Purification and characterization of a third cytosolic component of the superoxide-generating NADPH oxidase of macrophages.

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.