THE GDP-BOUND FORM OF THE SMALL G-PROTEIN RAC1 P21 IS A POTENT ACTIVATOR OF THE SUPEROXIDE-FORMING NADPH OXIDASE OF MACROPHAGES

Phagocytes produce superoxide by the assembly of a multicomponent comp lex that utilizes NADPH for the reduction of molecular oxygen (NADPH o xidase). The components participating in the assembly are a membrane-b ound flavocytochrome and three cytosolic proteins, one of which was sh own to be a dimer of the small GTP-binding protein (G protein) Rac1 p2 1 or Rac2 p21 with GDP dissociation inhibitor for Rho (Rho GDI). We de termined the identity and quantity of the nucleotide bound to Rac1 p21 by high performance anion exchange chromatography of extracts prepare d from highly purified Rac1 p21-Rho GDI, isolated from guinea pig macr ophage cytosol. Rac1 p21 contained only GDP at a ratio of close to 1 m ol of GDP per mol of G protein. The GDP-bound form of Rac1 p21 complex ed to Rho GDI functioned as a potent activator of NADPB oxidase in a c ell-free system that contained no free GTP or ATP. We propose that the GDP-bound form of Rac1 p21 might be the physiological activator of NA DPH oxidase in macrophages, following its dissociation from Rho GDI, a nd that nucleotide exchange or conversion to GTP is not necessarily in volved.