Role of the rac1 p21‐GDP‐dissociation inhibitor for rho heterodimer in the activation of the superoxide‐forming NADPH oxidase of macrophages

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Abstract
Activation of the superoxide (O−2)‐generating NADPH oxidase of phagocytes requires the interaction of membrane‐associated cytochrome b559 with three cytosolic components; p47‐phox, p67‐phox and σ1. We proposed that σ1 was a heterodimer composed of proteins of 22 kDa and 24 kDa that were tentatively identified as the small GTP‐binding protein (G protein) rac1 p21 and GDP‐dissociation inhibitor for rho (rho GDI). We now describe a modified procedure for the rapid purification of σ1 and demonstrate that the NADPH‐oxidase‐activating capacity is associated, throughout the purification sequence, with a protein binding 35S‐labelled guanosine 5′‐[3‐O‐thio]tri‐phosphate. SDS/PAGE analysis confirmed the absolute association of σ1 activity with the presence of both the 22 kDa and 24 kDa proteins. Immunoblotting with a battery 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 anti anti‐(rap1 p21). Free rac1 p21 (not in complex with rho GDI) was not detected at any stage of cytosol fractionation. The proteins comprising the σ1 heterodimer could be separated by reverse‐phase chromatography and amino acid sequencing was performed on peptides derived by trypsin digestion of each of the isolated proteins. This demonstrated the identity of the 22‐kDa protein with rac1 p21 and that of the 24‐kDa protein with rho GDI. Purified heterodimeric σ1 did not require exogenous GTP for activity under conditions that assured the absence of free nucleotides. Treatment of the σ1 heterodimer with 1% sodium cholate, followed by gel filtration or anion‐exchange chromatography in the presence of 1% sodium cholate, effectively separated rac1 p21 from rho GDI. Monomeric rac1 p21, obtained by these procedures, was able to stimulate cell‐free O−2 generation. Artificial heterodimeric σ1, capable of NADPH oxidase activation, could be reconstituted in vitro by recombining purified monomeric rac1 p21 and rho GDI and removing the sodium cholate used to dissociate the native σ1 dimer. Monomeric rac1 p21 exhibited an almost absolute dependence on exogenous GTP following removal of the endogenous nuclcotide in low Mg2+ solution. Under similar conditions, heterodimeric σ1 was resistant to nucleotide exchange. We propose that rac1 p21 is present in cytosol exclusively 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.

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