EVIDENCE FOR A READILY DISSOCIABLE COMPLEX OF P47PHOX AND P67PHOX IN CYTOSOL OF UNSTIMULATED HUMAN NEUTROPHILS

We explored the association between two cytosolic components of the ph agocyte respiratory burst oxidase, p47phox and p67phox. Both of these proteins bound to immobilized GTP or 2',5'-ADP, but not to GDP or ATP. Similarly, triazine dye-ligand chromatography demonstrated coisolatio n of p47phox and p67phox. Binding of p67phox to GTP was less avid than that of p47phox. Each of the proteins in whole neutrophil cytosol bou nd separately to GTP in the absence of the other, whereas studies with recombinant proteins showed binding of p47phox but not p67phox. These data suggest that p47phox and p67phox exist as a complex that very li kely involves at least one additional cytosolic protein. Sequential pr ecipitation in graded concentrations of ammonium sulfate demonstrated similar profiles for p47phox and p67phox. Charge-based separations usi ng either an anion or cation exchange resin resulted in coelution of t he two cytosolic oxidase components, in spite of their widely differin g charges as shown by separate analysis of the recombinant proteins. M oreover, preparative isoelectric focusing showed that a portion of the p47phox and p67phox in cytosol coisolated at a pi (7.3-8.3) midway be tween those of the separate proteins. Despite this, each protein sedim ented independently in sucrose density gradients. The data indicate th at the complex of p47phox and p67phox was dissociated by increased tem perature, high osmolarity, or prolonged incubation. We suggest that un der the physiologic conditions in the cytosol of intact phagocytes p47 phox, p67phox and probably other proteins exist as a macromolecular co mplex, the function of which may be to permit en bloc translocation of oxidase components to the plasma membrane.