Activation of liver alcohol dehydrogenase by glycosylation.
Ontology highlight
ABSTRACT:
SUBMITTER: Tsai CS
PROVIDER: S-EPMC1154095 | biostudies-other | 1983 Feb
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Daqu Alcohol dehydrogenase Raw sequence reads
| PRJNA759401 | ENA
Project description:A method for obtaining electrophoretically homogeneous rat liver alcohol dehydrogenase (EC 1.1.1.1) at a specific activity of 2-2.5 mumol/min per mg of protein is presented. Anti-sera prepared against the purified enzyme inhibit alcohol dehydrogenase by up to 75% and cause precipitation of virtually all the enzyme. The antisera were shown by immunoelectrophoresis of a partially purified liver homogenate to be specifically directed against alcohol dehydrogenase and were used to demonstrate that the alcohol dehydrogenases of rat brain and liver share common antigens. The total activity of alcohol dehydrogenase in rat brain homogenates is normally quite low, with as much as 10% of the total activity attributable to the activity in the blood contained within the brain; in cases of severe liver damage (induced experimentally with carbon tetrachloride) this contribution may rise to as much as 60%.
| S-EPMC1172622 | biostudies-other
Project description:During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2'-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme-NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ?1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.
| S-EPMC5518280 | biostudies-literature
Project description:The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 Å resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.
| S-EPMC3103598 | biostudies-literature
Project description:UNLABELLED:The important roles of retinols and their metabolites have recently been emphasized in the interactions between hepatic stellate cells (HSCs) and natural killer (NK) cells. Nevertheless, the expression and role of retinol metabolizing enzyme in both cell types have yet to be clarified. Thus, we investigated the expression of retinol metabolizing enzyme and its role in liver fibrosis. Among several retinol metabolizing enzymes, only alcohol dehydrogenase (ADH) 3 expression was detected in isolated HSCs and NK cells, whereas hepatocytes express all of them. In vitro treatment with 4-methylpyrazole (4-MP), a broad ADH inhibitor, or depletion of the ADH3 gene down-regulated collagen and transforming growth factor-?1 (TGF-?1) gene expression, but did not affect ?-smooth muscle actin gene expression in cultured HSCs. Additionally, in vitro, treatments with retinol suppressed NK cell activities, whereas inhibition of ADH3 enhanced interferon-? (IFN-?) production and cytotoxicity of NK cells against HSCs. In vivo, genetic depletion of the ADH3 gene ameliorated bile duct ligation- and carbon tetrachloride-induced liver fibrosis, in which a higher number of apoptotic HSCs and an enhanced activation of NK cells were detected. Freshly isolated HSCs from ADH3-deficient mice showed reduced expression of collagen and TGF-?1, but enhanced expression of IFN-? was detected in NK cells from these mice compared with those of control mice. Using reciprocal bone marrow transplantation of wild-type and ADH3-deficient mice, we demonstrated that ADH3 deficiency in both HSCs and NK cells contributed to the suppressed liver fibrosis. CONCLUSION:ADH3 plays important roles in promoting liver fibrosis by enhancing HSC activation and inhibiting NK cell activity, and could be used as a potential therapeutic target for the treatment of liver fibrosis.
| S-EPMC4867000 | biostudies-literature
Project description:The Michael hydratase - alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of ?,?-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.
| S-EPMC4052635 | biostudies-literature
Project description: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.
| S-EPMC4414724 | biostudies-literature
Project description:1. Yeast alcohol dehydrogenase was used to determine ethanol in the portal and hepatic veins and in the contents of the alimentary canal of rats given a diet free from ethanol. Measurable amounts of a substance behaving like ethanol were found. Its rate of interaction with yeast alcohol dehydrogenase and its volatility indicate that the substance measured was in fact ethanol. 2. The mean alcohol concentration in the portal blood of normal rats was 0.045mm. In the hepatic vein, inferior vena cava and aorta it was about 15 times lower. 3. The contents of all sections of the alimentary canal contained measurable amounts of ethanol. The highest values (average 3.7mm) were found in the stomach. 4. Infusion of pyrazole (an inhibitor of alcohol dehydrogenase) raised the alcohol concentration in the portal vein 10-fold and almost removed the difference between portal and hepatic venous blood. 5. Addition of antibiotics to the food diminished the ethanol concentration of the portal blood to less than one-quarter and that of the stomach contents to less than one-fortieth. 6. The concentration of alcohol in the alimentary canal and in the portal blood of germ-free rats was much decreased, to less than one-tenth in the alimentary canal and to one-third in the portal blood, but detectable quantities remained. These are likely to arise from acetaldehyde formed by the normal pathways of degradation of threonine, deoxyribose phosphate and beta-alanine. 7. The results indicate that significant amounts of alcohol are normally formed in the gastro-intestinal tract. The alcohol is absorbed into the circulation and almost quantitatively removed by the liver. Thus the function, or a major function, of liver alcohol dehydrogenase is the detoxication of ethanol normally present. 8. The alcohol concentration in the stomach of alloxan-diabetic rats was increased about 8-fold. 9. The activity of liver alcohol dehydrogenase is generally lower in carnivores than in herbivores and omnivores, but there is no strict parallelism between the capacity of liver alcohol dehydrogenase and dietary habit. 10. The activity of alcohol dehydrogenase of gastric mucosa was much decreased in two out of the three germ-free rats tested. This is taken to indicate that the enzyme, like gastric urease, may be of microbial origin. 11. When the body was flooded with ethanol by the addition of 10% ethanol to the drinking water the alcohol concentration in the portal vein rose to 15mm and only a few percent of the incoming ethanol was cleared by the liver.
| S-EPMC1179260 | biostudies-other
Project description:Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.
| S-EPMC1178266 | biostudies-other
Project description:1. The activity of liver alcohol dehydrogenase with cyclohexanol and cyclohexanone as substrates was studied, and the initial-rate parameters were determined from measurements at low substrate concentrations. In contrast with aliphatic ketones, cyclohexanone is a fairly good substrate, although less active than aliphatic aldehydes. The Michaelis constant for cyclohexanol is of the same order as that for ethanol, and the maximum rate and Michaelis constant for NAD(+) obtained with cyclohexanol are very similar to those obtained with primary aliphatic alcohols. The data for this substrate at low concentrations are therefore consistent with a compulsory-order mechanism in which ternary complexes are not rate-limiting. 2. With large concentrations of NAD(+), substrate activation is observed with increasing concentrations of cyclohexanol, whereas with small NAD(+) concentrations substrate inhibition is observed. This complex behaviour is explained by a mechanism previously proposed for this enzyme, which also satisfactorily described the kinetics of oxidation of primary and secondary aliphatic alcohols and aldehydes, including the substrate inhibition exhibited by primary alcohols, and the reduction of aldehydes. The activation with large concentrations of both NAD(+) and cyclohexanol is attributed to the formation of an abortive complex, E.NADH.ROH, from which NADH dissociates more rapidly than from the normal product complex E.NADH. Substrate inhibition in the presence of small NAD(+) concentrations is attributed to the formation of an active complex E.ROH, with which NAD(+) reacts more slowly than with the free enzyme. 3. Some support for these mechanisms of substrate activation and inhibition is obtained by approximate theoretical calculations, and their applicability to other two-substrate reactions that exhibit complex initial-rate behaviour, as a more likely alternative to the postulate of a second binding site for the substrate, is suggested.
| S-EPMC1265162 | biostudies-other