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.

Inhibitory Action of NoxA1 on Dual Oxidase Activity in Airway Cells

“…This might reflect the importance of the Duox C terminus as control mechanism for Ca 2ϩ binding or for termination of the electron flow. Limited proteolysis of thyroid NADPH oxidase by ␣-chymotrypsin results in irreversible, calcium-independent H 2 O 2 production (40). These data together with our observation support the idea that Duox activity might be regulated via the displacement or conformational change of an inhibitory domain.…”

Solubilization and Characterization of a Thyroid Ca2+‐Dependent and NADPH‐Dependent K3Fe(CN)6 Reductase

“…We previously reported that Ca2+ controls NADPH oxidase activity by acting directly on an autoinhibitory domain, or inhibitory protein component [27]. This study shows that the enzymatic activity remains Ca'+ dependent, despite treatment with detergent and 2 M KC1.…”

Structure, regulation and evolution of Nox‐family NADPH oxidases that produce reactive oxygen species

“…Regulation of Duox by Ca 2+ probably occurs via its paired EF‐hand motif. It has been reported that limited proteolysis with α‐chymotrypsin renders thyroid NADPH oxidase fully and irreversibly active independently of Ca 2+ [65]. This implies that the Ca 2+ ‐binding EF‐hands of Duox serve as an autoinhibitory domain, whereas those of Nox5 function as an activation domain [66].…”