The Ca2+/NADPH-dependent H2O2 generator in thyroid plasma membrane: inhibition by diphenyleneiodonium.
The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the peroxidase-catalysed biosynthesis of thyroid hormones. The electron transfer from NADPH to O2 catalysed by this system was studied by using diphenyleneiodonium (DPI), an inhibitor of flavo- and haemo-proteins. The prosthetic group of the H2O2 generator was removed by incubation with 5 mM CHAPS at 40 degrees C, and an active holoenzyme was reconstituted with FAD, but not with FMN. The H2O2-generating system also had an intrinsic Ca(2+)-dependent NADPH:ferricyanide reductase activity which is probably linked to its flavodehydrogenase component (or domain). Both activities, H2O2 production and ferricyanide reductase activity, were inhibited by DPI, with similar K1/2 (2.5 nmol/mg of protein). DPI only inhibited a system reduced with NADPH in the presence of Ca2+. NADPH could not be replaced by NADP+, NADH or sodium dithionite, suggesting the need for specific mild reduction of a redox centre in a particular conformation. Ferricyanide protected both activities against inhibition by DPI; the NADPH:ferricyanide reductase activity was completely protected at a ferricyanide concentration 20 times lower than that needed to protect the H2O2 formation, implying at least two target sites for DPI. One might be the flavodehydrogenase component; the other was beyond, on the entity which transfers the electrons to O2. This second site has not been identified.
Dème
D
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INSERM U. 96, Le Kremlin-Bicêtre, France.
Doussiere
J
J
De Sandro
V
V
Dupuy
C
C
Pommier
J
J
Virion
A
A
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004579:Electron Transport / Q000187:drug effects; D005292:Ferricyanides / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors; D009249:NADP / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D013552:Swine; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism*
D005292:Ferricyanides; D009861:Onium Compounds; C007931:hexacyanoferrate III; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C007517:diphenyleneiodonium; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; C014227:ferricyanide reductase; D002118:Calcium