Photolabeling of a O2-. generating protein in bovine polymorphonuclear neutrophils by an arylazido NADP+ analog.
A plasma membrane fraction of bovine polymorphonuclear neutrophils enriched in NADPH-dependent, O2-. generating oxidase activity, and a number of fractions solubilized in detergent, recovered during the course of the purification of this oxidase have been tested for their ability to react with radiolabeled N-4-azido-2-nitrophenyl aminobutyryl NADP+ (arylazido NADP+ or NAP4-NADP+). In the dark, NAP4-NADP+ and its reduced form NAP4-NADPH, were found to inhibit competitively the NADPH-dependent O2-. generating oxidase activity of the plasma membrane fraction of bovine neutrophils activated by phorbol myristate acetate. The nitrene derivative formed upon photoirradiation of NAP4-NADP(H) bound covalently to different proteins of the plasma membrane. Photolabeling of these proteins was prevented by preincubation with an excess of NADPH. Photolabeling of a protein of 65,000 Mr was decreased by omission of phorbol myristate acetate as activating agent of the respiratory burst in neutrophils or by addition of micromolar amounts of Cibacron Blue and mersalyl which are known to inhibit the production of O2-. by the plasma membrane fraction. During the course of the purification procedure, the 65000 Mr protein emerged as the preferentially photolabeled protein. These data, in agreement with previous findings concerning the purification of an NADPH-dependent, O2-. generating oxidase protein of Mr 65000 from bovine neutrophils (Doussière, J. and Vignais, P.V. (1985) Biochemistry 24, 7231-7239), strongly suggest that a single protein of Mr 65000, located in the plasma membrane fraction of bovine neutrophils, is able to act both as an NADPH deshydrogenase and as an oxygen reductase to generate O2-.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002417:Cattle; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis* / Q000302:isolation & purification; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010777:Photochemistry; D013053:Spectrophotometry
D000345:Affinity Labels; D001386:Azides; C050081:N-4-azido-2-nitrophenyl aminobutyryl NADP; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme