NADPH OXIDASE ACTIVITY IS INDEPENDENT OF P47(PHOX) IN-VITRO

The neutrophil superoxide generating NADPH oxidase is activated by the assembly of cytosolic protein components with a membrane-associated f lavocytochrome. The activity can be reconstituted in vitro using purif ied cytosolic factors p47(phox), p67(phox), and Rac plus the phospholi pid-reconstituted flavocytochrome b(558). Here, we demonstrate that ac tivity is reconstituted in the absence of p47(phox) when high concentr ations of p67(phox) and Rac are used. V-max values were the same in th e presence or absence of p47(phox), yet p47(phox) increases the affini ty of both p67(phox) and Rac for the oxidase complex by nearly 2 order s of magnitude, p67(phox)-(1-246), a truncated form of the protein whi ch eliminates SH3 domains involved in binding to p47(phox), also suppo rts superoxide generation, both in the presence and absence of p47(pho x), 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 tha t the C terminus contains a region which masks binding to the oxidase complex. Results indicate that p47(phox) does not play a direct role i n regulating electron transfer. Rather, ifs function is to serve as an adaptor protein to enhance the assembly of the other cytosolic compon ents with the flavocytochrome and possibly to unmask a binding region in the N terminus of p67(phox) by binding to its C-terminal domains. p 67(phox) and/or Rac play a more direct role in regulating electron tra nsfer.