ROLE OF THE RAC1 P21-GDP-DISSOCIATION INHIBITOR FOR RHO HETERODIMER IN THE ACTIVATION OF THE SUPEROXIDE-FORMING NADPH OXIDASE OF MACROPHAGES

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocy tes requires the interaction of membrane-associated cytochrome b559 wi th three cytosolic components; p47-phox, p67-phox and sigma1. We propo sed that sigma1 was a heterodimer composed of proteins of 22 kDa and 2 4 kDa that were tentatively identified as the small GTP-binding protei n (G protein) rac1 p21 and GDP-dissociation inhibitor for rho (rho GDI ). We now describe a modified procedure for the rapid purification of sigma1 and demonstrate that the NADPH-oxidase-activating capacity is a ssociated, throughout the purification sequence, with a protein bindin g S-35-labelled guanosine 5'-[3-O-thio]triphosphate. SDS/PAGE analysis confirmed the absolute association of sigma1 activity with the presen ce of both the 22 kDa and 24 kDa proteins. Immunoblotting with a batte ry of antibodies against the small G proteins demonstrated that the 22 -kDa protein was only recognized by antibodies reacting with rac1 p21; no reaction was found with anti-(rac2 p21), anti-[v-ras(H) p21] and a nti anti-(rap1 p21). Free rac1 p21 (not in complex with rho GDI) was n ot detected at any stage of cytosol fractionation. The proteins compri sing the sigma1 heterodimer could be separated by reverse-phase chroma tography and amino acid sequencing was performed on peptides derived b y trypsin digestion of each of the isolated proteins. This demonstrate d the identity of the 22-kDa protein with rac1 p21 and that of the 24- kDa protein with rho GDI. Purified heterodimeric sigma1 did not requir e exogenous GTP for activity under conditions that assured the absence of free nucleotides. Treatment of the sigma1 heterodimer with 1% sodi um cholate, followed by gel filtration or anion-exchange chromatograph y in the presence of 1% sodium cholate, effectively separated rac1 p21 from rho GDI. Monomeric rac1 p21, obtained by these procedures, was a ble to stimulate cell-free O2- generation. Artificial heterodimeric si gma1 capable of NADPH oxidase activation, could be reconstituted in vi tro by recombining purified monomeric rac1 p21 and rho GDI and removin g the sodium cholate used to dissociate the native sigma1 dimer. Monom eric rac1 p21 exhibited an almost absolute dependence on exogenous GTP following removal of the endogenous nucleotide in low Mg2+ Solution. Under similar conditions, heterodimeric sigma1 was resistant to nucleo tide exchange. We propose that rac1 p21 is present in cytosol exclusiv ely as a heterodimer with rho GDI and that activation of NADPH oxidase involves the dissociation of the dimer and the subsequent interaction of free rac1 p21 with another component participating in the assembly of the enzyme complex.