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• W1991488963 abstract "Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochromeb 558 with the cytosolic components p47 phox, p67 phox, and the small GTPase Rac. Herein, the interaction between Rac and p67 phox is explored using functional and physical methods. Mutually facilitated binding (EC50) of Rac1 and p67 phox within the NADPH oxidase complex was demonstrated using steady state kinetic methods measuring NADPH-dependent superoxide generation. Direct binding of Rac1 and Rac2 to p67 phox was shown using a fluorescent analog of GTP (methylanthraniloyl guanosine-5′-[β,γ-imido]triphosphate) bound to Rac as a reporter group. An increase in the methylanthraniloyl fluorescence was seen with added p67 phox but not p47 phox, and the emission maximum shifted from 445 to 440 nm. Rac1 and Rac2 bound to p67 phox with a 1:1 stoichiometry and with K d values of 120 and 60 nm, respectively. Mutational studies (Freeman, J., Kreck, M., Uhlinger, D. J., and Lambeth, J. D. (1994) Biochemistry 33, 13431–13435; Freeman, J. L., Abo, A., and Lambeth, J. D. (1996)J. Biol. Chem. 271, 19794–19801) previously identified two regions in Rac1 that are important for activity: the “effector region” (residues 26–45) and the “insert region” (residues 124–135). Proteins mutated in the effector region (Rac1(N26H), Rac1(I33N), and Rac1(D38N)) showed a marked increase in both the K d and the EC50, indicating that mutations in this region affect activity by inhibiting Rac binding to p67 phox. Insert region mutations (Rac1(K132E) and L134R), while showing markedly elevated EC50 values, bound with normal affinity to p67 phox. The structure of Rac1 determined by x-ray crystallography reveals that the effector region and the insert region are located in defined sectors on the surface of Rac1. A model is discussed in which the Rac1 effector region binds to p67 phox, the C terminus binds to the membrane, and the insert region interacts with a different protein component, possibly cytochrome b 558. Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochromeb 558 with the cytosolic components p47 phox, p67 phox, and the small GTPase Rac. Herein, the interaction between Rac and p67 phox is explored using functional and physical methods. Mutually facilitated binding (EC50) of Rac1 and p67 phox within the NADPH oxidase complex was demonstrated using steady state kinetic methods measuring NADPH-dependent superoxide generation. Direct binding of Rac1 and Rac2 to p67 phox was shown using a fluorescent analog of GTP (methylanthraniloyl guanosine-5′-[β,γ-imido]triphosphate) bound to Rac as a reporter group. An increase in the methylanthraniloyl fluorescence was seen with added p67 phox but not p47 phox, and the emission maximum shifted from 445 to 440 nm. Rac1 and Rac2 bound to p67 phox with a 1:1 stoichiometry and with K d values of 120 and 60 nm, respectively. Mutational studies (Freeman, J., Kreck, M., Uhlinger, D. J., and Lambeth, J. D. (1994) Biochemistry 33, 13431–13435; Freeman, J. L., Abo, A., and Lambeth, J. D. (1996)J. Biol. Chem. 271, 19794–19801) previously identified two regions in Rac1 that are important for activity: the “effector region” (residues 26–45) and the “insert region” (residues 124–135). Proteins mutated in the effector region (Rac1(N26H), Rac1(I33N), and Rac1(D38N)) showed a marked increase in both the K d and the EC50, indicating that mutations in this region affect activity by inhibiting Rac binding to p67 phox. Insert region mutations (Rac1(K132E) and L134R), while showing markedly elevated EC50 values, bound with normal affinity to p67 phox. The structure of Rac1 determined by x-ray crystallography reveals that the effector region and the insert region are located in defined sectors on the surface of Rac1. A model is discussed in which the Rac1 effector region binds to p67 phox, the C terminus binds to the membrane, and the insert region interacts with a different protein component, possibly cytochrome b 558. Neutrophils and macrophages reduce molecular oxygen with NADPH to produce superoxide (O·̄2) and secondarily derived reactive oxygen species (H2O2, HOCl, OH·), which function to kill phagocytosed microorganisms (1Badwey J.A. Karnovsky M.L. Annu. Rev. Biochem. 1980; 49: 695-726Crossref PubMed Scopus (839) Google Scholar, 2Chanock S. El Benna J. Smith R. Babior B. J. Biol. Chem. 1994; 269: 24519-24522Abstract Full Text PDF PubMed Google Scholar, 3Segal A.W. Abo A. Trends Biochem. Sci. 1993; 18: 43-47Abstract Full Text PDF PubMed Scopus (562) Google Scholar, 4Clark R.A. J. Infect. Dis. 1990; 161: 1140-1147Crossref PubMed Scopus (177) Google Scholar). Superoxide generation is catalyzed by an NADPH oxidase (also called the respiratory burst oxidase), which is dormant in resting cells but becomes active upon exposure to bacteria or to a variety of soluble stimuli. The enzyme consists of both cytosolic and plasma membrane-associated protein factors. Flavocytochromeb 558 is a membrane-associated heterodimer (5Segal A.W. Nature. 1987; 326: 88-91Crossref PubMed Scopus (205) Google Scholar, 6Nakamura M. Sendo S. van Zwieten R. Koga T. Roos D. Kanegasaki S. Blood. 1988; 72: 1550-1552Crossref PubMed Google Scholar, 7Parkos C.A. Dinauer M.C. Walker L.E. Rodger A.A. Jesaitis A.J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3319-3323Crossref PubMed Scopus (242) Google Scholar) that contains putative binding sites for NADPH, FAD, and heme (8Rotrosen D. Yeung C.L. Leto T.L. Malech H.L. Kwong C.H. Science. 1992; 256: 1459-1462Crossref PubMed Scopus (313) Google Scholar, 9Segal A.W. West I. Wientjes F. Nugent J.H.A. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrace G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (289) Google Scholar, 10Sumimoto H. Sakamoto N. Nozaki M. Sakaki Y. Takeshige K. Minakami S. Biochem. Biophys. Res. Commun. 1992; 186: 1368-1375Crossref PubMed Scopus (111) Google Scholar, 11Nishimoto Y. Otsuka-Murakami H. Lambeth D. J. Biol. Chem. 1995; 270: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and therefore represents the enzymatic component of the NADPH oxidase. Three cytosolic components (p47 phox, p67 phox, and a small molecular weight GTP-binding protein, Rac), activate superoxide generation and can be considered to be regulatory subunits of the flavocytochrome. p47 phox, while not essential for activity, functions as a regulated adaptor protein that increases the binding of p67 phox to the oxidase 100-fold (12Freeman J.L. Lambeth J.D. J. Biol. Chem. 1996; 271: 22578-22582Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). p47 phox appears to act by coupling p67 phox to the 22-kDa subunit of the flavocytochrome b 558 (13de Mendez I. Garrett M.C. Adams A.G. Leto T. J. Biol. Chem. 1994; 269: 16326-16332Abstract Full Text PDF PubMed Google Scholar, 14Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (254) Google Scholar, 15De Leo F. Ulman K. Davis A. Jutila K. Quinn M. J. Biol. Chem. 1996; 271: 17013-17020Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). p47 phoxand p67 phox exist in the cytosol of resting cells as a complex along with a third component, p40 phox (16Someya A. Nagaoka I. Yamashita T. FEBS Lett. 1993; 330: 215-218Crossref PubMed Scopus (72) Google Scholar, 17Wientjes F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (256) Google Scholar), which may function as an inhibitory protein. p47 phox and p67 phoxtranslocate to the plasma membrane (18Heyworth 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 (305) Google Scholar, 19Lomax K.J. Leto T.L. Nunoi H. Gallin J.I. Malech H.L. Science. 1989; 245: 409-412Crossref PubMed Scopus (233) Google Scholar, 20Leto 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 (296) Google Scholar, 21Tyagi S.R. Neckelmann N. Uhlinger D.J. Burnham D.N. Lambeth J.D. Biochemistry. 1992; 31: 2765-2774Crossref PubMed Scopus (42) Google Scholar), and this correlates with cell activation. In a cell-free system, p47 phox and p67 phox form a 1:1:1 complex with flavocytochromeb 558 (22Uhlinger 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). The small molecular weight GTP-binding protein, Rac, occurs as two isoforms (Rac1 and Rac2) that are 92% identical in amino acid sequence (23Didsbury J. Weber R.F. Bokoch G.M. Evans T. Snyderman R. J. Biol. Chem. 1989; 264: 16378-16382Abstract Full Text PDF PubMed Google Scholar). The two isoforms differ primarily in their C termini; Rac1 but not Rac2 contains a polybasic C terminus. In resting cells, Rac is located in a cytosolic complex with an inhibitory protein, RhoGDI (24Hiraoka K. Kaibuchi K. Ando S. Musha T. Takaishi K. Mizuno T. Asada M. Menard L. Tomhave E. Didsbury J. Snyderman R. Takai Y. Biochem. Biophys. Res. Commun. 1992; 182: 921-930Crossref PubMed Scopus (81) Google Scholar, 25Abo A. Pick E. Hall A. Totty N. Teahan C.G. Segal A.W. Nature. 1991; 353: 668-670Crossref PubMed Scopus (756) Google Scholar, 26Kwong C.H. Malech H.L. Rotrosen D. Leto T.L. Biochemistry. 1993; 32: 5711-5717Crossref PubMed Scopus (93) Google Scholar, 27Chuang T. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1993; 268: 26206-26211Abstract Full Text PDF PubMed Google Scholar). Upon cell activation, Rac2, the more abundant isoform in neutrophils (28Quinn M.T. Evans T. Loetterle L.R. Jesaitis A.J. Bokoch G.M. J. Biol. Chem. 1993; 268: 20983-20987Abstract Full Text PDF PubMed Google Scholar, 29El Benna J. Ruedi J.M. Babior B.M. J. Biol. Chem. 1994; 269: 6729-6734Abstract Full Text PDF PubMed Google Scholar, 30Abo A. Webb M.R. Grogan A. Segal A. Biochem. J. 1994; 298: 585-591Crossref PubMed Scopus (151) Google Scholar), becomes associated with the plasma membrane (28Quinn M.T. Evans T. Loetterle L.R. Jesaitis A.J. Bokoch G.M. J. Biol. Chem. 1993; 268: 20983-20987Abstract Full Text PDF PubMed Google Scholar), and translocation correlates with NADPH oxidase activity (28Quinn M.T. Evans T. Loetterle L.R. Jesaitis A.J. Bokoch G.M. J. Biol. Chem. 1993; 268: 20983-20987Abstract Full Text PDF PubMed Google Scholar, 30Abo A. Webb M.R. Grogan A. Segal A. Biochem. J. 1994; 298: 585-591Crossref PubMed Scopus (151) Google Scholar,31Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1993; 268: 8624-8631Abstract Full Text PDF PubMed Google Scholar). 1However, two studies (32Le Cabec V. Mohn H. Gacon G. Maridonneau-Parini I. Biochem. Biophys. Res. Commun. 1994; 198: 1216-1224Crossref PubMed Scopus (16) Google Scholar, 33Philips M. Feoktistov A. Pillinger M. Abramson S. J. Biol. Chem. 1995; 270: 11514-11521Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) have concluded that activation fails to correlate with translocation. 1However, two studies (32Le Cabec V. Mohn H. Gacon G. Maridonneau-Parini I. Biochem. Biophys. Res. Commun. 1994; 198: 1216-1224Crossref PubMed Scopus (16) Google Scholar, 33Philips M. Feoktistov A. Pillinger M. Abramson S. J. Biol. Chem. 1995; 270: 11514-11521Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) have concluded that activation fails to correlate with translocation. Translocation of Rac requires neither p47 phox nor p67 phox and occurs with different kinetics than these other cytosolic components (34Heyworth P. Bohl B. Bokoch G. Curnutte J. J. Biol. Chem. 1994; 269: 30749-30752Abstract Full Text PDF PubMed Google Scholar, 35Dorseuil O. Quinn M.T. Bokoch G.M. J. Leukocyte Biol. 1995; 58: 108-113Crossref PubMed Scopus (88) Google Scholar, 36Kleinberg M.E. Malech H.L. Mital D.A. Leto T.L. Biochemistry. 1994; 33: 2490-2495Crossref PubMed Scopus (26) Google Scholar, 37Dusi S. Donini M. Rossi F. Biochem. J. 1996; 314: 409-412Crossref PubMed Scopus (110) Google Scholar), suggesting that the binding of Rac to the plasma membrane is regulated by mechanisms that are distinct from those that regulate p47 phox/p67 phoxassembly. In their isoprenylated forms, both Rac1 and Rac2 can activate superoxide generation in a cell-free system (38Heyworth P.G. Knaus U.G. Xu X. Uhlinger D.J. Conroy L. Bokoch G.M. Curnutte J.T. Mol. Biol. Cell. 1993; 4: 261-269Crossref PubMed Scopus (111) Google Scholar, 39Escriou V. LaPorte F. Garin J. Brandolin G. Vignais P. J. Biol. Chem. 1994; 269: 14007-14014Abstract Full Text PDF PubMed Google Scholar, 40Ando S. Kaibuchi K. Sasaki T. Hiraoka K. Nishiyama T. Mizuno T. Asada M. Nunoi H. Matsuda I. Matsuura Y. Polakis P. McCormick F. Takai Y. J. Biol. Chem. 1992; 267: 25709-25713Abstract Full Text PDF PubMed Google Scholar). Kinetic characterization and binding studies are complicated by the presence of the isoprenyl group, which limits the solubility and requires the presence of a detergent. The proteins in their nonisoprenylated forms can be expressed in and purified from bacteria. However, in their nonisoprenylated forms, only Rac1 activates efficiently. This is because membrane association is essential for optimal function of Rac, and nonisoprenylated Rac1 can interacts with the membrane via its polybasic C terminus (41Kreck M.L. Freeman J.L. Abo A. Lambeth J.D. Biochemistry. 1996; 35: 15683-15692Crossref PubMed Scopus (92) Google Scholar). Bacterially expressed versions of Rac1 have therefore been used for most of the studies described herein. We have previously characterized two regions on Rac1 that are important for its ability to activate the NADPH oxidase. These are the effector region (within the range of residues 26–45) and the insert region (residues 124–135). The effector region shows homology to a region on Ras that has been characterized as mediating the GTP-dependent binding to the Ras effector Raf-1, a member of the MAP kinase cascade (42Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Abstract Full Text PDF PubMed Scopus (193) Google Scholar, 43Warne P.H. viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (579) Google Scholar). The effector region of Ras includes residues the conformation of which changes significantly depending on whether GTP or GDP is bound. The insert region on Rac has no counterpart in Ras. Mutation of residues in both the effector region and the insert region of Rac1 results in a marked decrease in the ability to support cell-free superoxide generation (44Freeman J.L.R. Kreck M.L. Uhlinger D.J. Lambeth J.D. Biochemistry. 1994; 33: 13431-13435Crossref PubMed Scopus (39) Google Scholar, 45Xu X. Barry D. Settleman J. Schwartz M. Bokoch G. J. Biol. Chem. 1994; 269: 23569-23576Abstract Full Text PDF PubMed Google Scholar, 46Diekmann D. Abo A. Johnson C. Segal A. Hall A. Science. 1994; 265: 531-533Crossref PubMed Scopus (344) Google Scholar), and the primary effect was decreased affinity of Rac1, based upon an increase in the EC50 (44Freeman J.L.R. Kreck M.L. Uhlinger D.J. Lambeth J.D. Biochemistry. 1994; 33: 13431-13435Crossref PubMed Scopus (39) Google Scholar, 47Freeman J.L. Abo A. Lambeth J.D. J. Biol. Chem. 1996; 271: 19794-19801Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Rac1 has been shown to bind to immobilized p67 phox (48Prigmore E. Ahmed S. Best A. Kozma R. Manser E. Segal A. Lim L. J. Biol. Chem. 1995; 270: 10717-10722Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 49Dorseuil O. Reibel L. Bokoch G. Camonis J. Gacon G. J. Biol. Chem. 1996; 271: 83-88Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and the interaction has been demonstrated using yeast two-hybrid analysis, but it is not clear whether Rac also interacts with other targets. In addition, no quantitative information on Rac binding to p67 phox is available (e.g. affinity, stoichiometry). Herein, steady state kinetic analysis was used to demonstrate the functional linkage between Rac and p67 phox. In addition, Rac binding to p67 phox was measured directly, making use of a fluorescent GTP analog that binds tightly to Rac as a reporter group. The Rac-associated analog undergoes an increase in fluorescence upon interaction of Rac with p67 phox, and this fluorescence change was used to quantify the Rac binding to p67 phox. This method, used in conjunction with mutational analysis, reveals that the effector region participates in binding to p67 phox, while the insert region does not. Mapping of residues the mutation of which lowers activity onto the structure of Rac1 (recently determined by x-ray crystallography; Ref. 50Hirshberg M. Stockley R.W. Dodson G. Webb M.R. Nat. Struct. Biol. 1997; 4: 147-152Crossref PubMed Scopus (185) Google Scholar) reveals that the effector region and the insert region are located in distinct sectors of the protein, consistent with a model in which these two regions bind to distinct targets within the NADPH oxidase complex. Cytochrome c (type VI), NADPH,n-octyl glucoside, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, andN-α-tosyl-l-lysine chloromethylketone were from Sigma. GTPγS was purchased from Boehringer Mannheim. HESPAN (6% hetastarch in 0.9% NaCl) was from American Hospital Supply Corp., and lymphocyte separation medium (6.2% Ficoll, 9.4% sodium diatrizoate) was obtained from Organon Tekniker. Superoxide dismutase and dithiothreitol were from Wako Pure Chemical Co. Heparin-Sepharose CL-6B, DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, ω-aminooctyl-agarose, and glutathione-Sepharose were purchased from Pharmacia LKB. l-α-phosphatidylcholine (bovine brain),l-α-phosphatidylethanolamine (bovine brain),l-α-phosphatidylinositol (bovine brain), and sphingomyelin (bovine erythrocyte) were from Sigma. All other reagents were of the highest grade available commercially. Methyl isotoic anhydride was purchased from Molecular Probes (Eugene, OR), and mant-GppNHp 2The abbreviations used are: mant-GppNHp, methylanthraniloyl guanosine-5′-[β,γ-imido] triphosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate; mant, methylanthraniloyl. was synthesized as described previously (51Hiratsuka T. Biochim. Biophys. Acta. 1983; 742: 496-508Crossref PubMed Scopus (394) Google Scholar). Recombinant p67 phox was expressed and purified from baculovirus-infected Hi5 insect cells as described (22Uhlinger 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, 31Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1993; 268: 8624-8631Abstract Full Text PDF PubMed Google Scholar). Wild type and mutant Rac1 were expressed in Escherichia coli as the glutathioneS-transferase fusion proteins, purified using glutathione-Sepharose beads, and cleaved from the glutathioneS-transferase domain with thrombin (52Kreck M.L. Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1994; 269: 4161-4168Abstract Full Text PDF PubMed Google Scholar). All recombinant proteins were purified to greater than 95% homogeneity. Human neutrophils were obtained from peripheral blood of normal healthy donors after obtaining informed consent. Erythrocytes were sedimented with HESPAN, and the mononuclear cells were removed from the resulting supernatant by centrifugation through lymphocyte separation medium (53Pember S.O. Barnes K.C. Brandt S.J. Kinkade Jr., J.M. Blood. 1983; 61: 1105-1115Crossref PubMed Google Scholar). The resulting cells were greater than 95% neutrophilic granulocytes. Neutrophils were resuspended in cavitation buffer (25 mm HEPES, pH 7.4, containing 100 mmKCl, 3 mm NaCl, 5 mm MgCl2, 6 μm diisopropyl fluorophosphate, 0.5 mmphenylmethylsulfonyl fluoride, 2 μm each leupeptin, pepstatin, and aprotinin). Cells (6 × 109) in 20 ml of ice-cold buffer were disrupted by nitrogen cavitation after being pressurized at 500 p.s.i. for 20 min at 4 °C, and plasma membranes were prepared as described (54Burnham D.N. Uhlinger D.J. Lambeth J.D. J. Biol. Chem. 1990; 265: 17550-17559Abstract Full Text PDF PubMed Google Scholar). Plasma membrane was solubilized in the presence of 40 mm octyl glucoside and 0.5% sodium cholate (11Nishimoto Y. Otsuka-Murakami H. Lambeth D. J. Biol. Chem. 1995; 270: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Detergent-solubilized cytochrome b 558 was purified as described previously (9Segal A.W. West I. Wientjes F. Nugent J.H.A. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrace G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (289) Google Scholar) with some modifications (11Nishimoto Y. Otsuka-Murakami H. Lambeth D. J. Biol. Chem. 1995; 270: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Purified FAD-depleted cytochrome b 558 (15.6 nmol of heme/mg of protein) was incubated in 50 mm Tris acetate buffer, pH 7.45, containing 5 mm KCl, 10% glycerol, 1 mm dithiothreitol, 1 mm EGTA, 1 mmphenylmethylsulfonyl fluoride, 2 μm each leupeptin, pepstatin, and aprotinin (buffer B), and phospholipids (l-α-phosphatidylcholine/l-α-phosphatidylethanolamine/l-α-phosphatidylinositol/sphingomyelin/cholesterol = 4:2:1:3:3 (w/w/w/w/w); lipid/protein = 100, w/w) were added along with a 10-fold excess of FAD over heme. After incubating at 4 °C for 2 h, the mixture was dialyzed against two changes of buffer B to remove free FAD. The FAD-reconstituted material typically contained a FAD:heme ratio of 0.4–0.5 (11Nishimoto Y. Otsuka-Murakami H. Lambeth D. J. Biol. Chem. 1995; 270: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The heme content of cytochromeb 558 reconstituted with FAD and phospholipids was determined by reduced minus oxidized difference spectroscopy at 424–440 nm using an extinction coefficient of 161 mm−1·cm−1 (55Lutter R. van Schaik M.L.J. van Zwieten R. Wever R. Roos D. Hamers M.N. J. Biol. Chem. 1985; 260: 2237-2244Abstract Full Text PDF PubMed Google Scholar). The flavin content of FAD-reconstituted cytochrome b 558 was estimated by the fluorimetric method (11Nishimoto Y. Otsuka-Murakami H. Lambeth D. J. Biol. Chem. 1995; 270: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Fluorescence spectra were recorded with a Hitachi model F-3000 spectrofluorimeter, and routine fluorescence measurements were made with a Perkin-Elmer LS-5B spectrofluorimeter. Samples (mant-GppNHp, Rac 1, cytochromeb 558, and cytosolic factors) were incubated at 20 °C in 0.3 ml of 20 mm Tris acetate buffer, pH 7.45, containing 3 mm NaCl, 50 mm KCl, and 0.1 μm MgCl2. Preloading of Racs with mant-GppNHp was carried out for 15–20 min, at which point the fluorescence change due to the guanine nucleotide binding was stable. Low MgCl2concentration was essential to facilitate complete guanine nucleotide exchange. Titrations were carried out by adding p67 phox and recording fluorescence readings until three successive stable readings, at least 45 s apart, were obtained. Fluorescence changes induced by p67 phox occurred rapidly (within 1–2 min) and did not change further with prolonged incubation. Spectral resolution was 5 nm for both the excitation and emission paths, respectively. Superoxide generation was measured by superoxide dismutase-inhibitable reduction of cytochrome c as described previously (54Burnham D.N. Uhlinger D.J. Lambeth J.D. J. Biol. Chem. 1990; 265: 17550-17559Abstract Full Text PDF PubMed Google Scholar) using a Thermomax kinetic microplate reader (Molecular Devices, Menlo Park, CA). Rac, preloaded with a 5-fold molar excess of GTPγS for 15 min at 25 °C in the absence of MgCl2 (52Kreck M.L. Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1994; 269: 4161-4168Abstract Full Text PDF PubMed Google Scholar), was combined with p47 phox, p67 phox, 10 nm cytochrome b558, and 1 μm FAD followed by activation with 40 μm arachidonate in 50 mm NaCl, 4 mm MgCl2, 1.25 mm EGTA, 20 mm Tris-HCl, pH 7.0, as described (56Rotrosen D. Yeung C.L. Katkin J.P. J. Biol. Chem. 1993; 268: 14256-14260Abstract Full Text PDF PubMed Google Scholar). The mixture was incubated at 25 °C for 5 min followed by the addition of 200 μm NADPH and 200 μm cytochromec. An extinction coefficient at 550 nm of 21 mm−1 cm−1 was used to calculate the quantity of cytochrome c reduced (57Lambeth J.D. Burnham D.N. Tyagi S.R. J. Biol. Chem. 1988; 263: 3818-3822Abstract Full Text PDF PubMed Google Scholar). The theoretical lines through the data shown in Figs. 1 and 2were calculated using a nonlinear least squares fit of the data using the Michaelis-Menten equation and were plotted using Sigma Plot. Kinetic constants are reported as V max and EC50 (effective concentration at 50% of V max). Fluorescence titrations were fit to a single site binding equation as described previously (58Nomanbhoy T.K. Cerione R.A. J. Biol. Chem. 1996; 271: 10003-10009Abstract Full Text Full Text PDF Scopus (103) Google Scholar) as follows,ΔF=ΔFmax((Kd+LT+RT)−((Kd+LT+RT) 2−4LTRT) 1/2/2RT)Equation 1 where ΔF is the fluorescence change after each addition of p67 phox, ΔF max is the maximal fluorescence change at infinite (extrapolated) p67 phox,K d is the dissociation constant,L T is the concentration of p67 phox, andR T is the total concentration of Rac(mant-GppNHp). Sigma plot was used to generate a nonlinear least squares fit of the data, solving for K d and ΔF max, constraining the fit to the actual concentration of Rac(mant-GppNHp) used in the experiment. For the Rac(D38N) mutation, it was necessary to assume the ΔF max to be the same as that of the wild type, since binding was weak and it was not feasible to approach saturation.Figure 5Comparison of binding of Rac1, Rac2, and Rac1 point mutants to p67phox. Titrations were carried out as in Fig. 4. The concentration of Rac or Rac mutants in various experiments shown ranged from 0.25 to 0.6 μm, and the corresponding concentration of mant-GppNHp was 70% of the Rac concentration. The Rac form used is indicated in each panel. The lines shown are derived from theoretical fits of the data, as described under “Experimental Procedures.” Thearrowheads on the y axes indicate the maximal fluorescence change, which was obtained either by extrapolation according to Equation 1 (top panels and lower right panel) or in a parallel titration using Rac1 (remaining panels) in cases where an accurate ΔF maxcould not be obtained by extrapolation. K d values obtained in these and other titrations are shown in Table I.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Effect of p67 phox on the fluorescence of the mant-GppNHp·Rac complex. A, the solid line shows the fluorescence emission spectrum (excitation wavelength, 355 nm) of mant-GppNHp (0.12 μm) in 20 mm Tris-HCl buffer, pH 7.45, containing 3 mmNaCl, 50 mm KCl, and 0.1 μm MgCl2(solid line). After adding 0.25 μm Rac 1 and incubating for 20 min at 20 °C, the fluorescence spectrum was recorded (dotted line, middle spectrum). Fifteen minutes after the addition of 0.30 μm p67 phox the emission spectrum (broken line, upper spectrum) was recorded. B, after recording a spectrum of the Rac(mant-GppNHp) complex (solid line), 0.30 μmp47 phox was incubated with the mixture for 20 min, and then the spectrum (dotted line, no change from solid line) was recorded. p67 phox (0.30 μm) was then added, and the emission spectrum (broken line) was recorded as above. C, the solid line shows the emission spectrum of mant-GppNHp (0.12 μm) in the absence of added Rac1. Thesuperimposed dotted line shows the spectrum 20 min after the addition of p67 phox (0.3 μm), again in the absence of Rac1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Quantitation of p67 phox binding to Rac1 using the increase in fluorescence of mant-GppNHp bound to Rac1. Rac1 (1.2 μm) was preincubated with 0.85 μm mant-GppNHp for 15–20 min as above to form the Rac·mant-GppNHp complex. This was then titrated with either p67 phox (filled circles) or p47 phox(filled triangles), and the fluorescence was recorded after each addition (excitation and emission wavelengths were 355 and 440 nm, respectively) until a stable value was achieved (about 3 min). The increase in fluorescence intensity is shown as a function of the concentration of the added component. The observed fluorescence was corrected for volume changes. The line shown is a theoretical fit of the data, calculated as described under “Experimental Procedures.” The K d value describing the theoretical line is 0.17 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Comparison of mant-GppNHp and GTPγS in the activation of superoxide generation by Rac1 and point-mutated Rac1. An aliquot of cytochrome b 558 (10 nm final concentration) reconstituted with phospholipid and FAD was preincubated at 25 °C with 0.25 mm arachidonate, recombinant p47 phox (0.2 μm), p67 phox(0.1 μm), and Rac or the indicated point-mutated Rac (0.5 μm), which had been “preloaded” with either GTPγS (solid bars) or mant-GppNHp (hatched bars).W.T., wild type Rac1; N26H, Rac1(N26H);D38N, Rac1(D38N). Superoxide generation was assayed as described under “Experimental Procedures.” The average and S.E. of a minimum of three determinations are" @default.
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• W1991488963 date "1997-07-01" @default.
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• W1991488963 title "Rac Binding to p67" @default.
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