Identification of the NADPH-binding protein of the neutrophil superoxide-generating oxidase of guinea pigs.

A cell-free system prepared from polymorphonuclear neutrophils is capable of NADPH-dependent generation of superoxide anion, but requires the simultaneous presence of plasma membranes, cytosol, arachidonate and guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The isolated membranes from such a preparation are able to catalyse NADPH-dependent superoxide formation independently of added cytosol and activators. Such activated membranes, activated in the cell-free system, must consequently contain all of the essential components required by the oxidase 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, is shown to act in the dark as a substrate for the oxidase activity in the activated membranes and an irreversible photodependent inhibitor following photoirradiation. The photoaffinity probe has been used to identify the specific NADPH-binding component of the oxidase in the activated membranes. In contrast with the sensitivity of the activated membranes, photoirradiation of arylazido-beta-alanyl-[32P]-NADPH under identical conditions, but with non-activated membranes, did not prevent subsequent activation of the treated membranes by cytosol, arachidonate and GTP[S]. However, photoirradiation of the cytosolic fraction in the presence of arylazido-beta-alanyl-[32P]NADPH resulted in an inhibition of the cytosol's ability to activate superoxide generation upon subsequent 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 associates with the plasma membrane upon activation. The superoxide-generating activity of the activated membranes was inhibited irreversibly in a concentration- and photo-dependent manner by arylazido-beta-alanyl-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-beta-alanyl-NADPH. Specificity of labelling was indicated by a lack of labelling of the 55.6 kDa region in the non-activated membranes and protection 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 NADPH-binding protein of the neutrophil superoxide-generating oxidase.