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 phagocyte 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 coisolation of p47phox and p67phox. Binding of p67phox to GTP was less avid than that of p47phox. Each of the proteins in whole neutrophil cytosol bound 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 likely involves at least one additional cytosolic protein. Sequential precipitation in graded concentrations of ammonium sulfate demonstrated similar profiles for p47phox and p67phox. Charge-based separations using either an anion or cation exchange resin resulted in coelution of the two cytosolic oxidase components, in spite of their widely differing charges as shown by separate analysis of the recombinant proteins. Moreover, preparative isoelectric focusing showed that a portion of the p47phox and p67phox in cytosol coisolated at a pI (7.3-8.3) midway between those of the separate proteins. Despite this, each protein sedimented independently in sucrose density gradients. The data indicate that the complex of p47phox and p67phox was dissociated by increased temperature, high osmolarity, or prolonged incubation. We suggest that under the physiologic conditions in the cytosol of intact phagocytes p47phox, p67phox and probably other proteins exist as a macromolecular complex, the function of which may be to permit en bloc translocation of oxidase components to the plasma membrane.
Iyer
S S
SS
Department of Medicine, University of Iowa, Iowa City.
Pearson
D W
DW
Nauseef
W M
WM
Clark
R A
RA
D000645:Ammonium Sulfate; D000818:Animals; D016367:Baculoviridae; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate; D006801:Humans; D007525:Isoelectric Focusing; D008024:Ligands; D046911:Macromolecular Substances; D009036:Moths; D009252:NADPH Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins
D008024:Ligands; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011994:Recombinant Proteins; C079789:neutrophil cytosol factor 67K; D006160:Guanosine Triphosphate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D000645:Ammonium Sulfate