Abstract
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.