P21RAC DOES NOT PARTICIPATE IN THE EARLY INTERACTION BETWEEN P47-PHOXAND CYTOCHROME B(558) THAT LEADS TO PHAGOCYTE NADPH OXIDASE ACTIVATION IN-VITRO

The phagocyte superoxide-generating NADPH oxidase, a multicomponent, m embrane-bound electron transport chain, consists of cytochrome b(558), p47-phox, p67-phox, and p21rac2 or p21rac2. The mechanisms of oxidase assembly are poorly understood. In previous studies using a cell-free NADPH oxidase system, we showed that preincubation of neutrophil memb rane with neutrophil cytosol containing p47-phox, but not p67-phox, le d to formation of a long-lived NADPH oxidase intermediate. This sugges ted that p47-phox interacted with cytochrome b(558) in the early stage s of oxidase assembly while p67-phox participated in a later stage. Pe ptides containing the sequence RGVHFIF (corresponding to amino acids 5 59-565 of the 91-kDa subunit of cytochrome b(558)) inhibit NADPH oxida se activity by blocking the early interaction between p47-phox and cyt ochrome b(558) In the present study, we examined whether p21rac facili tated the interaction between p47-phox and cytochrome b(558) We preinc ubated pure recombinant p47-phox with neutrophil membrane containing c ytochrome b(558) in the cell-free system. Superoxide-generating activi ty was subsequently reconstituted by adding pure rp67-phox and partial ly purified p21rac. RGVHFIF inhibited superoxide production if added t o the cell-free system during preincubation of rp47-phox with membrane . RGVHFIF was markedly less inhibitory if added to the cell-free syste m after membrane was preincubated with pure rp47-phox. In contrast to p47-phox, preincubation of membrane with either p21rac or rp67-phox co nferred no protection from inhibition of superoxide-generating activit y by RGVHFIF added after preincubation. We conclude that p21rac does n ot facilitate interaction of p47-phox with cytochrome b(558) and that p47-phox is the first cytosol protein to associate with cytochrome b(5 58) during oxidase assembly.