TRANSLOCATION OF GUINEA-PIG P40-PHOX DURING ACTIVATION OF NADPH OXIDASE

The superoxide-producing NADPH oxidase consists of membrane-associated cytochrome b(558) and cytosolic components, p47-phox and p67-phox. Re cently, we have found a novel cytosolic component, p40-phox, which is tightly associated with p67-phox. In this study, we examined the trans location of p40-phox during activation of NADPH oxidase in a cell-free system using the membrane and the purified p47-phox/p67-phox/p40-phox complex. p40-phox was translocated to the membrane by arachidonic aci d in a dose-dependent manner. The translocation pattern of p40-phox wa s similar to those of p47-phox and p67-phox. However, immunoprecipitat ion assay revealed that p40-phox was dissociated from p47-phox and p67 -phox during activation. The translocation of three cytosolic componen ts was not affected by the deletion of GTP-gamma-s from the reaction m ixture. Interestingly, a synthetic peptide corresponding to carboxyl-t erminus of p40-phox inhibited the activation of NADPH oxidase and tran slocation of p40-phox, p47-phox and p67-phox, suggesting that p40-phox might play a role in the activation of NADPH oxidase. These observati ons suggest that p40-phox is dissociated from p67-phox during activati on, and translocates to the membrane by GTP-gamma-s-independent mechan ism.