Involvement of p40(phox) in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67(phox)

Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91(phox) and p22(phox) subunits) and two cytosolic factors (p47(phox) and p67(phox)), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40(phox), as a molecule that associates with p67(phox) in human neutrophils. Although it has been found that this p40(phox) protein is defective in patients with chronic granulomatous disease (CGD) who lack p67(phox), evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40(phox) as well as against the COOH-terminal polypeptide of p67(phox) to examine the mode of interaction between p40(phox) and p67(phox) in a complex. The antibody against the COOH terminus of p67(phox) was able to coimmunoprecipitate g40(phox) in conjunction with p67(phox) itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40(phox) completely dissociated the p67(phox) molecule from the p40(phox)-p67(phox) complex unit without any detectable coimmunoprecipitation of p67(phox), despite their tight association, whereas that against the NH2 terminus of p40(phox) had absolutely no dissociation effect. Similar results were found regarding their effects on the O-2(-)-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40(phox). However, this dissociation did not affect the translocation of the cytosolic components including p47(phox) to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40(phox)-p67(phox) complex. These results Provide the first demonstration that p40(phox) is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67(phox).