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Inhibition of the catalytic activity of alcohol dehydrogenase by nitric oxide is associated with S nitrosylation and the release of zinc.
Biochemistry. 1996 Dec 17; 35(50):16186-94.B

Abstract

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.

Authors+Show Affiliations

Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8973191

Citation

Gergel, D, and A I. Cederbaum. "Inhibition of the Catalytic Activity of Alcohol Dehydrogenase By Nitric Oxide Is Associated With S Nitrosylation and the Release of Zinc." Biochemistry, vol. 35, no. 50, 1996, pp. 16186-94.
Gergel D, Cederbaum AI. Inhibition of the catalytic activity of alcohol dehydrogenase by nitric oxide is associated with S nitrosylation and the release of zinc. Biochemistry. 1996;35(50):16186-94.
Gergel, D., & Cederbaum, A. I. (1996). Inhibition of the catalytic activity of alcohol dehydrogenase by nitric oxide is associated with S nitrosylation and the release of zinc. Biochemistry, 35(50), 16186-94.
Gergel D, Cederbaum AI. Inhibition of the Catalytic Activity of Alcohol Dehydrogenase By Nitric Oxide Is Associated With S Nitrosylation and the Release of Zinc. Biochemistry. 1996 Dec 17;35(50):16186-94. PubMed PMID: 8973191.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Inhibition of the catalytic activity of alcohol dehydrogenase by nitric oxide is associated with S nitrosylation and the release of zinc. AU - Gergel,D, AU - Cederbaum,A I, PY - 1996/12/17/pubmed PY - 1996/12/17/medline PY - 1996/12/17/entrez SP - 16186 EP - 94 JF - Biochemistry JO - Biochemistry VL - 35 IS - 50 N2 - 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8973191/Inhibition_of_the_catalytic_activity_of_alcohol_dehydrogenase_by_nitric_oxide_is_associated_with_S_nitrosylation_and_the_release_of_zinc_ L2 - https://doi.org/10.1021/bi962043r DB - PRIME DP - Unbound Medicine ER -