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J. Biol. Chem., Vol. 266, Issue 35, 23577-23585, Dec, 1991
A Abo and E Pick
Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes
in a cell-free system by anionic amphiphiles requires the participation of
both membrane and cytosolic components. We reported that ammonium sulfate
fractionation (Pick, E., Kroizman, T., and Abo, A. (1989) J. Immunol. 143,
4180-4187) and affinity chromatography on 2',5'-ADP-agarose (Shaag, D., and
Pick, E. (1990) Biochim. Biophys. Acta 1037, 405-412) permit separation of
cytosol in two fractions (sigma 1 and sigma 2) that support O2- production
by solubilized membrane synergistically. We now describe the purification
of sigma 1 to near homogeneity and demonstrate that it represents a
cytosolic component distinct from p47-phox and p67-phox, that are both
found in fraction sigma 2. Sigma 1 was absolutely required for the full
expression of amphiphile-activated NADPH-oxidase activity. This requirement
was evident whether sigma 1 was added to cell-free systems composed of: (a)
solubilized membrane and a sigma 2-enriched cytosolic fraction, or (b)
purified cytochrome b559, incorporated in liposomes, and purified sigma 2.
Sigma 1 was purified by a sequence comprising ammonium sulfate
fractionation, hydrophobic chromatography on phenyl- Superose, absorption
with CM-Sepharose, anion exchange chromatography on DEAE-Sepharose, and gel
filtration on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis analysis of sigma 1 of maximal purity, under both reducing
and nonreducing conditions, demonstrated the presence of two proteins, of
24 and 22 kDa. On gel filtration, sigma 1 was eluted as a symmetrical peak
of 46 kDa that by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
analysis revealed the presence of both 24- and 22-kDa bands. We suggest
that, in its native form, sigma 1 might represent a complex of the 24- and
22- kDa proteins. The specific roles of each molecule in NADPH oxidase
function remain to be determined.
Purification and characterization of a third cytosolic component of the superoxide-generating NADPH oxidase of macrophages
Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Israel.
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