IDENTIFICATION OF THE NADPH-BINDING PROTEIN OF THE NEUTROPHIL SUPEROXIDE-GENERATING OXIDASE OF GUINEA-PIGS

A cell-free system prepared from polymorphonuclear neutrophils is capa ble of NADPH-dependent generation of superoxide anion, but requires th e simultaneous presence of plasma membranes, cytosol, arachidonate and guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The isolated membrane s from such a preparation are able to catalyse NADPH-dependent superox ide formation independently of added cytosol and activators. Such acti vated membranes, activated in the cell-free system, must consequently contain all of the essential components required by the oxidase for su peroxide formation, including the NADPH-binding component. Arylazido-b eta-alanyl-[P-32]NADPH (3'-O-{3-[N-(4-azido-2-nitrophenyl)-amino] prop ionyl}-[P-32]NADPH), an NADPH analogue and photoaffinity probe, is sho wn to act in the dark as a substrate for the oxidase activity in the a ctivated membranes and an irreversible photodependent inhibitor follow ing photoirradiation. The photoaffinity probe has been used to identif y the specific NADPH-binding component of the oxidase in the activated membranes. In contrast with the sensitivity of the activated membrane s, photoirradiation of arylazido-beta-alanyl-[P-32]-NADPH under identi cal conditions, but with non-activated membranes, did not prevent subs equent activation of the treated membranes by cytosol, arachidonate an d GTP[S]. However, photoirradiation of the cytosolic fraction in the p resence of arylazido-beta-alanyl-[P-32]NADPH resulted in an inhibition of the cytosol's ability to activate superoxide generation upon subse quent incubation with plasma membranes in the presence of arachidonate and GTP[S]. These observations are taken as a strong indication that the NADPH-binding protein of the oxidase is a cytosolic factor which a ssociates with the plasma membrane upon activation. The superoxide-gen erating activity of the activated membranes was inhibited irreversibly in a concentration- and photo-dependent manner by arylazido-beta-alan yl-NADPH. The arylazido-beta-alanyl-NADPH-photodependent inhibition of superoxide generation in the activated membranes correlated with the photodependent labelling of a protein of 55.6 kDa by the arylazido-bet a-alanyl-NADPH. Specificity of labelling was indicated by a lack of la belling of the 55.6 kDa region in the non-activated membranes and prot ection of the photodependent inhibition and labelling of the 55.6 kDa protein by NADPH. It is proposed that the arylazido-beta-alanyl-NADPH- labelled 55.6 kDa protein present on the activated membranes is the NA DPH-binding protein of the neutrophil superoxide-generating oxidase.