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

The phagocyte superoxide-generating NADPH oxidase, a multicomponent, membrane-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 membrane with neutrophil cytosol containing p47-phox, but not p67-phox, led to formation of a long-lived NADPH oxidase intermediate. This suggested that p47-phox interacted with cytochrome b(558) in the early stages of oxidase assembly while p67-phox participated in a later stage. Peptides containing the sequence RGVHFIF (corresponding to amino acids 559-565 of the 91-kDa subunit of cytochrome b(558)) inhibit NADPH oxidase activity by blocking the early interaction between p47-phox and cytochrome b(558) In the present study, we examined whether p21rac facilitated the interaction between p47-phox and cytochrome b(558) We preincubated pure recombinant p47-phox with neutrophil membrane containing cytochrome b(558) in the cell-free system. Superoxide-generating activity was subsequently reconstituted by adding pure rp67-phox and partially purified p21rac. RGVHFIF inhibited superoxide production if added to the cell-free system during preincubation of rp47-phox with membrane. RGVHFIF was markedly less inhibitory if added to the cell-free system after membrane was preincubated with pure rp47-phox. In contrast to p47-phox, preincubation of membrane with either p21rac or rp67-phox conferred no protection from inhibition of superoxide-generating activity by RGVHFIF added after preincubation. We conclude that p21rac does not facilitate interaction of p47-phox with cytochrome b(558) and that p47-phox is the first cytosol protein to associate with cytochrome b(558) during oxidase assembly.