title: Kinetic study of the activation of the neutrophil NADPH oxidase by arachidonic acid. Antagonistic effects of arachidonic acid and phenylarsine oxide.
abstract: The O(2)(-) generating NADPH oxidase complex of neutrophils comprises two sets of components, namely a membrane-bound heterodimeric flavocytochrome b which contains the redox centers of the oxidase and water-soluble proteins of cytosolic origin which act as activating factors of the flavocytochrome. The NADPH oxidase can be activated in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils in the presence of GTPgammaS and arachidonic acid. NADPH oxidase activation is inhibited by phenylarsine oxide (PAO), a sulfhydryl reagent for vicinal or proximal thiol groups. The site of action of PAO was localized by photolabeling in the beta-subunit of flavocytochrome b [Doussière, J., Poinas, A, Blais, C., and Vignais, P. V. (1998) Eur. J. Biochem. 251, 649-658]. Moreover, the spin state of heme b is controlled by interaction of arachidonic acid with the flavocytochrome b [Doussière, J., Gaillard, J., and Vignais, P. V. (1996) Biochemistry 35, 13400-13410]. Here we report that the promoting effect of arachidonic acid on the activation of NADPH oxidase is due to specific binding of arachidonic acid to flavocytochrome b. Elicitation of NADPH oxidase activity by arachidonic acid is in part associated with an increased affinity of flavocytochrome b for O(2), an effect that was counteracted by the methyl ester of arachidonic acid. On the other hand, the affinity for NADPH was not affected by arachidonic acid. We further demonstrate that PAO antagonizes the effect of arachidonic acid on oxidase activation by decreasing the affinity of the oxidase for O(2), but not for NADPH. PAO induced a change in the spin state of heme b, as arachidonic acid does, with, however, some differences in the constraints imposed to the heme. It is concluded that the opposite effects of arachidonic acid and PAO are exerted on the beta-subunit of flavocytochrome b at two different interacting sites.
authors.affiliation: Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR 314 CEA-CNRS), Département de Biologie Moléculaire et Structurale, Grenoble, France.
authors.forename: J
authors.initials: J
authors.lastname: Doussiere
authors.forename: F
authors.initials: F
authors.lastname: Bouzidi
authors.forename: A
authors.initials: A
authors.lastname: Poinas
authors.forename: J
authors.initials: J
authors.lastname: Gaillard
authors.forename: P V
authors.initials: PV
authors.lastname: Vignais
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D001152:Arsenicals / Q000494:pharmacology*; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004952:Esters; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D013997:Time Factors
chemical_list: D001152:Arsenicals; D004791:Enzyme Inhibitors; D004952:Esters; C029341:oxophenylarsine; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009249:NADP; D000255:Adenosine Triphosphate; D019255:NADPH Oxidases; D010100:Oxygen