SummaryThe NADPH-binding site of the respiratory burst oxidase system of neutrophils has been proposed to be either at a cytosolic component or at the ~-subunit of cytochrome b558. In this study, affinity labeling of resting and stimulated membranes, the latter having been assembled by all of the oxidase components from both membrane and cytosol, was carried out using [32p]NADPH dialdehyde (oNADPH). Stimulation of human neutrophils with PMA greatly increased O2--generating activity and caused considerable translocation of the cytosolic components p47ehox and p67vho~. Nevertheless, PMA stimulation did not produce a labeled band which included positions at 47, 67, and ,x,32 kD. The most intense band reflected a molecular mass of 84 kD regardless of the state of activation, but a labeled band was never found near the/3-subunit (91 kD) of cytochrome b558. This 84-kD protein was further confirmed in neutrophils of 14 patients with gp91vh~ X-linked chronic granulomatous disease. These results indicate that the NADPH-binding component is not recruited from the cytosol, and also, that a membranous redox component besides cytochrome b558 must be involved in the NADPH oxidase system. U 'pon stimulation, phagocytes (neutrophils, monocytes, and eosinophils) generate superoxide anion and other oxygen intermediates to kill microbes (for reviews see references 1-4). This respiratory burst is caused by activation of the O2--generating NADPH oxidase system, the components of which are distributed in cell membrane and cytosol in the resting state (5). Chronic granulomatous disease (CGD), in which the respiratory burst is hampered by genetic dysfunction, has contributed greatly to the discovery of the indispensable constituents of this system i.e., determination of the membranous components p22Vhox (ot-subunit) and gp91Vhox (~-subunit) of cytochrome b558 in X-linked CGD and determination of the cytosolic components p47p h~ and p67ehox in autosomal recessive CGD (1, 2, 4). Rac p21s, which are regulated by their stimulator and inhibitor for GDP dissociation, have recently been proposed to be involved as modulatory components in this NADPH oxidase system (6, 7). However, CGD caused by their absence has not been identified.In contrast to the above constituents, the NADPH-binding component of this oxidase system still remains controversial. The NADPH-binding site has been proposed to be located in a cytosolic protein and transferred upon cell stimulation (1,8,9). On the other hand, another recent hypothesis proposed that cytochrome b558 in membrane, but not in cytosol, is a flavoprotein containing both Flavin Adenine Dinucleotide (FAD)-and NADPH-binding sites, based only on the amino acid homology between p91ehox and the putative NADP* -and FAD-binding sites of the ferredoxin reductase (FNR) family (10-12). In one report (11), direct elucidation was attempted by photoaffinity labeling using [32p]2-azido-NADP +; however, binding to gp91Vhox was extremely difficult to judge. Furthermore, a recent report (13) has demonstrated that a membr...
