NADPH-binding protein of the neutrophil superoxide-generating oxidase of guinea pigs.
Polymorphonuclear neutrophil membranes, activated in a cell-free system, contain all of the essential components required for superoxide formation including the NADPH-binding component. Arylazido-beta-alanyl- [32P]NADPH--3'-O-(3-[N-(4-azido-2-nitrophenyl)amino] propionyl)-[32P]NADPH--an NADPH analogue and photoaffinity probe, has been used to identify the specific NADPH binding component of the oxidase in activated membranes. A protein of about 52 kDa was photodependently labeled in the activated membranes by arylazido-beta-alanyl-[32P]NADPH. Specificity of labeling was indicated by the absence of such labeling in nonactivated membranes. The 52-kDa-labeled protein was the only isotopically labeled protein extracted from the labeled membranes with the chaotrope sodium perchlorate. Sodium perchlorate extraction of the 52-kDa protein from activated membranes correlates with the loss of the membranes' superoxide-generating capability. Reconstitution of the lost activity for sodium perchlorate-extracted membranes was accomplished by reincubating the extracted membranes with cytosol. It is proposed that the arylazido- beta-alanyl-[32P]NADPH-labeled protein of 52- to 57-kDa present on the activated membranes is the NADPH-binding protein of the neutrophil superoxide-generating oxidase.
Ge
F
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Department of Biochemistry and Biophysics, John A. Burns School of Medicine, University of Hawaii, Honolulu 96822.
Guillory
R J
RJ
D000345:Affinity Labels; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006168:Guinea Pigs; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010777:Photochemistry; D011485:Protein Binding; D016897:Respiratory Burst; D013481:Superoxides / Q000378:metabolism
D000345:Affinity Labels; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases