Abstract

matic oxidation of NADPH in the presence of super-oxide anion by means of an isotopic assay for measure-ment of the oxidation of NADPH to NADP+. Human polymorphonuclear leukocyte granule NADPH oxidase activity was evaluated in the absence of Mn2 " and was found to be higher in granules from phagocytizing cells than in granules from resting cells. The drug phorbol myristate acetate, which affects the oxidative metabolism of the neutrophil like phagocytosis, was found to acti-vate granule NADPH oxidase activity. Superoxide dis-mutase was shown to inhibit NADPH oxidase activity both in the presence and absence of added Mn2'. The NADPH oxidase reaction in the absence of Mn2 " was optimal at pH 5.5, and was more linear with increasing time and protein concentration than in the presence of Mn2+. No activity was measurable in granules isolated from resting cells until the level of NADPH added was above 0.25 mM. Activity was present in granules iso-lated from cells challenged with opsonized zymosan, even at 0.05 mM NADPH, and was higher than the ac-tivity found in granule fractions from resting cells at all levels of NADPH tested. The addition of as little as 0.1 /AM NADH to the reaction mixture was found to inhibit granular NADPH oxidase activity, indicating a possible regulatory role for NADH. These results sug-gest that NADPH oxidase may be the enzyme that ini-tiates the metabolic events accompanying phagocytosis.