Jw. Park et al., ISOLATION OF A COMPLEX OF RESPIRATORY BURST OXIDASE COMPONENTS FROM RESTING NEUTROPHIL CYTOSOL, Biochemistry, 33(10), 1994, pp. 2907-2911
The respiratory burst oxidase of neutrophils is a multicomponent enzym
e, dormant in resting cells, that catalyzes the reduction of oxygen to
O2(-) at the expense of NADPH. In the resting neutrophil, some of the
components of the oxidase, including proteins p47(phox) and p67(phox)
are in the cytosol, while the rest are in a fraction that usually cop
urifies with plasma membrane. Recent evidence has suggested that at le
ast some of the cytosolic oxidase components exist as a complex. We ha
ve now purified such a complex from the cytoplasm of resting neutrophi
ls using an affinity column prepared with an antibody that recognizes
the C-terminal decapeptide of p47(phox). Immunoblotting showed that th
e complex contained both p47(phox) and p67(phox). When supplemented wi
th recombinant p67(phox), the complex displayed considerable activity
in a cell-free oxidase-activating system, and even without added p67(p
hox), the complex could more than double O(2)(-)production in an oxida
se-activating system supplemented with suboptimal amounts of cytosol.
Isolation of the complex was blocked by preincubating the affinity col
umn with CFSTKRKLASAV, the peptide against which the antibody was rais
ed. On gel filtration, the complex migrated with a molecular weight of
240-300K, similar to that observed with whole neutrophil cytosol. The
p47(phox)/p67(phox) ratio in the gel-filtered complex was approximate
ly 1 to 1. These results indicate that in resting neutrophil cytosol,
p47(phox) and p67(phox) exist as a complex.