Activation of the Superoxide Forming NADPH Oxidase in a Cell - free System by Sodium Dodecyl Sulfate

Sodium dodecyl sulfate (SDS) was shown to elicit NADPH-dependent superoxide (0;) production by a cell-free system derived from sonically disrupted resting guinea pig macrophages (Bromberg, Y., and Pick, E. (1985) J. Biol. Chem. 260, 13539-13545). 0; production was absolutely dependent on the cooperation between a membrane-associated component, sedimenting with the 48,000 % g pellet and a cytosolic factor, nonsedimentable at 265,000 % g. The present report describes the solubilization and characterization of the membrane-associated component of the SDS-activable 0;-forming NADPH oxidase (operationally termed A). Treatment of the 48,000 X g pellet with 30 mM octyl glucoside resuIted in complete transfer of A to the soluble fraction. The solubilized pellet produced an average of 0.92 pmol of O;/mg of protein/min upon reduction of octyl glucoside content below the critical micellar concentration and in the presence of cytosol, 100 p~ SDS, and 0.2 mM NADPH. The activity of solubilized pellet-cytosol combinations was also expressed as NADPH-dependent, azide-resistant oxygen consumption and hydrogen peroxide production. A was inactivated by the sulfhydryl reagent p-chloromercuribenzoate. Solubilized pellet contained spectroscopically detectable cytochrome bGb9 (225.6 f 15.0 pmol/ mg protein). Both A and cytochrome bb59 were bound by Cibacron Blue Sepharose and could be eluted by a gradient of octyl glucoside (0-30 m ~ ) in the presence of 1 M KCl. On high performance gel filtration on Superose 12, both A and cytochrome bG69 eluted in the excluded volume; when 25 mM octyl glucoside was present in the elution buffer, A was partially dissociated from cytochrome bSb9. Sequential purification of A on Blue Sepharose followed by gel filtration on Superose 12 in the presence of 25 mM octyl glucoside lead to complete resolution of A from cytochrome bG5@ ( A was found in the M, = 28,000-1 1,000 range while the bulk of cytochrome bs59 eluted in the M, = 113,000-7 1,000 range). We propose that A is distinct from cytochrome bbBB and represents a membrane-associated component in an amphiphile-activated electron transport chain from NADPH to oxygen.