Regulation of the thyroid NADPH-dependent H2O2 generator by Ca2+: studies with phenylarsine oxide in thyroid plasma membrane.
Pig thyroid plasma membranes contain a Ca(2+)-dependent NADPH:O2 oxidoreductase, the thyroid NADPH-dependent H2O2 generator. This provided the H2O2 for the peroxidase-catalysed synthesis of thyroid hormones. The effect of the tervalent arsenical, phenylarsine oxide (PAO), on the NADPH oxidase was studied. PAO caused two directly related dose-dependent effects with similar half-effect concentrations of PAO (3 nmol of PAO/mg of protein): (i) partial inactivation of H2O2 formation by the Ca(2+)-stimulated enzyme, and (ii) desensitization of the enzyme activity to Ca2+. PAO had no effect on membranes that had been Ca(2+)-desensitized by alpha-chymotrypsin treatment. The NADPH oxidase in membranes treated with excess PAO had the same Vmax with and without Ca2+. This value was half the Vmax of the native enzyme. However, the K(m) for NADPH determined with Ca2+ (18 microM, identical with that of the native enzyme) was approx, one-third of the K(m) measured without Ca2+, showing the direct action of Ca2+ on the PAO-enzyme complex. PAO had the same effects, partial inactivation and Ca2+ desensitization, on the NADPH: ferricyanide oxidoreductase activity of the NADPH oxidase, suggesting that PAO acts on the flavodehydrogenase entity of the enzyme. Both partial inactivation and Ca2+ desensitization were completely and specifically reversed by 2.3-dimercaptopropanol, partly reversed by dithiothreitol and not reversed by 2-mercaptoethanol, indicating that PAO binds to vicinal thiol groups. These results suggest that thiol groups are involved in the control of thyroid NADPH oxidase by Ca2+; PAO bound to vicinal thiols might alter the structure of the enzyme so that electron transfer occurs without Ca2+ but more slowly.
Gorin
Y
Y
INSERM Unité 96, Le Kremlin-Bicĕtre, France.
Leseney
A M
AM
Ohayon
R
R
Dupuy
C
C
Pommier
J
J
Virion
A
A
Dème
D
D
D000818:Animals; D001152:Arsenicals / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D005292:Ferricyanides / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism* / Q000502:physiology; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013552:Swine; D013961:Thyroid Gland / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*
D001152:Arsenicals; D005292:Ferricyanides; D013438:Sulfhydryl Compounds; C029341:oxophenylarsine; C007931:hexacyanoferrate III; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; D002118:Calcium