Activation of the O–2‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Abstract

The neutrophil NADPH oxidase activation factors, p47, p67 and the small guanosine-nucleotide-binding regulatory (G) protein Rac1, were expressed in a baculovirus/insect cell system and purified. In coinfection experiments in which Sf9 cells overexpressed concomitantly p47, p67 and Rac1, the latter was not detected in the p47–p67 complex. The propensity of p47 and p67 to associate together was used to purify recombinant p67 from baculovirus–infected Sf9 cells. 20% of the overexpressed Rac1 in infected Sf9 cells was prenylated and was extracted with low doses of detergent from membranes. Elicitation of full oxidase activity on crude neutrophil membranes using a cell–free system required addition of recombinant p47 and p67, but not that of Rac. In contrast, in the case of KCl-washed membranes, addition of Rac, prenylated or unprocessed, together with p47 and p67 was found to enhance oxidase activation up to fivefold. In all experiments, the amount of added arachidonic acid was optimized. In contrast to prenylated Rac, non-prenylated Rac had to be loaded with guanosine 5′-(3-thiotriphosphate) (GTP[SJ]) to exhibit full activation efficiency. In the cell-free system used, Rac was shown to be the mediator of the GTP[S] effect. The results suggest that the plasma membrane of resting neutrophils contains a sufficient amount of prenylated Rac for efficient oxidase activation. We therefore propose that Rac has a membrane-associated role and helps to dock and position p47 and p67 on the flavocytochrome b component of the oxidase complex.