Inhibition of the catalytic activity of alcohol dehydrogenase by nitric oxide is associated with S nitrosylation and the release of zinc.
Nitric oxide (NO) reacts with the sulfhydryl groups of proteins to form nitroso thiols. Alcohol dehydrogenase (ADH) plays an important role in the metabolism of ethanol. Chronic alcohol administration stimulates NO formation in the liver, and production of NO is increased in alcohol liver injury. The effect of exogenous and endogenous NO on rat or horse ADH activity was evaluated. Incubation of intact rat hepatocytes or cytosol isolated from hepatocytes with S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, resulted in a decrease in ADH activity. Endogenous NO synthesis was induced in rat hepatocytes by incubation with a mixture of cytokines and endotoxin in the presence of L-arginine. As NO production in hepatocytes increased over a 24 h time period, a significant decrease in ADH activity was observed. This effect was blocked by the competitive inhibitor of NO synthesis, N omega-nitro-L-arginine methyl ester, indicating that ADH was also inactivated by endogenously generated NO. The decreased activity of ADH was not related to lowering of the ADH content as shown by Western blot analysis. To evaluate the mechanism of inhibition, purified ADH from equine liver was incubated with gaseous NO or NO released from NO donors such as the diethylamine/nitric oxide complex (DEA/NO) and SNAP. NO donors inactivated ADH in a dose- and time-dependent manner. Trapping of NO with hemoglobin resulted in protection of ADH against inactivation by NO. There was no effect by analogues of the NO donors which do not release NO. NAD afforded some protection against the NO inactivation of ADH. Measurements of thiol oxidation, S nitrosylation, and zinc release were used to assess the effect of NO on ADH activity. Thiol oxidation, S-nitroso thiol formation, and zinc release correlated with inactivation of ADH by NO, indicating that disruption of the zinc/thiolate active center due to S nitrosylation of ADH results in zinc release, followed by inactivation of the enzyme. Recovery experiments were performed by incubating the NO-treated enzyme with dithiothreitol (DTT) and/or Zn2+. The inhibitory effect by NO was reversible since, after the nitrosylated enzyme was reduced with DTT followed by incubation with ZnCl2 to allow reincorporation of Zn2+, ADH activity was increased from 20% of control values to 70%. These results suggest that cysteine residues contained within the zinc/thiolate active center may be primary sites of NO interaction with ADH. NO may modulate the metabolism of ethanol and influence metabolic actions of ethanol via interaction with ADH.
Gergel
D
D
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Cederbaum
A I
AI
D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000494:pharmacology*; D016207:Cytokines / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D004229:Dithiothreitol / Q000494:pharmacology; D006454:Hemoglobins / Q000494:pharmacology; D006736:Horses; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis / Q000494:pharmacology*; D009573:Nitrites / Q000032:analysis; D009603:Nitroso Compounds; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D026423:S-Nitroso-N-Acetylpenicillamine; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D017967:Zinc Compounds / Q000494:pharmacology*
D002712:Chlorides; D016207:Cytokines; D006454:Hemoglobins; D009573:Nitrites; D009603:Nitroso Compounds; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D017967:Zinc Compounds; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D007371:Interferon-gamma; C016837:zinc chloride; D001120:Arginine; D000426:Alcohol Dehydrogenase; D010396:Penicillamine; D004229:Dithiothreitol; D019331:NG-Nitroarginine Methyl Ester