NAD(P)H oxidase activity in human neutrophils stimulated by phorbol myristate acetate

Abstract

Phorbol myristate acetate activated in normal human neutrophils a single enzymatic entity that was dormant in unstimulated cells, optimally active at pH 7.0, and capable of oxidizing either NADH or NADPH, producing NAD(P)+ and superoxide (O2-). Comparative fluorometric and spectrophotometric measurements supported the stoichiometry NAD(P)H + 202 → NAD(P)+ 2O2- + H+. The seemingly considerable NAD(P)+ production at pH 5.5 and 6.0 was due largely to monoenzymatic oxidation of NAD(P)H by chain reactions initiated by HO2- (perhydroxyl radical), the conjugate acid of O2-. This artifact, responsible for earlier erroneous assignments of an acid pH optimum for NAD(P)H oxidase, was prevented by including superoxide dismutase in fluorometric assays. NAD(P)H oxidase was more active towards NADPH (K(m) = 0.15±0.03 mM) than NADH (K(m) = 0.68±0.2 mM). No suggestion that oxidase activity was allosterically regulated by NAD(P)H was seen. Phorbol myristate acetate-induced O2- production was noted to be modulated by pH in intact neutrophils, suggesting that NAD(P)H oxidase is localized in the plasma membrane where its activity may be subject to (autoregulation by local H+ concentrations.