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

Activation of the superoxide (O^-₂)-generating NADPH oxidase of phagocytes in a cell-free system by anionic amphiphiles requires the partcipation 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 (σ₁ and σ₂) that support O^-₂ production by solubilized membrane synergistically. We now describe the purification of σ₁ to near homogeneity and demonstrate that it represents a cytosolic component distinct from p47-phox and p67-phox, that are both found in fraction σ₂. σ₁ was absolutely required for the full expression of amphiphile-activated NADPH-oxidase activity. This requirement was evident whether σ₁ was added to cell-free systems composed of: (a) solubilized membrane and a σ₂-enriched cytosolic fraction, or (b) purified cytochrome b₅₅₉, incorporated in liposomes, and purified σ₂. σ₁ 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 σ₁ of maximal purity, under both reducing and nonreducing conditions, demonstrated the presence of two proteins, of 24 and 22 kDa. On gel filtration, σ₁ 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, σ₁ 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.