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.