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(Received for publication, April 19, 1996, and in revised form, June 6, 1996)
From the Department of Biochemistry, Emory University Medical
School, Atlanta, Georgia 30322
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
p47phox, p67phox, and Rac plus the
phospholipid-reconstituted flavocytochrome
b558. Here, we demonstrate that activity is
reconstituted in the absence of p47phox when high
concentrations of p67phox and Rac are used.
Vmax values were the same in the presence or
absence of p47phox, yet p47phox increases the affinity
of both p67phox and Rac for the oxidase complex by nearly 2 orders of magnitude. p67phox-(1-246), a truncated form of the
protein which eliminates SH3 domains involved in binding to
p47phox, also supports superoxide generation, both in the
presence and absence of p47phox, providing further evidence for
p47phox independent activity. In the absence of
p47phox, p67phox-(1-246) binds to the NADPH oxidase
complex 3-fold more tightly than does native p67phox,
indicating that the C terminus contains a region which masks binding to
the oxidase complex. Results indicate that p47phox 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 p67phox by binding
to its C-terminal domains. p67phox and/or Rac play a more
direct role in regulating electron transfer.
Volume 271, Number 37,
Issue of September 13, 1996
pp. 22578-22582
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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