title: Activation of the NADPH-dependent H2O2-generating system in pig thyroid particulate fraction by limited proteolysis and Zn2+ treatment.
abstract: The NADPH-dependent H2O2-generating system in a pig thyroid particulate fraction requires micromolar concentrations of Ca2+ for activity. The H2O2 generator could be Ca(2+)-desensitized (i.e. made fully active in the absence of Ca2+) by limited proteolysis with alpha-chymotrypsin or by treatment with ZnCl2. The Zn2+ effect was temperature- and dose-dependent with an apparent half-maximum concentration of 0.15 mM at 40 degrees C. Ca2+ desensitization was not reversed by adding the Zn2+ chelators, 1,10-phenanthroline and EGTA, but about one-third of the Ca(2+)-sensitivity was recovered after addition of 10 mM-dithiothreitol. The proteolysed enzyme and the Zn(2+)-treated enzyme had different Km values for NADPH. The Zn2+ effect did not seem to involve proteolysis or membrane fusion. These results indicate that Ca2+ regulation occurs via an autoinhibitory domain or inhibitory protein component of the H2O2-generator system. Its inhibitory effect may be removed by proteolysis or conformational changes, making the catalytic site accessible to the substrate NADPH and/or enabling electrons to be transferred from NADPH to O2.
authors.affiliation: Unité 96 I.N.S.E.R.M., Le Kremlin-Bicêtre, France.
authors.forename: C
authors.initials: C
authors.lastname: Dupuy
authors.forename: A
authors.initials: A
authors.lastname: Virion
authors.forename: V
authors.initials: V
authors.lastname: De Sandro
authors.forename: R
authors.initials: R
authors.lastname: Ohayon
authors.forename: J
authors.initials: J
authors.lastname: Kaniewski
authors.forename: J
authors.initials: J
authors.lastname: Pommier
authors.forename: D
authors.initials: D
authors.lastname: Dème
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002614:Chelating Agents / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology*; D002918:Chymotrypsin / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D011480:Protease Inhibitors / Q000494:pharmacology; D013552:Swine; D013816:Thermodynamics; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology*; D017967:Zinc Compounds
chemical_list: D002614:Chelating Agents; D002712:Chlorides; D011480:Protease Inhibitors; D017967:Zinc Compounds; D009249:NADP; C016837:zinc chloride; D006861:Hydrogen Peroxide; D010744:Phosphoric Monoester Hydrolases; D002918:Chymotrypsin; D015032:Zinc; D002118:Calcium; D004229:Dithiothreitol