Key points are not available for this paper at this time.
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 b 559), representing the redox element responsible for the NADPH-dependent reduction of oxygen to superoxide (O·̄2), 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 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 b 559 combined with total cytosol or a mixture of recombinant p47phox, p67phox, 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 b 559, 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 p47phox and, to a lesser degree, p67phox. AEBSF also prevented the amphiphile-elicited translocation of p47phox and p67phox to the membrane. These results are interpreted as indicating that AEBSF interferes with the binding of p47phox and/or p67phox to cytochromeb 559, probably by a direct effect on cytochromeb 559. 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 b 559), representing the redox element responsible for the NADPH-dependent reduction of oxygen to superoxide (O·̄2), 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 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 b 559 combined with total cytosol or a mixture of recombinant p47phox, p67phox, 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 b 559, 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 p47phox and, to a lesser degree, p67phox. AEBSF also prevented the amphiphile-elicited translocation of p47phox and p67phox to the membrane. These results are interpreted as indicating that AEBSF interferes with the binding of p47phox and/or p67phox to cytochromeb 559, probably by a direct effect on cytochromeb 559. The production of reactive oxygen radicals represents the major microbicidal mechanism of phagocytes (1Klebanoff S.J. Gallin J.I. Goldstein I.M. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. Raven Press, New York1992: 541-588Google Scholar). Oxygen radicals are also generated, in lesser amounts, by some nonphagocytic cells, sharing the enzymatic machinery characteristic of phagocytes (2Cross A.R. Jones O.T.G. Biochim. Biophys. Acta. 1991; 1057: 281-298Crossref PubMed Scopus (455) Google Scholar), and, under certain conditions, by plant cells (3Dwyer S.C. Legendre L. Low P.S. Leto T.L. Biochim. Biophys. Acta. 1996; 1289: 231-237Crossref PubMed Scopus (113) Google Scholar). Interest in reactive oxygen species has also been stimulated by accumulating evidence for their involvement in the pathogenesis of diseases, ranging from respiratory distress syndrome to ischemia-reperfusion injury in several organs (4Halliwell B. FASEB J. 1987; 1: 358-364Crossref PubMed Scopus (1027) Google Scholar). The primordial oxygen radical produced by phagocytes is superoxide (O·̄2). 1The abbreviations used are: O·̄2, superoxide; GTPγS, guanosine 5′-3-O-(thio)triphosphate; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; AEBSNH2, 4-(2-aminoethyl)-benzenesulfonamide; AEBSAc, 4-(2-aminoethyl)-benzenesulfonic acid; MAEBSF, 4-(2-N-methylaminoethyl)-benzenesulfonyl fluoride; pABSF, 4-(amidino)-benzenesulfonyl fluoride; octyl glucoside,n-octyl-β-d-glucopyranoside; PMA, phorbol 12-myristate 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. It is generated, in response to appropriate stimuli, by NADPH-derived reduction of a by a flavocytochrome (cytochrome b in S.J. J. J. PubMed Google Biochim. Biophys. Acta. PubMed Scopus Google PubMed Scopus Google Scholar). b two redox and and electron from NADPH to oxygen in by the of cytochrome with two cytosolic p47phox and p67phox, and the small or in J. PubMed Scopus Google Scholar). the intact this is by the translocation of the cytosolic components to the to the assembly of is as the NADPH oxidase of NADPH in O·̄2 in by a cell-free consisting of and cytosol PubMed Scopus Google J. PubMed Google or of a mixture of purified or recombinant components, to a critical of an amphiphile or Leto T.L. PubMed Scopus Google J. PubMed Google Scholar). A of inhibitors of the O·̄2 NADPH oxidase been in A.R. PubMed Scopus Google Scholar). The for inhibitors is by two to as for the and mechanism of activation of the NADPH and their as in with production of oxygen radicals at an or inhibitors were for an effect on intact The of an is that not a an effect on membrane and and a direct effect on the components of the NADPH oxidase on the effect of inhibitors on from stimulated cells or on NADPH oxidase activation in the cell-free These the of inhibitors with a direct effect on the complex or on components of the NADPH oxidase the as a direct of NADPH oxidase is A.R. Jones O.T.G. J. PubMed Scopus Google Scholar). the that 4-(2-aminoethyl)-benzenesulfonyl fluoride also as as an irreversible serine protease inhibitor, the activation of the O·̄2 NADPH oxidase in intact stimulated macrophages and in cell-free The effect is by some related indicating that are with a of NADPH oxidase is in of the that AEBSF interferes with the of p47phox and/or p67phox with cytochrome b 559, probably by of cytochrome b 559. AEBSF lithium dodecyl and the were from was from 4-(2-N-methylaminoethyl)-benzenesulfonyl fluoride and 4-(amidino)-benzenesulfonyl fluoride were by was a of was from was from The were from NADPH superoxide oxidase phorbol 12-myristate phenylmethanesulfonyl fluoride 4-(amidino)-phenylmethanesulfonyl fluoride chloromethyl and inhibitors were in at a concentration at than the concentration used in NADPH oxidase the of PMSF, and DCIC, were in and were from the of with The cells were and as J. PubMed Google Scholar), and in for intact The membrane and cytosol were from cells by as J. PubMed Scopus Google Scholar), with some J. PubMed Google Scholar). were in in J. PubMed Scopus Google J. PubMed Google and solubilized in as J. PubMed Google Scholar). in the cell-free solubilized membrane was in to the concentration of octyl to b 559 was purified from the solubilized membrane and by a PubMed Scopus Google of J. 1991; PubMed Google Scholar). analysis of cytochrome b 559 was as in J. 1991; PubMed Google for p47phox and p67phox were of L. Leto of of cells in were with the recombinant and The recombinant were as by Leto T.L. J. 1991; PubMed Google Scholar). the cells were by three in a and at for and the at for The recombinant were purified by of the to as containing p47phox, was to a with with and was with a in the at a of containing p67phox, was to a with with and with a in the at a of recombinant were by p47phox was p67phox was was from with the of L. of as by Leto T.L. PubMed Scopus Google Scholar). the of the was by by three of and the was by at for The was from the on and was from the by was by on was with GTPγS, as 1991; PubMed Scopus Google Scholar). in a of were to of with in of an oxidative burst were in a of and the in the of a O·̄2 production was by the of superoxide at and the was from the of the in PubMed Scopus Google Scholar). amphiphile O·̄2 production in was by reduction in consisting of solubilized membrane and total cytosol or a of p47phox, p67phox, and as J. PubMed Google J. PubMed Google Scholar). some the membrane was by purified cytochrome b 559. were by the in activation by and NADPH-dependent O·̄2 production or by the J. PubMed Scopus Google at of the activation and of the NADPH by cell-free of a to in the O·̄2 production was as in J. Biophys. PubMed Scopus Google and NADPH-dependent oxygen by cell-free consisting of membrane and recombinant cytosolic components, was with the of a oxygen as R. J. 1987; PubMed Google Scholar). The translocation of p47phox and the membrane fraction of macrophages was in the to were with total cytosol to in a total of of NADPH oxidase assay J. PubMed Google Scholar), not containing and for at in the or presence of concentration found to O·̄2 production under The mixture was at at and, of the the membrane was in of NADPH oxidase assay and at for of the the were in of containing at for and to and as J. 1991; PubMed Google Scholar). The were with a mixture of p47phox and p67phox T.L. J. 1991; PubMed Google of L. of by The reactive were by by and to O·̄2 production in macrophages by a of PubMed Scopus Google Scholar). We three fMLP, and the to NADPH oxidase in intact cells by and used at concentrations found to O·̄2 in cells in at the presence of AEBSF, a of O·̄2 production by was AEBSF was the of PMA, the was to was at the AEBSF also a total of O·̄2 production by or not was with three AEBSF as a of on a by three and has a direct effect on the NADPH oxidase We the effect of AEBSF on the ability to O·̄2 by the oxidase system, the reduction AEBSF did not interfere with the of O·̄2 by and oxidase the O·̄2 in the or presence of AEBSF to was than of the NADPH oxidase in a cell-free by certain a mechanism the active in the intact PubMed Scopus Google J. PubMed Google Scholar). the effect of AEBSF on O·̄2 production in several of cell-free activation The of solubilized and from AEBSF was found to a inhibitory on O·̄2 with an of We the effect of AEBSF on a cell-free Leto T.L. PubMed Scopus Google J. PubMed Google Scholar), consisting of solubilized membrane or purified cytochrome b 559, in combined with recombinant p47phox, p67phox, and AEBSF was found to as an of O·̄2 production in this system, of purified NADPH oxidase components in the of other the of the of an in the inhibitory potency of AEBSF was not found in the cell-free consisting of solubilized membrane and of this in the presence of a of membrane or purified cytochrome b 559, the inhibitory effect of AEBSF was inversely related to the concentration of A more of the mechanism of this is in the that by AEBSF of O·̄2 production in the cell-free is to a direct effect on NADPH the of AEBSF on two of NADPH oxidase These were NADPH J. Biophys. PubMed Scopus Google and and NADPH-dependent oxygen R. J. 1987; PubMed Google Scholar), by of membrane and cytosol or membrane and recombinant cytosolic components. We found that AEBSF NADPH in a cell-free were in by the of AEBSF were in the two at AEBSF oxygen by We that AEBSF by with or more of the components of the NADPH oxidase The O·̄2 NADPH oxidase has a for NADPH of Biochim. Biophys. Acta. PubMed Scopus Google and the by amphiphile under cell-free the PubMed Scopus Google J. PubMed Google Scholar). The effect of AEBSF on NADPH oxidase activation in the cell-free was at concentrations of NADPH varying from to and by demonstrated that of AEBSF was with and of cytochromeb 559 results in a of from the cytochrome Leto T.L. PubMed Scopus Google J. 1991; PubMed Google PubMed Scopus Google Scholar), the of NADPH oxidase in the cell-free system, on PubMed Scopus Google Scholar). AEBSF with the of cytochrome b 559, by the effect of varying the concentration of on the inhibitory effect of We found that the of by AEBSF was of the concentration of to the that AEBSF with the activating amphiphile for with a of the NADPH oxidase We found that the concentration of LiDS, from the activating of to to did not or the inhibitory effect of AEBSF also of NADPH in intact cells and in is to the of the assembly of the components of the a complex in J. PubMed Scopus Google Scholar), this by the of electron the 559 The and in a cell-free activation assay J. PubMed Scopus Google Scholar). of a mixture of NADPH oxidase components to an concentration of activating amphiphile in a small for a in the of by and the of electron by of The results in a reduction in the concentration of amphiphile to a and the of assembly of the We this to the by the inhibitor, by AEBSF to the activation mixture at or the of in AEBSF was a more the assembly of the NADPH oxidase complex than at the of The that AEBSF was not at the of step is probably to the that some assembly in the of the The ability of AEBSF to to the components of the NADPH oxidase in the of electron also that mechanism of is from that of acted as an under conditions, as by NADPH oxidase J. J. PubMed Scopus Google Scholar). was of the cell-free assay for of NADPH oxidase activation by AEBSF was this activation were in the and presence of a concentration of AEBSF and with an concentration of for the were in assay to the amphiphile and or not to an activating concentration of for The results in that to of with AEBSF the O·̄2 production by activation with AEBSF was of that of of the to to of O·̄2 of that in untreated AEBSF was as an irreversible serine protease Google Scholar). It essential to inhibitory effect on NADPH oxidase activation was related to the ability of serine protease inhibitors to affect NADPH oxidase activation in the cell-free These were the irreversible inhibitors and the irreversible inhibitor, and the inhibitors and of an inhibitory effect on NADPH oxidase We the that related to the concentration of activating in PMSF, and did not an inhibitory effect at concentration AEBSF was a the of amphiphile the PMSF, and an effect on NADPH oxidase at concentrations of for We the of several analogues of AEBSF to interfere with the activation of NADPH oxidase. group of and MAEBSF, to AEBSF for the presence of an AEBSAc, a of of AEBSF, found to as a protease and AEBSNH2, in an group the fluoride found in AEBSF, also to lack protease inhibitory activity Google Scholar). in and were an inhibitory potency to that of AEBSF The group of amidinobenzene derivatives. was to a irreversible serine protease R. PubMed Scopus Google Scholar), and as a J. PubMed Scopus Google Scholar). in was a of NADPH oxidase was than that of We also the of an of the two and AEBSNH2, sharing the with AEBSF, on of NADPH oxidase activation by of the two at was of the inhibitory effect of AEBSF, at concentrations ranging from to These results that a sulfonyl fluoride to the is essential for inhibitory activity and that aminoalkylbenzene are more inhibitors than amidinobenzene We AEBSF and a NADPH oxidase The of was to or NADPH oxidase components to AEBSF for a in a small by assay containing the untreated fraction or a mixture of the untreated NADPH oxidase components, activating and resulted in a reduction in AEBSF concentration to effect to the to the We the effect of AEBSF on total total cytochrome b 559, and recombinant p47phox, p67phox, and of was or not to AEBSF in the or presence of LiDS, the concentration of amphiphile not to that the of NADPH oxidase upon complementation with untreated components. in of total solubilized membrane or purified and cytochromeb 559 to AEBSF, in the presence but not in the of LiDS, resulted in marked of their to support O·̄2 production upon complementation with cytosol and activation by cytosol as as p47phox and were to by a effect of AEBSF on p67phox was but this was to of and NADPH oxidase components, p67phox was the to by of components of the NADPH oxidase complex with AEBSF on their ability to support O·̄2 production in the cell-free or with AEBSF of NADPH oxidase activation in the cell-free in the of in the presence of solubilized membrane of cytochromeb was to AEBSF in the or presence of It was with cytosol to in to in was to AEBSF in the or presence of It was with solubilized membrane of cytochrome b in cytochromeb cytochrome b 559 in was to AEBSF in the or presence of It was with cytosol to in in was to AEBSF in the or presence of It was with the components, in at the solubilized membrane b p67phox and in was to AEBSF in the or presence of It was with the components in at the solubilized membrane b p47phox and in was to AEBSF in the or presence of It was with the components in at the solubilized membrane b 559), p47phox and p67phox components, at the concentrations in the were with AEBSF for at in a of in the or presence of LiDS, at concentrations in the was by the of the untreated NADPH oxidase components at concentration in of assay containing a concentration of LiDS, to in a concentration of not to AEBSF, were at this with AEBSF, as a for an effect of AEBSF on electron flow. was by the of are as of NADPH oxidase activation by the activity of containing a with AEBSF to that of containing untreated and of for each were O·̄2 production by a mixture containing a with AEBSF that of the mixture containing the untreated membrane of cytochromeb was to AEBSF in the or presence of It was with cytosol to in to in was to AEBSF in the or presence of It was with solubilized membrane of cytochrome b in cytochrome b 559 in was to AEBSF in the or presence of It was with cytosol to in p47phox in was to AEBSF in the or presence of It was with the components, in at the solubilized membrane b p67phox and p67phox in was to AEBSF in the or presence of It was with the components in at the solubilized membrane b p47phox and in was to AEBSF in the or presence of It was with the components in at the solubilized membrane b 559), p47phox and p67phox in a components, at the concentrations in the were with AEBSF for at in a of in the or presence of LiDS, at concentrations in the was by the of the untreated NADPH oxidase components at concentration in of assay containing a concentration of LiDS, to in a concentration of not to AEBSF, were at this with AEBSF, as a for an effect of AEBSF on electron flow. was by the of are as of NADPH oxidase activation by the activity of containing a with AEBSF to that of containing untreated and of for each were O·̄2 production by a mixture containing a with AEBSF that of the mixture containing the untreated The ability of AEBSF to cytochromeb 559 was in more by the of the of solubilized membrane by AEBSF, in the presence and of in AEBSF the of the membrane to support O·̄2 production in the presence of LiDS, with an of of was as an of the in the presence of a of cytochrome b 559, the inhibitory effect of AEBSF by the concentration of cytosolic The is that an essential in the assembly of the NADPH oxidase complex is the binding of p47phox to cytochromeb 559 in J. PubMed Scopus Google Scholar). of the results in the that AEBSF with p47phox for with cytochromeb 559. the of varying the concentration of p47phox on the of NADPH oxidase activation by AEBSF, in the presence of a of membrane and concentrations of p67phox and the concentrations p67phox and in the presence of concentrations of the other two cytosolic components. three concentrations of cytosolic were two of were on the of the a The were by the of a of NADPH oxidase and of AEBSF for each concentration of cytosolic were in p47phox was in were and for AEBSF with concentrations of of AEBSF were by the concentration of p67phox and not by varying the concentration of These results are with the that AEBSF interferes with the binding of p47phox to cytochromeb 559 but the that AEBSF also a direct p67phox and cytochrome b 559 The that AEBSF as a of p47phox was by a analysis of the effect of AEBSF on the activation of NADPH oxidase at concentrations of p47phox and the of the in the in AEBSF, in the concentration of to as a with to concentrations of AEBSF a or of was analysis of the effect of AEBSF on NADPH oxidase activation at varying concentrations of p67phox or or of NADPH oxidase in intact cells is by translocation of of p47phox and p67phox to the membrane J. PubMed Scopus Google Scholar). The of cytosolic components to the membrane is on the presence of cytochrome b 559 J. 1991; PubMed Scopus Google Scholar). translocation of p67phox to the membrane in the presence of p47phox J. 1991; PubMed Scopus Google Scholar), that at of the of p47phox is to as an for p67phox. It of to the effect of AEBSF on the translocation of cytosolic components to the membrane in the cell-free J. Google Scholar). in the of AEBSF to of and by a concentration of to in NADPH-dependent O·̄2 the translocation of p47phox and p67phox to the membrane. of the from cell-free in the and presence of AEBSF, for O·̄2 that lack of translocation was by an of NADPH oxidase in translocation of p47phox and p67phox by was on the presence of and was not the of of cytosolic components by J. PubMed Scopus Google Scholar). We found that AEBSF, as a irreversible serine protease of interferes with the activation of the O·̄2 NADPH oxidase in intact macrophages and in cell-free in a A of this is that of cell-free from consisting of total membrane and to of purified cytochromeb 559 and three recombinant cytosolic components. this was in of the by and PubMed Google Scholar), the inhibitory effect of AEBSF on O·̄2 production by in response to and AEBSF was found to of for O·̄2 production in response to and but was not to affect the response of cells, in the concentration Google Scholar). is a of the elicitation of an oxidative burst to the activation of in J. PubMed Scopus Google Scholar). of the evidence for a is on the effect of or protease serine protease on oxygen radical production by intact of to interpreted with that the of protease inhibitors on O·̄2 production were to their activity J. PubMed Scopus Google J. J. PubMed Google Biochim. Biophys. PubMed Scopus Google L. J. 1991; Google Scholar). results that AEBSF NADPH oxidase at in cell-free by a mechanism to protease inhibitory is by the other serine protease inhibitors DCIC, and were found to under is of that AEBSF, was found to O·̄2 production by intact PubMed Google Scholar), did not NADPH oxidase activation in the cell-free protease related to AEBSF and was NADPH oxidase activation inhibitory and and AEBSF was inhibitory in a cell-free of purified and recombinant and, of The inhibitory effect of AEBSF on NADPH oxidase activation a A of of and to AEBSF is were to as serine protease inhibitors J. PubMed Scopus Google Scholar). The presence of a reactive sulfonyl fluoride group on the compounds of a with their Google J. PubMed Scopus Google Scholar). We found compounds sharing an moiety AEBSAc, AEBSNH2, and AEBSF and MAEBSF, a reactive sulfonyl fluoride were three amidinobenzene pAPMSF, and pABSF, a sulfonyl fluoride group to the NADPH oxidase inhibitory and two serine protease inhibitors to AEBSF, but the group in AEBSF, were found to in the cell-free We that two are the for the of NADPH oxidase inhibitory a moiety and a reactive sulfonyl fluoride The for the of AEBSF to the that this cytochrome b 559. is on the that of total membrane fraction or purified and cytochromeb 559 with AEBSF, in the presence of a critical concentration of LiDS, resulted in a reduction in their ability to with untreated components in the assembly of NADPH oxidase. total cytosol of the three cytosolic components were to AEBSF to a A of this effect is on the presence of an The amphiphile the cytochrome b to AEBSF or a in cytochrome b 559 with the of but also to of an for a direct effect of at NADPH oxidase activating on cytochromeb 559 was by the that and of cytochromeb 559 with the NADPH oxidase inhibitory J. J. 1996; PubMed Scopus Google Scholar). The results of of the effect of varying the concentration of each of the three cytosolic components on the of AEBSF support the that AEBSF the binding of p47phox to cytochrome b interferes with the assembly of a active NADPH oxidase a mechanism is also in with the analysis of the of NADPH oxidase activation by AEBSF at concentrations of is evidence the small T.L. PubMed Scopus Google PubMed Scopus Google and J. PubMed Google J. PubMed Google L. PubMed Scopus Google of cytochromeb 559. also in p47phox, a from to or of represents a of with cytochromeb 559 J. PubMed Google J. PubMed Scopus Google J. 1996; PubMed Scopus Google Scholar). is in and is that with on of the two of cytochrome b 559. the of AEBSF is also to a lesser degree, by the concentration of p67phox. effect by that p67phox to cytochromeb 559 and that AEBSF interferes with this evidence for a direct p67phox and cytochromeb 559 J. 1996; PubMed Scopus Google J. 1996; PubMed Scopus Google Scholar). by an effect of varying the concentration of p67phox on the binding of p47phox to cytochromeb 559. on an of p47phox binding to cytochromeb 559 by p67phox was demonstrated J. PubMed Google Scholar), and a marked reduction in the translocation of p47phox to the membrane in of was in J. 1996; PubMed Scopus Google Scholar). that AEBSF the translocation of p67phox to the membrane by on the binding of each of the two cytosolic components to cytochrome b 559 or by an effect on the binding of the binding of the other as a protease inhibitor, AEBSF as a Google Scholar). The moiety as a of with and related of to the reactive sulfonyl fluoride group the with the of a with the Google J. PubMed Scopus Google J. PubMed Scopus Google Scholar). We that AEBSF also as a of NADPH oxidase binding by the of to an on of the of cytochromeb 559. step is by the of an irreversible by the sulfonyl fluoride The in the AEBSF as a inhibitor, with to p47phox, but that this was by a at that the of with concentrations of this was under of a on by that AEBSF, under certain at with other than serine These to and and the of the mechanism of of NADPH oxidase by AEBSF in and by in is of and to a of the of the assembly of the NADPH oxidase complex and to the of to used in with the production of oxygen is on the of the of found in the cell-free to the in intact the that AEBSF is a and NADPH oxidase and an for the of We L. Leto of for for p47phox and p67phox and in and for
Diatchuk et al. (Thu,) studied this question.