title: Further characterization of NADPH oxidase activity of human polymorphonuclear leukocytes.
abstract: Mn2+ was shown to catalyze a nonenzymatic oxidation of NADPH in the presence of superoxide anion by means of an isotopic assay for measurement 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 activate granule NADPH oxidase activity. Superoxide dismutase 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 isolated from cells challenged with opsonized zymosan, even at 0.05 mM NADPH, and was higher than the activity found in granule fractions from resting cells at all levels of NADPH tested. The addition of as little as 0.1 muM NADH to the reaction mixture was found to inhibit granular NADPH oxidase activity, indicating a possible regulatory role for NADH. These results suggest that NADPH oxidase may be the enzyme that initiates the metabolic events accompanying phagocytosis.
mesh_terms: D006801:Humans; D008345:Manganese; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction; D010587:Phagocytosis; D010704:Phorbols / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: D010704:Phorbols; D008345:Manganese; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D009247:NADH, NADPH Oxidoreductases; D013755:Tetradecanoylphorbol Acetate