Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method.
The ultrastructural localization of NADH oxidase, a possible enzyme in the increased oxidative activity of polymorphonuclear leukocytes (PMN) during phagocytosis, was studied. A new cytochemical technique for the localization of H2O2, a product of NADH oxidase activity, was developed. Cerous ions, in the presence of peroxide, form an electron-dense precipitate. Resting and phagocytically stimulated PMN were exposed to cerous ions at pH 7.5 to demonstrate sites of NADH-dependent, cyanide-insensitive H2O2 production. Resting PMN exhibites slight activity on the plasma membrane; phagocytizing PMN had extensive deposits of reaction product localized within the phagosome and on the plasma membrane. Peroxide involvement was demonstrated by the inhibitory effect of catalase on cerium precipitation; the surface localization of the enzyme responsible was confirmed by using nonpenetrating inhibitors of enzymatic activity. A correlative study was performed with an NADH-dependent, tetrazolium-reduction system. As with cerium, formazan deposition on the surface of the cell was NADH dependent, cyanide insensitive, and stimulated by phagocytosis. Superoxide dismutase did not inhibit tetrazolium reduction, as observed cytochemically, indicating direct enzymatic dye reduction without superoxide interposition. These findings, combined with oxygen consumption studies on resting and stimulated PMN in the presence or absence of NADH, indicate that NADH oxidase is a surface enzyme in human PMN. It is internalized during phagocytosis and retains its peroxide-generating capacity within the phagocytic vacuole.
D000818:Animals; D002374:Catalase / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002563:Cerium / Q000378:metabolism; D003486:Cyanides / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000648:ultrastructure; D005557:Formaldehyde / Q000494:pharmacology; D005976:Glutaral / Q000494:pharmacology; D006168:Guinea Pigs; D006651:Histocytochemistry / Q000379:methods*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D009243:NAD / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects; D010587:Phagocytosis / Q000187:drug effects; D011188:Potassium; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014617:Vacuoles / Q000648:ultrastructure
D003486:Cyanides; D008070:Lipopolysaccharides; D013439:Sulfhydryl Reagents; D009243:NAD; D005557:Formaldehyde; D009580:Nitroblue Tetrazolium; D002563:Cerium; D009249:NADP; D006861:Hydrogen Peroxide; D002374:Catalase; D009247:NADH, NADPH Oxidoreductases; D011188:Potassium; D005976:Glutaral