels are low. To test if 2-propanol acts as a selective force on Adh-2 genotype, or whether some other yet undetermined genetic factor is responsible, mature males of D. mojavensis lines derived from the Grand Canyon (race A) and Santa Catalina Island (race C), each with individuals homozygous for Adh-2F and Adh-2S, were exposed to 2-propanol for 24 h and ADH-2 specific activity was then determined on each genotype. Flies from five other localities homozygous for either the fast or slow allele also were examined. Results for all reported races of D. mojavensis were obtained. 2-propanol exposure inhibited ADH-2 specific activity in both genotypes from all localities, but inhibition was significantly less in two populations of race BII flies homozygous for Adh-2F. When F/F and S/S genotypes in flies from the same locality were compared, both genotypes showed high 2-propanol inhibition that was not statistically different, indicating that the F/F genotype alone does not provide a benefit against the inhibitory effects of 2-propanol. ADH-1 activity in female ovaries was inhibited less by 2-propanol than ADH-2. These results do not support the hypothesis that 2-propanol acts as a selective factor favoring the Adh-2F allele. PMID:15726639

  13. STRUCTURE TOXICITY IN RELATIONSHIPS FOR A,B-UNSATURATED ALCOHOLS IN FISH

    EPA Science Inventory

    Previous toxicity testing with fathead minnows (Pimephales promelas) indicated that some unsaturated acetylenic and allylic alcohols can be metabolically activated, via alcohol dehydrogenase, to highly toxic a,B-unsaturated aldehydes and ketones or allene derivatives. lthough sev...

  14. Reexamination of alcohol dehydrogenase structural mutants in Drosophila using protein blotting

    SciTech Connect

    Hollocher, H.; Place, A.R.

    1987-06-01

    Using protein blotting and an immuno-overlay procedure, the authors have reexamined the cross-reacting material produced by ADH null-activity mutants generated with ethyl methanesulfonate (EMS). Of the 13 mutants, 11 have an immunodetectable polypeptide of wild-type size. The native and urea denatured isoelectric points (pI) establish that 7 of 13 of the mutations have no effect on protein charge. The electrophoretic mobilities of each variant on increasing percent acrylamide gels (Ferguson analysis), reveal that 9 of the 11 immunodetectable mutations have retained the ability form dimers under native conditions. None of the inactive mutant proteins has the ability to form the adduct-bound isozyme. The authors have found no correlation between protein pI and i vivo stability. The observed frequencies of specific charge class alterations do not dispute the propensity of G:A transitions previously found for EMS mutagenesis.

  15. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes. PMID:26990764

  16. Impact of chronic low to moderate alcohol consumption on blood lipid and heart energy profile in acetaldehyde dehydrogenase 2-deficient mice

    PubMed Central

    Fan, Fan; Cao, Quan; Wang, Cong; Ma, Xin; Shen, Cheng; Liu, Xiang-wei; Bu, Li-ping; Zou, Yun-zeng; Hu, Kai; Sun, Ai-jun; Ge, Jun-bo

    2014-01-01

    Aim: To investigate the roles of acetaldehyde dehydrogenase 2 (ALDH2), the key enzyme of ethanol metabolism, in chronic low to moderate alcohol consumption-induced heart protective effects in mice. Methods: Twenty-one male wild-type (WT) or ALDH2-knockout (KO) mice were used in this study. In each genotype, 14 animals received alcohol (2.5%, 5% and 10% in week 1–3, respectively, and 18% in week 4–7), and 7 received water for 7 weeks. After the treatments, survival rate and general characteristics of the animals were evaluated. Serum ethanol and acetaldehyde levels and blood lipids were measured. Metabolomics was used to characterize the heart and serum metabolism profiles. Results: Chronic alcohol intake decreased the survival rate of KO mice by 50%, and significantly decreased their body weight, but did not affect those of WT mice. Chronic alcohol intake significantly increased the serum ethanol levels in both WT and KO mice, but KO mice had significantly higher serum acetaldehyde levels than WT mice. Chronic alcohol intake significantly increased the serum HDL cholesterol levels in WT mice, and did not change the serum HDL cholesterol levels in KO mice. After chronic alcohol intake, WT and KO mice showed differential heart and serum metabolism profiles, including the 3 main energy substrate types (lipids, glucose and amino acids) and three carboxylic acid cycles. Conclusion: Low to moderate alcohol consumption increases HDL cholesterol levels and improves heart energy metabolism profile in WT mice but not in ALDH2-KO mice. Thus, preserved ALDH2 function is essential for the protective effect of low to moderate alcohol on the cardiovascular system. PMID:24998256

  17. Activation of hepatic branched-chain 2-oxoacid dehydrogenase by rat liver cytosolic supernatant.

    PubMed

    Hauschildt, S

    1986-10-29

    Hepatic branched-chain 2-oxoacid dehydrogenase is inactivated by nutritional alterations. Reactivation occurs during preincubation of intact mitochondria in the presence of rat liver cytosolic supernatant. Cytosolic supernatant contains two factors capable of reactivating the enzyme. On gel-filtration (Sephadex G-100), one factor (AF1) elutes in the molecular range of 35,000-40,000 and the other factor (AF2) elutes slightly later than inorganic phosphate. AF2 is stable against heat denaturation and treatment with proteinases. It is destroyed by alkaline phosphatase and in the presence of Ap5A, atractyloside, CaCl2 and NaF its stimulatory effect on branched-chain 2-oxoacid dehydrogenase activity is abolished. Inhibition of activation by NaF suggests that a phosphatase might be involved in the activation process. PMID:3768411

  18. DFT study of the active site of the XoxF-type natural, cerium-dependent methanol dehydrogenase enzyme.

    PubMed

    Bogart, Justin A; Lewis, Andrew J; Schelter, Eric J

    2015-01-19

    Rare-earth metal cations have recently been demonstrated to be essential co-factors for the growth of the methanotrophic bacterium Methylacidiphilum fumariolicum SolV. A crystal structure of the rare-earth-dependent methanol dehydrogenase (MDH) includes a cerium cation in the active site. Herein, the Ce-MDH active site has been analyzed through DFT calculations. The results show the stability of the Ce(III)-pyrroloquinoline quinone (PQQ) semiquinone configuration. Calculations on the active oxidized form of this complex indicate a 0.81 eV stabilization of the PQQ(0) LUMO at cerium versus calcium, supporting the observation that the cerium cation in the active site confers a competitive advantage to Methylacidiphilum fumariolicum SolV. Using reported aqueous electrochemical data, a semi-empirical correlation was established based on cerium(IV/III) redox potentials. The correlation allowed estimation of the cerium oxidation potential of +1.35 V versus saturated calomel electrode (SCE) in the active site. The results are expected to guide the design of functional model complexes and alcohol-oxidation catalysts based on lanthanide complexes of biologically relevant quinones. PMID:25421364

  19. In vitro characterization of an enzymatic redox cascade composed of an alcohol dehydrogenase, an enoate reductases and a Baeyer–Villiger monooxygenase

    PubMed Central

    Oberleitner, Nikolin; Peters, Christin; Rudroff, Florian; Bornscheuer, Uwe T.; Mihovilovic, Marko D.

    2014-01-01

    An artificial enzyme cascade composed of an alcohol dehydrogenase, an enoate reductase and a Baeyer–Villiger monooxygenase was investigated in vitro to gain deeper mechanistic insights and understand the assets and drawbacks of this multi-step biocatalysis. Several substrates composed of different structural motifs were examined and provided access to functionalized chiral compounds in high yields (up to >99%) and optical purities (up to >99%). Hence, the applicability of the presented enzymatic cascade was exploited for the synthesis of biorenewable polyesters. PMID:24746588

  20. Species-specific differences in tissue-specific expression of alcohol dehydrogenase are under the control of complex cis-acting loci: Evidence from Drosophila hybrids

    SciTech Connect

    Ranganayakulu, G.; Reddy, A.R. ); Kirkpatrick, R.B.; Martin, P.F. )

    1991-12-01

    Differences in the expression of alcohol dehydrogenase in the hindgut and testis of adult Drosophila virilis, D. texana, D. novamexicana and D. borealis flies were observed. These heritable differences do not arise due to chromosomal rearrangements, since the polytene chromosome banding patterns did not reveal any such gross chromosomal rearrangements near the Adh locus in any of the tested species. Analysis of the interspecific hybrids revealed that these differences are controlled by complex cis-acting genetic loci. Further, the cis-acting locus controlling the expression of ADH in testis was found to be separable by crossing-over.

  1. Multichannel Simultaneous Determination of Activities of Lactate Dehydrogenase

    SciTech Connect

    Ma, L.

    2000-09-12

    It is very important to find the best conditions for some enzymes to do the best catalysis in current pharmaceutical industries. Based on the results above, we could say that this set-up could be widely used in finding the optimal condition for best enzyme activity of a certain enzyme. Instead of looking for the best condition for enzyme activity by doing many similar reactions repeatedly, we can complete this assignment with just one run if we could apply enough conditions.

  2. The dihydrolipoamide dehydrogenase of Aeromonas caviae ST exhibits NADH-dependent tellurite reductase activity.

    PubMed

    Castro, Miguel E; Molina, Roberto; Díaz, Waldo; Pichuantes, Sergio E; Vásquez, Claudio C

    2008-10-10

    Potassium tellurite (K(2)TeO(3)) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of TeO32- in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 (aceE), E2 (aceF), and E3 (lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms. PMID:18675788

  3. Alcohol Usage and Abrupt Cessation Modulate Diurnal Activity

    PubMed Central

    Norrell, Stacy; Reyes-Vasquez, Cruz; Burau, Keith; Dafny, Nachum

    2010-01-01

    Alcohol has many effects throughout the body. The effect on circadian rhythms and the correlation of these effects to withdrawal effects of alcohol present interesting findings. By measuring 3 planes of activity of female Sprague-Dawley rats during alcohol usage and continuing study through the first two days following withdrawal of alcohol allow for the observation of a drastic modulation of the circadian pattern of activity. PMID:20615456

  4. Succinate dehydrogenase activity and soma size of motoneurons innervating different portions of the rat tibialis anterior

    NASA Technical Reports Server (NTRS)

    Ishihara, A.; Roy, R. R.; Edgerton, V. R.

    1995-01-01

    The spatial distribution, soma size and oxidative enzyme activity of gamma and alpha motoneurons innervating muscle fibres in the deep (away from the surface of the muscle) and superficial (close to the surface of the muscle) portions of the tibialis anterior in normal rats were determined. The deep portion had a higher percentage of high oxidative fibres than the superficial portion of the muscle. Motoneurons were labelled by retrograde neuronal transport of fluorescent tracers: Fast Blue and Nuclear Yellow were injected into the deep portion and Nuclear Yellow into the superficial portion of the muscle. Therefore, motoneurons innervating the deep portion were identified by both a blue fluorescent cytoplasm and a golden-yellow fluorescent nucleus, while motoneurons innervating the superficial portion were identified by only a golden-yellow fluorescent nucleus. After staining for succinate dehydrogenase activity on the same section used for the identification of the motoneurons, soma size and succinate dehydrogenase activity of the motoneurons were measured. The gamma and alpha motoneurons innervating both the deep and superficial portions were located primarily at L4 and were intermingled within the same region of the dorsolateral portion of the ventral horn in the spinal cord. Mean soma size was similar for either gamma or alpha motoneurons in the two portions of the muscle. The alpha motoneurons innervating the superficial portion had a lower mean succinate dehydrogenase activity than those innervating the deep portion of the muscle. An inverse relationship between soma size and succinate dehydrogenase activity of alpha, but not gamma, motoneurons innervating both the deep and superficial portions was observed. Based on three-dimensional reconstructions within the spinal cord, there were no apparent differences in the spatial distribution of the motoneurons, either gamma or alpha, associated with the deep and superficial compartments of the muscle. The data

  5. Catalyst Activity Comparison of Alcohols over Zeolites

    SciTech Connect

    Ramasamy, Karthikeyan K.; Wang, Yong

    2013-01-01

    Alcohol transformation to transportation fuel range hydrocarbon on HZSM-5 (SiO2 / Al2O3 = 30) catalyst was studied at 360oC and 300psig. Product distributions and catalyst life were compared using methanol, ethanol, 1-propanol or 1-butanol as a feed. The catalyst life for 1-propanol and 1-butanol was more than double compared to that for methanol and ethanol. For all the alcohols studied, the product distributions (classified to paraffin, olefin, napthene, aromatic and naphthalene compounds) varied with time on stream (TOS). At 24 hours TOS, liquid product from 1-propanol and 1-butanol transformation primarily contains higher olefin compounds. The alcohol transformation process to higher hydrocarbon involves a complex set of reaction pathways such as dehydration, oligomerization, dehydrocyclization, and hydrogenation. Compared to ethylene generated from methanol and ethanol, oligomerization of propylene and butylene has a lower activation energy and can readily take place on weaker acidic sites. On the other hand, dehydrocyclization of propylene and butylene to form the cyclic compounds requires the sits with stronger acid strength. Combination of the above mentioned reasons are the primary reasons for olefin rich product generated in the later stage of the time on stream and for the extended catalyst life time for 1 propanol and 1 butanol compared to methanol and ethanol conversion over HZSM-5.

  6. Structural of the class II enzyme of human liver alcohol dehydrogenase: combined cDNA and protein sequence determination of the. pi. subunit

    SciTech Connect

    Hoeoeg, J.O.; von Bahr-Lindstroem, H.; Heden, L.O.; Holmquist, B.; Larsson, K.; Hempel, J.; Vallee, B.L.; Joernvall, H.

    1987-04-07

    The class II enzyme of human liver alcohol dehydrogenase was isolated, carboxymethylated, and cleaved with CNBr and proteolytic enzymes. Sequence analysis of peptides established structures corresponding to the ..pi.. subunit. Two segments from the C-terminal region unique to ..pi.. were selected for synthesis of oligodeoxyribonucleotide probes to screen a human liver cDNA library constructed in plasmid pT4. Sequence analysis of two identical hybridization-positive clones with cDNA inserts of about 2000 nucleotides gave the entire coding region of the ..pi.. subunit, a 61-nucleotide 5' noncoding region and a 741-nucleotide 3' noncoding region containing four possible polyadenylation sites. Translation of the coding region yields a 391-residue polypeptide, which in all regions except the C-terminal segment corresponds to the protein structure as determined directly by peptide analysis. With the class I numbering system, the exception concerns a residue exchange at position 368, the actual C-terminus which is Phe-374 by peptide data but a 12 residue extension by cDNA data, and possibly two further residue exchanges at positions 303 and 312. The size difference might indicate the existence of posttranslational modifications of the mature protein or, in combination with the residue exchanges, the existence of polymorphism at the locus for class II subunits. The ..pi.. subunit analyzed directly results in a 379-residue polypeptide and is the only class II size thus far known to occur in the mature protein. Comparison of the ..pi.. structure with those of the class I subunits (..cap alpha.., ..beta.., and ..gamma..) reveals a homology with extensive differences. Large variations in segments affecting relationships at the active site and the area of subunit interactions account for the significant alterations of enzymatic specificities and other properties that differentiate class II from class I enzymes.

  7. Rice alcohol dehydrogenase 1 promotes survival and has a major impact on carbohydrate metabolism in the embryo and endosperm when seeds are germinated in partially oxygenated water

    PubMed Central

    Takahashi, Hirokazu; Greenway, Hank; Matsumura, Hideo; Tsutsumi, Nobuhiro; Nakazono, Mikio

    2014-01-01

    Background and Aims Rice (Oryza sativa) has the rare ability to germinate and elongate a coleoptile under oxygen-deficient conditions, which include both hypoxia and anoxia. It has previously been shown that ALCOHOL DEHYDROGENASE 1 (ADH1) is required for cell division and cell elongation in the coleoptile of submerged rice seedlings by means of studies using a rice ADH1-deficient mutant, reduced adh activity (rad). The aim of this study was to understand how low ADH1 in rice affects carbohydrate metabolism in the embryo and endosperm, and lactate and alanine synthesis in the embryo during germination and subsequent coleoptile growth in submerged seedlings. Methods Wild-type and rad mutant rice seeds were germinated and grown under complete submergence. At 1, 3, 5 and 7 d after imbibition, the embryo and endosperm were separated and several of their metabolites were measured and compared. Key results In the rad embryo, the rate of ethanol fermentation was halved, while lactate and alanine concentrations were 2·4- and 5·7- fold higher in the mutant than in the wild type. Glucose and fructose concentrations in the embryos increased with time in the wild type, but not in the rad mutant. The rad mutant endosperm had lower amounts of the α-amylases RAMY1A and RAMY3D, resulting in less starch degradation and lower glucose concentrations. Conclusions These results suggest that ADH1 is essential for sugar metabolism via glycolysis to ethanol fermentation in both the embryo and endosperm. In the endosperm, energy is presumably needed for synthesis of the amylases and for sucrose synthesis in the endosperm, as well as for sugar transport to the embryo. PMID:24431339

  8. A functionally critical single nucleotide polymorphism in the gene encoding the membrane-bound alcohol dehydrogenase found in ethanol oxidation-deficient Gluconobacter thailandicus.

    PubMed

    Charoenyingcharoen, Piyanat; Matsutani, Minenosuke; Yakushi, Toshiharu; Theeragool, Gunjana; Yukphan, Pattaraporn; Matsushita, Kazunobu

    2015-08-10

    The Gluconobacter thailandicus strains NBRC3254, NBRC3255, NBRC3256, NBRC3257, and NBRC3258 are naturally deficient in the ethanol-oxidizing respiratory chain because they do not produce the cytochrome subunit of the membrane-bound alcohol dehydrogenase (ADH). Draft genomes of G. thailandicus strains NBRC3255 and NBRC3257 indicated that the adhB gene encoding the cytochrome subunit contains four base differences when compared to a closely related gene in the public database One of the nucleotide differences results in an Opal codon at the -19th tryptophan (Trp) in the signal sequence for translocation to the periplasmic space (here, the position of +1st residue is assigned to the N-terminal amino acid residue after signal peptide cleavage), while the other differences result in one missense and two silent amino acid alterations. All five of the G. thailandicus strains were shown to have the Trp(-19)Opal alteration. Ethanol oxidation and ADH activities in NBRC3255 were restored by transformation with a derivative of the endogenous adhB gene, of which the -19th Opal codon was altered to encode Trp. These results indicate that this sequence is a functionally critical single nucleotide polymorphism in the cytochrome subunit. Comparative genomic analyses between the draft genomes of NBRC3255 and NBRC3257 revealed that although the two genomes are closely related, they both have a significant number of unique open reading frames. We suggest that the closely related NBRC3255 and NBRC3257 diverged from a common ancestor having the mutation in the adhB gene, whereas no additional functionally critical mutation occurred in the adhB pseudogene over the course of evolution. PMID:25943635

  9. CvADH1, a member of short-chain alcohol dehydrogenase family, is inducible by gibberellin and sucrose in developing watermelon seeds.

    PubMed

    Kim, Joonyul; Kang, Hong-Gyu; Jun, Sung-Hoon; Lee, Jinwon; Yim, Jieun; An, Gynheung

    2003-01-01

    To understand the molecular mechanisms that control seed formation, we selected a seed-preferential gene (CvADH1) from the ESTs of developing watermelon seeds. RNA blot analysis and in situ localization showed that CvADH1 was preferentially expressed in the nucellar tissue. The CvADH1 protein shared about 50% homology with short-chain alcohol dehydrogenase including ABA2 in Arabidopsis thaliana, stem secoisolariciresinol dehydrogenase in Forsythia intermedia, and 3beta-hydroxysterol dehydrogenase in Digitalis lanata. We investigated gene-expression levels in seeds from both normally pollinated fruits and those made parthenocarpic via N-(2-chloro-4-pyridyl)-N'-phenylurea treatment, the latter of which lack zygotic tissues. Whereas the transcripts of CvADH1 rapidly started to accumulate from about the pre-heart stage in normal seeds, they were not detectable in the parthenocarpic seeds. Treating the parthenogenic fruit with GA(3) strongly induced gene expression, up to the level accumulated in pollinated seeds. These results suggest that the CvADH1 gene is induced in maternal tissues by signals made in the zygotic tissues, and that gibberellin might be one of those signals. We also observed that CvADH1 expression was induced by sucrose in the parthenocarpic seeds. Therefore, we propose that the CvADH1 gene is inducible by gibberellin, and that sucrose plays an important role in the maternal tissues of watermelon during early seed development. PMID:12552151

  10. Evolutionary origins of retinoid active short-chain dehydrogenases/reductases of SDR16C family.

    PubMed

    Belyaeva, Olga V; Chang, Chenbei; Berlett, Michael C; Kedishvili, Natalia Y

    2015-06-01

    Vertebrate enzymes that belong to the 16C family of short-chain dehydrogenases/reductases (SDR16C) were shown to play an essential role in the control of retinoic acid (RA) levels during development. To trace the evolution of enzymatic function of SDR16C family, and to examine the origins of the pathway for RA biosynthesis from vitamin A, we identified putative SDR16C enzymes through the extensive search of available genome sequencing data in a subset of species representing major metazoan phyla. The phylogenetic analysis revealed that enzymes from protostome, non-chordate deuterostome and invertebrate chordate species are found in three clades of SDR16C family containing retinoid active enzymes, which are retinol dehydrogenase 10 (RDH10), retinol dehydrogenases E2 (RDHE2) and RDHE2-similar, and dehydrogenase reductase (SDR family) member 3 (DHRS3). For the initial functional analysis, we cloned RDH10- and RDHE2-related enzymes from the early developmental stages of a non-chordate deuterostome, green sea urchin Lytechinus variegatus, and an invertebrate chordate, sea squirt Ciona intestinalis. In situ hybridization revealed that these proteins are expressed in a pattern relevant to development, while assays performed on proteins expressed in mammalian cell culture showed that they possess retinol-oxidizing activity as their vertebrate homologs. The existence of invertebrate homologs of DHRS3 was inferred from the analysis of phylogeny and cofactor-binding residues characteristic of preference for NADP(H). The presence of invertebrate homologs in the DHRS3 group of SDR16C is interesting in light of the complex mutually activating interaction, which we have recently described for human RDH10 and DHRS3 enzymes. Further functional analysis of these homologs will establish whether this interaction evolved to control retinoid homeostasis only in vertebrates, or is also conserved in pre-vertebrates. PMID:25451586

  11. Genetic improvement of Escherichia coli for ethanol production: Chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II

    SciTech Connect

    Ohta, Kazuyoshi; Beall, D.S.; Mejia, J.P.; Shanmugam, K.T.; Ingram, L.O. )

    1991-04-01

    Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high levels of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).

  12. Sulfur-rich zinc chemistry: new tris(thioimidazolyl)hydroborate ligands and their zinc complex chemistry related to the structure and function of alcohol dehydrogenase.

    PubMed

    Tesmer, M; Shu, M; Vahrenkamp, H

    2001-07-30

    The 1-substituted tris(2-thioimidazolyl)hydroborate ligands Tt(R) were prepared as the potassium salts from KBH(4) and the corresponding 1-R-2-thioimidazole for R = t-Bu and C(6)H(4)-p-CH(CH(3))(2) (Cum). Their reactions with zinc salts yielded the tetrahedral complexes Tt(R)Zn-X with X = F, Cl, ONO(2) and (Tt(t)()(-)(Bu))(2)Zn. With zinc perchlorate the labile perchlorate complexes Tt(R)Zn-OClO(3) were obtained. They served as starting materials for the incorporation of substrates which are relevant for the chemistry of horse liver alcohol dehydrogenase: Ethanol led to [Tt(t)()(-Bu)Zn.EtOH] ClO(4).EtOH, p-nitrophenol (NitOH) yielded Tt(Cum)Zn-ONit. Pyridine-2-carbaldehyde and salicylic aldehyde were incorporated as N(pyridine) and O(phenolate) coligands with possible additional O(aldehyde) coordination. Substituted pyridyl methanols (R-PyCH(2)OH) yielded the trinuclear complexes [(Tt(t)()(-Bu))(2)Zn(3)(R-PyCH(2)O)(2)] (ClO(4))(2) with bridging Tt and pyridylmethoxide ligands. Preliminary experiments on the functional modeling of alcohol dehydrogenase have shown that TtZn complexes promote both the dehydrogenation of 2-propanol and the hydrogenation of pentafluorobenzaldehyde. PMID:11466063

  13. Escherichia coli d-Malate Dehydrogenase, a Generalist Enzyme Active in the Leucine Biosynthesis Pathway*

    PubMed Central

    Vorobieva, Anastassia A.; Khan, Mohammad Shahneawz; Soumillion, Patrice

    2014-01-01

    The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of d-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on d-malate as a carbon source, the d-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB− strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (l(+)-tartrate, d-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution. PMID:25160617

  14. Modulation of pyruvate dehydrogenase kinase activity in cultured hepatocytes by glucagon and n-octanoate.

    PubMed Central

    Fatania, H R; Vary, T C; Randle, P J

    1986-01-01

    The activity of pyruvate dehydrogenase kinase in extracts of mitochondria from rat hepatocytes cultured for 21 h in medium 199 was increased 2.5-fold by the presence of 55 nM-glucagon and 1 mM-sodium n-octanoate in the culture medium. The change was comparable with that induced in vivo by 48 h starvation. The potential contribution of branched-chain complex to estimates of PDH-complex activity in rat liver mitochondria has been defined. PMID:3707545

  15. Differential effects of acute and chronic fructose administration on pyruvate dehydrogenase activity and lipogenesis

    SciTech Connect

    Wilson, L.

    1988-01-01

    These studies were undertaken to distinguish between the acute and chronic effects of fructose administration. In vivo, liver lipogenesis, as measured by {sup 3}H{sub 2}O incorporation, was greater in rats fed 60% fructose than in their glucose fed controls. Both fructose feeding, and fructose feeding plus intraperitoneal fructose injection increased the activities of 6-phosphogluconate dehydrogenase and malic enzyme. Liver PDH activity was increased by fructose feeding, and was increased even more by fructose feeding and injection of fructose, but this was not associated with any changes in hepatic ATP concentrations.

  16. Bacteriostatic activities of monoacyl sugar alcohols against thermophilic sporeformers.

    PubMed

    Piao, Junkui; Kawahara-Aoyama, Yumiko; Inoue, Takashi; Adachi, Shuji

    2006-01-01

    The bacteriostatic activities of monoacyl sugar alcohols with different acyl chains and hydrophilic heads were examined against some thermophilic sporeformers. Monomyristoyl erythritol and xylitol efficaciously suppressed their spore development. The number and orientation of the hydroxyl groups also played important roles in this activity, and monomyristoyl xylitol exhibited the highest activity among the monomyristoyl sugar alcohols. PMID:16428845

  17. Why are the 2-oxoacid dehydrogenase complexes so large? Generation of an active trimeric complex.

    PubMed

    Marrott, Nia L; Marshall, Jacqueline J T; Svergun, Dmitri I; Crennell, Susan J; Hough, David W; van den Elsen, Jean M H; Danson, Michael J

    2014-11-01

    The four-component polypeptides of the 2-oxoacid dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2-oxoacids derived from leucine, isoleucine and valine, and with pyruvate. The dihydrolipoyl acyl-transferase (E2) core of the complex is composed of identical trimer-forming units that assemble into a novel 42-mer structure comprising octahedral and icosahedral geometric aspects. From our previously determined structure of this catalytic core, the inter-trimer interactions involve a tyrosine residue near the C-terminus secured in a hydrophobic pocket of an adjacent trimer like a ball-and-socket joint. In the present study, we have deleted the five C-terminal amino acids of the E2 polypeptide (IIYEI) and shown by equilibrium centrifugation that it now only assembles into a trimeric enzyme. This was confirmed by SAXS analysis, although this technique showed the presence of approximately 20% hexamers. The crystal structure of the trimeric truncated E2 core has been determined and shown to be virtually identical with the ones observed in the 42-mer, demonstrating that removal of the C-terminal anchor does not significantly affect the individual monomer or trimer structures. The truncated E2 is still able to bind both 2-oxoacid decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components to give an active complex with catalytic activity similar to the native multienzyme complex. This is the first report of an active mini-complex for this enzyme, and raises the question of why all 2-oxoacid dehydrogenase complexes assemble into such large structures. PMID:25088564

  18. Fatty acid alcohol ester-synthesizing activity of lipoprotein lipase.

    PubMed

    Tsujita, T; Sumiyoshi, M; Okuda, H

    1999-12-01

    The fatty acid alcohol ester-synthesizing activity of lipoprotein lipase (LPL) was characterized using bovine milk LPL. Synthesizing activities were determined in an aqueous medium using oleic acid or trioleylglycerol as the acyl donor and equimolar amounts of long-chain alcohols as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with LPL, palmityl oleate was synthesized, in a time- and dose-dependent manner. Apo-very low density lipoprotein (apoVLDL) stimulated LPL-catalyzed palmityl oleate synthesis. The apparent equilibrium ratio of fatty acid alcohol ester/oleic acid was estimated using a high concentration of LPL and a long (20 h) incubation period. The equilibrium ratio was affected by the incubation pH and the alcohol chain length. When the incubation pH was below pH 7.0 and long chain fatty acyl alcohols were used as substrates, the fatty acid alcohol ester/free fatty acid equilibrium ratio favored ester formation, with an apparent equilibrium ratio of fatty acid alcohol ester/fatty acid of about 0.9/0.1. The equilibrium ratio decreased sharply at alkaline pH (above pH 8.0). The ratio also decreased when fatty alcohols with acyl chains shorter than dodecanol were used. When a trioleoylglycerol/fatty acyl alcohol emulsion was incubated with LPL, fatty acid alcohol esters were synthesized in a dose- and time-dependent fashion. Fatty acid alcohol esters were easily synthesized from trioleoylglycerol when fatty alcohols with acyl chains longer than dodecanol were used, but synthesis was decreased with fatty alcohols with acyl chain lengths shorter than decanol, and little synthesizing activity was detected with shorter-chain fatty alcohols such as butanol or ethanol. PMID:10578059

  19. Fate of free and linear alcohol-ethoxylate-derived fatty alcohols in activated sludge.

    PubMed

    Federle, Thomas W; Itrich, Nina R

    2006-05-01

    Pure homologues of [1-14C] C12, C14, and C16 alcohols and the linear alcohol ethoxylates, AE [1-14C alkyl] C13E9 and C16E9 were tested in a batch-activated sludge die-away system to assess their biodegradation kinetics and to predict levels of free alcohol derived from AE biodegradation in treated effluent. First-order rates for primary biodegradation were similar for all alcohols (86-113 h(-1)) and were used to predict removal under typical treatment conditions. Predicted removals of fatty alcohols ranged from 99.76% to 99.85%, consistent with published field data. During the biodegradation of the AE homologues, lower than expected levels of fatty alcohol based upon the assumption that biodegradation occurs through central fission were observed. Rather than fatty alcohols, the major metabolites were polar materials resulting from omega oxidation of the alkyl chain prior to or concurrent with central cleavage. The amounts of free fatty alcohols that were formed from AEs in influent and escape into effluent were negligible due both to their rapid degradation and to the finding that formation of free alcohol through central cleavage is only a minor degradation pathway in activated sludge. PMID:16026837

  20. [Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

    PubMed

    Voloshchuk, O N; Kopylchuk, G P

    2016-01-01

    Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage. PMID:27143375

  1. Novel biohybrids of layered double hydroxide and lactate dehydrogenase enzyme: Synthesis, characterization and catalytic activity studies

    NASA Astrophysics Data System (ADS)

    Djebbi, Mohamed Amine; Braiek, Mohamed; Hidouri, Slah; Namour, Philippe; Jaffrezic-Renault, Nicole; Ben Haj Amara, Abdesslem

    2016-02-01

    The present work introduces new biohybrid materials involving layered double hydroxides (LDH) and biomolecule such as enzyme to produce bioinorganic system. Lactate dehydrogenase (Lac Deh) has been chosen as a model enzyme, being immobilized onto MgAl and ZnAl LDH materials via direct ion-exchange (adsorption) and co-precipitation methods. The immobilization efficiency was largely dependent upon the immobilization methods. A comparative study shows that the co-precipitation method favors the immobilization of great and tunable amount of enzyme. The structural behavior, chemical bonding composition and morphology of the resulting biohybrids were determined by X-ray diffraction (XRD) study, Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM), respectively. The free and immobilized enzyme activity and kinetic parameters were also reported using UV-Visible spectroscopy. However, the modified LDH materials showed a decrease in crystallinity as compared to the unmodified LDH. The change in activity of the immobilized lactate dehydrogenase was considered to be due, to the reduced accessibility of substrate molecules to the active sites of the enzyme and the partial conformational change of the Lac Deh molecules as a result of the immobilization way. Finally, it was proven that there is a correlation between structure/microstructure and enzyme activity dependent on the immobilization process.

  2. Regulation of carbon monoxide dehydrogenase and hydrogenase in Rhodospirillum rubrum: Effects of CO and oxygen on synthesis and activity

    SciTech Connect

    Bonam, D.; Lehman, L.; Roberts, G.P.; Ludden, P.W.

    1989-06-01

    Exposure of the photosynthetic bacterium Rhodospirillum rubrum to carbon monoxide led to increased carbon monoxide dehydrogenase and hydrogenase activities due to de novo protein synthesis of both enzymes. Two-dimensional gels of (/sup 35/S)methionine-pulse-labeled cells showed that induction of CO dehydrogenase synthesis was rapidly initiated (less than 5 min upon exposure to CO) and was inhibited by oxygen. Both CO dehydrogenase and the CO-induced hydrogenase were inactivated by oxygen in vivo and in vitro. In contrast to CO dehydrogenase, the CO-induced hydrogenase was 95% inactivated by heating at 70 degrees C for 5 min. Unlike other hydrogenases, this CO-induced hydrogenase was inhibited only 60% by a 100% CO gas phase.

  3. Effects of Al(III) and nano-Al13 species on malate dehydrogenase activity.

    PubMed

    Yang, Xiaodi; Cai, Ling; Peng, Yu; Li, Huihui; Chen, Rong Fu; Shen, Ren Fang

    2011-01-01

    The effects of different aluminum species on malate dehydrogenase (MDH) activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT) modified glass carbon electrode (GCE). The results showed that Al(III) and Al(13) can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III) and Al(13) concentration increase. Our study also found that the effects of Al(III) and Al(13) on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules. PMID:22163924

  4. 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 ...

  5. The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity

    PubMed Central

    2011-01-01

    Background Escherichia coli synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O) and two of the hydrogenases (Hyd-1 and Hyd-2) have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H2: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity. Results Here we report the identification of a new H2: benzyl viologen oxidoreductase enzyme activity in E. coli that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of E. coli mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes. Conclusions The related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of Escherichia coli have hydrogen-oxidizing activity. These findings demonstrate that the

  6. Nonsense mutations in the alcohol dehydrogenase gene of Drosophila melanogaster correlate with an abnormal 3' end processing of the corresponding pre-mRNA.

    PubMed Central

    Brogna, S

    1999-01-01

    From bacteria to mammals, mutations that generate premature termination codons have been shown to result in the reduction in the abundance of the corresponding mRNA. In mammalian cells, more often than not, the reduction happens while the RNA is still associated with the nucleus. Here, it is reported that mutations in the alcohol dehydrogenase gene (Adh) of Drosophila melanogaster that generate premature termination codons lead to reduced levels of cytoplasmic and nuclear mRNA. Unexpectedly, it has been found that the poly(A) tails of Adh mRNAs and pre-mRNAs that carry a premature termination codon are longer than in the wild-type transcript. The more 5' terminal the mutation is, the longer is the poly(A) tail of the transcript. These findings suggest that the integrity of the coding region may be required for accurate mRNA 3' end processing. PMID:10199572

  7. Inhibitory effects of ofloxacin and cefepime on enzyme activity of 6-phosphogluconate dehydrogenase from chicken liver.

    PubMed

    Erat, Mustafa; Sakiroğlu, Halis

    2007-01-01

    In this study, effects of some antibiotics, namely, ofloxacin, cefepime, cefazolin, and ampicillin on the in vitro enzyme activity of 6-phosphogluconate dehydrogenase have been investigated. For this purpose, 6-phosphogluconate dehydrogenase was purified from chicken liver 535-fold with a yield of 18% by using ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. In order to check the purity of the enzyme, SDS polyacylamide gel electrophoresis (SDS-PAGE) was performed. This analysis revealed a highly pure enzyme band on the gel. Among the antibiotics, ofloxacin and cefepime exhibited inhibitory effects, but cefazolin and ampicillin showed neither important inhibitory nor activatory effects on the enzyme activity. The measured I(50) values by plotting activity percent vs. inhibitor concentration, [I(50)] were 0.1713 mM for ofloxacin and 6.0028 mM for cefepime. Inhibition constants, K(i), for ofloxacin and cefepime were also calculated as 0.2740 +/- 0.1080 mM and 12.869 +/- 16.6540 mM by means of Lineweaver-Burk graphs, and inhibition types of the antibiotics were found out to be non-competitive and competitive, respectively. It has been understood from the calculated inhibitory parameters that the purified chicken enzyme has been quite inhibited by these two antimicrobials. PMID:17305608

  8. Peroxisome proliferator-activated receptor ɣ activation induces 11β-hydroxysteroid dehydrogenase type 1 activity in human alternative macrophages

    PubMed Central

    Chinetti-Gbaguidi, Giulia; Bouhlel, Mohamed Amine; Copin, Corinne; Duhem, Christian; Derudas, Bruno; Neve, Bernardette; Noel, Benoit; Eeckhoute, Jerome; Lefebvre, Philippe; Seckl, Jonathan R.; Staels, Bart

    2012-01-01

    Objectives 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyses the intracellular reduction of inactive cortisone to active cortisol, the natural ligand activating the glucocorticoid receptor (GR). Peroxisome Proliferator-Activated Receptor gamma (PPARγ) is a nuclear receptor controlling inflammation, lipid metabolism and the macrophage polarization state. In this study, we investigated the impact of macrophage polarization on the expression and activity of 11β-HSD1 and the role of PPAR therein. Methods and Results 11β-HSD1 gene expression is higher in pro-inflammatory M1 and anti-inflammatory M2 macrophages than in resting macrophages (RM), whereas its activity is highest in M2 macrophages. Interestingly, PPARγ activation induces 11β-HSD1 enzyme activity in M2 macrophages, but not in RM or M1 macrophages. Consequently, human M2 macrophages displayed enhanced responsiveness to the 11β-HSD1 substrate cortisone, an effect amplified by PPAR -induction of 11β-HSD1 activity, as illustrated by an increased expression of GR target genes. Conclusions Our data identify a positive cross-talk between PPARγ and GR in human M2 macrophages via the induction of 11β-HSD1 expression and activity. PMID:22207732

  9. Alcohol

    MedlinePlus

    ... How Can I Help a Friend Who Cuts? Alcohol KidsHealth > For Teens > Alcohol Print A A A ... you can make an educated choice. What Is Alcohol? Alcohol is created when grains, fruits, or vegetables ...

  10. Decreased succinate dehydrogenase activity of gamma and alpha motoneurons in mouse spinal cords following 13 weeks of exposure to microgravity.

    PubMed

    Ishihara, Akihiko; Nagatomo, Fumiko; Fujino, Hidemi; Kondo, Hiroyo; Ohira, Yoshinobu

    2013-10-01

    Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm(2)) gamma and medium-sized (300-700 μm(2)) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm(2)) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity. PMID:23943522

  11. Cytoplasm-to-myonucleus ratios and succinate dehydrogenase activities in adult rat slow and fast muscle fibers

    NASA Technical Reports Server (NTRS)

    Tseng, B. S.; Kasper, C. E.; Edgerton, V. R.

    1994-01-01

    The relationship between myonuclear number, cellular size, succinate dehydrogenase activity, and myosin type was examined in single fiber segments (n = 54; 9 +/- 3 mm long) mechanically dissected from soleus and plantaris muscles of adult rats. One end of each fiber segment was stained for DNA before quantitative photometric analysis of succinate dehydrogenase activity; the other end was double immunolabeled with fast and slow myosin heavy chain monoclonal antibodies. Mean +/- S.D. cytoplasmic volume/myonucleus ratio was higher in fast and slow plantaris fibers (112 +/- 69 vs. 34 +/- 21 x 10(3) microns3) than fast and slow soleus fibers (40 +/- 20 vs. 30 +/- 14 x 10(3) microns3), respectively. Slow fibers always had small volumes/myonucleus, regardless of fiber diameter, succinate dehydrogenase activity, or muscle of origin. In contrast, smaller diameter (< 70 microns) fast soleus and plantaris fibers with high succinate dehydrogenase activity appeared to have low volumes/myonucleus while larger diameter (> 70 microns) fast fibers with low succinate dehydrogenase activity always had large volume/myonucleus. Slow soleus fibers had significantly greater numbers of myonuclei/mm than did either fast soleus or fast plantaris fibers (116 +/- 51 vs. 55 +/- 22 and 44 +/- 23), respectively. These data suggest that the myonuclear domain is more limited in slow than fast fibers and in the fibers with a high, compared to a low, oxidative metabolic capability.

  12. Lipid-mediated unfolding of 3-beta hydroxysteroid dehydrogenase2 is essential for steroidogenic activity

    PubMed Central

    Rajapaksha, Maheshinie; Thomas, James L.; Streeter, Michael; Prasad, Manoj; Whittal, Randy M.; Bell, John D.; Bose, Himangshu S.

    2011-01-01

    For inner mitochondrial membrane (IMM) proteins that do not undergo N-terminal cleavage, their activity may occur in the absence of a receptor present in the mitochondrial membrane. One such protein is human 3-beta hydroxysteroid dehydrogenase-2 (3βHSD2), the IMM resident protein responsible for catalyzing two key steps in steroid metabolism: the conversion of pregnenolone to progesterone and dehydroepiandrosterone (DHEA) to androstenedione. Conversion requires that 3βHSD2 serves as both a dehydrogenase and isomerase. The dual functionality of 3βHSD2 results from a conformational change, but the trigger for this change remains unknown. Using Fluorescence Resonance Energy Transfer (FRET), we found that 3βHSD2 interacted strongly with a mixture of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC). 3βHSD2 became less stable when incubated with the individual lipids, as indicated by the decrease in thermal denaturation (Tm), from 42° C to 37° C. DPPG, alone or in combination with DPPC, led to a decrease in α-helical content without affecting the β-sheet conformation. With the exception of the N-terminal 20 amino acids, mixed vesicles protected 3βHSD2 from trypsin digestion. However, protein incubated with DPPC was only partially protected. The lipid-mediated unfolding completely supports the model in which a cavity forms between the α-helix and β-sheet. As 3βHSD2 lacks a receptor, opening the conformation may activate the protein. PMID:22106846

  13. Distribution of Pyruvate Dehydrogenase Complex Activities between Chloroplasts and Mitochondria from Leaves of Different Species.

    PubMed Central

    Lernmark, U.; Gardestrom, P.

    1994-01-01

    Protoplasts from barley (Hordeum vulgare), pea (Pisum sativum), wheat (Triticum aestivum), and spinach (Spinacia oleracea) leaves were fractionated into chloroplast- and mitochondrion-enriched fractions. Pyruvate dehydrogenase complex capacities in mitochondria (mtPDC) and chloroplasts (cpPDC) were measured in appropriate fractions under conditions optimal for each isozyme. The total cellular capacity of PDC was similar in barley and pea but about 50% lower in wheat and spinach. In pea a distribution of 87% mtPDC and 13% cpPDC was found on a cellular basis. In barley, wheat, and spinach the subcellular distribution was the opposite, with about 15% mtPDC and 85% cpPDC. cpPDC activity was constant at about 0.1 nmol cell-1 h-1 in cells from different regions along the developing barley leaf and showed no correlation with developmental patterns of photosynthetic parameters, such as increasing Chl and NADP-glyceraldehyde-3-phosphate dehydrogenase activity. Similarly, the capacity of the mitochondrial isoform did not change during barley leaf development and had a developmental pattern similar to that of citrate synthase and fumarase. Differences in subcellular distribution of PDCs in barley and pea are proposed to be due to differences in regulation, not to changes in isozyme proportions during leaf development or to species-specific differences in phosphorylation state of mtPDC after organelle separation. PMID:12232437

  14. SIRT3 DEACETYLATES AND INCREASES PYRUVATE DEHYDROGENASE ACTIVITY IN CANCER CELLS

    PubMed Central

    Wagner, Brett A.; Song, Ha Yong; Zhu, Yueming; Vassilopoulos, Athanassios; Jung, Barbara; Buettner, Garry R.; Gius, David

    2015-01-01

    Pyruvate dehydrogenase E1 alpha (PDHE1α or PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex (PDC) that transforms pyruvate, via pyruvate decarboxylation, into acetyl-CoA that is subsequently used by both the citric acid cycle and oxidative phosphorylation to generate ATP. As such, PDH links glycolysis and oxidative phosphorylation in normal as well as cancer cells. Herein we report that SIRT3 interacts with PDHA1 and directs its enzymatic activity via changes in protein acetylation. SIRT3 deacetylates PDHA1 lysine 321 (K321) and a PDHA1 mutant, mimicking a deacetylated lysine (PDHA1K321R) increases in PDH activity, as compared to the K321 acetylation mimic (PDHA1K321Q) or wild-type PDHA1. Finally, PDHA1K321Q exhibited a more transformed in vitro cellular phenotype as compared to PDHA1K321R. These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyl-lysine suggesting that the acetylome, as well as the kinome, links glycolysis to respiration. PMID:25152236

  15. Dosage Compensation in DROSOPHILA MELANOGASTER Triploids. II. Glucose-6-Phosphate Dehydrogenase Activity

    PubMed Central

    Maroni, Gustavo; Plaut, Walter

    1973-01-01

    The level of activity of the enzyme glucose-6-phosphate dehydrogenase was determinel in flies having seven different chromosomic constitutions. All those having an integral number of chromosomes [XAA, XXAA, XAAA, XXAAA, and XXXAAA (X=X chromosome, A=set of autosomes)] were found to have similar units of enzyme activity/mg live weight, while diploid females with a duplication and triploid females with a deficiency showed dosage effect. The amount of enzyme activity per cell, on the other hand, is also independent of the number of X's present but appears roughly proportional to the number of sets of autosomes.—It is proposed that dosage-compensated sex-linked genes are controlled by a positively acting regulatory factor(s) of autosomal origin. With this hypothesis it is possible to explain dosage compensation as a consequence of general regulatory mechanisms without invoking a special device which applies only to the X chromosomes. PMID:17248620

  16. Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells

    SciTech Connect

    Durand, Fabien; Stines-Chaumeil, Claire; Flexer, Victoria; Andre, Isabelle; Mano, Nicolas

    2010-11-26

    Research highlights: {yields} A new mutant of PQQ-GDH designed for glucose biosensors application. {yields} First mutant of PQQ-GDH with higher activity for D-glucose than the Wild type. {yields} Position N428 is a key point to increase the enzyme activity. {yields} Molecular modeling shows that the N428 C mutant displays a better interaction for PQQ than the WT. -- Abstract: We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to a better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.

  17. Plasma lactic dehydrogenase activities in men during bed rest with exercise training

    NASA Technical Reports Server (NTRS)

    Greenleaf, J. E.; Juhos, L. T.; Young, H. L.

    1985-01-01

    Peak oxygen uptake and the activity of lactic dehydrogenase (LDH-T) and its five isoenzymes were measured by spectrophotometer in seven men before, during, and after bed rest and exercise training. Exercise training consisted of isometric leg exercises of 250 kcal/hr for a period of one hour per day. It is found that LDH-T was reduced by 0.05 percent in all three regimens by day 10 of bed rest, and that the decrease occurred at different rates. The earliest reduction in LDH-T activity in the no-exercise regimen was associated with a decrease in peak oxygen uptake of 12.3 percent. It is concluded that isometric (aerobic) muscular strength training appear to maintain skeletal muscle integrity better during bed rest than isotonic exercise training. Reduced hydrostatic pressure during bed rest, however, ultimately counteracts the effects of both moderate isometric and isotonic exercise training, and may result in decreased LDH-T activity.

  18. [Safety problems of occupational activity during alcoholic intoxication].

    PubMed

    Ushakov, I B; Popov, V I

    1999-01-01

    It has been established that work efficiency of persons occupied by machine operating duties degrades markedly under the action of an acute alcoholic intoxication--less on real machines and more on simulators and when operating simple technical devices. Work efficiency degradation is manifested by the task execution time increase (to a lesser degree) and by erroneous action quantity increase after taking alcohol, i.e. by work reliability decease. Most heavily it is manifested during the first 0.7-1.5 hr. After taking alcohol. And direct work efficiency index is usually improved 12-16 hr. after taking alcohol. Increase of erroneous action quantity is caused probably by a series of physiological, psychophysiological and psychological changes in the condition of a man under the action of alcohol. Time of the day when the activity takes place after taking that dose of alcohol is practically non valid for changing work efficiency index. PMID:11965736

  19. Phenylbutyrate increases pyruvate dehydrogenase complex activity in cells harboring a variety of defects

    PubMed Central

    Ferriero, Rosa; Boutron, Audrey; Brivet, Michele; Kerr, Douglas; Morava, Eva; Rodenburg, Richard J; Bonafé, Luisa; Baumgartner, Matthias R; Anikster, Yair; Braverman, Nancy E; Brunetti-Pierri, Nicola

    2014-01-01

    Objective Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. PDHC deficiency is genetically heterogenous and most patients have defects in the X-linked E1-α gene but defects in the other components of the complex encoded by PDHB, PDHX, DLAT, DLD genes or in the regulatory enzyme encoded by PDP1 have also been found. Phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of pyruvate dehydrogenase kinases and thus, has potential for therapy of patients with PDHC deficiency. In the present study, we investigated response to phenylbutyrate of multiple cell lines harboring all known gene defects resulting in PDHC deficiency. Methods Fibroblasts of patients with PDHC deficiency were studied for their enzyme activity at baseline and following phenylbutyrate incubation. Drug responses were correlated with genotypes and protein levels by Western blotting. Results Large deletions affecting PDHA1 that result in lack of detectable protein were unresponsive to phenylbutyrate, whereas increased PDHC activity was detected in most fibroblasts harboring PDHA1 missense mutations. Mutations affecting the R349-α residue were directed to proteasome degradation and were consistently unresponsive to short-time drug incubation but longer incubation resulted in increased levels of enzyme activity and protein that may be due to an additional effect of phenylbutyrate as a molecular chaperone. Interpretation PDHC enzyme activity was enhanced by phenylbutyrate in cells harboring missense mutations in PDHB, PDHX, DLAT, DLD, and PDP1 genes. In the prospect of a clinical trial, the results of this study may allow prediction of in vivo response in patients with PDHC deficiency harboring a wide spectrum of molecular defects. PMID:25356417

  20. Effect of different mulch materials on the soil dehydrogenase activity (DHA) in an organic pepper crop

    NASA Astrophysics Data System (ADS)

    Moreno, Marta M.; Peco, Jesús; Campos, Juan; Villena, Jaime; González, Sara; Moreno, Carmen

    2016-04-01

    The use biodegradable materials (biopolymers of different composition and papers) as an alternative to conventional mulches has increased considerably during the last years mainly for environmental reason. In order to assess the effect of these materials on the soil microbial activity during the season of a pepper crop organically grown in Central Spain, the soil dehydrogenase activity (DHA) was measured in laboratory. The mulch materials tested were: 1) black polyethylene (PE, 15 μm); black biopolymers (15 μm): 2) Mater-Bi® (corn starch based), 3) Sphere 4® (potato starch based), 4) Sphere 6® (potato starch based), 5) Bioflex® (polylactic acid based), 6) Ecovio® (polylactic acid based), 7) Mimgreen® (black paper, 85 g/m2). A randomized complete block design with four replications was adopted. The crop was drip irrigated following the water demand of each treatment. Soil samples (5-10 cm depth) under the different mulches were taken at different dates (at the beginning of the crop cycle and at different dates throughout the crop season). Additionally, samples of bare soil in a manual weeding and in an untreated control were taken. The results obtained show the negative effect of black PE on the DHA activity, mainly as result of the higher temperature reached under the mulch and the reduction in the gas interchange between the soil and the atmosphere. The values corresponding to the biodegradable materials were variable, although highlighting the low DHA activity observed under Bioflex®. In general, the uncovered treatments showed higher values than those reached under mulches, especially in the untreated control. Keywords: mulch, biodegradable, biopolymer, paper, dehydrogenase activity (DHA). Acknowledgements: the research was funded by Project RTA2011-00104-C04-03 from the INIA (Spanish Ministry of Economy and Competitiveness).

  1. Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula.

    PubMed

    Zhao, Qiao; Tobimatsu, Yuki; Zhou, Rui; Pattathil, Sivakumar; Gallego-Giraldo, Lina; Fu, Chunxiang; Jackson, Lisa A; Hahn, Michael G; Kim, Hoon; Chen, Fang; Ralph, John; Dixon, Richard A

    2013-08-13

    There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure. PMID:23901113

  2. Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula

    PubMed Central

    Zhao, Qiao; Tobimatsu, Yuki; Zhou, Rui; Pattathil, Sivakumar; Gallego-Giraldo, Lina; Fu, Chunxiang; Jackson, Lisa A.; Hahn, Michael G.; Kim, Hoon; Chen, Fang; Ralph, John; Dixon, Richard A.

    2013-01-01

    There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure. PMID:23901113

  3. Immobilization of Pichia pastoris cells containing alcohol oxidase activity

    PubMed Central

    Maleknia, S; Ahmadi, H; Norouzian, D

    2011-01-01

    Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

  4. Impulsivity moderates the association between physical activity and alcohol consumption

    PubMed Central

    Leasure, J. Leigh; Neighbors, Clayton

    2016-01-01

    Mounting evidence indicates that physical activity and alcohol consumption are positively associated, but potential moderators of this relationship remain unclear. Both physical activity and alcohol drinking are potentially reinforcing and may be more strongly associated among individuals who tend to be higher in reward seeking and related processes governed by the prefrontal cortex. Thus, behaviors linked to the prefrontal cortex, such as impulsivity, may influence the association between physical activity and alcohol intake. The present study therefore evaluated dimensions of impulsivity as moderators of the association between physical activity and alcohol consumption. We surveyed 198 undergraduate students and obtained self-reports of their drinking habits, physical activity, and dimensions of impulsivity. We found that moderate but not vigorous physical activity was positively associated with drinking. Linear regression analyses were used to evaluate dimensions of impulsivity as moderators of the association between physical activity (vigorous or moderate) and drinks per week. Results revealed a consistent pattern of interactions between the positive urgency and sensation seeking dimensions of impulsivity and moderate physical activity on number of drinks per week. For both interactions, there was a significant positive association between moderate physical activity and drinking at higher but not lower levels of impulsivity. We conclude that impulsivity moderates the positive association between physical activity and alcohol consumption. These results have significant implications for the develop ment of prevention and treatment programs for alcohol use disorders. PMID:24525252

  5. Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia.

    PubMed Central

    Jonas, S. K.; Benedetto, C.; Flatman, A.; Hammond, R. H.; Micheletti, L.; Riley, C.; Riley, P. A.; Spargo, D. J.; Zonca, M.; Slater, T. F.

    1992-01-01

    The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected]. Images Figure 1 PMID:1637668

  6. Alcohol

    MedlinePlus

    ... Text Size: A A A Listen En Español Alcohol Wondering if alcohol is off limits with diabetes? Most people with diabetes can have a moderate amount of alcohol. Research has shown that there can be some ...

  7. Alcohol

    MedlinePlus

    If you are like many Americans, you drink alcohol at least occasionally. For many people, moderate drinking ... risky. Heavy drinking can lead to alcoholism and alcohol abuse, as well as injuries, liver disease, heart ...

  8. Regulation of pyruvate dehydrogenase activity and glucose metabolism in post-ischaemic myocardium.

    PubMed

    Schöder, H; Knight, R J; Kofoed, K F; Schelbert, H R; Buxton, D B

    1998-02-27

    Pyruvate dehydrogenase (PDH) is regulated both by covalent modification and through modulation of the active enzyme by metabolites. In the isolated heart, post-ischaemic inhibition of PDH, leading to uncoupling of glycolysis and glucose oxidation and a decrease in cardiac efficiency, has been described. In vivo, post-ischaemic reperfusion leads to metabolic abnormalities consistent with PDH inhibition, but the effects of ischaemia/reperfusion on PDH are not well characterized. We therefore investigated PDH regulation following transient ischaemia in vivo. In 33 open-chest dogs, the left anterior descending (LAD) was occluded for 20 min followed by 4 h reperfusion. In 17 dogs, dichloroacetate (DCA) was injected prior to reperfusion, while 16 dogs served as controls. In dogs without DCA, glucose oxidation and lactate uptake were lower in reperfused than in remote tissue, suggesting reduced flux through PDH. However, percent active and total PDH measured in myocardial biopsies were similar in both territories, excluding covalent enzyme modification or loss of functional enzyme. DCA activated PDH activity similarly in both regions and abolished differences in glucose oxidation and lactate uptake. Thus, decreased PDH flux in reperfused myocardium does not result from covalent modification or loss of total enzyme activity, but more likely from metabolite inhibition of the active enzyme. DCA leads to essentially complete activation of PDH, increases overall glucose utilization and abolishes post-ischaemic inhibition of glucose oxidation. PMID:9545535

  9. Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity

    DOE PAGESBeta

    Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar; Gollapalli, Deviprasad R.; Cuny, Gregory D.; Joachimiak, Andrzej; Hedstrom, Lizbeth

    2015-04-21

    Inosine 5´-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment of Cryptosporidium infections. Here, the structure of C. parvum IMPDH (CpIMPDH) in complex with inosine 5´-monophosphate (IMP) and P131, an inhibitor with in vivo anticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO2 moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is a new strategymore » for the further optimization of C. parvum inhibitors for both antiparasitic and antibacterial applications.« less

  10. The Hydrogenase Activity of the Molybdenum/Copper-containing Carbon Monoxide Dehydrogenase of Oligotropha carboxidovorans*

    PubMed Central

    Wilcoxen, Jarett; Hille, Russ

    2013-01-01

    The reaction of the air-tolerant CO dehydrogenase from Oligotropha carboxidovorans with H2 has been examined. Like the Ni-Fe CO dehydrogenase, the enzyme can be reduced by H2 with a limiting rate constant of 5.3 s−1 and a dissociation constant Kd of 525 μm; both kred and kred/Kd, reflecting the breakdown of the Michaelis complex and the reaction of free enzyme with free substrate in the low [S] regime, respectively, are largely pH-independent. During the reaction with H2, a new EPR signal arising from the Mo/Cu-containing active site of the enzyme is observed which is distinct from the signal seen when the enzyme is reduced by CO, with greater g anisotropy and larger hyperfine coupling to the active site 63,65Cu. The signal also exhibits hyperfine coupling to at least two solvent-exchangeable protons of bound substrate that are rapidly exchanged with solvent. Proton coupling is also evident in the EPR signal seen with the dithionite-reduced native enzyme, and this coupling is lost in the presence of bicarbonate. We attribute the coupled protons in the dithionite-reduced enzyme to coordinated water at the copper site in the native enzyme and conclude that bicarbonate is able to displace this water from the copper coordination sphere. On the basis of our results, a mechanism for H2 oxidation is proposed which involves initial binding of H2 to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center. PMID:24165123

  11. Lipid-mediated unfolding of 3β-hydroxysteroid dehydrogenase 2 is essential for steroidogenic activity.

    PubMed

    Rajapaksha, Maheshinie; Thomas, James L; Streeter, Michael; Prasad, Manoj; Whittal, Randy M; Bell, John D; Bose, Himangshu S

    2011-12-27

    For inner mitochondrial membrane (IMM) proteins that do not undergo N-terminal cleavage, the activity may occur in the absence of a receptor present in the mitochondrial membrane. One such protein is human 3β-hydroxysteroid dehydrogenase 2 (3βHSD2), the IMM resident protein responsible for catalyzing two key steps in steroid metabolism: the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione. Conversion requires that 3βHSD2 serve as both a dehydrogenase and an isomerase. The dual functionality of 3βHSD2 results from a conformational change, but the trigger for this change remains unknown. Using fluorescence resonance energy transfer, we found that 3βHSD2 interacted strongly with a mixture of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC). 3βHSD2 became less stable when incubated with the individual lipids, as indicated by the decrease in thermal denaturation (T(m)) from 42 to 37 °C. DPPG, alone or in combination with DPPC, led to a decrease in α-helical content without an effect on the β-sheet conformation. With the exception of the 20 N-terminal amino acids, mixed vesicles protected 3βHSD2 from trypsin digestion. However, protein incubated with DPPC was only partially protected. The lipid-mediated unfolding completely supports the model in which a cavity forms between the α-helix and β-sheet. As 3βHSD2 lacks a receptor, opening the conformation may activate the protein. PMID:22106846

  12. Effect of various chemicals on the aldehyde dehydrogenase activity of the rat liver cytosol.

    PubMed

    Marselos, M; Vasiliou, V

    1991-01-01

    The cytosolic activity of aldehyde dehydrogenase (ALDH) was studied in the rat liver, after acute administration of various carcinogenic and chemically related compounds. Male Wistar rats were treated with 27 different chemicals, including polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, azo dyes, as well as with some known direct-acting carcinogens. The cytosolic ALDH activity of the liver was determined either with propionaldehyde and NAD (P/NAD), or with benzaldehyde and NADP (B/NADP). The activity of ALDH remained unaffected after treatment with 1-naphthylamine, nitrosamines and also with the direct-acting chemical carcinogens tested. On the contrary, polycyclic aromatic hydrocarbons, polychlorinated biphenyls (Arochlor 1254) and 2-naphthylamine produced a remarkable increase of ALDH. In general, the response to the effectors was disproportionate between the two types of enzyme activity, being much in favour for the B/NADP activity. This fact resulted to an inversion of the ratio B/NADP vs. P/NAD, which under constitutive conditions is lower than 1. In this respect, the most potent compounds were found to be polychlorinated biphenyls, 3-methylcholanthrene, benzo(a)pyrene and 1,2,5,6-dibenzoanthracene. Our results suggest that the B/NADP activity of the soluble ALDH is greatly induced after treatment with compounds possessing aromatic ring(s) in their molecule. It is not known, if this response of the hepatocytes is related with the process of chemical carcinogenesis. PMID:2060039

  13. Muscular cholinesterase and lactate dehydrogenase activities in deep-sea fish from the NW Mediterranean.

    PubMed

    Koenig, Samuel; Solé, Montserrat

    2014-03-01

    Organisms inhabiting submarine canyons can be potentially exposed to higher inputs of anthropogenic chemicals than their counterparts from the adjacent areas. To find out to what extend this observation applies to a NW Mediterranean canyon (i.e. Blanes canyon) off the Catalan coast, four deep-sea fish species were collected from inside the canyon (BC) and the adjacent open slope (OS). The selected species were: Alepocephalus rostratus, Lepidion lepidion, Coelorinchus mediterraneus and Bathypterois mediterraneus. Prior to the choice of an adequate sentinel species, the natural variation of the selected parameters (biomarkers) in relation to factors such as size, sex, sampling depth and seasonality need to be characterised. In this study, the activities of cholinesterases (ChEs) and lactate dehydrogenase (LDH) enzymes were determined in the muscle of the four deep-sea fish. Of all ChEs, acetylcholinesterase (AChE) activity was dominant and selected for further monitoring. Overall, AChE activity exhibited a significant relationship with fish size whereas LDH activity was mostly dependent on the sex and gonadal development status, although in a species-dependent manner. The seasonal variability of LDH activity was more marked than for AChE activity, and inside-outside canyon (BC-OS) differences were not consistent in all contrasted fish species, and in fact they were more dependent on biological traits. Thus, they did not suggest a differential stress condition between sites inside and outside the canyon. PMID:24296242

  14. Recipient pretransplant inosine monophosphate dehydrogenase activity in nonmyeloablative hematopoietic cell transplantation.

    PubMed

    Bemer, Meagan J; Risler, Linda J; Phillips, Brian R; Wang, Joanne; Storer, Barry E; Sandmaier, Brenda M; Duan, Haichuan; Raccor, Brianne S; Boeckh, Michael J; McCune, Jeannine S

    2014-10-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5'-monophosphate to xanthosine 5'-monophosphate (XMP). We developed a highly sensitive liquid chromatography-mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  15. PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSIS OF INOSINE MONOPHOSPHATE DEHYDROGENASE (IMPDH) ACTIVITY IN MMF-TREATED HCT RECIPIENTS

    PubMed Central

    Li, Hong; Mager, Donald E.; Sandmaier, Brenda M.; Storer, Barry E.; Boeckh, Michael J.; Bemer, Meagan J.; Phillips, Brian R.; Risler, Linda J.; McCune, Jeannine S.

    2014-01-01

    A novel approach to personalizing postgrafting immunosuppression in hematopoietic cell transplant (HCT) recipients is evaluating inosine monophosphate dehydrogenase (IMPDH) activity as a drug-specific biomarker of mycophenolic acid (MPA)-induced immunosuppression. This prospective study evaluated total MPA, unbound MPA, and total MPA glucuronide plasma concentrations and IMPDH activity in peripheral blood mononuclear cells (PMNC) at five time points after the morning dose of oral mycophenolate mofetil (MMF) on day +21 in 56 nonmyeloablative HCT recipients. Substantial interpatient variability in the pharmacokinetics and pharmacodynamics was observed and accurately characterized by the population pharmacokinetic/dynamic model. IMPDH activity decreased with increasing MPA plasma concentration, with maximum inhibition coinciding with maximum MPA concentration in most patients. The overall relationship between MPA concentration and IMPDH activity was described by a direct inhibitory Emax model with an IC50 = 3.23 mg/L total MPA and 57.3 ng/mL unbound MPA. The day +21 IMPDH area under the effect curve (AUEC) was associated with cytomegalovirus reactivation, non-relapse mortality, and overall mortality. In conclusion, a pharmacokinetic/dynamic model was developed that relates plasma MPA concentrations with PMNC IMPDH activity after an MMF dose in HCT recipients. Future studies should validate this model and confirm that day +21 IMPDH AUEC is a predictive biomarker. PMID:24727337

  16. Active-Loop Dynamics within the Michaelis Complex of Lactate Dehydrogenase from Bacillus stearothermophilus.

    PubMed

    Nie, Beining; Lodewyks, Kara; Deng, Hua; Desamero, Ruel Z B; Callender, Robert

    2016-07-12

    Laser-induced temperature-jump relaxation spectroscopy was used to study the active site mobile-loop dynamics found in the binding of the NADH nucleotide cofactor and oxamate substrate mimic to lactate dehydrogenase in Bacillus stearothermophilus thermophilic bacteria (bsLDH). The kinetic data can be best described by a model in which NADH can bind only to the open-loop apoenzyme, oxamate can bind only to the bsLDH·NADH binary complex in the open-loop conformation, and oxamate binding is followed by closing of the active site loop preventing oxamate unbinding. The open and closed states of the loop are in dynamic equilibrium and interconvert on the submillisecond time scale. This interconversion strongly accelerates with an increase in temperature because of significant enthalpy barriers. Binding of NADH to bsLDH results in minor changes of the loop dynamics and does not shift the open-closed equilibrium, but binding of the oxamate substrate mimic shifts this equilibrium to the closed state. At high excess oxamate concentrations where all active sites are nearly saturated with the substrate mimic, all active site mobile loops are mainly closed. The observed active-loop dynamics for bsLDH is very similar to that previously observed for pig heart LDH. PMID:27319381

  17. Cytochrome b5 augments 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase activity.

    PubMed

    Goosen, Pierre; Storbeck, Karl-Heinz; Swart, Amanda C; Conradie, Riaan; Swart, Pieter

    2011-11-01

    During adrenal steroidogenesis the competition between 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD) and cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1) for Δ(5) steroid intermediates greatly influences steroidogenic output. Cytochrome-b(5) (Cyt-b(5)), a small electron transfer hemoprotein, known to augment the lyase activity of CYP17A1, has been shown to alter the steroidogenic outcome of this competition. In this study, the influence of Cyt-b(5) on 3βHSD activity was investigated. In COS-1 cells, Cyt-b(5) was shown to significantly increase the activity of both caprine and ovine 3βHSD towards pregnenolone, 17-OH pregnenolone and dehydroepiandrosterone in a substrate and species specific manner. Furthermore, kinetic studies revealed Cyt-b(5) to have no influence on the K(m) values while significantly increasing the V(max) values of ovine 3βHSD for all its respective substrates. In addition, the activity of ovine 3βHSD in microsomal preparations was significantly influenced by the addition of either purified Cyt-b(5) or anti-Cyt-b(5) IgG. The results presented in this study indicate that Cyt-b(5) augments 3βHSD activity and represents the first documentation of such augmentation in any species. PMID:21930205

  18. Influence of spaceflight on succinate dehydrogenase activity and soma size of rat ventral horn neurons

    NASA Technical Reports Server (NTRS)

    Ishihara, A.; Ohira, Y.; Roy, R. R.; Nagaoka, S.; Sekiguchi, C.; Hinds, W. E.; Edgerton, V. R.

    1996-01-01

    Succinate dehydrogenase (SDH) activities and soma cross-sectional areas (CSA) of neurons in the dorsolateral region of the ventral horn at the L5 segmental level of the spinal cord in the rat were determined after 14 days of spaceflight and after 9 days of recovery on earth. The results were compared to those in age-matched ground-based control rats. Spinal cords were quick-frozen, and the SDH activity and CSA of a sample of neurons with a visible nucleus were determined using a digitizer and a computer-assisted image analysis system. An inverse relationship between CSA and SDH activity of neurons was observed in all groups of rats. No change in mean CSA or mean SDH activity or in the size distribution of neurons was observed following spaceflight or recovery. However, there was a selective decrease in the SDH activity of neurons with soma CSA between 500 and 800 microns2 in the flight rats, and this effect persisted for at least 9 days following return to 1 g. It remains to be determined whether the selected population of motoneurons or the specific motor pools affected by spaceflight may be restricted to specific muscles.

  19. Effect of feeding and of DDT on the activity of hepatic glucose 6- phosphate dehydrogenase in two salmonids

    USGS Publications Warehouse

    Buhler, Donald R.; Benville, P.

    1969-01-01

    The specific activity of liver glucose 6-phosphate dehydrogenase in yearling rainbow trout remained unchanged when the fish were starved for periods as long as 8 weeks and when starved animals were fed diets of various compositions. Injection of insulin concurrently with refeeding also failed to alter the specific activity of the enzyme in trout. The absence of a dietary or insulin influence on the teleost enzyme system is to be contrasted with studies in mammals in which the activity of hepatic glucose 6-P dehydrogenase was markedly stimulated after refeeding starved animals or injection of insulin.Ingestion of the pesticide DDT by juvenile coho salmon or adult rainbow trout also had no effect on the specific activity of liver glucose 6-P dehydrogenase and DDT failed to inhibit the rainbow trout enzyme in vitro. These results also differ considerably from those found in higher animals.These results suggest that the glucose 6-P dehydrogenase enzyme in teleosts may be under a different type of regulatory control from that found in mammals.

  20. Periplasmic nitrate reductase and formate dehydrogenase: similar molecular architectures with very different enzymatic activities.

    PubMed

    Cerqueira, Nuno M F S A; Gonzalez, Pablo J; Fernandes, Pedro A; Moura, José J G; Ramos, Maria João

    2015-11-17

    It is remarkable how nature has been able to construct enzymes that, despite sharing many similarities, have simple but key differences that tune them for completely different functions in living cells. Periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) from the DMSOr family are representative examples of this. Both enzymes share almost identical three-dimensional protein foldings and active sites, in terms of coordination number, geometry and nature of the ligands. The substrates of both enzymes (nitrate and formate) are polyatomic anions that also share similar charge and stereochemistry. In terms of the catalytic mechanism, both enzymes have a common activation mechanism (the sulfur-shift mechanism) that ensures a constant coordination number around the metal ion during the catalytic cycle. In spite of these similarities, they catalyze very different reactions: Nap abstracts an oxygen atom from nitrate releasing nitrite, whereas FdH catalyzes a hydrogen atom transfer from formate and releases carbon dioxide. In this Account, a critical analysis of structure, function, and catalytic mechanism of the molybdenum enzymes periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) is presented. We conclude that the main structural driving force that dictates the type of reaction, catalyzed by each enzyme, is a key difference on one active site residue that is located in the top region of the active sites of both enzymes. In both enzymes, the active site is centered on the metal ion of the cofactor (Mo in Nap and Mo or W in Fdh) that is coordinated by four sulfur atoms from two pyranopterin guanosine dinucleotide (PGD) molecules and by a sulfido. However, while in Nap there is a Cys directly coordinated to the Mo ion, in FdH there is a SeCys instead. In Fdh there is also an important His that interacts very closely with the SeCys, whereas in Nap the same position is occupied by a Met. The role of Cys in Nap and SeCys in FdH is similar in both

  1. Effects of low molecular-weight organic acids and dehydrogenase activity in rhizosphere sediments of mangrove plants on phytoremediation of polycyclic aromatic hydrocarbons.

    PubMed

    Wang, Yuanyuan; Fang, Ling; Lin, Li; Luan, Tiangang; Tam, Nora F Y

    2014-03-01

    This work evaluated the roles of the low-molecular-weight organic acids (LMWOAs) from root exudates and the dehydrogenase activity in the rhizosphere sediments of three mangrove plant species on the removal of mixed PAHs. The results showed that the concentrations of LMWOAs and dehydrogenase activity changed species-specifically with the levels of PAH contamination. In all plant species, the concentration of citric acid was the highest, followed by succinic acid. For these acids, succinic acid was positively related to the removal of all the PAHs except Chr. Positive correlations were also found between the removal percentages of 4-and 5-ring PAHs and all LMWOAs, except citric acid. LMWOAs enhanced dehydrogenase activity, which positively related to PAH removal percentages. These findings suggested that LMWOAs and dehydrogenase activity promoted the removal of PAHs. Among three mangrove plants, Bruguiera gymnorrhiza, the plant with the highest root biomass, dehydrogenase activity and concentrations of LMWOAs, was most efficient in removing PAHs. PMID:24287262

  2. Alcohol

    MedlinePlus

    ... Got Homework? Here's Help White House Lunch Recipes Alcohol KidsHealth > For Kids > Alcohol Print A A A Text Size What's in ... What Is Alcoholism? Say No en español El alcohol Getting the Right Message "Hey, who wants a ...

  3. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

    PubMed Central

    Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

    2015-01-01

    ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

  4. Glutamate dehydrogenase in brain mitochondria: do lipid modifications and transient metabolon formation influence enzyme activity?

    PubMed Central

    McKenna, Mary C.

    2011-01-01

    Metabolism of glutamate, the primary excitatory neurotransmitter in brain, is complex and of paramount importance to overall brain function. Thus, understanding the regulation of enzymes involved in formation and disposal of glutamate and related metabolites is crucial to understanding glutamate metabolism. Glutamate dehydrogenase (GDH) is a pivotal enzyme that links amino acid metabolism and TCA cycle activity in brain and other tissues. The allosteric regulation of GDH has been extensively studied and characterized. Less is known about the influence of lipid modifications on GDH activity, and the participation of GDH in transient heteroenzyme complexes (metabolons) that can greatly influence metabolism by altering kinetic parameters and lead to channeling of metabolites. This review summarizes evidence for palmitoylation and acylation of GDH, information on protein binding, and information regarding the participation of GDH in transient heteroenzyme complexes. Recent studies suggest that a number of other proteins can bind to GDH altering activity and overall metabolism. It is likely that these modifications and interactions contribute additional levels of regulation of GDH activity and glutamate metabolism. PMID:21771624

  5. Determination of beta-hydroxyacyl CoA-dehydrogenase activity in meat by electrophoretically mediated microanalysis.

    PubMed

    Vallejo-Cordoba, Belinda; Mazorra-Manzano, Miguel A; González-Córdova, Aarón F

    2003-01-01

    The combined use of an in-tube enzyme assay and capillary electrophoresis for determining beta-hydroxyacyl CoA-dehydrogenase (beta-HADH) activity in meat was investigated. Beta-HADH is a significant mitochondrial enzyme in food muscle; thus, the determination of its activity is important in food analysis. The enzymatic assay and the separation of the reaction products were carried out by electrophoretically mediated microanalysis (EMMA) using a plug-plug reaction mode at variable potential. For the quantification of beta-HADH activity, the rate of conversion of reduced beta-nicotinamide adenine dinucleotide (NADH) to beta-nicotinamide adenine dinucleotide (NAD+) was calculated by determining NAD+ at 260 nm. A calibration curve for NAD+ concentration versus normalized areas showed a highly significant (p < 0.001) linear relationship (R2 = 0.993). Accurate quantification of beta-HADH activity was achieved since on-line monitoring allowed us to account for the NAD+ produced from NADH degradation by applying a correction factor. An average reaction time of 0.66 +/- 0.06 sec was determined for a protein concentration in the range of 0.1-0.5 mg protein/mL. The assay was reproducible since coefficients of variation of less than 6.2% were calculated for triplicate analyses. PMID:14596340

  6. Unexpected Histone H3 Tail-clipping Activity of Glutamate Dehydrogenase*

    PubMed Central

    Mandal, Papita; Verma, Naveen; Chauhan, Sakshi; Tomar, Raghuvir S.

    2013-01-01

    Clipping of histone tails has been reported in several organisms. However, the significance and regulation of histone tail clipping largely remains unclear. According to recent discoveries H3 clipping has been found to be involved in regulation of gene expression and chromatin dynamics. Earlier we had provided evidence of tissue-specific proteolytic processing of histone H3 in White Leghorn chicken liver nuclei. In this study we identify a novel activity of glutamate dehydrogenase (GDH) as a histone H3-specific protease in chicken liver tissue. This protease activity is regulated by divalent ions and thiol-disulfide conversion in vitro. GDH specifically clips H3 in its free as well as chromatin-bound form. Furthermore, we have found an inhibitor that inhibits the H3-clipping activity of GDH. Like previously reported proteases, GDH too may have the potential to regulate/modulate post-translational modifications of histone H3 by removing the N-terminal residues of the histone. In short, our findings identify an unexpected proteolytic activity of GDH specific to histone H3 that is regulated by redox state, ionic concentrations, and a cellular inhibitor in vitro. PMID:23673664

  7. Pyruvate dehydrogenase activity and quantity decreases after coronary artery bypass grafting: a prospective observational study

    PubMed Central

    Andersen, Lars W.; Liu, Xiaowen; Peng, Teng J.; Giberson, Tyler A.; Khabbaz, Kamal R.; Donnino, Michael W.

    2014-01-01

    Introduction Pyruvate dehydrogenase (PDH) is a key gatekeeper enzyme in aerobic metabolism. The main purpose of this study was to determine if PDH activity is affected by major stress in the form of coronary artery bypass grafting (CABG) which has previously been used as a model of critical illness. Methods We conducted a prospective, observational study of patients undergoing CABG at an urban, tertiary care hospital. We included adult patients undergoing CABG with or without concomitant valve surgery. Measurements of PDH activity and quantity and thiamine were obtained prior to surgery, at the completion of surgery, and 6 hours post-surgery. Results Fourteen patients were enrolled (age: 67 ± 10 years, 21 % female). Study subjects had a mean 41.7 % (SD: 27.7) reduction in PDH activity after surgery and a mean 32.0% (SD: 31.4) reduction 6 hours after surgery (p < 0.001). Eight patients were thiamine deficient (≤ 7 nmol/L) after surgery compared to none prior to surgery (p = 0.002). Thiamine level was a significantly associated with PDH quantity at all time points (p = 0.01). Post-surgery lactate levels were inversely correlated with post-surgery thiamine levels (r = −0.58 and p = 0.04). Conclusion The stress of major surgery causes decreased PDH activity and quantity, and depletion of thiamine levels. PMID:25526377

  8. Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling

    PubMed Central

    Gomes, Katia M.S.; Campos, Juliane C.; Bechara, Luiz R.G.; Queliconi, Bruno; Lima, Vanessa M.; Disatnik, Marie-Helene; Magno, Paulo; Chen, Che-Hong; Brum, Patricia C.; Kowaltowski, Alicia J.; Mochly-Rosen, Daria; Ferreira, Julio C.B.

    2014-01-01

    Aims We previously demonstrated that pharmacological activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) protects the heart against acute ischaemia/reperfusion injury. Here, we determined the benefits of chronic activation of ALDH2 on the progression of heart failure (HF) using a post-myocardial infarction model. Methods and results We showed that a 6-week treatment of myocardial infarction-induced HF rats with a selective ALDH2 activator (Alda-1), starting 4 weeks after myocardial infarction at a time when ventricular remodelling and cardiac dysfunction were present, improved cardiomyocyte shortening, cardiac function, left ventricular compliance and diastolic function under basal conditions, and after isoproterenol stimulation. Importantly, sustained Alda-1 treatment showed no toxicity and promoted a cardiac anti-remodelling effect by suppressing myocardial hypertrophy and fibrosis. Moreover, accumulation of 4-hydroxynonenal (4-HNE)-protein adducts and protein carbonyls seen in HF was not observed in Alda-1-treated rats, suggesting that increasing the activity of ALDH2 contributes to the reduction of aldehydic load in failing hearts. ALDH2 activation was associated with improved mitochondrial function, including elevated mitochondrial respiratory control ratios and reduced H2O2 release. Importantly, selective ALDH2 activation decreased mitochondrial Ca2+-induced permeability transition and cytochrome c release in failing hearts. Further supporting a mitochondrial mechanism for ALDH2, Alda-1 treatment preserved mitochondrial function upon in vitro aldehydic load. Conclusions Selective activation of mitochondrial ALDH2 is sufficient to improve the HF outcome by reducing the toxic effects of aldehydic overload on mitochondrial bioenergetics and reactive oxygen species generation, suggesting that ALDH2 activators, such as Alda-1, have a potential therapeutic value for treating HF patients. PMID:24817685

  9. Asp295 stabilizes the active-site loop structure of pyruvate dehydrogenase, facilitating phosphorylation of Ser292 by pyruvate dehydrogenase-kinase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed an invitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thaliana a2b2-hetero tetrameric pyruvate dehydrogenase (E1) plus A.thaliana E1-kinase (AtPDK). Upon addition of MgATP...

  10. Persistence of the effect of insulin on pyruvate dehydrogenase activity in rat white and brown adipose tissue during the preparation and subsequent incubation of mitochondria.

    PubMed

    Denton, R M; McCormack, J G; Marshall, S E

    1984-01-15

    Increases in the amount of the active non-phosphorylated form of pyruvate dehydrogenase in rat epididymal adipose tissue, as a result of incubation with insulin, persist not only during the preparation of mitochondria but also during subsequent incubation of coupled mitochondria in the presence of respiratory substrates. No effect on insulin was found if the hormone was added directly to mitochondria in the presence or absence of added plasma membranes. Concentrations of several possible regulators of pyruvate dehydrogenase kinase (ATP, ADP, NADH, NAD+, acetyl-CoA, CoA and potassium) were measured in rat epididymal-adipose-tissue mitochondria incubated under conditions where differences in pyruvate dehydrogenase activity persist as a result of insulin action. No alterations were found, and it is suggested that inhibition of the kinase is not the principal means by which insulin activates pyruvate dehydrogenase. The intramitochondrial concentration of magnesium was also unaffected. Differences in pyruvate dehydrogenase activity in interscapular brown adipose tissue associated with manipulation of plasma insulin concentrations of cold-adapted rats were also shown to persist during the preparation and subsequent incubation of mitochondria in the presence or absence of GDP. It is pointed out that the persistence of the effect of insulin on pyruvate dehydrogenase in incubated mitochondria will facilitate the recognition of the mechanism of this action of the hormone. Evidence that the short-term action of insulin involves an increase in pyruvate dehydrogenase phosphate phosphatase activity rather than inhibition of that of pyruvate dehydrogenase kinase is discussed. PMID:6320807

  11. Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity

    PubMed Central

    Leopold, Jane A.; Dam, Aamir; Maron, Bradley A.; Scribner, Anne W.; Liao, Ronglih; Handy, Diane E.; Stanton, Robert C.; Pitt, Bertram; Loscalzo, Joseph

    2013-01-01

    Hyperaldosteronism is associated with impaired vascular reactivity; however, the mechanism by which aldosterone promotes endothelial dysfunction remains unknown. Glucose-6-phosphate dehydrogenase (G6pd), the principal source of Nadph, modulates vascular function by limiting oxidant stress to preserve bioavailable nitric oxide (NO•). In these studies, we show that aldosterone (10−9-10−7 mol/l) decreases endothelial G6pd expression and activity in vitro resulting in increased oxidant stress and decreased cGMP levels similar to what is observed in G6pd-deficient cells. Aldosterone decreases G6pd expression by protein kinase A activation to increase expression of Crem, which interferes with Creb binding to the G6pd promoter. In vivo, infusion of aldosterone decreases vascular G6pd expression and impairs vascular reactivity. These effects are abrogated by spironolactone or vascular gene transfer of G6pd. These studies demonstrate that aldosterone induces a G6pd-deficient phenotype to impair endothelial function; aldosterone antagonism or gene transfer of G6pd improves vascular reactivity by restoring G6pd activity. PMID:17273168

  12. Myricetin is a novel inhibitor of human inosine 5'-monophosphate dehydrogenase with anti-leukemia activity.

    PubMed

    Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang; Lu, Weiqiang; Huang, Jin

    2016-09-01

    Human inosine 5'-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC50 values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. PMID:27378425

  13. 2-Butanol and butanone production in Saccharomyces cerevisiae through combination of a B12 dependent dehydratase and a secondary alcohol dehydrogenase using a TEV-based expression system.

    PubMed

    Ghiaci, Payam; Norbeck, Joakim; Larsson, Christer

    2014-01-01

    2-Butanol and its chemical precursor butanone (methyl ethyl ketone--MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuteri), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions. PMID:25054226

  14. Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure.

    PubMed

    Anderson, Nickolas A; Tobimatsu, Yuki; Ciesielski, Peter N; Ximenes, Eduardo; Ralph, John; Donohoe, Bryon S; Ladisch, Michael; Chapple, Clint

    2015-08-01

    Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content. PMID:26265762

  15. Characterization of two novel alcohol short-chain dehydrogenases/reductases from Ralstonia eutropha H16 capable of stereoselective conversion of bulky substrates.

    PubMed

    Magomedova, Zalina; Grecu, Andreea; Sensen, Christoph W; Schwab, Helmut; Heidinger, Petra

    2016-03-10

    Biocatalysis has significant advantages over organic synthesis in the field of chiral molecule production and several types of stereoselective enzymes are already in use in industrial biotechnology. However, there is still a high demand for new enzymes capable of transforming bulky molecules with sufficient operability. In order to reveal novel high-potential biocatalysts, the complete genome of the β-proteobacterium Ralstonia eutropha H16 was screened for potential short-chain dehydrogenases/reductases (SDRs). We were able to identify two (S)-enantioselective SDRs named A5 and B3. These showed clear preference towards long-chain and aromatic secondary alcohols, aldehydes and ketones, with diaryl diketone benzil as one of the best substrates. In addition the phylogenetic analysis of all enzyme types, which are known to facilitate benzil reduction, revealed at least two separate evolutionary clusters. Our results indicate the biotechnological potential of SDRs A5 and B3 for the production of chiral compounds with potential commercial value. PMID:26812656

  16. 2-Butanol and Butanone Production in Saccharomyces cerevisiae through Combination of a B12 Dependent Dehydratase and a Secondary Alcohol Dehydrogenase Using a TEV-Based Expression System

    PubMed Central

    Ghiaci, Payam; Norbeck, Joakim; Larsson, Christer

    2014-01-01

    2-Butanol and its chemical precursor butanone (methyl ethyl ketone – MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuterii), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions. PMID:25054226

  17. Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure

    PubMed Central

    Anderson, Nickolas A.; Tobimatsu, Yuki; Ciesielski, Peter N.; Ximenes, Eduardo; Ralph, John; Donohoe, Bryon S.; Ladisch, Michael; Chapple, Clint

    2015-01-01

    Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content. PMID:26265762

  18. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    SciTech Connect

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; Westpheling, Janet; Elkins, James G.

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important component in making biofuels production from lignocellulosic biomass feasible.

  19. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    DOE PAGESBeta

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; Westpheling, Janet; Elkins, James G.

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important componentmore » in making biofuels production from lignocellulosic biomass feasible.« less

  20. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers

    PubMed Central

    Nemeria, Natalia S.; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-01-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4′-aminopyrimidine N1′ atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu571, Glu235, and Glu237) and Arg606 resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu235 makes no direct contact with the cofactor. The role of the conserved Glu571 residue in both catalysis and cofactor orientation is revealed by the combined results for the first time. PMID:20106967

  1. Validation of the in vivo assessment of pyruvate dehydrogenase activity using hyperpolarised 13C MRS.

    PubMed

    Atherton, Helen J; Schroeder, Marie A; Dodd, Michael S; Heather, Lisa C; Carter, Emma E; Cochlin, Lowri E; Nagel, Simon; Sibson, Nicola R; Radda, George K; Clarke, Kieran; Tyler, Damian J

    2011-02-01

    Many diseases of the heart are characterised by changes in substrate utilisation, which is regulated in part by the activity of the enzyme pyruvate dehydrogenase (PDH). Consequently, there is much interest in the in vivo evaluation of PDH activity in a range of physiological and pathological states to obtain information on the metabolic mechanisms of cardiac diseases. Hyperpolarised [1-(13)C]pyruvate, detected using MRS, is a novel technique for the noninvasive evaluation of PDH flux. PDH flux has been assumed to directly reflect in vivo PDH activity, although to date this assumption remains unproven. Control animals and animals undergoing interventions known to modulate PDH activity, namely high fat feeding and dichloroacetate infusion, were used to investigate the relationship between in vivo hyperpolarised MRS measurements of PDH flux and ex vivo measurements of PDH enzyme activity (PDH(a)). Further, the plasma concentrations of pyruvate and other important metabolites were evaluated following pyruvate infusion to assess the metabolic consequences of pyruvate infusion during hyperpolarised MRS experiments. Hyperpolarised MRS measurements of PDH flux correlated significantly with ex vivo measurements of PDH(a), confirming that PDH activity influences directly the in vivo flux of hyperpolarised pyruvate through cardiac PDH. The maximum plasma concentration of pyruvate reached during hyperpolarised MRS experiments was approximately 250 µM, equivalent to physiological pyruvate concentrations reached during exercise or with dietary interventions. The concentrations of other metabolites, including lactate, glucose and β-hydroxybutyrate, did not vary during the 60 s following pyruvate infusion. Hence, during the 60-s data acquisition period, metabolism was minimally affected by pyruvate infusion. PMID:20799252

  2. Stabilization of a formate dehydrogenase by covalent immobilization on highly activated glyoxyl-agarose supports.

    PubMed

    Bolivar, Juan M; Wilson, Lorena; Ferrarotti, Susana Alicia; Fernandez-Lafuente, Roberto; Guisan, Jose M; Mateo, Cesar

    2006-03-01

    Formate dehydrogenase (FDH) is a stable enzyme that may be readily inactivated by the interaction with hydrophobic interfaces (e.g., due to strong stirring). This may be avoided by immobilizing the enzyme on a porous support by any technique. Thus, even if the enzyme is going to be used in an ultra-membrane reactor, the immobilization presents some advantages. Immobilization on supports activated with bromocianogen, polyethylenimine, glutaraldehyde, etc., did not promote any stabilization of the enzyme under thermal inactivation. However, the immobilization of FDH on highly activated glyoxyl agarose has permitted increasing the enzyme stability against any distorting agent: pH, T, organic solvent, etc. The time of support-enzyme reaction, the temperature of immobilization, and the activation of the support need to be optimized to get the optimal stability-activity properties. Optimized biocatalyst retained 50% of the offered activity and became 50 times more stable at high temperature and neutral pH. Moreover, the quaternary structure of this dimeric enzyme becomes stabilized by immobilization under optimized conditions. Thus, at acidic pH (conditions where the subunit dissociation is the first step in the enzyme inactivation), the immobilization of both subunits of the enzyme on glyoxyl-agarose has allowed the enzyme to be stabilized by hundreds of times. Moreover, the optimal temperature of the enzyme has been increased (even by 10 degrees C at pH 4.5). Very interestingly, the activity with NAD(+)-dextran was around 60% of that observed with free cofactor. PMID:16529396

  3. Mitochondrial Dihydrolipoyl Dehydrogenase Activity Shapes Photosynthesis and Photorespiration of Arabidopsis thaliana.

    PubMed

    Timm, Stefan; Wittmiß, Maria; Gamlien, Sabine; Ewald, Ralph; Florian, Alexandra; Frank, Marcus; Wirtz, Markus; Hell, Rüdiger; Fernie, Alisdair R; Bauwe, Hermann

    2015-07-01

    Mitochondrial dihydrolipoyl dehydrogenase (mtLPD; L-protein) is an integral component of several multienzyme systems involved in the tricarboxylic acid (TCA) cycle, photorespiration, and the degradation of branched-chain α-ketoacids. The majority of the mtLPD present in photosynthesizing tissue is used for glycine decarboxylase (GDC), necessary for the high-flux photorespiratory glycine-into-serine conversion. We previously suggested that GDC activity could be a signal in a regulatory network that adjusts carbon flux through the Calvin-Benson cycle in response to photorespiration. Here, we show that elevated GDC L-protein activity significantly alters several diagnostic parameters of cellular metabolism and leaf gas exchange in Arabidopsis thaliana. Overexpressor lines displayed markedly decreased steady state contents of TCA cycle and photorespiratory intermediates as well as elevated NAD(P)(+)-to-NAD(P)H ratios. Additionally, increased rates of CO2 assimilation, photorespiration, and plant growth were observed. Intriguingly, however, day respiration rates remained unaffected. By contrast, respiration was enhanced in the first half of the dark phase but depressed in the second. We also observed enhanced sucrose biosynthesis in the light in combination with a lower diel magnitude of starch accumulation and breakdown. These data thus substantiate our prior hypothesis that facilitating flux through the photorespiratory pathway stimulates photosynthetic CO2 assimilation in the Calvin-Benson cycle. They furthermore suggest that this regulation is, at least in part, dependent on increased light-capture/use efficiency. PMID:26116608

  4. Evaluation on the inhibition of pyrrol-2-yl ethanone derivatives to lactate dehydrogenase and anticancer activities

    NASA Astrophysics Data System (ADS)

    Lu, Na-Na; Weng, Zhao-Yue; Chen, Qiu-Yun; Boison, Daniel; Xiao, Xin-Xin; Gao, Jing

    2016-08-01

    Lactate dehydrogenase A (LDH-A) is a potentially important metabolic target for the inhibition of the highly activated glycolysis pathway in cancer cells. In order to develop bifunctional compounds as inhibitor of LDH-A and anticancer agents, two pyrrol-2-yl methanone (or ethanone) derivatives (PM1 and PM2) were synthesized and evaluated as inhibitors of LDH-A based on the enzyme assay and cell assay by spectroscopy analysis. Fluorescence and CD spectra results demonstrated that both the change of second structure of LDH-A and the affinity interaction for compounds to LDH-A gave great effect on the activity of LDH-A. In particular, low concentration of compounds (1 μμ-25 μμ) could change the level of pyruvate in cancer cells. Moreover, the in vitro assay results demonstrated that pyrrol-2-yl ethanone derivatives can inhibit the proliferation of cancer cells. Therefore, pyrrol-2-yl ethanone derivatives (PM2) can be both LDH-A inhibitor and anticancer agents.

  5. Rotenone Decreases Intracellular Aldehyde Dehydrogenase Activity: Implications for the Pathogenesis of Parkinson Disease

    PubMed Central

    Goldstein, David S.; Sullivan, Patti; Cooney, Adele; Jinsmaa, Yunden; Kopin, Irwin J.; Sharabi, Yehonatan

    2015-01-01

    Repeated systemic administration of the mitochondrial complex I inhibitor rotenone produces a rodent model of Parkinson disease (PD). Mechanisms of relatively selective rotenone-induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the “catecholaldehyde hypothesis,” buildup of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F-labeled catechols were measured in PC12 cells incubated with rotenone (0-1000 nM, 180 minutes), without or with F-dopamine (2 μM) to track vesicular uptake and catecholamine metabolism. Rotenone dose-dependently increased DOPAL, F-DOPAL, and 3,4-dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD+ was supplied, whereas the direct-acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective rotenone-induced toxicity in dopaminergic neurons. PMID:25645689

  6. Hypoxic repression of pyruvate dehydrogenase activity is necessary for metabolic reprogramming and growth of model tumours

    PubMed Central

    Golias, Tereza; Papandreou, Ioanna; Sun, Ramon; Kumar, Bhavna; Brown, Nicole V.; Swanson, Benjamin J.; Pai, Reetesh; Jaitin, Diego; Le, Quynh-Thu; Teknos, Theodoros N.; Denko, Nicholas C.

    2016-01-01

    Tumour cells fulfil the bioenergetic and biosynthetic needs of proliferation using the available environmental metabolites. Metabolic adaptation to hypoxia causes decreased mitochondrial function and increased lactate production. This work examines the biological importance of the hypoxia-inducible inhibitory phosphorylations on the pyruvate dehydrogenase E1α subunit. Pancreatic cancer cell lines were genetically manipulated to alter the net phosphorylation of PDH E1α through reduced kinase expression or enhanced phosphatase expression. The modified cells were tested for hypoxic changes in phosphorylated E1α, mitochondrial metabolism and growth as xenografted tumours. Even though there are four PDHK genes, PDHK1 is essential for inhibitory PDH phosphorylation of E1α at serine 232, is partially responsible for modification of serines 293 and 300, and these phosphorylations are necessary for model tumour growth. In order to determine the clinical relevance, a cohort of head and neck cancer patient biopsies was examined for phosphorylated E1α and expression of PDHK1. Patients with detectable 232 phosphorylation or expression of PDHK1 tend to have worse clinical outcome. These data show that PDHK1 activity is unique and non-redundant in the family of PHDK enzymes and a PDHK1 specific inhibitor would therefore have anti-cancer activity with reduced chance of side effects from inhibition of other PDHKs. PMID:27498883

  7. Hypoxic repression of pyruvate dehydrogenase activity is necessary for metabolic reprogramming and growth of model tumours.

    PubMed

    Golias, Tereza; Papandreou, Ioanna; Sun, Ramon; Kumar, Bhavna; Brown, Nicole V; Swanson, Benjamin J; Pai, Reetesh; Jaitin, Diego; Le, Quynh-Thu; Teknos, Theodoros N; Denko, Nicholas C

    2016-01-01

    Tumour cells fulfil the bioenergetic and biosynthetic needs of proliferation using the available environmental metabolites. Metabolic adaptation to hypoxia causes decreased mitochondrial function and increased lactate production. This work examines the biological importance of the hypoxia-inducible inhibitory phosphorylations on the pyruvate dehydrogenase E1α subunit. Pancreatic cancer cell lines were genetically manipulated to alter the net phosphorylation of PDH E1α through reduced kinase expression or enhanced phosphatase expression. The modified cells were tested for hypoxic changes in phosphorylated E1α, mitochondrial metabolism and growth as xenografted tumours. Even though there are four PDHK genes, PDHK1 is essential for inhibitory PDH phosphorylation of E1α at serine 232, is partially responsible for modification of serines 293 and 300, and these phosphorylations are necessary for model tumour growth. In order to determine the clinical relevance, a cohort of head and neck cancer patient biopsies was examined for phosphorylated E1α and expression of PDHK1. Patients with detectable 232 phosphorylation or expression of PDHK1 tend to have worse clinical outcome. These data show that PDHK1 activity is unique and non-redundant in the family of PHDK enzymes and a PDHK1 specific inhibitor would therefore have anti-cancer activity with reduced chance of side effects from inhibition of other PDHKs. PMID:27498883

  8. Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis.

    PubMed

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)(+) dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP(+) yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  9. Aldehyde Dehydrogenase Activity Identifies a Population of Human Skeletal Muscle Cells With High Myogenic Capacities

    PubMed Central

    Vauchez, Karine; Marolleau, Jean-Pierre; Schmid, Michel; Khattar, Patricia; Chapel, Alain; Catelain, Cyril; Lecourt, Séverine; Larghéro, Jérôme; Fiszman, Marc; Vilquin, Jean-Thomas

    2009-01-01

    Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM), umbilical cord blood (UCB), and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study, we have identified ALDH+ cells within human skeletal muscles, and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH, Aldefluor, identified brightly stained (ALDHbr) cells with low side scatter (SSClo), in enzymatically dissociated muscle biopsies, thereafter abbreviated as SMALD+ (for skeletal muscle ALDH+) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD+/CD34− and SMALD+/CD34+ cells. These sub-populations did not initially express endothelial (CD31), hematopoietic (CD45), and myogenic (CD56) markers. Upon sorting, however, whereas SMALD+/CD34+ cells developed in vitro as a heterogeneous population of CD56− cells able to differentiate in adipoblasts, the SMALD+/CD34− fraction developed in vitro as a highly enriched population of CD56+ myoblasts able to form myotubes. Moreover, only the SMALD+/CD34− population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. PMID:19738599

  10. Mitochondrial Dihydrolipoyl Dehydrogenase Activity Shapes Photosynthesis and Photorespiration of Arabidopsis thaliana

    PubMed Central

    Timm, Stefan; Wittmiß, Maria; Gamlien, Sabine; Ewald, Ralph; Florian, Alexandra; Frank, Marcus; Wirtz, Markus; Hell, Rüdiger; Fernie, Alisdair R.; Bauwe, Hermann

    2015-01-01

    Mitochondrial dihydrolipoyl dehydrogenase (mtLPD; L-protein) is an integral component of several multienzyme systems involved in the tricarboxylic acid (TCA) cycle, photorespiration, and the degradation of branched-chain α-ketoacids. The majority of the mtLPD present in photosynthesizing tissue is used for glycine decarboxylase (GDC), necessary for the high-flux photorespiratory glycine-into-serine conversion. We previously suggested that GDC activity could be a signal in a regulatory network that adjusts carbon flux through the Calvin-Benson cycle in response to photorespiration. Here, we show that elevated GDC L-protein activity significantly alters several diagnostic parameters of cellular metabolism and leaf gas exchange in Arabidopsis thaliana. Overexpressor lines displayed markedly decreased steady state contents of TCA cycle and photorespiratory intermediates as well as elevated NAD(P)+-to-NAD(P)H ratios. Additionally, increased rates of CO2 assimilation, photorespiration, and plant growth were observed. Intriguingly, however, day respiration rates remained unaffected. By contrast, respiration was enhanced in the first half of the dark phase but depressed in the second. We also observed enhanced sucrose biosynthesis in the light in combination with a lower diel magnitude of starch accumulation and breakdown. These data thus substantiate our prior hypothesis that facilitating flux through the photorespiratory pathway stimulates photosynthetic CO2 assimilation in the Calvin-Benson cycle. They furthermore suggest that this regulation is, at least in part, dependent on increased light-capture/use efficiency. PMID:26116608

  11. Modulation of glyceraldehyde-3-phosphate dehydrogenase activity by surface functionalized quantum dots.

    PubMed

    Ghosh, Srabanti; Ray, Manju; Das, Mahua Rani; Chakrabarti, Adrita; Khan, Ali Hossain; Sarma, D D; Acharya, Somobrata

    2014-03-21

    Enzymatic regulation is a fast and reliable diagnosis tool via identification and design of inhibitors for modulation of enzyme function. Previous reports on quantum dots (QDs)-enzyme interactions reveal a protein-surface recognition ability leading to promising applications in protein stabilization, protein delivery, bio-sensing and detection. However, the direct use of QDs to control enzyme inhibition has never been revealed to date. Here we show that a series of biocompatible surface-functionalized metal-chalcogenide QDs can be used as potent inhibitors for malignant cells through the modulation of enzyme activity, while normal cells remain unaffected. The in vitro activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme involved critically in the glycolysis of cancer cells, is inactivated selectively in a controlled way by the QDs at a significantly low concentration (nM). Cumulative kinetic studies delineate that the QDs undergo both reversible and irreversible inhibition mechanisms owing to the site-specific interactions, enabling control over the inhibition kinetics. These complementary loss-of-function probes may offer a novel route for rapid clinical diagnosis of malignant cells and biomedical applications. PMID:24496476

  12. Gossypol enantiomers potently inhibit human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase activities.

    PubMed

    Dong, Yaoyao; Mao, Baiping; Li, Linxi; Guan, Hongguo; Su, Ying; Li, Xiaoheng; Lian, Qingquan; Huang, Ping; Ge, Ren-Shan

    2016-03-01

    Gossypol is a chemical isolated from cotton seeds. It exists as (+) or (-) enantiomer and has been tested for anticancer, abortion-inducing, and male contraception. Progesterone formed from pregnenolone by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and estradiol from androgen by aromatase (CYP19A1) are critical for the maintenance of pregnancy or associated with some cancers. In this study we compared the potencies of (+)- and (-)-gossypol enantiomers in the inhibition of HSD3B1 and aromatase activities as well as progesterone and estradiol production in human placental JEG-3 cells. (+) Gossypol showed potent inhibition on human placental HSD3B1 with IC50 value of 2.3 μM, while (-) gossypol weakly inhibited it with IC50 over 100 μM. In contrast, (-) gossypol moderately inhibited CYP19A1 activity with IC50 of 23 μM, while (+) gossypol had no inhibition when the highest concentration (100 μM) was tested. (+) Gossypol enantiomer competitively inhibited HSD3B1 against substrate pregnenolone and showed mixed mode against NAD(+). (-) Gossypol competitively inhibited CYP19A1 against substrate testosterone. Gossypol enantiomers showed different potency related to their inhibition on human HSD3B1 and CYP19A1. Whether gossypol enantiomer is used alone or in combination relies on its application and beneficial effects. PMID:26709042

  13. Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

    PubMed Central

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P.; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V.

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  14. Evaluation on the inhibition of pyrrol-2-yl ethanone derivatives to lactate dehydrogenase and anticancer activities.

    PubMed

    Lu, Na-Na; Weng, Zhao-Yue; Chen, Qiu-Yun; Boison, Daniel; Xiao, Xin-Xin; Gao, Jing

    2016-08-01

    Lactate dehydrogenase A (LDH-A) is a potentially important metabolic target for the inhibition of the highly activated glycolysis pathway in cancer cells. In order to develop bifunctional compounds as inhibitor of LDH-A and anticancer agents, two pyrrol-2-yl methanone (or ethanone) derivatives (PM1 and PM2) were synthesized and evaluated as inhibitors of LDH-A based on the enzyme assay and cell assay by spectroscopy analysis. Fluorescence and CD spectra results demonstrated that both the change of second structure of LDH-A and the affinity interaction for compounds to LDH-A gave great effect on the activity of LDH-A. In particular, low concentration of compounds (1μμ-25μμ) could change the level of pyruvate in cancer cells. Moreover, the in vitro assay results demonstrated that pyrrol-2-yl ethanone derivatives can inhibit the proliferation of cancer cells. Therefore, pyrrol-2-yl ethanone derivatives (PM2) can be both LDH-A inhibitor and anticancer agents. PMID:27104676

  15. Acyl carrier protein-specific 4'-phosphopantetheinyl transferase activates 10-formyltetrahydrofolate dehydrogenase.

    PubMed

    Strickland, Kyle C; Hoeferlin, L Alexis; Oleinik, Natalia V; Krupenko, Natalia I; Krupenko, Sergey A

    2010-01-15

    4'-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4'-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G(1) arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions. PMID:19933275

  16. The role of cysteine in the alteration of bovine liver dihydrodiol dehydrogenase 3 activity.

    PubMed Central

    Nanjo, H; Adachi, H; Aketa, M; Mizoguchi, T; Nishihara, T; Terada, T

    1995-01-01

    Bovine liver NADP(+)-dependent dihydrodiol dehydrogenase (DD3) is extremely sensitive to SH reagents such as N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid). NEM produced time- and concentration-dependent inactivation of DD3 in a pseudo-first-order reaction manner. This inactivation was prevented by NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, 2',5'-ADP and 2'-AMP but not by substrates, NAD+, nicotinamide mononucleotide or 5'-ADP.DD3 was absorbed by an affinity column of thiopropyl-Sepharose 6B, but enzyme incubated with both NEM and NADP+ was not. Moreover, one [14C]NEM molecule was incorporated into a cysteine of DD3 in the presence, and two cysteines of DD3 in the absence, of NADP+. These results suggested that two cysteine residues were modified per enzyme molecule by NEM, one was protected by NADP+ and the other had no significant function for the enzyme activity. Two radiolabelled peptides (P1 and P2) produced by the digestion with lysyl endopeptidase of [14C]NEM-modified DD3 could be separated by reverse-phase HPLC. P1, which was radiolabelled by [14C]NEM only in the absence of NADP+, showed the following sequence; H2N-Tyr-Lys-Pro-Val-Xaa-Asn-Gln-Val-Glu- NEM.Cys-His-Pro-Tyr-Phe-Asn-Gln-Ser-Lys-COOH (Xaa indicates a possible cysteine residue). This sequence was very similar to that of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD/DD) (residues 184 to 201) and was also highly conserved in the aldo-keto reductase superfamily. The sequence of P2, which had radioactivity in both the absence and presence of NADP+, also contained an NEM-modified cysteine and was similar in sequence to the regions located in loop A of rat 3 alpha-HSD/DD. The present study suggests that P1, which may have a cysteine residue corresponding to Cys-193 of rat 3 alpha-HSD/DD, functions in the alteration of DD3 activity depending on the modulation of NADP(+)-binding ability through a thiol/disulphide exchange reaction similar to that of

  17. Cellular recovery of glyceraldehyde-3-phosphate dehydrogenase activity and thiol status after exposure to hydroperoxides

    SciTech Connect

    Brodie, A.E.; Reed, D.J. )

    1990-01-01

    The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.

  18. Functional response of the isolated, perfused normoxic heart to pyruvate dehydrogenase activation by dichloroacetate and pyruvate

    PubMed Central

    Jaimes, Rafael; Kuzmiak-Glancy, Sarah; Brooks, Daina M.; Swift, Luther M.; Posnack, Nikki G.; Kay, Matthew W.

    2015-01-01

    Dichloroacetate (DCA) and pyruvate activate pyruvate dehydrogenase (PDH), a key enzyme that modulates glucose oxidation and mitochondrial NADH production. Both compounds improve recovery after ischemia in isolated hearts. However, the action of DCA and pyruvate in normoxic myocardium is incompletely understood. We measured the effect of DCA and pyruvate on contraction, mitochondrial redox state, and intracellular calcium cycling in isolated rat hearts during normoxic perfusion. Normalized epicardial NADH fluorescence (nNADH) and left ventricular developed pressure (LVDP) were measured before and after administering DCA (5 mM) or pyruvate (5 mM). Optical mapping of Rhod-2AM was used to measure cytosolic calcium kinetics. DCA maximally activated PDH, increasing the ratio of active to total PDH from 0.48±0.03 to 1.03 ±0.03. Pyruvate sub-maximally activated PDH to a ratio of 0.75±0.02. DCA and pyruvate increased LVDP. When glucose was the only exogenous fuel, pyruvate increased nNADH by 21.4±2.9 % while DCA reduced nNADH by 21.4±6.1 % and elevated the incidence of premature ventricular contractions (PVCs). When lactate, pyruvate, and glucose were provided together as exogenous fuels, nNADH increased with DCA, indicating that PDH activation with glucose as the only exogenous fuel depletes PDH substrate. Calcium transient time-to-peak was shortened by DCA and pyruvate and SR calcium re-uptake was 30 % longer. DCA and pyruvate increased SR calcium load in myocyte monolayers. Overall, during normoxia when glucose is the only exogenous fuel, DCA elevates SR calcium, increases LVDP and contractility, and diminishes mitochondrial NADH. Administering DCA with plasma levels of lactate and pyruvate mitigates the drop in mitochondrial NADH and prevents PVCs. PMID:26142699

  19. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

    PubMed Central

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Zhang, Dong-Jie; Guo, Zhen-Hua; Guo, Yun-Yun; Zhu, Meng; Bai, Jing

    2015-01-01

    The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (−) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes. PMID:26580437

  20. Alcohol

    MedlinePlus

    ... as well as injuries, liver disease, heart disease, cancer, and other health problems. It can also cause problems at home, at work, and with friends. NIH: National Institute on Alcohol Abuse and Alcoholism

  1. Report of National Collegiate Alcohol Awareness Week Activities Fall 1987.

    ERIC Educational Resources Information Center

    Owens, Ann English; And Others

    This document presents a report of the education and prevention activities recognizing National Collegiate Alcohol Awareness (NCAA) Week undertaken at Central Michigan University in Mt. Pleasant, Michigan during October and early November, 1987. It begins with a brief review of the university's campus-wide programs, goals, and activities to reduce…

  2. Alcoholism.

    ERIC Educational Resources Information Center

    Caliguri, Joseph P., Ed.

    This extensive annotated bibliography provides a compilation of documents retreived from a computerized search of the ERIC, Social Science Citation Index, and Med-Line databases on the topic of alcoholism. The materials address the following areas of concern: (1) attitudes toward alcohol users and abusers; (2) characteristics of alcoholics and…

  3. Redox balance and mitochondrial glycerol phosphate dehydrogenase activity in trained rats.

    PubMed

    Casimiro-Lopes, Gustavo; Ramos, Dionizio; Sorenson, Martha M; Salerno, Verônica P

    2012-11-01

    Free radical production is increased in many disease states and during exercise, but in the latter the concurrent stimulation of the antioxidant defense system seems to protect the organism from excessive production of reactive oxygen species. Chronic exercise can exert negative effects on the activity of mitochondrial glycerol phosphate dehydrogenase (mGPdH), which may offer some explanation for the antioxidant effects of training, since this enzyme is a relevant producer of free radicals. To test this correlation, we compared mGPdH activity, two antioxidant defense markers and two markers of oxidative stress in sedentary and trained (Tr) rats. Training was through a swimming exercise 3 days a week. After 8 weeks, Tr rats lasted twice as long as controls in an acute swimming test with a 5% load. Forty-eight hours after the last exercise, the animals were killed to collect blood and tissues. Tr animals presented lower body weight and visceral fat mass with lower triglyceride content in visceral fat and plasma (p < 0.05). The specific activity of mGPdH in muscle mitochondria was reduced in Tr rats by 88% (p < 0.05). Total antioxidant capacity, lipid peroxidation and reduced glutathione (GSH) in liver and muscle were unaltered, while plasma GSH increased by 21% (p < 0.05). These data suggest a profile of successful redox equilibrium maintenance in Tr rats, with a potentially significant contribution from the lower level of mGPdH activity in muscle. This training protocol appears to be suitable for use in detailed studies of biochemical adaptations to oxidative stress. PMID:22391683

  4. Regulation of pyruvate dehydrogenase activity and citric acid cycle intermediates during high cardiac power generation

    PubMed Central

    Sharma, Naveen; Okere, Isidore C; Brunengraber, Daniel Z; McElfresh, Tracy A; King, Kristen L; Sterk, Joseph P; Huang, Hazel; Chandler, Margaret P; Stanley, William C

    2005-01-01

    A high rate of cardiac work increases citric acid cycle (CAC) turnover and flux through pyruvate dehydrogenase (PDH); however, the mechanisms for these effects are poorly understood. We tested the hypotheses that an increase in cardiac energy expenditure: (1) activates PDH and reduces the product/substrate ratios ([NADH]/[NAD+] and [acetyl-CoA]/[CoA-SH]); and (2) increases the content of CAC intermediates. Measurements were made in anaesthetized pigs under control conditions and during 15 min of a high cardiac workload induced by dobutamine (Dob). A third group was made hyperglycaemic (14 mm) to stimulate flux through PDH during the high work state (Dob + Glu). Glucose and fatty acid oxidation were measured with 14C-glucose and 3H-oleate. Compared with control, the high workload groups had a similar increase in myocardial oxygen consumption ( and cardiac power. Dob increased PDH activity and glucose oxidation above control, but did not reduce the [NADH]/[NAD+] and [acetyl-CoA]/[CoA-SH] ratios, and there were no differences between the Dob and Dob + Glu groups. An additional group was treated with Dob + Glu and oxfenicine (Oxf) to inhibit fatty acid oxidation: this increased [CoA-SH] and glucose oxidation compared with Dob; however, there was no further activation of PDH or decrease in the [NADH]/[NAD+] ratio. Content of the 4-carbon CAC intermediates succinate, fumarate and malate increased 3-fold with Dob, but there was no change in citrate content, and the Dob + Glu and Dob + Glu + Oxf groups were not different from Dob. In conclusion, compared with normal conditions, at high myocardial energy expenditure (1) the increase in flux through PDH is regulated by activation of the enzyme complex and continues to be partially controlled through inhibition by fatty acid oxidation, and (2) there is expansion of the CAC pool size at the level of 4-carbon intermediates that is largely independent of myocardial fatty acid oxidation. PMID:15550462

  5. Lack of Skeletal Muscle IL-6 Affects Pyruvate Dehydrogenase Activity at Rest and during Prolonged Exercise

    PubMed Central

    Gudiksen, Anders; Schwartz, Camilla Lindgren; Bertholdt, Lærke; Joensen, Ella; Knudsen, Jakob G.; Pilegaard, Henriette

    2016-01-01

    Pyruvate dehydrogenase (PDH) plays a key role in the regulation of skeletal muscle substrate utilization. IL-6 is produced in skeletal muscle during exercise in a duration dependent manner and has been reported to increase whole body fatty acid oxidation, muscle glucose uptake and decrease PDHa activity in skeletal muscle of fed mice. The aim of the present study was to examine whether muscle IL-6 contributes to exercise-induced PDH regulation in skeletal muscle. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (control) completed a single bout of treadmill exercise for 10, 60 or 120 min, with rested mice of each genotype serving as basal controls. The respiratory exchange ratio (RER) was overall higher (P<0.05) in IL-6 MKO than control mice during the 120 min of treadmill exercise, while RER decreased during exercise independent of genotype. AMPK and ACC phosphorylation also increased with exercise independent of genotype. PDHa activity was in control mice higher (P<0.05) at 10 and 60 min of exercise than at rest but remained unchanged in IL-6 MKO mice. In addition, PDHa activity was higher (P<0.05) in IL-6 MKO than control mice at rest and 60 min of exercise. Neither PDH phosphorylation nor acetylation could explain the genotype differences in PDHa activity. Together, this provides evidence that skeletal muscle IL-6 contributes to the regulation of PDH at rest and during prolonged exercise and suggests that muscle IL-6 normally dampens carbohydrate utilization during prolonged exercise via effects on PDH. PMID:27327080

  6. [Effect of temperature acclimation on glucose-6-phosphate dehydrogenase activity in various tissues of the Mullus barbatus ponticus].

    PubMed

    Rusinova, O S

    1997-01-01

    Acclimation of the Mullus barbatus ponticus to the temperature fall (from 16 to 8 degrees C) induces an increase in the activity of glucose-6-phosphate dehydrogenase both in summer and in autumn. These changes are more expressed in the liver and red muscle than in the white ones. The values of Km of glucose-6-phosphate (only in autumn) and NADP are also higher at 8 than at 16 degrees C. Actinomycin D does not prevent from cold activation of this enzyme in liver only in autumn, when the activity of glucose-6-phosphate dehydrogenase is 8-10 times higher than in summer, and in the red muscle in the both seasons, although this injection decreases the level of enzyme activity in this muscle. This injection does not change the values of Km for glucose-6-phosphate and NADP. It is supposed that the activation of glucose-6-phosphate dehydrogenase in the liver and red muscle during cold adaptation may be a result of change of substrate-binding ability and enzyme interaction with NADP without changes in the enzyme biosynthesis. PMID:9606826

  7. Estimating the number of viable animal cells in multi-well cultures based on their lactate dehydrogenase activities.

    PubMed

    Haslam, G; Wyatt, D; Kitos, P A

    2000-01-01

    A method is described for estimating the numbers ofanimal cells in multi-well culture by simultaneouslymeasuring the lactate dehydrogenase activity of thetotal culture and the medium. The difference betweenthe two reflects the dehydrogenase content of thecells and correlates with cell number. This LDH/INTmethod was tested using several lines of normal andtransformed suspension and adherent cells. Thelactate dehydrogenase activities of duplicate cultureswere determined colourimetrically using reactioncocktails containing lactate, NAD(+), diaphorase,and p-iodonitrotetrazolium violet, with and withoutTriton X-100. The difference in absorbance at 490 nm(DeltaA(490) = A(490, test) - A(490, control)) was used to calculate the lactatedehydrogenase activity of the total culture (+ Triton)and the medium (- Triton). The cellular lactatedehydrogenase activity (difference between totaland medium dehydrogenaseactivities) was proportional to viable cell number. The effects on cell growth of four metabolicinhibitors, sodium azide, actinomycin D,cycloheximide, and taxol, were determined using theLDH/INT assay and direct cell counting. The inhibitorconcentrations that caused decreases in the LDHactivity and cell number by 50% were similar. TheLDH/INT assay is quick and sensitive, works equallywell for adherent and suspension cells, and providesinformation about LDH activities of both the mediumand cells. It is particularly useful for screeningpotential cell-growth inhibitors. PMID:19002967

  8. Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity

    PubMed Central

    Capoccia, Benjamin J.; Robson, Debra L.; Levac, Krysta D.; Maxwell, Dustin J.; Hohm, Sarah A.; Neelamkavil, Marian J.; Bell, Gillian I.; Xenocostas, Anargyros; Link, Daniel C.; Piwnica-Worms, David; Nolta, Jan A.

    2009-01-01

    The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDHhi) activity, a progenitor cell function conserved between several lineages. BM ALDHhi cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDHhi cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDHhi cells, mice transplanted with purified ALDHhi cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDHhi cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDHhi cell engraftment stimulated endogenous revascularization. Thus, human BM ALDHhi cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans. PMID:19324906

  9. Not only osmoprotectant: betaine increased lactate dehydrogenase activity and L-lactate production in lactobacilli.

    PubMed

    Zou, Huibin; Wu, Zaiqiang; Xian, Mo; Liu, Hui; Cheng, Tao; Cao, Yujin

    2013-11-01

    Lactobacilli are commonly used for industrial production of polymer-grade L-lactic acid. The present study tested the Tween 80 alternative betaine in L-lactate production by several industrial lactobacilli. In flask fermentation of Lactobacillus casei, Lactobacillus buchneri, Lactobacillus lactis and Lactobacillus rhamnosus, the betaine addition (2g/l) had similar osmoprotectant effect with Tween 80 but had increased the lactate dehydrogenase activities and L-lactate production than Tween 80 control. In fed-batch fermentation of L. casei, betaine supplementation improved the L-lactic acid titer to 190 g/l, the yield to 95.5% (g L-lactic acid/g glucose), the productivity to 2.6g/lh, and the optical purity to 97.0%. The results demonstrated that supplementation of Tween 80 alternative - betaine in the fermentation medium is feasible for industrial l-lactic acid fermentation by lactobacilli, which will improve the lactate production but will not increase the process costs and modify any process conditions. PMID:24035452

  10. Estrogen-related receptor alpha modulates lactate dehydrogenase activity in thyroid tumors.

    PubMed

    Mirebeau-Prunier, Delphine; Le Pennec, Soazig; Jacques, Caroline; Fontaine, Jean-Fred; Gueguen, Naig; Boutet-Bouzamondo, Nathalie; Donnart, Audrey; Malthièry, Yves; Savagner, Frédérique

    2013-01-01

    Metabolic modifications of tumor cells are hallmarks of cancer. They exhibit an altered metabolism that allows them to sustain higher proliferation rates in hostile environment outside the cell. In thyroid tumors, the expression of the estrogen-related receptor α (ERRα), a major factor of metabolic adaptation, is closely related to the oxidative metabolism and the proliferative status of the cells. To elucidate the role played by ERRα in the glycolytic adaptation of tumor cells, we focused on the regulation of lactate dehydrogenases A and B (LDHA, LDHB) and the LDHA/LDHB ratio. Our study included tissue samples from 10 classical and 10 oncocytic variants of follicular thyroid tumors and 10 normal thyroid tissues, as well as samples from three human thyroid tumor cell lines: FTC-133, XTC.UC1 and RO82W-1. We identified multiple cis-acting promoter elements for ERRα, in both the LDHA and LDHB genes. The interaction between ERRα and LDH promoters was confirmed by chromatin immunoprecipitation assays and in vitro analysis for LDHB. Using knock-in and knock-out cellular models, we found an inverse correlation between ERRα expression and LDH activity. This suggests that thyroid tumor cells may reprogram their metabolic pathways through the up-regulation of ERRα by a process distinct from that proposed by the recently revisited Warburg hypothesis. PMID:23516535

  11. Alcohol and Alcohol Safety. Volume II of II. A Curriculum Manual for Elementary Level. A Teacher's Activities Guide.

    ERIC Educational Resources Information Center

    Finn, Peter; Platt, Judith

    This curriculum manual for the elementary school level is the first in a series on alcohol and alcohol safety and is designed as a teacher's activities guide. Each activity provided is a self-contained learning experience which requires varying numbers of class period and focuses on one or more objectives. Activities are numbered consecutively and…

  12. Regulation of the Activity of Lactate Dehydrogenases from Four Lactic Acid Bacteria*

    PubMed Central

    Feldman-Salit, Anna; Hering, Silvio; Messiha, Hanan L.; Veith, Nadine; Cojocaru, Vlad; Sieg, Antje; Westerhoff, Hans V.; Kreikemeyer, Bernd; Wade, Rebecca C.; Fiedler, Tomas

    2013-01-01

    Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs. PMID:23720742

  13. Cytotoxicity and characterization of an active metabolite of benzamide riboside, a novel inhibitor of IMP dehydrogenase.

    PubMed

    Gharehbaghi, K; Paull, K D; Kelley, J A; Barchi, J J; Marquez, V E; Cooney, D A; Monks, A; Scudiero, D; Krohn, K; Jayaram, H N

    1994-03-15

    Benzamide riboside exhibits significant cytotoxicity against a variety of human tumor cells in culture. On the basis of metabolic studies, the primary target of this drug's action appears to be IMP dehydrogenase (IMPDH). Incubation of human myelogenous leukemia K562 cells with an IC50 concentration of benzamide riboside resulted in an expansion of IMP pools (5.9-fold), with a parallel reduction in the concentration of GMP (90%), GDP (63%), GTP (55%) and dGTP (40%). On kinetic grounds, it was deduced that benzamide riboside (whose Ki versus IMPDH is 6.4 mM, while that of its 5'-monophosphate is 3.9 mM) or its 5'-monophosphate were unlikely to be responsible for inhibition of this target enzyme, IMPDH, since only micromolar concentrations of benzamide riboside were needed to exert potent inhibition of tumor-cell growth. Studies on the metabolism of this C-nucleoside have revealed the presence of a new peak eluting in the nucleoside diphosphate area on HPLC. Treatment of this peak with venom phosphodiesterase degraded it and concurrently nullified its inhibitory activity versus IMPDH; alkaline phosphatase, on the other hand, totally failed to digest the anabolite. These results suggest that the metabolite in question is the phosphodiester, benzamide adenine dinucleotide (BAD). Evidence that the inhibitor was an analog of NAD, wherein the nicotinamide moiety has been replaced by benzamide, was provided by both NMR and mass spectrometric analysis and confirmed by enzymatic synthesis. Further insight into the nature of the active principle was obtained from kinetic studies, which established that BAD competitively inhibited NAD utilization by partially purified IMPDH from K562 cells with a Ki of 0.118 microM. In concert, these studies establish that benzamide riboside exhibits potent antiproliferative activity by inhibiting IMPDH through BAD. PMID:7907081

  14. Enhanced aldehyde dehydrogenase activity by regenerating NAD+ in Klebsiella pneumoniae and implications for the glycerol dissimilation pathways.

    PubMed

    Li, Ying; Su, Mingyue; Ge, Xizhen; Tian, Pingfang

    2013-10-01

    In Klebsiella pneumoniae, 3-hydroxypropaldehyde is converted to 3-hydroxypropionic acid (3-HP) by aldehyde dehydrogenase (ALDH) with NAD(+) as a cofactor. Although ALDH overexpression stimulates the formation of 3-HP, it ceases to accumulate when NAD(+) is exhausted. Here we show that NAD(+) regeneration, together with ALDH overexpression, facilitates 3-HP production and benefits cell growth. Three distinct NAD(+)-regenerating enzymes: NADH oxidase and NADH dehydrogenase from K. pneumoniae, and glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae, were individually expressed in K. pneumoniae. In vitro assay showed their higher activities than that of the control, indicating their capacities to regenerate NAD(+). When they were respectively co-expressed with ALD4, an ALDH from S. cerevisiae, the activities of ALD4 were significantly elevated compared with that expressing ALD4 alone, suggesting that the regenerated NAD(+) enhanced the activity of ALD4. More interestingly, the growth rates of all NAD(+)-regenerating strains were prolonged in comparison with the control, indicating that NAD(+) regeneration stimulated cell proliferation. This study not only reveals the reliance of ALD4 activity on NAD(+) availability but also provides a method for regulating the dha regulon. PMID:23794046

  15. Isolation and characterization of a Chinese hamster ovary cell line deficient in fatty alcohol:NAD sup + oxidoreductase activity

    SciTech Connect

    James, P.F.; Lee, J. ); Rizzo, W.B.; Zoeller, R.A. )

    1990-08-01

    The authors have isolated a mutant Chinese hamster ovary cell line that is defective in long-chain fatty alcohol oxidation. The ability of the mutant cells to convert labeled hexadecanol to the corresponding fatty acid in vivo was reduced to 5% of the parent strain. Whole-cell homogenates from the mutant strain, FAA.1, were deficient in long-chain fatty alcohol:NAD{sup +} oxidoreductase activity, which catalyzes the oxidation of hexadecanol to hexadecanoic acid, although the intermediate fatty aldehyde was formed normally. A direct measurement of fatty aldehyde dehydrogenase showed that the FAA.1, strain was defective in this component of FAO activity. FAA.1 is a two-stage mutant that was selected from a previously described parent strain, ZR-82, which is defective in ether lipid biosynthesis and peroxisome assembly. Because of combined defects in ether lipid biosynthesis and fatty alcohol oxidation, the ability of the FAA.1 cells to incorporate hexadecanol into complex lipids was greatly impaired, resulting in a 60-fold increase in cellular fatty alcohol levels. As the FAO deficiency in FAA.1 cells appears to be identical to the defect associated with the human genetic disorder Sjoegren-Larsson syndrome, the FAA.1 cell line may be useful in studying this disease.

  16. Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars

    PubMed Central

    Bicho, Paul A.; Runnals, P. Lynn; Cunningham, J. Douglas; Lee, Hung

    1988-01-01

    The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates. PMID:16347538

  17. Effect of metals and other inorganic ions on soil microbial activity: soil dehydrogenase assay as a simple toxicity test

    SciTech Connect

    Rogers, J.E.; Li, S.W.

    1985-06-01

    The purpose of this report is to illustrate the utility of the soil dehydrogenase assay as an effective primary test for assessing the potential toxicity of chemicals to soil microbial activity. In this manuscript the authors describe their use of the soil dehydrogenase assay in determining the effects of a number of potential toxic inorganic ions on soil microbial activity. The ions include Cu/sup 2 +/, Mg/sup 2 +/, Ni/sup 2 +/, Zn/sup 2 +/, NH/sub 4//sup +/, Cd/sup 2 +/, Cr/sup 32/, F/sup -/, AsO/sub 4//sup 3 -/, BO/sub 3//sup 3 -/, and SO/sub 4//sup 2 -/.

  18. Structure-Activity Relationship Studies and Biological Characterization of Human NAD+-dependent 15-Hydroxyprostaglandin Dehydrogenase Inhibitors

    PubMed Central

    Duveau, Damien Y.; Yasgar, Adam; Wang, Yuhong; Hu, Xin; Kouznetsova, Jennifer; Brimacombe, Kyle R.; Jadhav, Ajit; Simeonov, Anton; Thomas, Craig J.; Maloney, David J.

    2014-01-01

    The structure-activity relationship (SAR) study of two chemotypes identified as inhibitors of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD, 15-PGDH) was conducted. Top compounds from both series displayed potent inhibition (IC50 <50 nM), demonstrate excellent selectivity towards HPGD and potently induce PGE2 production in A549 lung cancer and LNCaP prostate cancer cells. PMID:24360556

  19. Alcohol Dehydrogenase Protects against Endoplasmic Reticulum Stress-Induced Myocardial Contractile Dysfunction via Attenuation of Oxidative Stress and Autophagy: Role of PTEN-Akt-mTOR Signaling

    PubMed Central

    Pang, Jiaojiao; Fuller, Nathan D.; Hu, Nan; Barton, Linzi A.; Henion, Jeremy M.; Guo, Rui; Chen, Yuguo; Ren, Jun

    2016-01-01

    Background The endoplasmic reticulum (ER) plays an essential role in ensuring proper folding of the newly synthesized proteins. Aberrant ER homeostasis triggers ER stress and development of cardiovascular diseases. ADH is involved in catalyzing ethanol to acetaldehyde although its role in cardiovascular diseases other than ethanol metabolism still remains elusive. This study was designed to examine the impact of ADH on ER stress-induced cardiac anomalies and underlying mechanisms involved using cardiac-specific overexpression of alcohol dehydrogenase (ADH). Methods ADH and wild-type FVB mice were subjected to the ER stress inducer tunicamycin (1 mg/kg, i.p., for 48 hrs). Myocardial mechanical and intracellular Ca2+ properties, ER stress, autophagy and associated cell signaling molecules were evaluated. Results ER stress compromised cardiac contractile function (evidenced as reduced fractional shortening, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration and impaired intracellular Ca2+ homeostasis), oxidative stress and upregulated autophagy (increased LC3B, Atg5, Atg7 and p62), along with dephosphorylation of PTEN, Akt and mTOR, all of which were attenuated by ADH. In vitro study revealed that ER stress-induced cardiomyocyte anomaly was abrogated by ADH overexpression or autophagy inhibition using 3-MA. Interestingly, the beneficial effect of ADH was obliterated by autophagy induction, inhibition of Akt and mTOR. ER stress also promoted phosphorylation of the stress signaling ERK and JNK, the effect of which was unaffected by ADH transgene. Conclusions Taken together, these findings suggested that ADH protects against ER stress-induced cardiac anomalies possibly via attenuation of oxidative stress and PTEN/Akt/mTOR pathway-regulated autophagy. PMID:26807981

  20. Engineering of xylose reductase and overexpression of xylitol dehydrogenase and xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha

    PubMed Central

    Dmytruk, Olena V; Dmytruk, Kostyantyn V; Abbas, Charles A; Voronovsky, Andriy Y; Sibirny, Andriy A

    2008-01-01

    Background The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 – 48°C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved. Results Site-specific mutagenesis of H. polymorpha XYL1 gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of XYL1 gene under control of the strong constitutive HpGAP promoter was overexpressed on a Δxyl1 background. This resulted in significant increase in the KM for NADPH in the mutated xylose reductase (K341 → R N343 → D), while KM for NADH remained nearly unchanged. The recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on Δxyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48°C. A significant increase in ethanol productivity (up to 7.3 times) was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mg × (L × h)-1 as compared to 4.2 mg × (L × h)-1 for the wild type strain. Conclusion Recombinant strains of H. polymorpha engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation. PMID:18651968

  1. Cloning and overexpression of an NADH-dependent alcohol dehydrogenase gene from Candida maris involved in (R)-selective reduction of 5-acetylfuro[2,3-c]pyridine.

    PubMed

    Kawano, Shigeru; Yano, Miho; Hasegawa, Junzo; Yasohara, Yoshihiko

    2011-01-01

    5-((R)-1-Hydroxyethyl)-furo[2,3-c]pyridine ((R)-FPH) is a useful chiral building block in the synthesis of pharmaceuticals. An NADH-dependent alcohol dehydrogenase (AFPDH) isolated from Candida maris catalyzed the reduction of 5-acetylfuro[2,3-c]pyridine (AFP) to (R)-FPH with 100% enantiomeric excess. The gene encoding AFPDH was cloned and sequenced. The AFPDH gene comprises 762 bp and encodes a polypeptide of 27,230 Da. The deduced amino acid sequence showed a high degree of similarity to those of other members of the short-chain alcohol dehydrogenase superfamily. The AFPDH gene was overexpressed in Escherichia coli under the control of the lac promoter. One L of the cultured broth of an E. coli transformant coexpressing AFPDH and the glucose dehydrogenase (GDH) gene reduced 250 g of AFP to (R)-FPH in an organic solvent two-phase system. Under coupling with NADH regeneration using 2-propanol, 1 L of the cultured broth of an E. coli transformant expressing the AFPDH gene reduced 150 g of AFP to (R)-FPH. The optical purity of the (R)-FPH formed was 100% enantiomeric excess under both reaction conditions. PMID:22056439

  2. Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases: Synthesis, Structure–Activity Relationship, and Selective Antitumor Activity

    PubMed Central

    2015-01-01

    Mutations of isocitrate dehydrogenase 1 (IDH1) are frequently found in certain cancers such as glioma. Different from the wild-type (WT) IDH1, the mutant enzymes catalyze the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid (D2HG), leading to cancer initiation. Several 1-hydroxypyridin-2-one compounds were identified to be inhibitors of IDH1(R132H). A total of 61 derivatives were synthesized, and their structure–activity relationships were investigated. Potent IDH1(R132H) inhibitors were identified with Ki values as low as 140 nM, while they possess weak or no activity against WT IDH1. Activities of selected compounds against IDH1(R132C) were found to be correlated with their inhibitory activities against IDH1(R132H), as well as cellular production of D2HG, with R2 of 0.83 and 0.73, respectively. Several inhibitors were found to be permeable through the blood–brain barrier in a cell-based model assay and exhibit potent and selective activity (EC50 = 0.26–1.8 μM) against glioma cells with the IDH1 R132H mutation. PMID:25271760

  3. Inhibition of cancer-associated mutant isocitrate dehydrogenases: synthesis, structure-activity relationship, and selective antitumor activity.

    PubMed

    Liu, Zhen; Yao, Yuan; Kogiso, Mari; Zheng, Baisong; Deng, Lisheng; Qiu, Jihui J; Dong, Shuo; Lv, Hua; Gallo, James M; Li, Xiao-Nan; Song, Yongcheng

    2014-10-23

    Mutations of isocitrate dehydrogenase 1 (IDH1) are frequently found in certain cancers such as glioma. Different from the wild-type (WT) IDH1, the mutant enzymes catalyze the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid (D2HG), leading to cancer initiation. Several 1-hydroxypyridin-2-one compounds were identified to be inhibitors of IDH1(R132H). A total of 61 derivatives were synthesized, and their structure-activity relationships were investigated. Potent IDH1(R132H) inhibitors were identified with Ki values as low as 140 nM, while they possess weak or no activity against WT IDH1. Activities of selected compounds against IDH1(R132C) were found to be correlated with their inhibitory activities against IDH1(R132H), as well as cellular production of D2HG, with R(2) of 0.83 and 0.73, respectively. Several inhibitors were found to be permeable through the blood-brain barrier in a cell-based model assay and exhibit potent and selective activity (EC50 = 0.26-1.8 μM) against glioma cells with the IDH1 R132H mutation. PMID:25271760

  4. Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light.

    PubMed

    Ponc, R H; Carriazo, C S; Vermouth, N T

    2001-01-01

    During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were

  5. Cryopreservation of glucose-6-phosphate dehydrogenase activity inside red blood cells: developing a specimen repository in support of development and evaluation of glucose-6-phosphate dehydrogenase deficiency tests

    PubMed Central

    2013-01-01

    Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common human enzyme deficiency. It is characterized by abnormally low levels of G6PD activity. Individuals with G6PD deficiency are at risk of undergoing acute haemolysis when exposed to 8‒aminoquinoline-based drugs, such as primaquine. For this reason it is imperative to identify individuals with G6PD deficiency prior to administering these anti-malarial drugs. There is a need for the development and evaluation of point-of-care G6PD deficiency screening tests suitable for areas of the developing world where malarial treatments are frequently administered. The development and evaluation of new G6PD tests will be greatly assisted with the availability of specimen repositories. Methods Cryopreservation of erythrocytes was evaluated as a means to preserve G6PD activity. Blood specimens from 31 patients including ten specimens with normal G6PD activity, three with intermediate activity, and 18 with deficient activity were cryopreserved for up to six months. Results Good correlation in G6PD activity between fresh and cryopreserved specimens (R2 = 0.95). The cryopreserved specimens show an overall small drop in mean G6PD activity of 0.23 U/g Hb (P=0.23). Cytochemical staining showed that intracellular G6PD activity distribution within the red blood cell populations is preserved during cryopreservation. Furthermore, the mosaic composition of red blood cells in heterozygous women is also preserved for six months or more. The fluorescent spot and the BinaxNOW qualitative tests for G6PD deficiency also showed high concordance in G6PD status determination between cryopreserved specimens and fresh specimens. Conclusions A methodology for establishing a specimen panel for evaluation of G6PD tests is described. The approach is similar to that used in several malaria research facilities for the cryopreservation of parasites in clinical specimens and axenic cultures. Specimens stored in this manner will aid

  6. Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass.

    PubMed

    Mathieu, Yann; Piumi, Francois; Valli, Richard; Aramburu, Juan Carro; Ferreira, Patricia; Faulds, Craig B; Record, Eric

    2016-04-15

    Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol oxidases (AAO) and glucose oxidases, both of which are flavoenzymes. The recent study of thePycnoporus cinnabarinusCIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously inAspergillus niger Based on the enzyme's activity and specificity, it was assigned to the glucose dehydrogenases (PcinnabarinusGDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol oxidase fromPleurotus eryngii Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as aryl-alcohol:quinone oxidoreductases (AAQOs) with two enzymes possessing residual oxidase activity (PcAAQO2 andPcAAQO3). Structural comparison ofPcAAQO homology models withP. eryngiiAAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability ofPcAAQOs to reduce radical intermediates generated by laccase fromP. cinnabarinuswas demonstrated, shedding light on the ligninolytic system of this fungus. PMID:26873317

  7. Automatically-Activated Attitudes as Mechanisms for Message Effects: The Case of Alcohol Advertisements

    PubMed Central

    Goodall, Catherine E.; Slater, Michael D.

    2010-01-01

    Alcohol advertisements may influence impulsive, risky behaviors indirectly, via automatically-activated attitudes toward alcohol. Results from an experiment in which participants were exposed to either four alcohol advertisements, four control advertisements, or four drunk driving public service advertisements, suggested that alcohol advertisements had more measurable effects on implicit, than on explicit attitude measures. Moreover, there were significant indirect paths from alcohol advertisement exposure through automatically-activated alcohol attitudes on willingness to engage in risky alcohol-related behaviors, notably drinking and driving. A mechanism that may explain how these advertisements activate automatic, non-deliberative alcohol attitudes was investigated. Associative evidence was found supportive of an evaluative conditioning mechanism, in which positive responses to an alcohol advertisement may lead to more positive automatically-activated attitudes toward alcohol itself. PMID:21258609

  8. Mechanisms of mutagenesis: analysis through the use of alcohol dehydrogenase in Drosophila. Progress report, February 1984-January 1985

    SciTech Connect

    Sofer, W.H.

    1985-01-01

    A technique was developed for somatic transformation, making it possible to inject embryos with ADH genes in a variety of forms. A selection procedure was developed to chemically select strains of flies that are lacking beta - galactosidase activity. Two strains of Adh-negative flies whose lesions were generated with formaldehyde were characterized with respect to the ADH protein they manufacture, the rate at which they synthesize ADH, and the nature of the ADH mNRNA they make. (ACR)

  9. Escherichia coli Pyruvate Dehydrogenase Complex Is an Important Component of CXCL10-Mediated Antimicrobial Activity

    PubMed Central

    Schutte, Kirsten M.; Fisher, Debra J.; Burdick, Marie D.; Mehrad, Borna; Mathers, Amy J.; Mann, Barbara J.; Nakamoto, Robert K.

    2015-01-01

    Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains of Escherichia coli and report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enables E. coli to resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc, aceE. Resistance to CXCL10 also occurred following deletion of either aceF or lpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both the E. coli parent and aceE deletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine. PMID:26553462

  10. Pyruvate dehydrogenase complex of ascites tumour. Activation by AMP and other properties of potential significance in metabolic regulation.

    PubMed Central

    Lazo, P A; Sols, A

    1980-01-01

    1. AMP is an activator of the pyruvate dehydrogenase complex of the Ehrlich--Lettré ascites tumour, increasing its V up to 2-fold, with Ka of 40 microM at pH 7.4. This activation appears to be an allosteric effect on the decarboxylase subunit of the complex. 2. The pyruvate dehydrogenase complex has a Km for pyruvate within the range 17--36 microM depending on the pH, the optimum pH being approx. 7.4, with a V of approx. 0.1 unit/g of cells. The rate-limiting step is dependent on the transformation of the enzyme--substrate complex. The Km for CoA is 15 microM. The Km for NAD+ is 0.7 mM for both the complex and the lipoamide dehydrogenase. The complex is inhibited by acetyl-CoA competitively with CoA; the Ki is 60 microM. The lipoamide dehydrogenase is inhibited by NADH and NADPH competitively with NAD+, with Ki values of 80 and 90 microM respectively. In the reverse reaction the Km values for NADH and NADPH are essentially equal to their Ki values for the forward reaction, the V for the latter being 0.09 of that of the former. Hence the reaction rate of the complex in vivo is likely to be markedly affected by feedback isosteric inhibition by reduced nicotinamide nucleotides and possibly acetyl-CoA. PMID:7193456

  11. Circadian rhythm in plasma concentrations of gamma-hydroxybutyric acid in alcoholics.

    PubMed

    Hoes, M J; Vree, T B; Guelen, P J

    1981-08-01

    Gamma-hydroxybutyric acid (GHB) was orally administered to six alcoholics at 09.00 and 23.00 h. The plasma concentrations of GHB show a clear circadian pattern, the area under the curve in the daytime experiments being 61% of that in the night experiments. The significance of alcohol dehydrogenase, the catabolic enzyme of GHB, for the difference is discussed. It is concluded that, although the activity of alcohol dehydrogenase in alcoholics is quantitatively disturbed, it remains subject to physiologic circadian activation. PMID:7341501

  12. Virucidal activity of alcohol-based hand rub disinfectants.

    PubMed

    Iwasawa, Atsuo; Niwano, Yoshimi; Kohno, Masahiro; Ayaki, Masahiko

    2012-03-01

    We investigated the virucidal activity of commercially available alcohol-based hand rub products against coxsackievirus A7, B5, feline calicivirus F9, and human adenovirus type 3, type 7, type 8 using susceptible cell lines, Vero cells, CRFK cells, and A549 cells. Fifteen tested hand rub products were ethanol (EtOH) for disinfection (Japanese Pharmacopoeia Grade), two EtOH-based products, one povidone iode-containing product, one alkyldiaminoethylglycine hydrochloride-containing product, six benzalkonium chloride (BAK)-containing products, and four chlorohexidine gluconate (CHG)-containing products. Some active ingredients (BAK, benzetonium chloride, and CHG) were diluted with EtOH to make 0.5% and 0.2% solutions. Virus inactivation rates were calculated after contact with each hand rub product for 10 or 60 seconds. Of the hand rub products tested, only the povidone iode-based product showed antiviral activity superior to that of EtOH against all the strains. EtOH solutions of active ingredients (0.2% and 0.5%) also showed decreased antiviral activity. In conclusion, antiviral activity of all the commercially available alcohol-based hand rub products except that containing povidone idode was dependent on their active ingredients. The povidone idode-containing hand rub product kept its effectiveness even after the dilution with EtOH. Although alcohol-based hand rub products are convenient and suitable for the control of some microbes, they are not generally recommended for the control of viral infections. PMID:22451431

  13. Threonine-Insensitive Homoserine Dehydrogenase From Soybean: Genomic Organization, Kinetic Mechanism, and In vivo Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspartate kinase (AK) and homoserine dehydrogenase (HSD) functions as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback inhibited by threonine. In plants, the biochemical properties of AK and bifunctional AK-HSD enzymes have been characterized, but the mol...

  14. Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties, activity, and expression during tuber dormancy progression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in meristems isolated from field-g...

  15. Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

    SciTech Connect

    Doherty, R.E.; Haywood-Small, S.L.; Sisley, K.; Cross, N.A.

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Isolated ALDH{sup Hi} PC3 cells preferentially form primitive holoclone-type colonies. Black-Right-Pointing-Pointer Primitive holoclone colonies are predominantly ALDH{sup Lo} but contain rare ALDH{sup Hi} cells. Black-Right-Pointing-Pointer Holoclone-forming cells are not restricted to the ALDH{sup Hi} population. Black-Right-Pointing-Pointer ALDH phenotypic plasticity occurs in PC3 cells (ALDH{sup Lo} to ALDH{sup Hi} and vice versa). Black-Right-Pointing-Pointer ALDH{sup Hi} cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH{sup Lo} cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH{sup Hi} population, or whether all ALDH{sup Hi} cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH{sup Hi} cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH{sup Hi} cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH{sup Lo} population can develop ALDH{sup Hi} populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH{sup Hi} cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in

  16. The Identification of Perillyl Alcohol Glycosides with Improved Antiproliferative Activity

    PubMed Central

    2015-01-01

    A facile route to perillyl alcohol (POH) differential glycosylation and the corresponding synthesis of a set of 34 POH glycosides is reported. Subsequent in vitro studies revealed a sugar dependent antiproliferative activity and the inhibition of S6 ribosomal protein phosphorylation as a putative mechanism of representative POH glycosides. The most active glycoside from this cumulative study (4′-azido-d-glucoside, PG9) represents one of the most cytotoxic POH analogues reported to date. PMID:25121720

  17. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

    SciTech Connect

    Catalanotti, C.; Dubini, A.; Subramanian, V.; Yang, W. Q.; Magneschi, L.; Mus, F.; Seibert, M.; Posewitz, M. C.; Grossman, A. R.

    2012-02-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

  18. Activation of Melatonin Receptors Reduces Relapse-Like Alcohol Consumption.

    PubMed

    Vengeliene, Valentina; Noori, Hamid R; Spanagel, Rainer

    2015-12-01

    Melatonin is an endogenous synchronizer of biological rhythms and a modulator of physiological functions and behaviors of all mammals. Reduced levels of melatonin and a delay of its nocturnal peak concentration have been found in alcohol-dependent patients and rats. Here we investigated whether the melatonergic system is a novel target to treat alcohol addiction. Male Wistar rats were subjected to long-term voluntary alcohol consumption with repeated abstinence phases. Circadian drinking rhythmicity and patterns were registered with high temporal resolution by a drinkometer system and analyzed by Fourier analysis. We examined potential antirelapse effect of the novel antidepressant drug agomelatine. Given that agomelatine is a potent MT1 and MT2 receptor agonist and a 5-HT2C antagonist we also tested the effects of melatonin itself and the 5-HT2C antagonist SB242084. All drugs reduced relapse-like drinking. Agomelatine and melatonin administered at the end of the light phase led to very similar changes on all measures of the post-abstinence drinking behavior, suggesting that effects of agomelatine on relapse-like behavior are mostly driven by its melatonergic activity. Both drugs caused a clear phase advance in the diurnal drinking pattern when compared with the control vehicle-treated group and a reduced frequency of approaches to alcohol bottles. Melatonin given at the onset of the light phase had no effect on the circadian phase and very small effects on alcohol consumption. We conclude that targeting the melatonergic system in alcohol-dependent individuals can induce a circadian phase advance, which may restore normal sleep architecture and reduce relapse behavior. PMID:25994077

  19. NADP-dependent aromatic alcohol dehydrogenase in polyploid wheats and their diploid relatives. On the origin and phylogeny of polyploid wheats.

    PubMed

    Jaaska, V

    1978-09-01

    The three major isoenzymes of the NADP-dependent aromatic alcohol dehydrogenase (ADH-B), distinguished in polyploid wheats by means of polyacrylamide gel electrophoresis, are shown to be coded by homoeoalleles of the locus Adh-2 on short arms of chromosomes of the fifth homoeologous group. Essentially codominant expression of the Adh-2 homoeolleles of composite genomes was observed in young seedlings of hexaploid wheats (T. aestivum s.l.) and tetraploid wheats of the emmer group (T. turgidum s.l.), whereas only the isoenzyme characteristic of the A genome is present in the seedlings of the timopheevii-group tetraploids (T. timopheevii s.str. and T. araraticum).The slowest-moving B(3) isoenzyme of polyploid wheats, coded by the homoeoallele of the B genome, is characteristic of the diploid species Aegilops speltoides S.l., including both its awned and awnless forms, but was not encountered in Ae. bicornis, Ae. sharonensis and Ae. longissima. The last two diploids, as well as Ae. tauschii, Ae. caudata, Triticum monococcum s.str., T. boeoticum s.l. (incl. T. thaoudar) and T. urartu all shared a common isoenzyme coinciding electrophoretically with the band B(2) controlled by the A and D genome homoeoalleles in polyploid wheats. Ae. bicomis is characterized by the slowest isoenzyme, B(4), not found in wheats and in the other diploid Aegilops species studied.Two electrophoretic variants of ADH-B, B(1) and B(2), considered to be alloenzymes of the A genome homoeoallele, were observed in T. dicoccoides, T. dicoccon, T. turgidum. s.str. and T. spelta, whereas B(2) was characteristic of T. timopheevii s.l. and only B(1) was found in the remaining taxa of polyploid wheats. The isoenzyme B(1), not encountered among diploid species, is considered to be a mutational derivative which arose on the tetraploid level from its more ancestral form B(2) characteristic of diploid wheats.The implication of the ADH-B isoenzyme data to the problems of wheat phylogeny and gene evolution is

  20. Glycemic Allostasis during Mental Activities on Fasting in Non-alcohol Users and Alcohol Users with Different Durations of Abstinence.

    PubMed

    Welcome, Mo; Pereverzev, Va

    2014-09-01

    Glycemic allostasis is the process by which blood glucose stabilization is achieved through the balancing of glucose consumption rate and release into the blood stream under a variety of stressors. This paper reviews findings on the dynamics of glycemic levels during mental activities on fasting in non-alcohol users and alcohol users with different periods of abstinence. Referred articles for this review were searched in the databases of PubMed, Scopus, DOAJ and AJOL. The search was conducted in 2013 between January 20 and July 31. The following keywords were used in the search: alcohol action on glycemia OR brain glucose OR cognitive functions; dynamics of glycemia, dynamics of glycemia during mental activities; dynamics of glycemia on fasting; dynamics of glycemia in non-alcohol users OR alcohol users; glycemic regulation during sobriety. Analysis of the selected articles showed that glycemic allostasis during mental activities on fasting is poorly regulated in alcohol users even after a long duration of sobriety (1-4 weeks after alcohol consumption), compared to non-alcohol users. The major contributor to the maintenance of euglycemia during mental activities after the night's rest (during continuing fast) is gluconeogenesis. PMID:25364589

  1. Glycemic Allostasis during Mental Activities on Fasting in Non-alcohol Users and Alcohol Users with Different Durations of Abstinence

    PubMed Central

    Welcome, MO; Pereverzev, VA

    2014-01-01

    Glycemic allostasis is the process by which blood glucose stabilization is achieved through the balancing of glucose consumption rate and release into the blood stream under a variety of stressors. This paper reviews findings on the dynamics of glycemic levels during mental activities on fasting in non-alcohol users and alcohol users with different periods of abstinence. Referred articles for this review were searched in the databases of PubMed, Scopus, DOAJ and AJOL. The search was conducted in 2013 between January 20 and July 31. The following keywords were used in the search: alcohol action on glycemia OR brain glucose OR cognitive functions; dynamics of glycemia, dynamics of glycemia during mental activities; dynamics of glycemia on fasting; dynamics of glycemia in non-alcohol users OR alcohol users; glycemic regulation during sobriety. Analysis of the selected articles showed that glycemic allostasis during mental activities on fasting is poorly regulated in alcohol users even after a long duration of sobriety (1-4 weeks after alcohol consumption), compared to non-alcohol users. The major contributor to the maintenance of euglycemia during mental activities after the night's rest (during continuing fast) is gluconeogenesis. PMID:25364589

  2. 26 CFR 601.527 - Other provisions applied to representation in alcohol, tobacco, and firearms activities.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... alcohol, tobacco, and firearms activities. 601.527 Section 601.527 Internal Revenue INTERNAL REVENUE... Conference and Practice Requirements Requirements for Alcohol, Tobacco, and Firearms Activities § 601.527 Other provisions applied to representation in alcohol, tobacco, and firearms activities. The...

  3. 26 CFR 601.527 - Other provisions applied to representation in alcohol, tobacco, and firearms activities.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... alcohol, tobacco, and firearms activities. 601.527 Section 601.527 Internal Revenue INTERNAL REVENUE... Conference and Practice Requirements Requirements for Alcohol, Tobacco, and Firearms Activities § 601.527 Other provisions applied to representation in alcohol, tobacco, and firearms activities. The...

  4. 26 CFR 601.527 - Other provisions applied to representation in alcohol, tobacco, and firearms activities.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... alcohol, tobacco, and firearms activities. 601.527 Section 601.527 Internal Revenue INTERNAL REVENUE... Conference and Practice Requirements Requirements for Alcohol, Tobacco, and Firearms Activities § 601.527 Other provisions applied to representation in alcohol, tobacco, and firearms activities. The...

  5. 26 CFR 601.527 - Other provisions applied to representation in alcohol, tobacco, and firearms activities.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... alcohol, tobacco, and firearms activities. 601.527 Section 601.527 Internal Revenue INTERNAL REVENUE... Conference and Practice Requirements Requirements for Alcohol, Tobacco, and Firearms Activities § 601.527 Other provisions applied to representation in alcohol, tobacco, and firearms activities. The...

  6. 26 CFR 601.527 - Other provisions applied to representation in alcohol, tobacco, and firearms activities.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... alcohol, tobacco, and firearms activities. 601.527 Section 601.527 Internal Revenue INTERNAL REVENUE... Conference and Practice Requirements Requirements for Alcohol, Tobacco, and Firearms Activities § 601.527 Other provisions applied to representation in alcohol, tobacco, and firearms activities. The...

  7. Behaviour of mesotrione in maize and soil system and its influence on soil dehydrogenase activity.

    PubMed

    Kaczynski, Piotr; Lozowicka, Bozena; Hrynko, Izabela; Wolejko, Elzbieta

    2016-11-15

    The aim of this study was to investigate the dissipation of mesotrione and effect on dehydrogenase activity (DHA) in maize and soil system. The paper for the first time describes behaviour of this herbicide applied at various doses (separately or in mixture with other herbicide) in acidic and alkaline environment. The experiments were conducted using the method randomized blocks in four repetition cycles. Chemical application in seven variants at recommended doses of herbicide were performed. The sample preparation was performed by a modified QuEChERS method and the concentrations of mesotrione in maize and soil were determined by the liquid chromatography with tandem mass spectrometry (LC-MS/MS). The limit of detection was 0.0005mgkg(-1) and quantification 0.001mgkg(-1). The dissipation of mesotrione were described according to first-order (FO) kinetics equation with R(2) were between 0.8794 and 0.9934. The initial deposit of herbicide in soil and maize was higher in an acidic environment (0.06-0.18mgkg(-1)). A positive correlation between an alkaline pH and the rate of dissipation in soil was observed. The results showed that the time after which 50% (DT50) of substance has been degraded was different for both plant and soil. DT50 for soil was within the range 3.2-6.0days and 2.9-4.4days, for the maize 3.9-4.8days and 3.4-4.5days in an alkaline and an acidic environment, respectively. Concentration of mesotrione at applicable MRL level of 0.05mgkg(-1) in maize was achieved at 0.5-5.9days and at proposed MRL of 0.01mgkg(-1) at 8.8-15.8days. The results indicate that the application of mesotrione affected on DHA in the soil. One day after application this herbicide, concentration of DHA in soil was lower than in control plots, but after 21days was observed trend of increasing DHA. PMID:27492351

  8. Alcohol.

    ERIC Educational Resources Information Center

    Schibeci, Renato

    1996-01-01

    Describes the manufacturing of ethanol, the effects of ethanol on the body, the composition of alcoholic drinks, and some properties of ethanol. Presents some classroom experiments using ethanol. (JRH)

  9. Biological activity of pyrazole and imidazole-dehydroepiandrosterone derivatives on the activity of 17β-hydroxysteroid dehydrogenase.

    PubMed

    Cabeza, Marisa; Posada, Alejandro; Sánchez-Márquez, Araceli; Heuze, Yvonne; Moreno, Isabel; Soriano, Juan; Garrido, Mariana; Cortés, Francisco; Bratoeff, Eugene

    2016-01-01

    The enzyme type 5 17β-hydroxysteroid dehydrogenase 5 (17β-HSD5) catalyzes the transformation of androstenedione (4-dione) to testosterone (T) in the prostate. This metabolic pathway remains active in cancer patients receiving androgen deprivation therapy. Since physicians seek to develop advantageous and better new treatments to increase the average survival of these patients, we synthesized several different dehydroepiandrosterone derivatives. These compounds have a pyrazole or imidazole function at C-17 and an ester moiety at C-3 and were studied as inhibitors of 17β-HSD5. The kinetic parameters of this enzyme were determined for use in inhibition assays. Their pharmacological effect was also determined on gonadectomized hamsters treated with Δ(4)-androstenedione (4-dione) or testosterone (T) and/or the novel compounds. The results indicated that the incorporation of a heterocycle at C-17 induced strong 17β-HSD5 inhibition. These derivatives decreased flank organ diameter and prostate weight in castrated hamsters treated with T or 4-dione. Inhibition of 17β-HSD5 by these compounds could have therapeutic potential for the treatment of prostate cancer and benign prostatic hyperplasia. PMID:25690043

  10. Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells

    PubMed Central

    Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

    2015-01-01

    Background Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). Results/Methodology We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Conclusion Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease. PMID:25826140

  11. [Activity of NADP-dependent glyceraldehyde-phosphate dehydrogenase and phosphoenolpyruvate carboxylase in wheat leaves under water stress].

    PubMed

    Cherniad'ev, I I; Monakhova, O F

    2006-01-01

    The activities of NADP: glyceraldehyde-phosphate dehydrogenase (GAPDH), an enzyme complex comprising of phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.13), and phosphoenolpyruvate carboxylase (PEPK; EC 4.1.1.31) in seedlings and leaves of wheat (Triticum aestivum L.) plants of the cultivars Mironovskaya 808 and Lutescens 758 have been compared under conditions of normal water supply, water deficiency, and subsequent rehydration. GAPDH activity, which determines the carbohydrate route of photosynthetic metabolism at the initial stages, is decreased by water stress to a greater extent than that of PEPK, on the activity of which non-carbohydrate metabolic pathways depend. Pretreatment of seedlings and mature plants with natural (6-benzylaminopurine) and synthetic (tidiazuron, kartolin-2, and kartolin-4) cytokinins attenuates the loss of enzyme activities during drought and facilitates their recovery within the period of rehydration; both effects are underlain by augmentation of reparation processes. The relative intensification of non-carbohydrate pathways of photosynthetic metabolism, observed under conditions of water deficiency, is accompanied by an increase in the osmotic pressure of cell sap. Possible mechanisms of this protector effect of cytokinin preparations are discussed. PMID:16878554

  12. Mitigation of Radiation-Induced Dermatitis by Activation of Aldehyde Dehydrogenase 2 Using Topical Alda-1 in Mice1

    PubMed Central

    Ning, Shoucheng; Budas, Grant R.; Churchill, Eric N.; Chen, Che-Hong; Knox, Susan J.; Mochly-Rosen, Daria

    2012-01-01

    Ning, S., Budas, G. R., Churchill, E. N., Chen, C., Knox, S. J. and Mochly-Rosen, D. Mitigation of Radiation-Induced Dermatitis by Activation of Aldehyde Dehydrogenase 2 Using Topical Alda-1 in Mice. Radiation-induced dermatitis is a debilitating clinical problem in cancer patients undergoing cancer radiation therapy. It is also a possible outcome of exposure to high levels of radiation due to accident or hostile activity. We report that activation of aldehyde dehydrogenase 2 (ALDH2) enzymatic activity using the allosteric agonist, Alda-1, significantly reduced 4-hydroxynonenal adducts accumulation, delayed the onset of radiation dermatitis and substantially reduced symptoms in a clinically-relevant model of radiation-induced dermatitis. Importantly, Alda-1 did not radioprotect tumors in mice. Rather, it increased the sensitivity of the tumors to radiation therapy. This is the first report of reactive aldehydes playing a role in the intrinsic radiosensitivity of normal and tumor tissues. Our findings suggest that ALDH2 represents a novel target for the treatment of radiation dermatitis without reducing the benefit of radiotherapy. PMID:22404739

  13. The effect of five fasciolicides on malate dehydrogenase activity and mortality of Fasciola gigantica, Fasciolopsis buski and Paramphistomum explanatum.

    PubMed

    Probert, A J; Sharma, R K; Singh, K; Saxena, R

    1981-06-01

    The effect of oxyclozanide, hexachlorophene, nitroxynil, rafoxanide and diamphenethide on malate dehydrogenase activity of homogenates of Fasciola gigantica, Fasciolopsis buski and Paramphistomum explanatum was investigated. The ratio of oxaloacetate reduction to malate oxidation in homogenates of Fasciola gigantica, Fasciolopsis buski and P. explanatum was 4.5:1, 3.6:1 and 5.2:1 respectively. Oxyclozanide and rafoxanide at 10(-3) M inhibited enzyme activity by 100% in homogenates from all three species while hexachlorophene at 10(-3) M also caused 100% inhibition in homogenates from Fasciola gagantica and P. explanatum but only 65% of malate oxidation in Fasciolopsis buski homogenates. Nitroxynil at 10(-3) M produced 60% inhibition in F. buski homogenates yet had little effect at this concentration on preparations from the other species. Little inhibition was seen with diamphenethide, even at high concentrations. Rapid death of Fasicola gigantica and P. explanatum resulted in vitro when 10(-3) M oxyclozanide, hexachlorophene, nitroxynil or rafoxanide, were added to the incubation medium. Fasciolopsis buski was killed by 10(-3) M oxyclozanide but at this concentration the remaining compounds only caused reduced activity. Assay of malate dehydrogenase following drug treatment in vitro failed to show any appreciable reduction in enzyme activity in Fasciola gigantica and P. explanatum but oxyclozanide and hexachlorophene produced inhibition in Fasciolopsis buski. The mode of action of these compounds is discussed. PMID:7264272

  14. Purification and Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities.

    PubMed

    Adem, Sevki; Ciftci, Mehmet

    2016-06-01

    The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive. PMID:26820767

  15. A novel zinc-binding alcohol dehydrogenase 2 from Arachis diogoi, expressed in resistance responses against late leaf spot pathogen, induces cell death when transexpressed in tobacco.

    PubMed

    Kumar, Dilip; Rampuria, Sakshi; Singh, Naveen Kumar; Kirti, Pulugurtha B

    2016-03-01

    A novel zinc-binding alcohol dehydrogenase 2 (AdZADH2) was significantly upregulated in a wild peanut, Arachis diogoi treated with conidia of late leaf spot (LLS) pathogen, Phaeoisariopsis personata. This upregulation was not observed in a comparative analysis of cultivated peanut, which is highly susceptible to LLS. This zinc-binding alcohol dehydrogenase possessed a Rossmann fold containing NADB domain in addition to the MDR domain present in all previously characterized plant ADH genes/proteins. Transient over-expression of AdZADH2 under an estradiol inducible promoter (XVE) resulted in hypersensitive response (HR)-like cell death in tobacco leaf. However, the same level of cell death was not observed when the domains were transiently expressed individually. Cell death observed in tobacco was associated with overexpression of cell death related proteins, antioxidative enzymes such as SOD, CAT and APX and pathogenesis-related (PR) proteins. In A. diogoi, AdZADH2 expression was significantly upregulated in response to the plant signaling hormones salicylic acid, methyl jasmonate, and sodium nitroprusside. PMID:27047748

  16. Moderators of the Relationship between Physical Activity and Alcohol Consumption in College Students

    ERIC Educational Resources Information Center

    Buscemi, Joanna; Martens, Matthew P.; Murphy, James G.; Yurasek, Ali M.; Smith, Ashley E.

    2011-01-01

    Objective: Among college students, several studies have found a positive relationship between physical activity and alcohol use. The current study tested gender, Greek status, and ethnicity as potential moderators of the physical activity-alcohol use relationship. Participants: Participants were college freshmen (n = 310) endorsing alcohol/drug…

  17. 3D-QSAR Studies on a Series of Dihydroorotate Dehydrogenase Inhibitors: Analogues of the Active Metabolite of Leflunomide

    PubMed Central

    Li, Shun-Lai; He, Mao-Yu; Du, Hong-Guang

    2011-01-01

    The active metabolite of the novel immunosuppressive agent leflunomide has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. Self-organizing molecular field analysis (SOMFA), a simple three-dimensional quantitative structure-activity relationship (3D-QSAR) method is used to study the correlation between the molecular properties and the biological activities of a series of analogues of the active metabolite. The statistical results, cross-validated rCV2 (0.664) and non cross-validated r2 (0.687), show a good predictive ability. The final SOMFA model provides a better understanding of DHODH inhibitor-enzyme interactions, and may be useful for further modification and improvement of inhibitors of this important enzyme. PMID:21686163

  18. A low-molecular-mass protein from Methylococcus capsulatus (Bath) is responsible for the regulation of formaldehyde dehydrogenase activity in vitro.

    PubMed

    Tate, S; Dalton, H

    1999-01-01

    An 8.6 kDa protein, which the authors call a modifin, has been purified from Methylococcus capsulatus (Bath) and has been shown to alter the substrate specificity and kinetics of NAD+-linked formaldehyde dehydrogenase (FDH) isolated from the same organism. Purification methods for both the modifin and FDH are presented which reliably produced pure protein for further analysis. Analysis of the molecular mass and N-terminal sequence of both FDH and the modifin indicate that they are unique proteins and show no similarity to alcohol or aldehyde dehydrogenase enzymes isolated from methylotrophic bacteria. Substrate specificity studies demonstrated that FDH oxidized formaldehyde exclusively in the presence of the modifin; a diverse range of aldehydes and alcohols were oxidized by FDH in the absence of the modifin. No formaldehyde oxidation was detected in the absence of the modifin. Attempts to replace the modifin with glutathione or high concentrations of methanol to stimulate formaldehyde oxidation failed. With acetaldehyde as substrate, FDH showed standard Michaelis-Menten kinetics; interaction of FDH with the modifin using formaldehyde as substrate altered the kinetics of the reaction to sigmoidal. Kinetic analysis during turnover experiments indicated that the FDH may be associated with bound formaldehyde following enzyme isolation and that NAD may also be associated with the enzyme but in a form that is less tightly bound than found with the methanol dehydrogenase from Bacillus methanolicus. Data are presented which indicate that the modifin may play an important role in regulating formaldehyde concentration in vivo. PMID:10206695

  19. Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease

    PubMed Central

    Tsedensodnom, Orkhontuya; Vacaru, Ana M.; Howarth, Deanna L.; Yin, Chunyue; Sadler, Kirsten C.

    2013-01-01

    SUMMARY Secretory pathway dysfunction and lipid accumulation (steatosis) are the two most common responses of hepatocytes to ethanol exposure and are major factors in the pathophysiology of alcoholic liver disease (ALD). However, the mechanisms by which ethanol elicits these cellular responses are not fully understood. Recent data indicates that activation of the unfolded protein response (UPR) in response to secretory pathway dysfunction can cause steatosis. Here, we examined the relationship between alcohol metabolism, oxidative stress, secretory pathway stress and steatosis using zebrafish larvae. We found that ethanol was immediately internalized and metabolized by larvae, such that the internal ethanol concentration in 4-day-old larvae equilibrated to 160 mM after 1 hour of exposure to 350 mM ethanol, with an average ethanol metabolism rate of 56 μmol/larva/hour over 32 hours. Blocking alcohol dehydrogenase 1 (Adh1) and cytochrome P450 2E1 (Cyp2e1), the major enzymes that metabolize ethanol, prevented alcohol-induced steatosis and reduced induction of the UPR in the liver. Thus, we conclude that ethanol metabolism causes ALD in zebrafish. Oxidative stress generated by Cyp2e1-mediated ethanol metabolism is proposed to be a major culprit in ALD pathology. We found that production of reactive oxygen species (ROS) increased in larvae exposed to ethanol, whereas inhibition of the zebrafish CYP2E1 homolog or administration of antioxidants reduced ROS levels. Importantly, these treatments also blocked ethanol-induced steatosis and reduced UPR activation, whereas hydrogen peroxide (H2O2) acted as a pro-oxidant that synergized with low doses of ethanol to induce the UPR. Collectively, these data demonstrate that ethanol metabolism and oxidative stress are conserved mechanisms required for the development of steatosis and hepatic dysfunction in ALD, and that these processes contribute to ethanol-induced UPR activation and secretory pathway stress in hepatocytes. PMID

  20. Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants.

    PubMed

    Nunes-Nesi, Adriano; Carrari, Fernando; Lytovchenko, Anna; Smith, Anna M O; Loureiro, Marcelo Ehlers; Ratcliffe, R George; Sweetlove, Lee J; Fernie, Alisdair R

    2005-02-01

    Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the mitochondrial malate dehydrogenase gene in the antisense orientation and exhibiting reduced activity of this isoform of malate dehydrogenase show enhanced photosynthetic activity and aerial growth under atmospheric conditions (360 ppm CO2). In comparison to wild-type plants, carbon dioxide assimilation rates and total plant dry matter were up to 11% and 19% enhanced in the transgenics, when assessed on a whole-plant basis. Accumulation of carbohydrates and redox-related compounds such as ascorbate was also markedly elevated in the transgenics. Also increased in the transgenic plants was the capacity to use L-galactono-lactone, the terminal precursor of ascorbate biosynthesis, as a respiratory substrate. Experiments in which ascorbate was fed to isolated leaf discs also resulted in increased rates of photosynthesis providing strong indication for an ascorbate-mediated link between the energy-generating processes of respiration and photosynthesis. This report thus shows that the repression of this mitochondrially localized enzyme improves both carbon assimilation and aerial growth in a crop species. PMID:15665243

  1. The substrate tolerance of alcohol oxidases.

    PubMed

    Pickl, Mathias; Fuchs, Michael; Glueck, Silvia M; Faber, Kurt

    2015-08-01

    Alcohols are a rich source of compounds from renewable sources, but they have to be activated in order to allow the modification of their carbon backbone. The latter can be achieved via oxidation to the corresponding aldehydes or ketones. As an alternative to (thermodynamically disfavoured) nicotinamide-dependent alcohol dehydrogenases, alcohol oxidases make use of molecular oxygen but their application is under-represented in synthetic biotransformations. In this review, the mechanism of copper-containing and flavoprotein alcohol oxidases is discussed in view of their ability to accept electronically activated or non-activated alcohols and their propensity towards over-oxidation of aldehydes yielding carboxylic acids. In order to facilitate the selection of the optimal enzyme for a given biocatalytic application, the substrate tolerance of alcohol oxidases is compiled and discussed: Substrates are classified into groups (non-activated prim- and sec-alcohols; activated allylic, cinnamic and benzylic alcohols; hydroxy acids; sugar alcohols; nucleotide alcohols; sterols) together with suitable alcohol oxidases, their microbial source, relative activities and (stereo)selectivities. PMID:26153139

  2. Sirtuin 3 (SIRT3) protein regulates long-chain acyl-CoA dehydrogenase by deacetylating conserved lysines near the active site.

    PubMed

    Bharathi, Sivakama S; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E; Rardin, Matthew J; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W; Hirschey, Matthew D; Goetzman, Eric S

    2013-11-22

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

  3. Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

    PubMed Central

    Bharathi, Sivakama S.; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E.; Rardin, Matthew J.; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W.; Hirschey, Matthew D.; Goetzman, Eric S.

    2013-01-01

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

  4. The thiocarbamate disulphide drug, disulfiram induces osteopenia in rats by inhibition of osteoblast function due to suppression of acetaldehyde dehydrogenase activity.

    PubMed

    Mittal, Monika; Khan, Kainat; Pal, Subhashis; Porwal, Konica; China, Shyamsundar Pal; Barbhuyan, Tarun K; Baghel, Khemraj S; Rawat, Tara; Sanyal, Sabyasachi; Bhadauria, Smrati; Sharma, Vishnu L; Chattopadhyay, Naibedya

    2014-05-01

    Dithiocarbamates (DTC), a sulfhydryl group containing compounds, are extensively used by humans that include metam and thiram due to their pesticide properties, and disulfiram (DSF) as an alcohol deterrent. We screened these DTC in an osteoblast viability assay. DSF exhibited the highest cytotoxicity (IC50 488nM). Loss in osteoblast viability and proliferation was due to induction of apoptosis via G1 arrest. DSF treatment to osteoblasts reduced glutathione (GSH) levels and exogenous addition of GSH prevented DSF-induced reactive oxygen species generation and osteoblast apoptosis. DSF also inhibited osteoblast differentiation in vitro and in vivo, and the effect was associated with inhibition of aldehyde dehydrogenase (ALDH) activity. Out of various ALDH isozymes, osteoblasts expressed only ALDH2 and DSF downregulated its transcript as well as activity. Alda-1, a specific activator of ALDH2, stimulated osteoblast differentiation. Subcutaneous injection of DSF over the calvarium of new born rats reduced the differentiation phenotype of calvarial osteoblasts but increased the mRNA levels of Runx-2 and osteocalcin. DSF treatment at a human-equivalent dose of 30 mg/kg p.o. to adult Sprague Dawley rats caused trabecular osteopenia and suppressed the formation of mineralized nodule by bone marrow stromal cells. Moreover, DSF diminished bone regeneration at the fracture site. In growing rats, DSF diminished growth plate height, primary and secondary spongiosa, mineralized osteoid and trabecular strength. Substantial decreased bone formation was also observed in the cortical site of these rats. We conclude that DSF has a strong osteopenia inducing effect by impairing osteoblast survival and differentiation due to the inhibition of ALDH2 function. PMID:24496638

  5. Pyruvate Dehydrogenase Kinase-4 Structures Reveal a Metastable Open Conformation Fostering Robust Core-free Basal Activity

    SciTech Connect

    Wynn, R. Max; Kato, Masato; Chuang, Jacinta L.; Tso, Shih-Chia; Li, Jun; Chuang, David T.

    2008-10-21

    Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-{angstrom} crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, compared with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp{sup 394}-Trp{sup 395}) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core.

  6. Immunostimulating activity of polyhydric alcohol isolated from Taxus cuspidata.

    PubMed

    Lee, Choon Guen; Lee, Jisun; Lee, Da Gyung; Kim, Joo Won; Alnaeeli, Mawadda; Park, Yong Il; Park, Jae Kweon

    2016-04-01

    A polyhydric alcohol (PAL) was isolated from Taxus cuspidata and its immunostimulatory activities were assessed. The primary monosaccharide composition of the PAL was determined to be glucose, where HPAEC analysis showed no significant amount of any other sugars. However, glycerol and xylitol were identified as the main sugar alcohols. Fourier-transform infrared (FT-IR) analysis indicated that the purified PAL is a complex glycitol, which structurally contains significant amount of hydroxyl groups. MALDI-TOF mass spectroscopy also demonstrated that PAL is a complex glycitol built in hexose polymerization. Enzyme linked immunosorbent assay showed that the PAL stimulates the release of the proinflammatory cytokines TNF-α and IL-6 in a dose-dependent manner. Furthermore, treatment of RAW 264.7 cells with PAL for 24h remarkably increased the phosphorylation levels of ERK, p38 and JNK in a dose-dependent manner, whereas the total protein levels of ERK (t-ERK), p38 (t-p38) and JNK (t-JNK) remained unchanged. These results clearly demonstrate that PAL stimulates the immune response in RAW 264.7 cells through the activation of MAPKs (ERK, p38 and JNK) signaling pathway. To the best of our knowledge, this is the first study to demonstrate the primary structure and immune-stimulating activities of PAL from the fruit of T. cuspidata. PMID:26791584

  7. Phytochemical concentrations and biological activities of Sorghum bicolor alcoholic extracts.

    PubMed

    Dia, Vermont P; Pangloli, Philipus; Jones, Lynsey; McClure, Angela; Patel, Anjali

    2016-08-10

    Sorghum is an important cereal with reported health benefits. The objectives of this study were to measure the biological activities of alcoholic extracts of ten sorghum varieties and to determine the association between the color of the extracts and their biological activities. Variation on concentrations of bioactives among sorghum varieties was observed with ethanolic extracts giving higher concentrations than methanolic extracts. The color of the extracts significantly correlated with the concentrations of bioactives and with nitric oxide scavenging activity. Freeze-dried ethanol extract is more potent than freeze-dried methanol extract and caused cytotoxicity to A27801AP and PTX-10 OVCA with ED50 values of 0.69 and 1.29 mg mL(-1), respectively. Pre-treatment of OVCA with ethanol extract led to chemosensitization to paclitaxel and the proliferation and colony formation of OVCA cells were reduced by 14.7 to 44.6% and 36.4 to 40.1%, respectively. Sorghum is a potential source of colorants with health promoting properties. This is the first report on the capability of sorghum alcoholic extracts to cause cytotoxicity and chemosensitize ovarian cancer cells in vitro. PMID:27406291

  8. Bioinformatics based structural characterization of glucose dehydrogenase (gdh) gene and growth promoting activity of Leclercia sp. QAU-66

    PubMed Central

    Naveed, Muhammad; Ahmed, Iftikhar; Khalid, Nauman; Mumtaz, Abdul Samad

    2014-01-01

    Glucose dehydrogenase (GDH; EC 1.1. 5.2) is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p ≤ 0.05) promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities. PMID:25242947

  9. 20-alpha-Hydroxysteroid dehydrogenase from pseudopregnant rat ovary: obtention and characterization of a monoclonal antibody against the enzyme activity.

    PubMed

    De La Llosa-Hermier, M P; Nocart, M; Paly, J; Hermier, C

    1992-12-01

    The enzyme 20-alpha-hydroxysteroid dehydrogenase (20-alpha-HSD) was purified from pseudopregnant rat ovaries and used as antigen for the development of a monoclonal antibody by the hybridoma technique. Spleen cells of BALB/c mice immunized with purified 20-alpha-HSD were fused with SP2/0 mouse myeloma cells. Among the colonies of hybrid cells, one (designated mAb-HSD 11) was found to be secreting antibodies (IgM) able to inhibit 20-alpha-HSD activity. The antibody-secreting hybridome was amplified by ascitic fluid production and the monoclonal antibody purified by Bakerbond ABx procedure. Purified mAb-HSD 11 was able to inhibit 20-alpha-HSD activity in a dose-dependent manner. Studies of Michaelis constants of 20-alpha-HSD indicate that this monoclonal antibody increases the Km for 20-alpha-dihydroprogesterone and decreases the Vmax. PMID:1292619

  10. Bioinformatics based structural characterization of glucose dehydrogenase (gdh) gene and growth promoting activity of Leclercia sp. QAU-66.

    PubMed

    Naveed, Muhammad; Ahmed, Iftikhar; Khalid, Nauman; Mumtaz, Abdul Samad

    2014-01-01

    Glucose dehydrogenase (GDH; EC 1.1. 5.2) is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p ≤ 0.05) promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities. PMID:25242947

  11. Influence of hexaconazole, carbofuran and ethion on soil microflora and dehydrogenase activities in soil and intact cell.

    PubMed

    Kalam, A; Mukherjee, A K

    2001-01-01

    Total microbial count was highly affected (up to 61% at 1000 micrograms level) in presence of hexaconazole and persisted up to 21 days. Bacteria were more susceptible than actinomycetes. Carbofuran and ethion were moderately toxic to soil microflora. Inhibitory effects of all the three pesticides gradually decreased after 21 days as was evident by increase in total microbial count except in carbofuran. GDH activity in soil was also affected initially (up to 14 days) by all the three pesticides (60.3% in hexaconazole at 1000 micrograms level) and inhibition gradually decreased to zero except in carbofuran (15-20% toxicity persisted up to 35 days). GDH and LDH activity in presence of hexaconazole was strongly affected in intact cells of some standard culture of bacteria like Rhizobium sp. (host Dolichos sp., 32.1 and 72.5%), Bacillus subtilis Cohn (86.75 and 76.5%), Azotobacter sp. (36.9 and 55.4%) and B. sphaericus (67.6% GDH) respectively. Carbofuran inhibited the enzyme activity in B. subtilis (55.55 and 35.3%) and to some extent in B. sphaericus. Ethion moderately inhibited LDH activity in Rhodococcus sp. AK1 (17.1 and 33.3%), Rhizobium (27.6% LDH), E. coli HB 101 (34.2% LDH) as evidenced by formazan formation. From the result, it might be concluded that among the above three pesticides tested hexaconazole strongly inhibited the dehydrogenase system in bacteria including nitrogen fixing bacteria of soil and thus may affect soil fertility. It was concluded that hexaconazole was more toxic than ethion to dehydrogenase enzymes. PMID:11349536

  12. Activation of the nickel-deficient carbon monoxide dehydrogenase from Rhodospirillum rubrum: Kinetic characterization and reductant requirement

    SciTech Connect

    Ensign, S.A.; Campbell, M.J.; Ludden, P.W. )

    1990-02-27

    The requirements for and kinetics of the activation of the nickel-deficient (apo) CO dehydrogenase from Rhodospirillum rubrum by exogenous nickel have been investigated. The activation is strictly dependent upon the presence of a low-potential one-electron reductant. Sodium dithionite and reduced methylviologen are suitable reductants, whereas reduced indigo carmine and the two-electron reductants sodium borohydride, NADH, and dithiothreitol are ineffective in stimulating activation. The midpoint potential for activation was observed at approximately {minus}475 mV. The ability of a reductant to stimulate activation is correlated with the reduced state of the enzyme Fe{sub 4}-S{sub 4} centers. The activation follows apparent first-order kinetics in a saturable fashion, yielding a rate constant of 0.157 min{sup {minus}1} at saturating concentration of nickel. The initial rate at which the enzyme is activated by NiCl{sub 2} is also a saturable process, yielding a dissociation constant (K{sub D}) of 755 {mu}M for the initial association of nickel and enzyme. Cadmium(II), zinc(II), cobalt(II), and iron(II) are competitive inhibitors of nickel activation with inhibition constants of 2.44, 4.16, 175, and 349 {mu}M, respectively. Manganese(II), calcium(II), and magnesium(II) exhibit no inhibition of activation.

  13. Determination of Glutamate Dehydrogenase Activity and Its Kinetics in Mouse Tissues using Metabolic Mapping (Quantitative Enzyme Histochemistry)

    PubMed Central

    Botman, Dennis; Tigchelaar, Wikky

    2014-01-01

    Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)+ to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we assessed the effects of various glutamate concentrations in combination with either the coenzyme NAD+ or NADP+ on GDH activity in mouse liver cryostat sections using metabolic mapping. NAD+-dependent GDH Vmax was 2.5-fold higher than NADP+-dependent Vmax, whereas the Km was similar, 1.92 mM versus 1.66 mM, when NAD+ or NADP+ was used, respectively. With either coenzyme, Vmax was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a Ki of 12.2 and 3.95 for NAD+ and NADP+ used as coenzyme, respectively. NAD+- and NADP+-dependent GDH activities were examined in various mouse tissues. GDH activity was highest in liver and much lower in other tissues. In all tissues, the highest activity was found when NAD+ was used as a coenzyme. In conclusion, GDH activity in mice is highest in the liver with NAD+ as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 mM. PMID:25124006

  14. Ontogenetic changes in citrate synthase and lactate dehydrogenase activity in the jumping muscle of the American locust (Schistocerca americana).

    PubMed

    Kirkton, Scott D; Nyberg, Elizabeth T; Fox, Kristin M

    2011-10-01

    Intraspecific studies have repeatedly shown that muscle-specific oxidative enzyme activities scale negatively with body mass while muscle-specific glycolytic enzyme activities scale positively. However, most of these studies have not included juveniles. In this study, we examined how citrate synthase (CS, EC 2.3.3.1) and lactate dehydrogenase (LDH; EC 1.1.1.27) activity in the jumping muscle of Schistocerca americana grasshoppers varied with ontogeny across a 40-fold increase in body size. In contrast to the pattern observed when adult conspecifics are compared, we show that jumping muscle CS activity increased more than 2-fold from 2nd instars to adults, while jumping muscle LDH activity increased more than 5-fold. The increased LDH activity in older grasshoppers supports previous data that older grasshoppers have a reduced jumping endurance. The increased CS activity with age may help older grasshoppers efficiently produce aerobic ATP to bend cuticular springs for energy storage before a jump or alternatively recover from anaerobic metabolism after jumping. Metabolic changes in S. americana jumping muscle are similar to other developing taxa and highlight the importance of including juveniles within intraspecific studies. When compared to adults, juvenile locomotion may have increased selection pressure because of both greater energetic demands during growth and higher predation rates. PMID:21807111

  15. At5g50600 encodes a member of the short-chain dehydrogenase reductase superfamily with 11beta- and 17beta-hydroxysteroid dehydrogenase activities associated with Arabidopsis thaliana seed oil bodies.

    PubMed

    d'Andréa, S; Canonge, M; Beopoulos, A; Jolivet, P; Hartmann, M A; Miquel, M; Lepiniec, L; Chardot, T

    2007-02-01

    In a previous work, we presented evidence for the presence of a protein encoded by At5g50600 in oil bodies (OBs) from Arabidopsis thaliana [P. Jolivet, E. Roux, S. D'Andrea, M. Davanture, L. Negroni, M. Zivy, T. Chardot, Protein composition of oil bodies in Arabidopsis thaliana ecotype WS, Plant Physiol. Biochem. 42 (2004) 501-509]. Using specific antibodies and proteomic techniques, we presently confirm the existence of this protein, which is a member of the short-chain steroid dehydrogenase reductase superfamily. We have measured its activity toward various steroids (cholesterol, dehydroepiandrosterone, cortisol, corticosterone, estradiol, estrone) and NAD(P)(H), either within purified OBs or as a purified bacterially expressed chimera. Both enzymatic systems (OBs purified from A. thaliana seeds as well as the chimeric enzyme) exhibited hydroxysteroid dehydrogenase (HSD) activity toward estradiol (17beta-hydroxysteroid) with NAD+ or NADP+, NADP+ being the preferred cofactor. Low levels of activity were observed with cortisol or corticosterone (11beta-hydroxysteroids), but neither cholesterol nor DHEA (3beta-hydroxysteroids) were substrates, whatever the cofactor used. Similar activity profiles were found for both enzyme sources. Purified OBs were found to be also able to catalyze estrone reduction (17beta-ketosteroid reductase activity) with NADPH. The enzyme occurring in A. thaliana OBs can be classified as a NADP+-dependent 11beta-,17beta-hydroxysteroid dehydrogenase/17beta-ketosteroid reductase. This enzyme probably corresponds to AtHSD1, which is encoded by At5g50600. However, its physiological role and substrates still remain to be determined. PMID:17074428

  16. Peroxisome Proliferator-Activated Receptor γ Regulates Chronic Alcohol-Induced Alveolar Macrophage Dysfunction.

    PubMed

    Yeligar, Samantha M; Mehta, Ashish J; Harris, Frank L; Brown, Lou Ann S; Hart, C Michael

    2016-07-01

    Peroxisome proliferator-activated receptor (PPAR) γ is critical for alveolar macrophage (AM) function. Chronic alcohol abuse causes AM phagocytic dysfunction and susceptibility to respiratory infections by stimulating nicotinamide adenine dinucleotide oxidases (Nox), transforming growth factor-β1, and oxidative stress in the AM. Because PPARγ inhibits Nox expression, we hypothesized that alcohol reduces PPARγ, stimulating AM dysfunction. AMs were examined from: (1) patients with alcoholism or control patients; (2) a mouse model of chronic ethanol consumption; (3) PPARγ knockout mice; or (4) MH-S cells exposed to ethanol in vitro. Alcohol reduced AM PPARγ levels and increased Nox1, -2, and -4, transforming growth factor-β1, oxidative stress, and phagocytic dysfunction. Genetic loss of PPARγ recapitulated, whereas stimulating PPARγ activity attenuated alcohol-mediated alterations in gene expression and phagocytic function, supporting the importance of PPARγ in alcohol-induced AM derangements. Similarly, PPARγ activation in vivo reduced alcohol-mediated impairments in lung bacterial clearance. Alcohol increased levels of microRNA-130a/-301a, which bind to the PPARγ 3' untranslated region to reduce PPARγ expression. MicroRNA-130a/-301a inhibition attenuated alcohol-mediated PPARγ reductions and derangements in AM gene expression and function. Alcohol-induced Toll-like receptor 4 endocytosis was reversed by PPARγ activation. These findings demonstrate that targeting PPARγ provides a novel therapeutic approach for mitigating alcohol-induced AM derangements and susceptibility to lung infection. PMID:26677910

  17. The Protective Effects of Buzui on Acute Alcoholism in Mice

    PubMed Central

    Wen, Da-Chao; Gao, Shu-di; Hu, Xiao-yu; Yi, Cheng

    2016-01-01

    This study was designed to investigate the role of a traditional buzui recipe in anti-inebriation treatment. Buzui consists of Fructus Schisandrae Chinensis, Fructus Chebulae, Fructus Mume, Fructus Crataegi, Endothelium Corneum Gigeriae Galli, and Excrementum Bombycis. The buzui mixture was delivered by gavage, and ethanol was delivered subsequent to the final treatment. The effects of buzui on the righting reflex, inebriation rates, and the survival curve are depicted. Blood alcohol concentrations, alanine aminotransferase (ALT) levels, aspartate aminotransferase (AST) levels, and alkaline phosphatase (ALP) levels were recorded. The activities of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and superoxide dismutase (SOD), as well as malonaldehyde (MDA) levels, were also measured. Our results demonstrated that a traditional buzui recipe showed significant effects on promoting wakefulness and the prevention of acute alcohol intoxication, accelerating the metabolism of alcohol in the liver and reducing the oxidative damage caused by acute alcoholism. PMID:26884793

  18. Ergot alkaloid biosynthesis in Aspergillus fumigatus: conversion of chanoclavine-I to chanoclavine-I aldehyde catalyzed by a short-chain alcohol dehydrogenase FgaDH.

    PubMed

    Wallwey, Christiane; Matuschek, Marco; Li, Shu-Ming

    2010-02-01

    Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaDH has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaDH comprises 261 amino acids with a molecular mass of about 27.8 kDa and contains the conserved motifs of classical short-chain dehydrogenases/reductases (SDRs), but shares no worth mentioning sequence similarity with SDRs and other known proteins. The coding region of fgaDH consisting of two exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE60 and overexpressed in E. coli. The soluble tetrameric His(6)-FgaDH was purified to apparent homogeneity and characterized biochemically. It has been shown that FgaDH catalyzes the oxidation of chanoclavine-I in the presence of NAD(+) resulting in the formation of chanoclavine-I aldehyde, which was unequivocally identified by NMR and MS analyzes. Therefore, FgaDH functions as a chanoclavine-I dehydrogenase and represents a new group of short-chain dehydrogenases. K (M) values for chanoclavine-I and NAD(+) were determined at 0.27 and 1.1 mM, respectively. The turnover number was 0.38 s(-1). PMID:20039019

  19. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application. PMID:25743073

  20. Green Tea Polyphenols Control Dysregulated Glutamate Dehydrogenase in Transgenic Mice by Hijacking the ADP Activation Site

    SciTech Connect

    Li, Changhong; Li, Ming; Chen, Pan; Narayan, Srinivas; Matschinsky, Franz M.; Bennett, Michael J.; Stanley, Charles A.; Smith, Thomas J.

    2012-05-09

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic {beta}-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

  1. Novel Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Anti-malarial Activity in the Mouse Model

    SciTech Connect

    Booker, Michael L.; Bastos, Cecilia M.; Kramer, Martin L.; Barker, Jr., Robert H.; Skerlj, Renato; Sidhu, Amar Bir; Deng, Xiaoyi; Celatka, Cassandra; Cortese, Joseph F.; Guerrero Bravo, Jose E.; Crespo Llado, Keila N.; Serrano, Adelfa E.; Angulo-Barturen, Iñigo; Jiménez-Díaz, María Belén; Viera, Sara; Garuti, Helen; Wittlin, Sergio; Papastogiannidis, Petros; Lin, Jing-wen; Janse, Chris J.; Khan, Shahid M.; Duraisingh, Manoj; Coleman, Bradley; Goldsmith, Elizabeth J.; Phillips, Margaret A.; Munoz, Benito; Wirth, Dyann F.; Klinger, Jeffrey D.; Wiegand, Roger; Sybertz, Edmund

    2010-11-22

    Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED{sub 50} values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

  2. Locus coeruleus neuronal activity determines proclivity to consume alcohol in a selectively-bred line of rats that readily consumes alcohol.

    PubMed

    West, Charles H K; Boss-Williams, Katherine A; Ritchie, James C; Weiss, Jay M

    2015-11-01

    Sprague-Dawley rats selectively-bred for susceptibility to stress in our laboratory (Susceptible, or SUS rats) voluntarily consume large amounts of alcohol, and amounts that have, as shown here, pharmacological effects, which normal rats will not do. In this paper, we explore neural events in the brain that underlie this propensity to readily consume alcohol. Activity of locus coeruleus neurons (LC), the major noradrenergic cell body concentration in the brain, influences firing of ventral tegmentum dopaminergic cell bodies of the mesocorticolimbic system (VTA-DA neurons), which mediate rewarding aspects of alcohol. We tested the hypothesis that in SUS rats alcohol potently suppresses LC activity to markedly diminish LC-mediated inhibition of VTA-DA neurons, which permits alcohol to greatly increase VTA-DA activity and rewarding aspects of alcohol. Electrophysiological single-unit recording of LC and VTA-DA activity showed that in SUS rats alcohol decreased LC burst firing much more than in normal rats and as a result markedly increased VTA-DA activity in SUS rats while having no such effect in normal rats. Consistent with this, in a behavioral test for reward using conditioned place preference (CPP), SUS rats showed alcohol, given by intraperitoneal (i.p.) injection, to be rewarding. Next, manipulation of LC activity by microinfusion of drugs into the LC region of SUS rats showed that (a) decreasing LC activity increased alcohol intake and increasing LC activity decreased alcohol intake in accord with the formulation described above, and (b) increasing LC activity blocked both the rewarding effect of alcohol in the CPP test and the usual alcohol-induced increase in VTA-DA single-unit activity seen in SUS rats. An important ancillary finding in the CPP test was that an increase in LC activity was rewarding by itself, while a decrease in LC activity was aversive; consequently, effects of LC manipulations on alcohol-related reward in the CPP test were perhaps even

  3. Reprint of: Locus coeruleus neuronal activity determines proclivity to consume alcohol in a selectively-bred line of rats that readily consumes alcohol.

    PubMed

    West, Charles H K; Boss-Williams, Katherine A; Ritchie, James C; Weiss, Jay M

    2016-02-01

    Sprague-Dawley rats selectively-bred for susceptibility to stress in our laboratory (Susceptible, or SUS rats) voluntarily consume large amounts of alcohol, and amounts that have, as shown here, pharmacological effects, which normal rats will not do. In this paper, we explore neural events in the brain that underlie this propensity to readily consume alcohol. Activity of locus coeruleus neurons (LC), the major noradrenergic cell body concentration in the brain, influences firing of ventral tegmentum dopaminergic cell bodies of the mesocorticolimbic system (VTA-DA neurons), which mediate rewarding aspects of alcohol. We tested the hypothesis that in SUS rats alcohol potently suppresses LC activity to markedly diminish LC-mediated inhibition of VTA-DA neurons, which permits alcohol to greatly increase VTA-DA activity and rewarding aspects of alcohol. Electrophysiological single-unit recording of LC and VTA-DA activity showed that in SUS rats alcohol decreased LC burst firing much more than in normal rats and as a result markedly increased VTA-DA activity in SUS rats while having no such effect in normal rats. Consistent with this, in a behavioral test for reward using conditioned place preference (CPP), SUS rats showed alcohol, given by intraperitoneal (i.p.) injection, to be rewarding. Next, manipulation of LC activity by microinfusion of drugs into the LC region of SUS rats showed that (a) decreasing LC activity increased alcohol intake and increasing LC activity decreased alcohol intake in accord with the formulation described above, and (b) increasing LC activity blocked both the rewarding effect of alcohol in the CPP test and the usual alcohol-induced increase in VTA-DA single-unit activity seen in SUS rats. An important ancillary finding in the CPP test was that an increase in LC activity was rewarding by itself, while a decrease in LC activity was aversive; consequently, effects of LC manipulations on alcohol-related reward in the CPP test were perhaps even