Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Characterization of the membrane-associated component.
Sodium dodecyl sulfate (SDS) was shown to elicit NADPH-dependent superoxide (O2-) 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). O2- production was absolutely dependent on the cooperation between a membrane-associated component, sedimenting with the 48,000 X g pellet and a cytosolic factor, nonsedimentable at 265,000 X g. The present report describes the solubilization and characterization of the membrane-associated component of the SDS-activable O2(-)-forming NADPH oxidase (operationally termed pi). Treatment of the 48,000 X g pellet with 30 mM octyl glucoside resulted in complete transfer of pi to the soluble fraction. The solubilized pellet produced an average of 0.92 mumol of O2-/mg of protein/min upon reduction of octyl glucoside content below the critical micellar concentration and in the presence of cytosol, 100 microM 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. pi was inactivated by the sulfhydryl reagent p-chloromercuribenzoate. Solubilized pellet contained spectroscopically detectable cytochrome b559 (225.6 +/- 15.0 pmol/mg mg protein). Both pi and cytochrome b559 were bound by Cibacron Blue Sepharose and could be eluted by a gradient of octyl glucoside (0-30 mM) in the presence of 1 M KCl. On high performance gel filtration on Superose 12, both pi and cytochrome b559 eluted in the excluded volume; when 25 mM octyl glucoside was present in the elution buffer, pi was partially dissociated from cytochrome b559. Sequential purification of pi on Blue Sepharose followed by gel filtration on Superose 12 in the presence of 25 mM octyl glucoside lead to complete resolution of pi from cytochrome b559 (pi was found in the Mr = 28,000 - 11,000 range while the bulk of cytochrome b559 eluted in the Mr = 113,000 - 71,000 range). We propose that pi is distinct from cytochrome b559 and represents a membrane-associated component in an amphiphile-activated electron transport chain from NADPH to oxygen.
Pick
E
E
Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Israel.
Bromberg
Y
Y
Shpungin
S
S
Gadba
R
R
D000818:Animals; D002474:Cell-Free System; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D006168:Guinea Pigs; D008264:Macrophages / Q000201:enzymology; D008566:Membranes / Q000032:analysis; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D010101:Oxygen Consumption; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D013053:Spectrophotometry; D013481:Superoxides / Q000378:metabolism*
D002729:Chloromercuribenzoates; D013481:Superoxides; D012967:Sodium Dodecyl Sulfate; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases