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. 1996 Nov 15;320(Pt 1):33–38. doi: 10.1042/bj3200033

Stoichiometry of the subunits of flavocytochrome b558 of the NADPH oxidase of phagocytes.

T M Wallach 1, A W Segal 1
PMCID: PMC1217894  PMID: 8947464

Abstract

Flavocytochrome b558, the membrane-spanning component of the NADPH oxidase system of phagocytic cells, is composed of two subunits, p22phox and gp91phox (where phox stands for phagocyte oxidase). The stoichiometry of the subunits has been determined for purified flavocytochrome b556 by: (1) densitometry of Coomassie Blue-stained proteins separated by SDS/PAGE, (2) aromatic absorbance at 280 mm by the subunits after separation by gel filtration under denaturing conditions, (3) crosslinking studies with bis[sulphosuccinimidyl]suberate, where the molecular mass of the cross-linked complex was determined by Western blotting, and (4) radiolabelling of pure flavocytochrome b556 on lysine residues with 125I-labelled Bolton-Hunter reagent (N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate), followed by SDS/PAGE and determination of the radioactivity on each subunit. The ratio of p22phox to gp91phox in the purified flavocytochrome b556 was related back to that in the neutrophil membrane by quantitative Western and dot-blotting to ensure that the stoichiometry was maintained during purification. These measurements showed that the two subunits were present in neutrophil membranes in a molar ratio of 1:1.

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Selected References

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  1. Clark R. A. The human neutrophil respiratory burst oxidase. J Infect Dis. 1990 Jun;161(6):1140–1147. doi: 10.1093/infdis/161.6.1140. [DOI] [PubMed] [Google Scholar]
  2. Cross A. R., Curnutte J. T. The cytosolic activating factors p47phox and p67phox have distinct roles in the regulation of electron flow in NADPH oxidase. J Biol Chem. 1995 Mar 24;270(12):6543–6548. doi: 10.1074/jbc.270.12.6543. [DOI] [PubMed] [Google Scholar]
  3. Cross A. R., Rae J., Curnutte J. T. Cytochrome b-245 of the neutrophil superoxide-generating system contains two nonidentical hemes. Potentiometric studies of a mutant form of gp91phox. J Biol Chem. 1995 Jul 21;270(29):17075–17077. doi: 10.1074/jbc.270.29.17075. [DOI] [PubMed] [Google Scholar]
  4. Dinauer M. C., Pierce E. A., Bruns G. A., Curnutte J. T., Orkin S. H. Human neutrophil cytochrome b light chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease. J Clin Invest. 1990 Nov;86(5):1729–1737. doi: 10.1172/JCI114898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fujii H., Johnson M. K., Finnegan M. G., Miki T., Yoshida L. S., Kakinuma K. Electron spin resonance studies on neutrophil cytochrome b558. Evidence that low-spin heme iron is essential for O2-. generating activity. J Biol Chem. 1995 May 26;270(21):12685–12689. [PubMed] [Google Scholar]
  6. Fujii H., Kakinuma K. Electron paramagnetic resonance studies on cytochrome b-558 and peroxidases of pig blood granulocytes. Biochim Biophys Acta. 1992 Sep 9;1136(3):239–246. doi: 10.1016/0167-4889(92)90112-o. [DOI] [PubMed] [Google Scholar]
  7. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  8. Harper A. M., Dunne M. J., Segal A. W. Purification of cytochrome b-245 from human neutrophils. Biochem J. 1984 Apr 15;219(2):519–527. doi: 10.1042/bj2190519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Huang J., Hitt N. D., Kleinberg M. E. Stoichiometry of p22-phox and gp91-phox in phagocyte cytochrome b558. Biochemistry. 1995 Dec 26;34(51):16753–16757. doi: 10.1021/bi00051a024. [DOI] [PubMed] [Google Scholar]
  10. Hurst J. K., Loehr T. M., Curnutte J. T., Rosen H. Resonance Raman and electron paramagnetic resonance structural investigations of neutrophil cytochrome b558. J Biol Chem. 1991 Jan 25;266(3):1627–1634. [PubMed] [Google Scholar]
  11. Iizuka T., Kanegasaki S., Makino R., Tanaka T., Ishimura Y. Studies on neutrophil b-type cytochrome in situ by low temperature absorption spectroscopy. J Biol Chem. 1985 Oct 5;260(22):12049–12053. [PubMed] [Google Scholar]
  12. Jesaitis A. J. Structure of human phagocyte cytochrome b and its relationship to microbicidal superoxide production. J Immunol. 1995 Oct 1;155(7):3286–3288. [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Miki T., Fujii H., Kakinuma K. EPR signals of cytochrome b558 purified from porcine neutrophils. J Biol Chem. 1992 Sep 25;267(27):19673–19675. [PubMed] [Google Scholar]
  15. Nisimoto Y., Otsuka-Murakami H., Lambeth D. J. Reconstitution of flavin-depleted neutrophil flavocytochrome b558 with 8-mercapto-FAD and characterization of the flavin-reconstituted enzyme. J Biol Chem. 1995 Jul 7;270(27):16428–16434. doi: 10.1074/jbc.270.27.16428. [DOI] [PubMed] [Google Scholar]
  16. Nugent J. H., Gratzer W., Segal A. W. Identification of the haem-binding subunit of cytochrome b-245. Biochem J. 1989 Dec 15;264(3):921–924. doi: 10.1042/bj2640921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Parkos C. A., Allen R. A., Cochrane C. G., Jesaitis A. J. Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000. J Clin Invest. 1987 Sep;80(3):732–742. doi: 10.1172/JCI113128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Parkos C. A., Allen R. A., Cochrane C. G., Jesaitis A. J. The quaternary structure of the plasma membrane b-type cytochrome of human granulocytes. Biochim Biophys Acta. 1988 Jan 20;932(1):71–83. doi: 10.1016/0005-2728(88)90140-5. [DOI] [PubMed] [Google Scholar]
  19. Parkos C. A., Dinauer M. C., Jesaitis A. J., Orkin S. H., Curnutte J. T. Absence of both the 91kD and 22kD subunits of human neutrophil cytochrome b in two genetic forms of chronic granulomatous disease. Blood. 1989 May 1;73(6):1416–1420. [PubMed] [Google Scholar]
  20. Parkos C. A., Dinauer M. C., Walker L. E., Allen R. A., Jesaitis A. J., Orkin S. H. Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b. Proc Natl Acad Sci U S A. 1988 May;85(10):3319–3323. doi: 10.1073/pnas.85.10.3319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Quinn M. T., Mullen M. L., Jesaitis A. J. Human neutrophil cytochrome b contains multiple hemes. Evidence for heme associated with both subunits. J Biol Chem. 1992 Apr 15;267(11):7303–7309. [PubMed] [Google Scholar]
  22. Quinn M. T., Parkos C. A., Walker L., Orkin S. H., Dinauer M. C., Jesaitis A. J. Association of a Ras-related protein with cytochrome b of human neutrophils. Nature. 1989 Nov 9;342(6246):198–200. doi: 10.1038/342198a0. [DOI] [PubMed] [Google Scholar]
  23. Rotrosen D., Yeung C. L., Leto T. L., Malech H. L., Kwong C. H. Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase. Science. 1992 Jun 5;256(5062):1459–1462. doi: 10.1126/science.1318579. [DOI] [PubMed] [Google Scholar]
  24. Segal A. W. Absence of both cytochrome b-245 subunits from neutrophils in X-linked chronic granulomatous disease. Nature. 1987 Mar 5;326(6108):88–91. doi: 10.1038/326088a0. [DOI] [PubMed] [Google Scholar]
  25. Segal A. W., Geisow M., Garcia R., Harper A., Miller R. The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH. Nature. 1981 Apr 2;290(5805):406–409. doi: 10.1038/290406a0. [DOI] [PubMed] [Google Scholar]
  26. Segal A. W. Structure of the NADPH-oxidase: membrane components. Immunodeficiency. 1993;4(1-4):167–179. [PubMed] [Google Scholar]
  27. Segal A. W., West I., Wientjes F., Nugent J. H., Chavan A. J., Haley B., Garcia R. C., Rosen H., Scrace G. Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes. Biochem J. 1992 Jun 15;284(Pt 3):781–788. doi: 10.1042/bj2840781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Teahan C., Rowe P., Parker P., Totty N., Segal A. W. The X-linked chronic granulomatous disease gene codes for the beta-chain of cytochrome b-245. 1987 Jun 25-Jul 1Nature. 327(6124):720–721. doi: 10.1038/327720a0. [DOI] [PubMed] [Google Scholar]
  29. Thrasher A. J., Keep N. H., Wientjes F., Segal A. W. Chronic granulomatous disease. Biochim Biophys Acta. 1994 Oct 21;1227(1-2):1–24. doi: 10.1016/0925-4439(94)90100-7. [DOI] [PubMed] [Google Scholar]
  30. Yamaguchi T., Hayakawa T., Kaneda M., Kakinuma K., Yoshikawa A. Purification and some properties of the small subunit of cytochrome b558 from human neutrophils. J Biol Chem. 1989 Jan 5;264(1):112–118. [PubMed] [Google Scholar]

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