INHIBITION OF NADPH OXIDASE ACTIVATION BY 4-(2-AMINOETHYL)-BENZENESULFONYL FLUORIDE AND RELATED-COMPOUNDS

The elicitation of an oxidative burst in phagocytes rests on the assem bly of a multicomponental complex (NADPH oxidase) consisting of a memb rane-associated flavocytochrome (cytochrome b(559)), representing the redox element responsible for the NADPH-dependent reduction of oxygen to superoxide (O-2(radical anion)), two cytosolic components (p47(phox ), p67(phox)), and the small GTPase Rac (1 or 2), We found that 4-(2-a minoethyl)-benzenesulfonyl fluoride (AEBSF), an irreversible serine pr otease inhibitor, prevented the elicitation of O-2(radical anion) prod uction in intact macrophages and the amphiphile-dependent activation o f NADPH oxidase in a cell free system, consisting of solubilized membr ane or purified cytochrome b(559) combined with total cytosol or a mix ture of recombinant p47(phox), p67(phox), and Rad. AEBSF acted at the activation step and did not interfere with the ensuing electron flour, It did not scavenge oxygen radicals and did not affect assay reagents , Five other serine protease inhibitors (three irreversible and two re versible) were found to lack an inhibitory effect on cell-free activat ion of NADPH oxidase, A structure-function study of AEBSF analogues de monstrated that the presence of a sulfonyl fluoride group was essentia l for inhibitory activity and that compounds containing an aminoalkylb enzene moiety were more active than amidinobenzene derivatives, Exposu re of the membrane fraction or of purified cytochrome b(559), but not of cytosol or recombinant cytosolic components, to AEBSF, in the prese nce of a critical concentration of the activating amphiphile lithium d odecyl sulfate, resulted in a marked impairment of their ability to su pport cell-free NADPH oxidase activation upon complementation with unt reated cytosol or cytosolic components, Kinetic analysis of the effect of varying the concentration of each of the three cytosolic component s on the inhibitory potency of AEBSF indicated that this was inversely related to the concentrations of p47(phox) and, to a lesser degree, p 67(phox), AEBSF also prevented the amphiphile-elicited translocation o f p47(phox) and p67(phox) to the membrane, These results are interpret ed as indicating that AEBSF interferes with the binding of p47(phox) a nd/or p67(phox) to cytochrome b(559), probably by a direct effect on c ytochrome b(559).