Activation of the NADPH-dependent H2O2-generating system in pig thyroid particulate fraction by limited proteolysis and Zn2+ treatment.
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.
Dupuy
C
C
Unité 96 I.N.S.E.R.M., Le Kremlin-Bicêtre, France.
Virion
A
A
De Sandro
V
V
Ohayon
R
R
Kaniewski
J
J
Pommier
J
J
Dème
D
D
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
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