Activation by ATP of Calcium-Dependent NADPH-Oxidase Generating Hydrogen Peroxide in Thyroid Plasma Membranes1

Abstract

An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.

NADPH‐dependent H2O2 generation catalyzed by thyroid plasma membranes

“…Furthermore, none of these reductase activities were significantly inhibited by superoxide dismutase, as previously reported by Michot et al [17] and Nakamura et al [12]. However, a recent report from this group describes the formation of an 0, adduct of diacetyldcuterohemc-substituted horseradish peroxidase during incubation of the membrane fraction with NADPH [18].…”

“…We postulated that this inhibitor was associated with the NADPH oxidase in thc absence of Ca2 + , thus preventing NADPH oxidation. Nakamura et al [12] studied the NADPH-dependent H,02-generating system in thyroid plasma membranes, and found an NADPH-dependent cytochrome c and 2,6-dichloroindophenol reductase activity which was not influenced by C a Z f . They suggest that these two reductase activities are catalyzed by the Hz02-generating system and that Ca2+ acts beyond the NADPH oxidation step.…”

Ca2+ regulation of thyroid NADPH‐dependent H2O2 generation

“…The presence of such an H2Oz generator has been recently confirmed and studied by Nakamura et al [14] in a preparation of thyroid fragmented plasma membranes. However, the mode of Ca 2÷ action remains unknown.…”

“…Phorbol ester (TPA) has been reported to increase the generation of H202 obtained by stimulation of thyroid slices by subthreshold doses of ionophore A23187 [12]. Furthermore, ATP has been shown to enhance twice the activity of the Ca2+-stimulated H20~-generating enzyme [14]. Therefore the implication of protein kinase C and/or other protein kinases in the control by Ca 2+ of H202 generation cannot be excluded.…”

Dual Oxidase-2 Has an Intrinsic Ca2+-dependent H2O2-generating Activity

“…Thyroid NADPH oxidase requires the presence of micromolar concentrations of calcium to acquire a functional conformation and to generate H 2 O 2 (34,37,38). Duox1 and Duox2 contain two Ca 2ϩ -binding motifs (EF-hand), which could be involved in the direct activation of the H 2 O 2 generator by calcium.…”