NADPH oxidase activity is independent of p47phox in vitro.
The neutrophil superoxide generating NADPH oxidase is activated by the assembly of cytosolic protein components with a membrane-associated flavocytochrome. The activity can be reconstituted in vitro using purified cytosolic factors p47(phox), p67(phox), and Rac plus the phospholipid-reconstituted flavocytochrome b558. Here, we demonstrate that activity is reconstituted in the absence of p47(phox) when high concentrations of p67(phox) and Rac are used. Vmax values were the same in the presence or absence of p47(phox), yet p47(phox) increases the affinity of both p67(phox) and Rac for the oxidase complex by nearly 2 orders of magnitude. p67(phox)-(1-246), a truncated form of the protein which eliminates SH3 domains involved in binding to p47(phox), also supports superoxide generation, both in the presence and absence of p47(phox), providing further evidence for p47(phox) independent activity. In the absence of p47(phox), p67(phox)-(1-246) binds to the NADPH oxidase complex 3-fold more tightly than does native p67(phox), indicating that the C terminus contains a region which masks binding to the oxidase complex. Results indicate that p47(phox) does not play a direct role in regulating electron transfer. Rather, its function is to serve as an adaptor protein to enhance the assembly of the other cytosolic components with the flavocytochrome and possibly to unmask a binding region in the N terminus of p67(phox) by binding to its C-terminal domains. p67(phox) and/or Rac play a more direct role in regulating electron transfer.
Freeman
J L
JL
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322, USA.
Lambeth
J D
JD
D016718:Arachidonic Acid / Q000378:metabolism; D004579:Electron Transport; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology; D010750:Phosphoproteins / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D020744:rac GTP-Binding Proteins
D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D016718:Arachidonic Acid; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins