ACTIVATION OF THE O-2(-)-GENERATING NADPH OXIDASE IN A SEMI-RECOMBINANT CELL-FREE SYSTEM - ASSESSMENT OF THE FUNCTION OF RAC IN THE ACTIVATION PROCESS

The neutrophil NADPH oxidase activation factors, p47, p67 and the smal l guanosine-nucleotide-binding regulatory (G) protein Rac1, were expre ssed in a baculovirus/insect cell system and purified. In coinfection experiments in which Sf9 cells overexpressed concomitantly p47, p67 an d Rad, the latter was not detected in the p47-p67 complex. The propens ity of p47 and p67 to associate together was used to purify recombinan t p67 from baculovirus-infected Sf9 cells. 20% of the overexpressed Ra d in infected Sf9 cells was prenylated and was extracted with low dose s 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 unprocess ed, together with p47 and p67 was found to enhance oxidase activation up to fivefold. In all experiments, the amount of added arachidonic ac id was optimized. In contrast to prenylated Rac, non-prenylated Rac ha d to be loaded with guanosine 5'-(3-thiotriphosphate) (GTP[S]) to exhi bit full activation efficiency. In the cell-free system used, Rac was shown to be the mediator of the GTP[S] effect. The results suggest tha t the plasma membrane of resting neutrophils contains a sufficient amo unt of prenylated Rac for efficient oxidase activation. We therefore p ropose that Rac has a membrane-associated role and helps to dock and p osition p47 and p67 on the flavocytochrome b component of the oxidase complex.