P40(phox) associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67(phox) and P47(phox)

NADPH oxidase is an O-2 .(-)-generating enzyme found in phagocytes such as neutrophils, It is composed of a membrane-bound cytochrome b, the cytosolic proteins p67(phox), p47(phox), p40(phox), and the G-protein p21rac, The sy stem is dormant in resting cells but acquires catalytic activity on exposur e to appropriate stimuli. Cytochrome b, p67(phox), p47(phox), and rac2 asso ciate with the cytoskeleton and membrane skeleton of activated neutrophils, It is not known whether p40(phox) associates with the cytoskeleton. The pu rpose of this study was to analyze the subcellular distribution of p40(phox ). When resting neutrophils were lysed in Triton X-100 or octyl glucoside b uffer and separated into detergent-soluble and detergent-insoluble fraction s, p40(phox) and p67(phox) were mainly associated with the detergent-insolu ble fraction (defined as the cytoskeleton), whereas p47(phox) was mainly fo und in the soluble fraction. Neutrophil activation by phorbol myristate ace tate (PMA) induced p47(phox) translocation to the cytoskeleton but did not affect the distribution of p40(phox) or p67(phox). Using immunofluorescence confocal microscopy, we found that p40(phox) colocalized with filamentous actin, In neutrophils from a p67(phox)-deficient patient with detectable p4 0(phox), P40(phox) associated with the cytoskeleton only after activation b y PMA, A complex containing the three proteins was isolated from the cytosk eleton of activated neutrophils, When activated membranes were treated with Triton X-100 buffer; p40(phox), p47(phox), and p67(phox) were found in the membrane skeleton enriched in NADPH-oxidase activity; some p40(phox) and p 47(phox) was found in the soluble membrane fraction, but no p67(phox) was d etected. These findings show that p40(phox), like p67(phox) and p47(phox), binds to the cytoskeleton and membrane skeleton, In addition, p40(phox) can dissociate from p67(phox) in activated membranes.