Key points are not available for this paper at this time.
Activation of the human NADPH oxidase requires the interaction of at least four cytosolic proteins and one membrane-bound heterodimeric protein. Src homology 3 (SH3) domains and their proline-rich counterstructures have been shown to play an important role in protein-protein interactions. Because it was found that the cytosolic oxidase components p67phox, p47phox, and p40phox reside in a complex in resting neutrophils, we studied the role of SH3 domains in their interaction by use of an overlay technique. Wild-type and mutated 35S-labeled p67phox and p47phox were used to detect immobilized cytosolic proteins on a protein blot. A specific association of native p67phox to blotted p47phox and blotted p40phox was found. These interactions were not disturbed by deleting the only proline-rich region (amino acids 227–231) in p67phox. We also found a specific association of native p47phox with blotted p67phox. Deletions in a putative SH3-binding region of p47phox completely abrogated the interaction with p67phox. Other results suggest that the C terminus of p47phox exposes this SH3-binding domain for interaction with p67phox. Similar results were obtained when the binding of cytosolic p67phox to wild-type or mutated p47phox were studied in solution. Interestingly, mutants of p47phox unable to bind to p67phox were fully capable of supporting superoxide production under cell-free activation conditions. We conclude that an interaction between the C-terminal proline-rich region of p47phox and the second SH3 domain of p67phox is not required for oxidase activity in the cell-free assay. Activation of the human NADPH oxidase requires the interaction of at least four cytosolic proteins and one membrane-bound heterodimeric protein. Src homology 3 (SH3) domains and their proline-rich counterstructures have been shown to play an important role in protein-protein interactions. Because it was found that the cytosolic oxidase components p67phox, p47phox, and p40phox reside in a complex in resting neutrophils, we studied the role of SH3 domains in their interaction by use of an overlay technique. Wild-type and mutated 35S-labeled p67phox and p47phox were used to detect immobilized cytosolic proteins on a protein blot. A specific association of native p67phox to blotted p47phox and blotted p40phox was found. These interactions were not disturbed by deleting the only proline-rich region (amino acids 227–231) in p67phox. We also found a specific association of native p47phox with blotted p67phox. Deletions in a putative SH3-binding region of p47phox completely abrogated the interaction with p67phox. Other results suggest that the C terminus of p47phox exposes this SH3-binding domain for interaction with p67phox. Similar results were obtained when the binding of cytosolic p67phox to wild-type or mutated p47phox were studied in solution. Interestingly, mutants of p47phox unable to bind to p67phox were fully capable of supporting superoxide production under cell-free activation conditions. We conclude that an interaction between the C-terminal proline-rich region of p47phox and the second SH3 domain of p67phox is not required for oxidase activity in the cell-free assay. The NADPH oxidase of phagocytic cells is an electron transport system that catalyzes the one-electron reduction of oxygen to superoxide (O2⨪), a process essential for the host defense against invading micro-organisms (1Smith R.M. Curnutte J.T. Blood. 1991; 77: 673-686Crossref PubMed Google Scholar). For an active NADPH oxidase, at least five different proteins are required: the membrane-bound cytochrome b558 (consisting of the subunits gp91phox and p22phox) and three cytosolic proteins, p47phox, p67phox, and a low molecular weight GTP-binding protein, either Rac-1 (in macrophages) or Rac-2 (in neutrophils) (2Abo A. Pick E. Hall A. Totty N. Teahan C.G. Segal A.W. Nature. 1991; 353: 668-670Crossref PubMed Scopus (765) Google Scholar, 3Knaus U.G. Heyworth P.G. Kinsella B.T. Curnutte J.T. Bokoch G.M. J. Biol. Chem. 1992; 267: 23575-23582Abstract Full Text PDF PubMed Google Scholar). Recently, a fourth cytosolic oxidase component has been found, p40phox, which is probably involved in stabilizing p67phox (4Wientjes F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (260) Google Scholar, 5Tsunawaki S. Mizunari H. Nagata M. Tatsuzawa O. Kuratsuji T. Biochem. Biophys. Res. Commun. 1994; 199: 1378-1387Crossref PubMed Scopus (96) Google Scholar). Upon cell activation, all three cytosolic Phox proteins translocate to the plasma membrane to form the membrane-bound NADPH oxidase (4Wientjes F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (260) Google Scholar, 6Ambruso D.R. Bolscher B.G.J.M. Stokman P.M. Verhoeven A.J. Roos D. J. Biol. Chem. 1990; 265 (Correction): 924-930J. Biol. Chem. 1990; 265 (Correction): 19370-19371Abstract Full Text PDF PubMed Google Scholar, 7Clark R.A. Volpp B.D. Leidal K.G. Nauseef W.M. J. Clin. Invest. 1990; 85: 714-721Crossref PubMed Scopus (327) Google Scholar, 8Heyworth P.G. Curnutte J.T. Nauseef W.M. Volpp B.D. Pearson D.W. Rosen H. Clark R.A. J. Clin. Invest. 1991; 87: 352-356Crossref PubMed Scopus (308) Google Scholar). P40phox is not essential for oxidase activity, as is illustrated by a complete reconstitution of oxidase activity in a cell-free assay containing purified cytochrome b558, recombinant p47phox, p67phox, and Rac protein (9Uhlinger D.J. Inge K.L. Kreck M.L. Tyagi S.R. Neckelmann N. Lambeth J.D. Biochem. Biophys. Res. Commun. 1992; 186: 509-516Crossref PubMed Scopus (33) Google Scholar, 10Rotrosen D. Yeung C.L. Katkin J.P. J. Biol. Chem. 1993; 268: 14256-14260Abstract Full Text PDF PubMed Google Scholar). Both p47phox and p67phox contain two so-called Src homology (SH3) 1The abbreviations used are: SH3Src homology 3GTPγSguanosine 5′-3-O-(thio)triphosphatePAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferasePCRpolymerase chain reactionMES4-morpholineethanesulfonic acidCGDchronic granulomatous disease domains (11Volpp B.D. Nauseef W.M. Donelson J.E. Moser D.R. Clark R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7195-7199Crossref PubMed Scopus (229) Google Scholar, 12Leto T.L. Lomax K.J. Volpp B.D. Nunoi H. Sechler J.M.G. Nauseef W.M. Clark R.A. Gallin J.I. Malech H.L. Science. 1990; 248: 727-730Crossref PubMed Scopus (298) Google Scholar), which are known from several other proteins to be involved in protein-protein interactions (13Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1438) Google Scholar, 14Musacchio A. Gibson T. Lehto V.P. Saraste M. FEBS Lett. 1992; 307: 55-61Crossref PubMed Scopus (235) Google Scholar). P40phox contains one SH3 domain (4Wientjes F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (260) Google Scholar). In addition, p47phox and p67phox as well as p22phox possess proline-rich regions that are putative SH3 counterstructures (15Parkos C.A. Dinauer M.C. Walker L.E. Allen R.A. Jesaitis A.J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3319-3323Crossref PubMed Scopus (244) Google Scholar, 16Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1022) Google Scholar). It is known that the cytosolic components of the NADPH oxidase reside in a complex of 240–300 kDa in the cytosol of resting neutrophils (17Park J.-W. Ma M. Ruedi J.M. Smith R.M. Babior B.M. J. Biol. Chem. 1992; 267: 17327-17332Abstract Full Text PDF PubMed Google Scholar, 18Park J.-W. Benna J.E. Scott K.E. Christensen B.L. Chanock S.J. Babior B.M. Biochemistry. 1994; 33: 2907-2911Crossref PubMed Scopus (86) Google Scholar). Recently, Finan et al. (19Finan P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google Scholar) showed an interaction between the second SH3 domain of p67phox and p47phox that could be inhibited by peptides resembling the proline-rich region in p47phox. Other investigators have shown that neither of the two SH3 domains of p67phox are required in the cell-free oxidase system, but both are essential for restoration of oxidase activity in Epstein-Barr virus-transformed β lymphocytes that lack p67phox (20De Mendez I. Garrett M.C. Adams A.G. Leto T.L. J. Biol. Chem. 1994; 269: 16326-16332Abstract Full Text PDF PubMed Google Scholar) Src homology 3 guanosine 5′-3-O-(thio)triphosphate polyacrylamide gel electrophoresis glutathione S-transferase polymerase chain reaction 4-morpholineethanesulfonic acid chronic granulomatous disease Here, we report that the proline-rich region of p47phox is essential for the interaction with p67phox but not for oxidase activity in the cell-free system. In addition, the C-terminal 13 amino acids of p47phox seem to be important for exposing this proline-rich domain. The proline-rich region in p67phox is not required for interaction with either p47phox or p40phox. Materials—Guanosine 5′-3-O-(thio)triphosphate (GTP7S) and NADPH were purchased from Boehringer (Mannheim, Germany). Reagents and molecular weight markers for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were from Bio-Rad. BA84 nitrocellulose sheets for Western blotting were obtained from Schleicher 73: 1686-1694Crossref PubMed Google Scholar). Rabbit antisera specific for either p47phox or p67phox were raised against the C-terminal 12 residues of these proteins. The rabbit antiserum directed against p40phox was a kind gift of Dr. F. Wientjes (Division of Molecular Medicine of University College, London). Goat antiserum directed against glutathione S-transferase (GST) was purchased from Pharmacia (Uppsala, Sweden). Goat-anti-rabbit-IgG, conjugated to horseradish peroxidase, was produced in our institute (CLB, Amsterdam). Rabbit-anti-goat-IgG, conjugated to horseradish peroxidase, was obtained from Sigma. The ECL chemiluminescence kit with luminol was from Amersham International (Amersham, Buckinghamshire, United Kingdom). X-Omat AR diagnostic films were from Kodak. Taq polymerase and dNTPs for PCR were obtained from Promega. The TNT T7-coupled reticulocyte lysate system was purchased from Promega, and the “translational grade” 35Smethionine was from Amersham International. Isolation and Fractionation of Leukocytes—Human neutrophils were isolated from 30 buffy coats of 500-ml blood donations of healthy donors, as described (22Roos D. De Boer M. Methods Enzymol. 1986; 132: 225-243Crossref PubMed Scopus (142) Google Scholar). Subsequently, neutrophils were fractionated by sonication and sucrose-gradient centrifugation as described (23Bolscher B.G.J.M. Van Zwieten R. Kramer I-J.M. Weening R. Verhoeven A.J. Roos D. J. Clin. Invest. 1989; 83: 757-763Crossref PubMed Scopus (54) Google Scholar). For immunodetection, 10 μg of plasma-membrane protein and 20 μg of cytosol protein were dissolved in SDS sample buffer (62.5 mM Tris, pH 6.8, 10% (w/v) SDS, 5% (v/v) 2-mercaptoethanol) and were separated on a 10% polyacrylamide gel according to Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar) in a Bio-Rad Mini-Protean II gel apparatus. Western blotting was performed in a mini-Trans-Blot cell according to the manufacturer’s (Bio-Rad) recommendations. Detection of proteins was performed as previously described (25Leusen J.H.W. De Boer M. Bolscher B.G.J.M. Hilarius P.M. Weening R.S. Ochs H.D. Roos D. Verhoeven A.J. J. Clin. Invest. 1994; 95: 2120-2126Crossref Google Scholar). When the blot was stained for two sets of proteins, the nitrocellulose was stripped with 62.5 mM Tris, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol after the first immunodetection. Preparation of Rac-2-enriched Cytosol Fraction—Neutrophil cytosol was diluted 5-fold in 10 mM MES buffer, pH 6.8, containing 1 mM EGTA and the protease inhibitors leupeptin (100 μμ) and phenylmethylsulfonyl fluoride (20 μg/ml) and was incubated with ½ volume of CM-Sepharose for 1 at B.G.J.M. Verhoeven A.J. Roos D. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). The was used as a of Rac protein in the cell-free assay and was of p47phox and p67phox as by immunodetection. of p47phox and of from obtained from healthy donors, the of p47phox and p67phox was obtained by PCR and the which proteins with the in the regions in C 10% was in the PCR for p47phox. PCR to of the p47phox and for p67phox and to The in amino acid residues was when the was with and which at and The was by two PCR the first containing with and and the second containing with and of the amino acid an of amino acids was The protein with the C-terminal 13 amino acids was by with and which at and The was by and which to these and to a The and amino acids residues and were by with and which at and The were by and for and and for The of the proline-rich region in was by PCR of with and containing and The the of with and the PCR and the were were by with the system S. of proteins was performed in as previously described 1988; PubMed Scopus Google Scholar). The of the proteins was by activity with 1 mM and 1 mM glutathione in 100 mM buffer were at In and were the by use of the and For in 1 μg of was used in the TNT T7-coupled system was performed in the of at 30 for according to the manufacturer’s protein of the in was to by cytosol and plasma membrane or recombinant proteins were separated by and to The blot was with 5% (w/v) and 100 mM in for 1 and in and in oxidase buffer mM mM mM 1 mM mM and mM pH The in was diluted in oxidase buffer and to the blot at Subsequently, the blot was five in oxidase buffer, and to for of to or μg of were incubated in oxidase buffer with of (v/v) and or cytosol μg of in the or of 100 SDS and 10 for at volume the three with buffer, the proteins were from the with Laemmli sample buffer and to on a 10% and for p67phox as described (25Leusen J.H.W. De Boer M. Bolscher B.G.J.M. Hilarius P.M. Weening R.S. Ochs H.D. Roos D. Verhoeven A.J. J. Clin. Invest. 1994; 95: 2120-2126Crossref Google Scholar). of NADPH oxidase activity in cell-free was performed by the superoxide reduction of cytochrome in a The of in were and were at μg of were with a Rac-2-enriched cytosol and recombinant p47phox and recombinant p67phox The was incubated in of oxidase buffer containing cytochrome of was by of SDS (100 μμ) and NADPH μμ) was and the of cytochrome reduction was at of p47phox and p67phox with the cytosolic oxidase components p47phox and p67phox are known to with the membrane-bound cytochrome b558 R.A. Volpp B.D. Leidal K.G. Nauseef W.M. J. Clin. Invest. 1990; 85: 714-721Crossref PubMed Scopus (327) Google Scholar, 8Heyworth P.G. Curnutte J.T. Nauseef W.M. Volpp B.D. Pearson D.W. Rosen H. Clark R.A. J. Clin. Invest. 1991; 87: 352-356Crossref PubMed Scopus (308) Google Scholar), we to study this interaction with an overlay technique. plasma-membrane proteins were separated by and to The blot was incubated with in p47phox or p67phox, which 1 binding to gp91phox or p22phox was by either cytosolic protein A and that the cytosolic proteins not with cytochrome b558 in resting also plasma isolated from neutrophils were binding was with either of cytochrome b558 A and the overlay with p47phox showed a binding to a which also stained with an antiserum against p67phox The overlay with p67phox showed two at and kDa which also stained with antisera directed against p47phox and p40phox these are the cytosolic oxidase proteins, to the plasma membrane When cytosol of resting neutrophils was with this a was in the overlay with p47phox In addition, and were in the overlay with the antisera against the three cytosolic Phox components proteins with the with Cytosol from the of the proteins in the cytosol that p47phox and p67phox, we used cytosol from neutrophils of either p47phox or p67phox that p67phox to both and in cytosol from a healthy but only to a at kDa in cytosol from the In the cytosol of the the was but the to be with the that p40phox is or in (4Wientjes F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (260) Google Scholar, 5Tsunawaki S. Mizunari H. Nagata M. Tatsuzawa O. Kuratsuji T. Biochem. Biophys. Res. Commun. 1994; 199: 1378-1387Crossref PubMed Scopus (96) Google Scholar). When the binding of p47phox was studied in these cytosol a was found in the blotted cytosol of a healthy and of a p47phox but not of an the were by of Antibodies on the study the regions of the proteins important in these we first antisera directed against the 12 residues of p47phox and p67phox could 3 a blot of recombinant p47phox and p67phox as proteins with the and When blotted was with an antiserum and with p67phox, of the antiserum was when p47phox was with the binding to on the blot was completely In the antiserum not with the binding of p67phox to p47phox and the of recombinant protein in all the blot was also of of p47phox and the regions important for the binding between p47phox and p67phox in four mutants of p47phox and one of p67phox were a of the regions with to the SH3 domains and proline-rich that as SH3-binding The mutated proteins were as proteins to and to by protein The is shown in The in with the of Deletions in p47phox on with and mutants were with p67phox. binding of p67phox to and binding to and binding to and These results are in with the of Finan et al. (19Finan P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google Scholar) that the SH3-binding domain of p47phox to p67phox. β of that blotted bind to but not to with the by the in the C-terminal of p47phox is important in the binding to p67phox, when p47phox is in solution. Because the overlay requires that one of the proteins is we also performed binding with both p47phox and p67phox in solution. the binding of cytosolic p67phox to or in the or of SDS and these the results were to the overlay and a of p67phox, p67phox, and and not bind p67phox SDS and these two mutants showed an binding of p67phox, but this was also with the that of the p67phox in the cytosol was to cytosolic p47phox and not to in binding to the proteins, we the with with the results as described of of of of p67phox was in the overlay the is shown in A cytosol of healthy on a with or Both proteins to p47phox and p40phox. when and were blotted and with 35S-labeled p47phox, was between the interactions of p47phox with the or the of p67phox with the of superoxide production by the recombinant proteins and in a cell-free system with plasma and a cytosol as a of Rac-2 protein. The results are shown in a of the second SH3 showed a of superoxide production of the other mutants a It is well that so-called SH3 domains are involved in protein-protein interactions (13Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1438) Google Scholar, 14Musacchio A. Gibson T. Lehto V.P. Saraste M. FEBS Lett. 1992; 307: 55-61Crossref PubMed Scopus (235) Google Scholar). The for these SH3 domains has been as proline-rich found in proteins R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1022) Google Scholar). Both p47phox and p67phox of the NADPH oxidase contain two SH3 domains and one putative SH3-binding domain. the two membrane-bound cytochrome b558 only p22phox contains a putative SH3-binding residues We at the interaction of the cytosolic oxidase proteins with the membrane-bound cytochrome b558 by an overlay as described in P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google neither p47phox p67phox to either p22phox or gp91phox on a blot regions on both subunits of cytochrome b558 are required for a interaction with the cytosolic proteins. with the that in either of cytochrome b558 to of of p47phox and p67phox cell was shown for an with a in gp91phox (25Leusen J.H.W. De Boer M. Bolscher B.G.J.M. Hilarius P.M. Weening R.S. Ochs H.D. Roos D. Verhoeven A.J. J. Clin. Invest. 1994; 95: 2120-2126Crossref Google Scholar) and for an with a in a proline-rich region of p22phox J.H.W. Bolscher B.G.J.M. Hilarius P.M. Weening R.S. R.A. Roos D. Verhoeven A.J. J. 1994; PubMed Scopus Google Scholar). is by the results of et al. H. Y. Nunoi H. H. T. Y. M. S. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), found an interaction between the SH3 domains of p47phox and the proline-rich region of both as proteins with The this The cytosolic oxidase components p47phox and p67phox to reside in a complex of 240–300 kDa in the cytosol of resting neutrophils (17Park J.-W. Ma M. Ruedi J.M. Smith R.M. Babior B.M. J. Biol. Chem. 1992; 267: 17327-17332Abstract Full Text PDF PubMed Google Scholar, 18Park J.-W. Benna J.E. Scott K.E. Christensen B.L. Chanock S.J. Babior B.M. Biochemistry. 1994; 33: 2907-2911Crossref PubMed Scopus (86) Google Scholar). We studied this interaction by an overlay p47phox to a protein in blotted cytosol that was from p67phox and p67phox to a blotted and protein. The was in and the was described as the cytosolic oxidase component p40phox that is to play a role in stabilizing p67phox (4Wientjes F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (260) Google Scholar, 5Tsunawaki S. Mizunari H. Nagata M. Tatsuzawa O. Kuratsuji T. Biochem. Biophys. Res. Commun. 1994; 199: 1378-1387Crossref PubMed Scopus (96) Google Scholar). the regions of p47phox and p67phox that play a role in these we studied the of antibodies directed against the C of p47phox and and we mutants of these proteins. antiserum directed against the C terminus of p67phox but an antiserum directed against the 12 residues of p47phox the binding of p47phox to recombinant p67phox immobilized on a blot completely this antiserum on the binding of p67phox to blotted recombinant p47phox by the antiserum bind to this p47phox. that the C terminus of p47phox has in the binding to p67phox. was by the with the protein which the 13 p67phox to blotted but binding of to blotted was not of a of the second SH3 domain of p47phox in on the interaction with p67phox. the putative SH3-binding domain of p47phox is essential for the interaction with p67phox, as with and These mutated proteins lack a of the proline-rich region of p47phox, which was by Finan et al. (19Finan P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google Scholar), with use of peptides as the in p47phox of the C-terminal SH3 domain of p67phox. In addition, our results that the 13 amino acids of p47phox are required to proline-rich region for binding to p67phox. Similar results were obtained with an assay that interaction between p47phox and p67phox both in which that the proline-rich region of p47phox is the only important domain for interaction with p67phox The of SDS and as cell-free not the of the binding of to p67phox, a specific binding of p67phox was It has been by Wientjes et al. (4Wientjes F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (260) Google Scholar) that the interaction between p40phox and p67phox is on the SH3 domain of p40phox and a proline-rich region of p67phox. this we residues and used it in an overlay with blotted cytosol of this on the binding of p67phox to p40phox or p47phox was this on the of p67phox to superoxide production Because other proline-rich regions are in p67phox, the of the binding between p40phox and p67phox to be Interestingly, was found for the p47phox mutants between the to bind p67phox and the in the cell-free assay. For which p67phox only superoxide production be by a association with the SH3-binding domain in p22phox the second SH3 domain is in this p47phox For and the is not with p67phox, but a superoxide production in the cell-free assay the proline-rich region of p47phox, important for p67phox is not required in the NADPH that a complex of the cytosolic Phox proteins is not required for activity in the cell-free system. our not that activation of the NADPH oxidase regions that other interactions between p47phox and p67phox. In the interaction between the cytosolic Phox proteins be studied with the overlay as a for the resting in the human a complex of cytosolic Phox proteins not seem to be required for activity in the cell-free assay. it be to in cells the mutated p47phox proteins that have been found in this study to be in p67phox These are in our
Leusen et al. (Mon,) studied this question.