title: Respiratory burst of rabbit peritoneal neutrophils. Transition from an NADPH diaphorase activity to an .O2(-)-generating oxidase activity.
abstract: Superoxide (.O2-) production by the NADPH oxidase of a membrane fraction derived from rabbit peritoneal neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate (PMA) was studied at 25 degrees C under different conditions, and measured by the superoxide dismutase inhibitable reduction of cytochrome c. Whereas PMA-activated rabbit neutrophils incubated in a glucose-supplemented medium exhibited a substantial rate of production of .O2-, the membranes prepared by sonication of the activated neutrophils were virtually unable to generate .O2- in the presence of NADPH. Instead, they exhibited an NADPH-dependent diaphorase activity, measured by the superoxide-dismutase-insensitive reduction of cytochrome c. Upon addition of arachidonic acid, which is known to elicit oxidase activation, the NADPH diaphorase activity of the rabbit neutrophil membranes vanished and was stoichiometrically replaced by an NADPH oxidase activity. The emerging oxidase activity was fully sensitive to iodonium biphenyl, a potent inhibitor of the respiratory burst, whereas the diaphorase activity was not affected. Addition of 0.1% Triton X-100 or an excess of arachidonic acid, acting as detergent, resulted in the reappearance of the diaphorase activity at the expense of the oxidase activity. These results indicate that the diaphorase-oxidase transition is reversible. When the rabbit neutrophil membranes were supplemented with rabbit neutrophil cytosol, guanosine 5'-[gamma-thio]triphosphate and Mg2+, in addition to arachidonic acid, not only the NADPH diaphorase activity disappeared, but the emerging NADPH oxidase activity was markedly enhanced (about 10 times compared to that of membranes treated with arachidonic acid alone). The diaphorase-oxidase transition was accompanied by a 10-fold increase in the Km for NADPH, suggesting a change of conformation propagated to the NADPH-binding site during the transition. The treatment of PMA-activated rabbit neutrophils with cross-linking reagents, like glutaraldehyde or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, prevented the loss of the PMA-elicited oxidase activity upon disruption of the cells by sonication, suggesting that the interactions between the components of the oxidase complex are stabilized by cross-linking.
authors.affiliation: Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires, Grenoble, France.
authors.forename: F
authors.initials: F
authors.lastname: Laporte
authors.forename: J
authors.initials: J
authors.lastname: Doussiere
authors.forename: P V
authors.initials: PV
authors.lastname: Vignais
mesh_terms: D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D005292:Ferricyanides / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D066298:In Vitro Techniques; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism*; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010084:Oxidation-Reduction; D010529:Peritoneal Cavity / Q000166:cytology; D011817:Rabbits; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D001095:Arachidonic Acids; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D005292:Ferricyanides; D013481:Superoxides; D016718:Arachidonic Acid; D009580:Nitroblue Tetrazolium; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D009252:NADPH Dehydrogenase; D013755:Tetradecanoylphorbol Acetate; D005976:Glutaral