Abstract
The elicitation of an oxidative burst in phagocytes rests on the assembly of a multicomponental complex (NADPH oxidase) consisting of a membrane-associated flavocytochrome (cytochrome b559), representing the redox element responsible for the NADPH-dependent reduction of oxygen to superoxide (O-2), two cytosolic components (p47(phox), p67(phox)), and the small GTPase Rac (1 or 2). We found that 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), an irreversible serine protease inhibitor, prevented the elicitation of O-2 production in intact macrophages and the amphiphile-dependent activation of NADPH oxidase in a cell-free system, consisting of solubilized membrane or purified cytochrome b559 combined with total cytosol or a mixture of recombinant p47(phox), p67(phox), and Rac1. AEBSF acted at the activation step and did not interfere with the ensuing electron flow. It did not scavenge oxygen radicals and did not affect assay reagents. Five other serine protease inhibitors (three irreversible and two reversible) were found to lack an inhibitory effect on cell-free activation of NADPH oxidase. A structure-function study of AEBSF analogues demonstrated that the presence of a sulfonyl fluoride group was essential for inhibitory activity and that compounds containing an aminoalkylbenzene moiety were more active than amidinobenzene derivatives. Exposure of the membrane fraction or of purified cytochrome b559, but not of cytosol or recombinant cytosolic components, to AEBSF, in the presence of a critical concentration of the activating amphiphile lithium dodecyl sulfate, resulted in a marked impairment of their ability to support cell-free NADPH oxidase activation upon complementation with untreated cytosol or cytosolic components. Kinetic analysis of the effect of varying the concentration of each of the three cytosolic components on the inhibitory potency of AEBSF indicated that this was inversely related to the concentrations of p47(phox) and, to a lesser degree, p67(phox). AEBSF also prevented the amphiphile-elicited translocation of p47(phox) and p67(phox) to the membrane. These results are interpreted as indicating that AEBSF interferes with the binding of p47(phox) and/or p67(phox) to cytochrome b559, probably by a direct effect on cytochrome b559.
Highlights
The elicitation of an oxidative burst in phagocytes rests on the assembly of a multicomponental complex (NADPH oxidase) consisting of a membrane-associated flavocytochrome, representing the redox element responsible for the NADPH-dependent reduction of oxygen to superoxide (O2.), two cytosolic components (p47phox, p67phox), and the small GTPase Rac (1 or 2)
We found that 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), an irreversible serine protease inhibitor, prevented the elicitation of O2. production in intact macrophages and the amphiphile-dependent activation of NADPH oxidase in a cell-free system, consisting of solubilized membrane or purified cytochrome b559 combined with total cytosol or a mixture of recombinant p47phox, p67phox, and Rac1
In the present paper we report that 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF; known as Pefabloc SC), originally developed as an irreversible serine protease inhibitor, prevents the activation of the O2. generating NADPH oxidase in both intact stimulated macrophages and in cell-free systems
Summary
O2., superoxide; GTP␥S, guanosine 5Ј3-O-(thio)triphosphate; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; AEBSNH2, 4-(2-aminoethyl)-benzenesulfonamide; AEBSAc, 4-(2aminoethyl)-benzenesulfonic acid; MAEBSF, 4-(2-N-methylaminoethyl)-benzenesulfonyl fluoride; pABSF, 4-(amidino)-benzenesulfonyl fluoride; octyl glucoside, n-octyl--D-glucopyranoside; PMA, phorbol 12myristate 13-acetate; fMLP, N-formyl-Met-Leu-Phe; PMSF, phenylmethanesulfonyl fluoride; pAPMSF, 4-(amidino)-phenylmethanesulfonyl fluoride; TPCK, n-tosyl-L-phenylalanine chloromethyl ketone; DCIC, 3,4-dichloroisocoumarin; LiDS, lithium dodecyl sulfate. These latter approaches should allow the selection of inhibitors with a direct effect on the assembled complex or on individual components of the NADPH oxidase complex. The only compound having gained wide acceptance as a relatively specific direct inhibitor of NADPH oxidase is diphenylene iodonium [14]. In the present paper we report that 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF; known as Pefabloc SC), originally developed as an irreversible serine protease inhibitor, prevents the activation of the O2. The effect is shared by some structurally related compounds, indicating that we are dealing with a new class of NADPH oxidase inhibitors. Evidence is presented in favor of the proposal that AEBSF interferes with the interaction of p47phox and/or p67phox with cytochrome b559, probably by chemical modification of cytochrome b559
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