The NADPH oxidae and PI 3‐kinase: the role of p40phox

In response to bacterial infection, the neutrophil NADPH oxidase localizes to phagosomes and catalyzes the transfer of electrons from NADPH to molecular oxygen to form superoxide, which assists in bacterial killing. The roles of two of the cytosolic components, p67phox and p47phox, in facilitating electron transfer to the membrane‐bound cytochrome are relatively clear. However, the role of the third cytosolic component, p40phox, remains controversial. We recently identified the PX domains of p47phox and p40phox as phosphoinositide‐binding domains that recognize specific lipid products of PI 3‐kinase and have begun to dissect the roles of these domains and the function of p40phox in oxidase assembly and activation. The biochemical function of p40phox was then investigated using a permeabilized whole neutrophil model system. We show in that system that immuno‐depletion of p40phox from neutrophil cytosol prior to reconstitution of the permeabilized neutrophil “cores” significantly impairs intracellular superoxide production. This activity can be restored by the re‐addition of recombinant wild‐type p40phox, but not by the addition of p40phox containing either a deletion of or a mutation in the PtdIns(3)P‐binding pocket of the PX domain, or a mutation in the PB1 domain which interacts with p67phox, or a mutation in the SH3 domain, whose target is unknown. When the permeabilized PMN system was modified to allow separation of the oxidase assembly process from initial superoxide production, we observe a 75% reduction in Vmax apparent in the absence of p40phox with no significant change in the Km apparent for NADPH. Furthermore, re‐addition of p40phox to depleted NADPH oxidase reactions results in an instantaneous change in the ROS production rate, suggesting that p40phox directly stimulates catalytic activity of the holoenzyme complex.