Binding of FAD to Cytochrome b558 Is Facilitated during Activation of the Phagocyte NADPH Oxidase, Leading to Superoxide Production
2004; Elsevier BV; Volume: 279; Issue: 25 Linguagem: Inglês
10.1074/jbc.m309724200
ISSN1083-351X
AutoresShukichi Hashida, Satoru Yuzawa, Nobuo Suzuki, Yūko Fujioka, T Takikawa, Hideki Sumimoto, Fuyuhiko Inagaki, Hirotada Fujii,
Tópico(s)Immune Response and Inflammation
ResumoThe superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47phox, p67phox, p40phox, and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b558 (cyt b558) with different amounts of FAD. A remarkably high activity, over 100 μmol/s/μmol heme, was obtained in the oxidase with purified cyt b558, ternary complex (p47-p67-p40phox), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant Kd of FAD in cyt b558 of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b558 increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b558 recovered electron flow from NADPH to heme. From both Kd values of FAD and the amount of incorporated FAD in cyt b558 of reconstituted oxidase, in combination with spectra, we propose the model in which the Kd values of FAD in cyt b558 is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase. The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47phox, p67phox, p40phox, and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b558 (cyt b558) with different amounts of FAD. A remarkably high activity, over 100 μmol/s/μmol heme, was obtained in the oxidase with purified cyt b558, ternary complex (p47-p67-p40phox), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant Kd of FAD in cyt b558 of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b558 increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b558 recovered electron flow from NADPH to heme. From both Kd values of FAD and the amount of incorporated FAD in cyt b558 of reconstituted oxidase, in combination with spectra, we propose the model in which the Kd values of FAD in cyt b558 is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase. The phagocyte NADPH oxidase catalyzes the generation of superoxide anions (⋅NO and superoxide (O2˙−) in response to invading microorganisms. Superoxide anions are precursors of a variety of reactive oxygen species that are utilized in killing bacterial and fungal pathogens (1Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar, 2Lambeth J.D. J. Biochem. Mol. Biol. 2000; 33: 427-439Google Scholar). The physiological significance of the phagocyte NADPH oxidase in host defense is illustrated by the severe recurrent bacterial and fungal infections that occur in patients with chronic granulomatous disease whose phagocytes are unable to generate ⋅NO and superoxide (O2˙− (3Segal B.H. Leto T.L. Gallin J.I. Malech H.L. Holland S.M. Medicine (Baltimore). 2000; 79: 170-200Crossref PubMed Scopus (720) Google Scholar, 4Nauseef W.M. Proc. Assoc. Am. Physicians. 1999; 111: 373-382Crossref PubMed Scopus (85) Google Scholar). This NADPH oxidase complex consists of membrane-bound flavocytochrome b558, a heterodimer composed of gp91phox and p22phox, four cytosolic proteins, p40phox, p47phox, p67phox, and the small GTPase Rac. In the resting cells, p40phox, p47phox, and p67phox exist as a heterotrimeric complex that contains one copy of each protein (5Lapouge K. Smith S.J.M. Groemping Y. Rittinger K. J. Biol. Chem. 2002; 277: 10121-10128Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Activation of the oxidase is initiated by phosphorylation, which might induce the conformational changes by modulating intra- and intermolecular interactions in the p47-p67-p40phox complex. With these interactions, the activated oxidase is formed via assembly of the cytosolic regulatory proteins with cyt b558. 1The abbreviations used are: cyt b558, cytochrome b558; HTG, N-heptyl-β-thioglucoside; TC, ternary complex of recombinant cytosolic proteins p47-p67-p40phox; GTPγS, guanosine 5′-(γ-thio)triphosphate; PBS, phosphate-buffered saline; Ni-NTA, nickel-nitrilotriacetic acid; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; SH3, Src homolog 3; gp, glycoprotein.1The abbreviations used are: cyt b558, cytochrome b558; HTG, N-heptyl-β-thioglucoside; TC, ternary complex of recombinant cytosolic proteins p47-p67-p40phox; GTPγS, guanosine 5′-(γ-thio)triphosphate; PBS, phosphate-buffered saline; Ni-NTA, nickel-nitrilotriacetic acid; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; SH3, Src homolog 3; gp, glycoprotein.Activation of phagocyte NADPH oxidase can be mimicked in a cell-free system reconstituted with cyt b558, p47phox, p67phox p40phox, and Rac. These cytosolic proteins bind to cyt b558 to form an active oxidase complex, which can generate ⋅NO and superoxide (O2˙− in the presence of NADPH. The activated NADPH oxidase is highly labile because of the dissociation of each subunit of the active complex (6Tamura M. Takeshita M. Curnutte J.T. Uhlinger D.J. Lambeth J.D. J. Biol. Chem. 1992; 267: 7529-7538Abstract Full Text PDF PubMed Google Scholar). Recently, interactions among each subunit protein have been extensively studied; protein-protein interactions mediated by Src homolog 3 (SH3), tetratricopeptide repeat domain, and switch I have been reported (7Sumimoto H. Koga 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 (255) Google Scholar, 8Koga H. Terasawa H. Nunoi H. Takeshige K. Inagaki F. Sumimoto H. J. Biol. Chem. 1999; 274: 25051-25060Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 9Lapouge K. Smith S.J.M. Walker P.A. Gamblin S.J. Smerdon S.J. Rittinger K. Mol. Cell. 2000; 6: 899-907Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). In the cytoplasm of resting cells, p40phox, p47phox, and p67phox exist as a tight complex that can be purified by gel chromatography with an apparent molecular mass of 250–300 kDa (10Park J.W. Benna J.E. Scott K.E. Christensen B.L. Chanock S.J. Babior B.M. Biochemistry. 1994; 33: 2907-2911Crossref PubMed Scopus (85) Google Scholar, 11Wienties F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (256) Google Scholar). Sedimentation equilibrium and dynamic light-scattering experiments disclosed that the p47-p67-p40phox complex contains one copy of each protein, and the apparent high molecular weight of this complex, as estimated by gel filtration studies, is because of an extended, nonglobular shape (5Lapouge K. Smith S.J.M. Groemping Y. Rittinger K. J. Biol. Chem. 2002; 277: 10121-10128Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar).gp91phox in cyt b558 represents the only catalytic component in NADPH oxidase, containing both redox centers, FAD, and two nonidentical hemes. Many studies of the heme in cyt b558 have been reported as follows: (i) two hemes are located in gp91phox (12Yu L. Quinn M. Cross A. Dinauer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7993-7998Crossref PubMed Scopus (177) Google Scholar); (ii) two nonidentical hemes with midpoint redox potentials of -265 and -225 mV (13Cross A. Rae J. Curnutte J. J. Biol. Chem. 1995; 270: 17075-17077Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar); (iii) the heme has a low spin six coordination site. (14Fujii H. Johnson M.K. Finnegan M.G. Miki T. Yoshida L.S. Kakinuma K. J. Biol. Chem. 1995; 270: 12685-12689Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar); and (iv) the low spin state of the heme is essential for ⋅NO and superoxide (O2˙− generation in reconstituted NADPH oxidase (15Fujii H. Finnegan M.G. Johnson M.K. J. Biochem. (Tokyo). 1999; 126: 708-714Crossref PubMed Scopus (7) Google Scholar). In contrast to the extensive studies on the heme, a relatively small amount of information on FAD (16Koshkin V. Pick E. FEBS Lett. 1994; 338: 285-289Crossref PubMed Scopus (65) Google Scholar, 17Cross A.R. Curnutte J.T. J. Biol. Chem. 1995; 270: 6543-6548Abstract Full Text PDF PubMed Scopus (90) Google Scholar, 18Nisimoto Y. Otsuka-Murakami H. Lambeth D.J. J. Biol. Chem. 1995; 270: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) has been obtained, including such as the contents of flavins (FAD and FMN) in the oxidase and reflavination in flavocytochrome b558. One plausible model is that FAD is involved in ⋅NO and superoxide (O2˙− generation in NADPH oxidase and that exogenous FAD can restore ⋅NO and superoxide (O2˙− generating activities in the cell-free system reconstructed with FAD-depleted purified cyt b558 and cytosolic proteins. Detailed information on the behavior of FAD molecules at the FAD-binding site of the NADPH oxidase and its physiological status is requisite to elucidate the electron transfer reactions among the two redox centers, but the behavior of FAD in intact cells is still unclear. Therefore, a simplified in vitro experiment excluding unfavorable side reactions and thermal instability of participating proteins is desired and required.In the present work we describe the reconstitution of NADPH oxidase activity in a cell-free system with FAD-depleted purified cyt b558 and recombinant FAD-free cytosolic regulatory proteins, i.e. the ternary complex (p47-p67-p40phox) and Rac. We report the effects of exogenously added FAD on the reconstituted activity of NADPH oxidase, and we show that enhancement of the activity by FAD is dependent on the assembly process of the reconstituted oxidase. Furthermore, we show that electron transfer from NADPH to heme under anaerobiosis is restored after incorporating exogenous FAD into FAD-depleted cyt b558. These analyses provide the direct evidence that the electron flow from NADPH to heme via FAD observed is directly related to the ⋅NO and superoxide (O2˙−-generating reaction in phagocyte NADPH oxidase.EXPERIMENTAL PROCEDURESMaterials—Sodium myristate and diisopropyl fluorophosphates were obtained from Wako Pure Chemicals. DEAE-Sepharose CL-6B, CM-Sepharose, heparin-Sepharose, glutathione-Sepharose 4B, Superdex 75 column, Superdex 200 column, pGEX 6P, and PreScission protease were obtained from Amersham Bioscience. N-Heptyl-β-d-thioglucoside (HTG) and EGTA were purchased from Dojindo Laboratories. NADPH was from Oriental Yeast. Superoxide dismutase, FAD, FMN, GTPγS, glucose oxidase, arachidonic acid, phorbol 12-myristate 13-acetate, and cytochrome c (type VI from horse heart) were purchased from Sigma. Ni-NTA was obtained from Qiagen. pProEX HTb and TEV protease were purchased from Invitrogen. Centriprep YM-10 was obtained from Millipore. 4-(2-aminoethyl)-Benzenesulfonyl fluoride hydrochloride (AEBSF) was purchased from Nacalai Tesque. Kodakaraensis DNA polymerase for PCR and ligation high for subcloning were obtained from Toyobo. Restriction enzymes, EcoRI and NdeI, were purchased from Takara. The oligonucleotide primers were synthesized by Sigma-Genosys.Purification of Cyt b558—Neutrophils were obtained from pig blood, as described previously (19Fujii H. Kakinuma K. Biochim. Biophys. Acta. 1991; 1095: 201-209Crossref PubMed Scopus (19) Google Scholar), and then treated with 2 mm diisopropyl fluorophosphate for 20 min on ice. Cyt b558 was solubilized from the membrane fraction with HTG and was purified by using DEAE-Sepharose, CM-Sepharose, and heparin-Sepharose as described previously (14Fujii H. Johnson M.K. Finnegan M.G. Miki T. Yoshida L.S. Kakinuma K. J. Biol. Chem. 1995; 270: 12685-12689Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 20Miki T. Yoshida L.S. Kakinuma K. J. Biol. Chem. 1992; 267: 18695-18701Abstract Full Text PDF PubMed Google Scholar). The specific content of heme in the purified cyt b558 preparation was 10.6–12.4 nmol per mg of protein.Preparation of Recombinant Proteins—The hexahistidine tag was introduced into the vector pGEX-6p-1. Two oligonucleotides with the sequences 5′-TCGACTCCACCATCATCATCATCATTAATGC-3′ and 5′-GGCCGCATTAATGATGATGATGATGGTGGAG-3′ were annealed and phosphorylated. The product was ligated into SalI- and NotI-digested pGEX-6p-1 (pGEX-His).The full-length p47phox was constructed by PCR from the plasmid of human p47phox (7Sumimoto H. Koga 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 (255) Google Scholar, 21Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), using 5′-GGGAATTCATGGGGGACACCTTCATCCGTC-3′ as the forward primer and 5′-GGGTCGACCGACGGCAGACGCCAGCTTC-3′ as the reverse primer. The PCR product was digested with EcoRI and SalI, gel-purified, and ligated into EcoRI- and SalI-digested pGEX-His. The plasmids of full-length p47phox with an amino-terminal glutathione S-transferase-tagged and a carboxyl-terminal hexahistidine-tagged (His-tagged) construct were transformed in Escherichia coli BL21(DE3) and were overexpressed. The cells were disrupted by sonication at 4 °C in phosphate-buffered saline (PBS), pH 7.4, and 0.1 mm AEBSF. The protein was applied on a glutathione-Sepharose 4B column equilibrated with PBS buffer, pH 7.4. The bound protein was eluted with 25 mm Tris buffer, pH 8.0, and 25 mm reduced glutathione. The amino-terminal glutathione S-transferase tag in proteins were then removed by incubation with PreScission protease for 12 h at 4 °C, and the digested proteins were dialyzed against 2 liters of 25 mm Tris buffer, pH 7.8, and 500 mm NaCl. The dialyzed proteins with carboxyl-terminal His tag were applied on a Ni-NTA column equilibrated with 25 mm Tris buffer, pH 7.8, 500 mm NaCl, and 5 mm imidazole. The bound protein was eluted with 25 mm Tris buffer, pH 7.8, 500 mm NaCl, and 250 mm imidazole. Fractions containing proteins were purified on a Superdex 200 gel filtration column and eluted with 25 mm Tris buffer, pH 7.4, and 150 mm NaCl. The protein was concentrated by Centriprep YM-30 to about 10 mg/ml.The full-length p67phox was amplified by PCR from the plasmid of human p67phox (7Sumimoto H. Koga 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 (255) Google Scholar, 21Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), using 5′-GGGGATCCATGTCCCTGGTGGAGGCCATC-3′ as the forward primer and 5′-GGGTCGACCGACTTCTCTCCGAGTGCTTTC-3′ as the reverse primer. The PCR product was digested with BamHI and SalI, gel-purified, and ligated into BamHI- and SalI-digested pGEX-His. The plasmids of the full-length p67phox with amino-terminal glutathione S-transferase-tagged and carboxyl-terminal His-tagged constructs were transformed and overexpressed, as shown in the preparation for the full-length p47phox. From obtained cells, the full-length p67phox was similarly purified, as shown above.The full-length p40phox were amplified by PCR from the plasmid of human p40phox (22Nakamura R. Sumimoto H. Mizuki K. Hata K. Ago T. Kitajima S. Takeshige K. Sasaki Y. Ito T. Eur. J. Biochem. 1998; 251: 583-589Crossref PubMed Scopus (72) Google Scholar), using 5′-CCCCATGGCTGTGGCCCAGCAGCTGC-3′ as the forward primer and 5′-CCGAATTCATTATGGCATCGTGTTGTAGACCCTGTAGTTG-3′ as the reverse primer. The PCR product was digested with NcoI and EcoRI, gel-purified, and ligated into BamHI- and SalI-digested pProEX HTb (pPro-p40). The plasmid pT-Trx was a generous gift from Dr. S. Ishii (Laboratory of Molecular Genetics, The Institute of Physical and Chemical Research, RIKEN) (23Yasukawa T. Kanei-Ishii C. Maekawa T. Fujimoto J. Yamamoto T. Ishii S. J. Biol. Chem. 1995; 270: 25328-25331Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). These plasmids of pPro-p40 and pT-Trx were co-transformed in E. coli BL21(DE3) and were overexpressed. The cells were disrupted by sonication at 4 °C in 25 mm Tris buffer, pH 7.8, and 500 mm NaCl. The protein was applied on a Ni-NTA column equilibrated with 25 mm Tris buffer, pH 7.8, 500 mm NaCl, and 5 mm imidazole. The bound protein was eluted with 25 mm Tris buffer, pH 7.8, 500 mm NaCl, and 250 mm imidazole. Fractions containing proteins were purified on a Superdex 75 gel filtration column and eluted with 25 mm Tris buffer, pH 8.0, 150 mm NaCl. The amino-terminal His tag of p40phox was removed by incubation with TEV protease for 12 h at 4 °C. Further purification was carried out on a Superdex 75 gel filtration column eluted with 25 mm Tris buffer, pH 8.5, and 150 mm NaCl. The protein was concentrated by Centriprep YM-30 to around 10 mg/ml.The DNA encoding Rac2 constitutively active form was obtained, as described previously (8Koga H. Terasawa H. Nunoi H. Takeshige K. Inagaki F. Sumimoto H. J. Biol. Chem. 1999; 274: 25051-25060Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar), and was subcloned into pProEX HTb (pPro-Rac2). The plasmid pProRac2 was transformed in E. coli BL21(DE3) and was overexpressed. The cells were disrupted by sonication at 4 °C in PBS including 0.1 mm AEBSF, pH 7.4. The protein was applied on a Ni-NTA column equilibrated with PBS and 5 mm imidazole, pH 7.4. After washing with a 20-fold column volume of PBS and 10-fold column volume of 25 mm Tris buffer, pH 7.4, including 5 mm imidazole, the bound protein was eluted with 25 mm Tris buffer, pH 7.4, 150 mm NaCl, and 250 mm imidazole. Fractions containing proteins were purified on a Superdex 75 gel filtration column and eluted with 25 mm Tris buffer, pH 8.0, 150 mm NaCl.Preparation of Binary and Ternary Cytosolic Complexes—To obtain binary complexes of p47-p67phox and p67-p40phox, p67phox was mixed with a 2-fold molar excess of p47phox or p40phox. After the mixtures had been incubated for more than 10 min at 4 °C, the binary complexes were purified from excess uncomplexed protein (p47phox or p40phox) by gel filtration on a Superdex 200 column using a buffer containing 50 mm Tris buffer, pH 7.4, and 150 mm NaCl. Fractions containing the binary complexes were concentrated by Centriprep YM-50 to around 10 mg protein/ml.Ternary complex of p47-p67-p40phox was obtained by mixing the binary complex p47-p67phox with a 2-fold molar excess of p40phox. After the mixtures had been incubated for more than 10 min at 4 °C, purification of ternary complex from excess uncomplexed p40phox was performed by size exclusion chromatography on a Superdex 200 column with a buffer containing 25 mm Tris buffer, pH 7.4, 150 mm NaCl. The ternary complex was concentrated by Centriprep YM-50 to around 10 mg of protein/ml.Preparation of Truncated Binary Complex—Fusion protein between truncated p67phox-(1–242) and truncated p47phox-(151–286) (p67-p47phox truncated binary complex) was constructed by a two-step PCR technique. The first step was amplification of the truncated p67phox gene and the truncated p47phox gene. The DNA fragment encoding p67phox-(1–242) was amplified from the full-length p67phox gene by PCR using primers as follows: 5′-primer (primer A) was 5′-GGCCATATGTCCCTGGTGGAGGCCAATCAGC-3′, including NdeI digestion site at the 5′-end, and 3′-primer (primer B) was 5′-CAGGATGATGGGGCCGGTGATGTCCCCTTCCAGAGCCCTGAAGATCTC-3′, including 24 bases at the 5′-end of p47phox-(151–286). The DNA fragment encoding p47phox-(151–286) was amplified from the full-length p47phox gene with PCR using primers as follows: 5′-primer (primer C) was 5′-GACATCACCGGCCCCATCATCCTG-3′, and 3′-primer (primer D) was 5′-GCGCGAATTCATTAGTCTTGCCCCGACTTTTGCAGGTA-3′, including the stop codon TAA and the EcoRI site. Two PCR products had 24 overlapping base pairs (5′-end of p47phox-(151–286)). For fusion of p67phox-(1–242) gene and p47phox-(151–286) gene as the second step, five cycles of denaturation, annealing, and extension were carried out at 98 °C (15 s) and 74 °C (30 s). This fused gene was amplified by an additional 25 thermal cycles under the same conditions using primer A and D. The fused gene was then subcloned into the NdeI-EcoRI site of pET28a, and this plasmid was transformed in E. coli BL21(DE3), in which the truncated binary complex was overexpressed. The cells were disrupted by sonication at 4 °C in PBS, pH 7.4, and 0.1 mm AEBSF. The protein was applied on a Ni-NTA column equilibrated with PBS, pH 7.4. The bound protein was eluted with 25 mm Tris buffer, pH 7.4, 150 mm NaCl, and 250 mm imidazole. Fractions containing proteins were purified on a Superdex 75 gel filtration column.Reconstitution of NADPH Oxidase and Assay for ⋅NO and superoxide (O2˙− Generation— The ⋅NO and superoxide (O2˙− generating activity of reconstituted NADPH oxidase complex was determined by the superoxide dismutase-inhibitable reduction of cytochrome c, as described previously (19Fujii H. Kakinuma K. Biochim. Biophys. Acta. 1991; 1095: 201-209Crossref PubMed Scopus (19) Google Scholar). The standard reaction mixture contained partially or fully purified cyt b558 (1 pmol of heme), TC (250 nm), and Rac2 (400 nm) in a volume of 100 μl of the assay buffer (1 mm NaN3, 1 mm EGTA, 1 mm MgCl2, 50 mm phosphate buffer, pH 7.0), and it was treated with 20 μm GTPγS for 1 min. The reaction mixture above was supplemented with an appropriate amount of amphiphile, such as sodium myristate or arachidonic acid, and incubated at 25 °C for 5 min. Unless specified, the activation was carried out in the presence of FAD. The mixture was transferred to a cuvette containing cytochrome c (final concentration, 50 μm), and the ⋅NO and superoxide (O2˙−-generating reaction was started by the addition of 150 μm NADPH. ⋅NO and superoxide (O2˙− production was measured with a Hitachi dual-wavelength spectrophotometer (model 556) at 550 nm due to the reduction of cytochrome c.Reconstitution of Flavin-depleted Cyt b558 with Native FAD and Assay of Noncovalently bound FAD—Purified FAD-depleted cyt b558 (200 pmol) was mixed with TC (80 nmol) and Rac2 (40 nmol) in the presence or absence of 2 nmol of excess FAD and was incubated with sodium myristate (600 nmol) for 5 min at 25 °C. After the above treatment, the mixture was passed through a PD-10 column (Amersham Biosciences) equilibrated with 50 mm phosphate buffer, pH 7.4, 0.6% HTG, and 10% glycerol to separate FAD-reconstituted cyt b558 from excess free flavin. FAD-reconstituted cyt b558 fractions were pooled and concentrated by freeze-drying and then boiled at 100 °C for 15 min. After centrifugation at 15,000 rpm for 15 min, FAD in boiled extract of purified cyt b558 fractions was quantified either by fluorometry with a Wallac 1420 ARVOsx (24Faeder E.J. Siegel L.M. Anal. Biochem. 1973; 53: 332-336Crossref PubMed Scopus (221) Google Scholar, 25Kakinuma K. Kaneda M. Chiba T. Ohnishi T. J. Biol. Chem. 1986; 261: 9426-9432Abstract Full Text PDF PubMed Google Scholar) or the chemiluminescence method (26Yoshida L.S. Chiba T. Kakinuma K. Biochim. Biophys. Acta. 1992; 1135: 245-252Crossref PubMed Scopus (10) Google Scholar). Calibration was performed with standard solutions of FAD.Measurement of Electron Flux through Redox Centers under Anaerobic Conditions—The electron fluxes through two redox centers, FAD and heme, in cyt b558 were examined. The electron flux from NADPH to heme via FAD in reflavinated cyt b558 was measured by following the absorbance at 558 nm with Unisoku Biospectrophotometer US-401. The extent of reduction in cyt b558 was calculated by using a millimolar extinction coefficient of 21.6 at 558–540 nm (27Gabig T.G. Schervich E.W. Santinga J.T. J. Biol. Chem. 1982; 257: 4114-4119Abstract Full Text PDF PubMed Google Scholar). Spectral changes of cyt b558 were observed continuously over the 400–600 nm range. Strictly anaerobic conditions were achieved by including glucose/glucose oxidase (40 units/ml) in an airtight cuvette (19Fujii H. Kakinuma K. Biochim. Biophys. Acta. 1991; 1095: 201-209Crossref PubMed Scopus (19) Google Scholar).RESULTSTernary Complex Used in This Study—The soluble fulllength proteins of p47phox, p67phox, and p40phox were obtained in relatively high yields (>20 mg of protein/liter), as described under "Experimental Procedures." Fig. 1A shows the gel filtration chromatogram of purified proteins; binary (p47-p67phox) and ternary (p47-p67-p40phox) complexes were fractionated without contamination of monomer components. The molecular mass of purified ternary complex was calibrated by the analytical gel filtration, and the obtained value was around 250 kDa, which was similar to that in the previous report (5Lapouge K. Smith S.J.M. Groemping Y. Rittinger K. J. Biol. Chem. 2002; 277: 10121-10128Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Fig. 1B shows silver-stained SDS-PAGE analysis of purified proteins. The purity of each sample is demonstrated by SDS-PAGE; full-length p40phox (lane 1), p47phox (lane 2), p67phox (lane 3), binary (p47-p67phox), and ternary (p47-p67-p40phox) complexes are shown in Fig. 1B.Reconstituted Activity of NADPH Oxidase—Cyt b558 was purified from neutrophil membranes without any modification of the heme environment and was used for evaluation of ⋅NO and superoxide (O2˙− generating activity in the cell-free system. Heme and FAD associated with the cytochrome preparations at each purification step were measured and are summarized in Table I. Crude membrane contained the heme and FAD of cyt b558 in a molar ratio of about 2.5:1. During the purification, the specific content of FAD decreased, whereas that of the heme increased. These purified cyt b558 exhibited high ⋅NO and superoxide (O2˙− generating activity in the cell-free system with native cytosol, which shows that cyt b558 used in this study was highly purified without any irreversible protein denaturation.Table IPurification of cyt b588 from resting neutrophil membranesHemeFADHeme/FADActivityaSuperoxide generating activity was evaluated in a cell-free system reconstituted with native cytosol and membrane fractions or purified cyt b558 at 25 °Cpmol/mg proteinmol/s/mol hemeCrude membrane238 ± 15 (n = 15)95 ± 13 (n = 15)2.5114.2 ± 2.5 (n = 15)Crude extract1650 ± 123 (n = 15)432 ± 72 (n = 15)3.8264.5 ± 8.3 (n = 15)Purified cyt b55811,600 ± 950 (n = 12)201 ± 94 (n = 12)57.760.8 ± 6.7 (n = 12)a Superoxide generating activity was evaluated in a cell-free system reconstituted with native cytosol and membrane fractions or purified cyt b558 at 25 °C Open table in a new tab The ⋅NO and superoxide (O2˙− generating activity of the oxidase was reconstituted in the cell-free system with recombinant, full-length FAD-free cytosolic proteins (Rac2, p40phox, p47phox, and p67phox) and purified cyt b558. Incubation of the cytosolic component individually and in combination or the TC with purified cyt b558 was examined, and the purified cyt b558 showed cytosolic proteindependent superoxide production. In the cell-free reconstituted system, the activity was totally dependent on the addition of both FAD and anionic amphiphile activators (such as myristic acid or arachidonic acid). Replacement of FAD with FMN abolished the stimulation of superoxide production. The remarkably high ⋅NO and superoxide (O2˙− generating activity of the reconstituted oxidase, over 100 μmol of ⋅NO and superoxide (O2˙− /s/μmol of heme, was obtainable in the system with TC. The reconstituted oxidase activity with TC (p47-p67-p40phox) was compared with the binary complex p47-p67phox. The activity with TC was 9.9 ± 2.8% (p < 0.05) higher than with p47/p67phox, showing that p40phox is not essential for reconstitution of the activity. Previous findings (28Cross A. Biochem. J. 2000; 349: 113-117Crossref PubMed Google Scholar, 29Kuribayashi F. Nunoi H. Wakamatsu K. Tsunawaki S. Sato K. Ito T. Sumimoto H. EMBO J. 2002; 21: 6312-6320Crossref PubMed Scopus (122) Google Scholar) were also confirmed in the cell-free reconstituted system with the recombinant TC. In the case of the other binary complexes, p47-p40phox or p67-p40phox, any superoxide generating activity was not detected under similar reaction conditions.Stability of the Reconstituted NADPH Oxidase Activity—The purified ternary complex, p47-p67-p40phox, exists at a tight complex of 1:1:1 stoichiometry in solutions (5Lapouge K. Smith S.J.M. Groemping Y. Rittinger K. J. Biol. Chem. 2002; 277: 10121-10128Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) and is stable for at least 24 h without any dissociation of each component, as judged from the gel filtration experiment of purified ternary complex. Fig. 2 shows the stability of the oxidase activity reconstituted with TC at 25 °C
Referência(s)