Activation of the superoxide-forming NADPH oxidase of macrophages requires two cytosolic components - One of them is also present in certain nonphagocytic cells

The superoxide-forming NADPH oxidase of resting macrophages can be activated in a cell-free system by certain anionic amphiphiles, most notably SDS. Activation requires the cooperation of membrane-associated and cytosolic components. We now report that at least two cytosolic factors are required for SDS-elicited activation of NADPH oxidase of guinea pig macrophages. Treatment of cytosol with ammonium sulfate at 37% saturation led to the partition of the two factors in the supernatant and precipitate fractions (termed components σ₁ and σ₂, respectively). Although each fraction by itself was inactive, recombining them resulted in complete recovery of the original ability of native cytosol to support SDS-elicited superoxide production by octyl-glucoside solubilized macrophage membranes. Both components are proteins, as shown by their susceptibility to trypsin and proteinase K, and were inactivated by heating at 60°C. σ₂, but not σ₁, was inactivated by treatment with the covalent sulfhydryl reagent N-ethylmaleimide. On high-performance gel filtration, σ₁ was found to have a molecular mass of 30 to 52 kDa, whereas σ₂ eluted with molecules of 150 to 440 kDa. Component σ₁ was partially purified from the ammonium sulfate supernatant fraction of cytosol by hydrophobic interaction chromatography followed by gel filtration. A material behaving like σ₁ was also found to be present in the cytosol of guinea pig thymus cells, lymph node lymphocytes and brain and of the mouse myeloma cell line MOPC 315. However, σ₂ appears to be strictly phagocyte specific. The molecular characteristics of σ₁ components from nonphagocytic cells were similar to those of macrophage σ₁, as shown by their presence in the supernatant, after treatment of cytosol with ammonium sulfate at 37% saturation, a molecular mass close to 30 to 52 kDa and a similar behavior on hydrophobic interaction chromatography. These findings raise the possibility that cytosolic component σ₁ might be the bearer of a cellular function, more general than the one suggested by its role in the activation of NADPH oxidase of phagocytes.