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. 1984 Apr 15;219(2):519–527. doi: 10.1042/bj2190519

Purification of cytochrome b-245 from human neutrophils.

A M Harper, M J Dunne, A W Segal
PMCID: PMC1153509  PMID: 6331390

Abstract

The low potential cytochrome b (b-245) of the microbicidal oxidase of phagocytic cells has been purified from neutrophils from patients with chronic myeloid leukaemia. Cells were homogenized in the presence of proteinase inhibitors and centrifuged to remove the cytoplasm. The pellets containing membranes, granules and other organelles (15 mg/ml) were then washed with buffered sodium cholate (5 mg/ml). Residual pellets were subsequently solubilized with the non-ionic detergent Triton N 101 (10 mg/ml) which extracted about 60% of the cytochrome b. About 10% of the cytochrome b was of mitochondrial origin which was removed on a column of n-amino-octyl-Sepharose that did not adsorb cytochrome b-245. Cytochrome b-245 was chromatographed on a column of heparin-agarose and eluted with NaCl to give a peak specific content of 11-16 nmol of cytochrome b-245/mg of protein, representing a 140-200-fold purification with a recovery of 15%. This technique results in the purification of approx. 100-150 nmol of highly purified cytochrome b-245 from (3-5) X 10(11) cells within 4 days. The most purified material gave a broad band with an apparent Mr of between 68 000 and 78 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, but gel filtration indicated an aggregated form of the protein in Triton N101 . Purified protein (14 nmol of haem/mg of protein) did not contain FAD or FMN and had no NADPH-dependent O2--generating activity.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albert A. D., Litman B. J. Independent structural domains in the membrane protein bovine rhodopsin. Biochemistry. 1978 Sep 19;17(19):3893–3900. doi: 10.1021/bi00612a001. [DOI] [PubMed] [Google Scholar]
  2. Amrein P. C., Stossel T. P. Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate. Blood. 1980 Sep;56(3):442–447. [PubMed] [Google Scholar]
  3. Badwey J. A., Karnovsky M. L. Active oxygen species and the functions of phagocytic leukocytes. Annu Rev Biochem. 1980;49:695–726. doi: 10.1146/annurev.bi.49.070180.003403. [DOI] [PubMed] [Google Scholar]
  4. Bellavite P., Cross A. R., Serra M. C., Davoli A., Jones O. T., Rossi F. The cytochrome b and flavin content and properties of the O2- -forming NADPH oxidase solubilized from activated neutrophils. Biochim Biophys Acta. 1983 Jul 28;746(1-2):40–47. doi: 10.1016/0167-4838(83)90008-0. [DOI] [PubMed] [Google Scholar]
  5. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  6. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  7. Cross A. R., Higson F. K., Jones O. T., Harper A. M., Segal A. W. The enzymic reduction and kinetics of oxidation of cytochrome b-245 of neutrophils. Biochem J. 1982 May 15;204(2):479–485. doi: 10.1042/bj2040479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cross A. R., Jones O. T., Garcia R., Segal A. W. The association of FAD with the cytochrome b-245 of human neutrophils. Biochem J. 1982 Dec 15;208(3):759–763. doi: 10.1042/bj2080759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cross A. R., Jones O. T., Harper A. M., Segal A. W. Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. A possible oxidase in the respiratory burst. Biochem J. 1981 Feb 15;194(2):599–606. doi: 10.1042/bj1940599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duong L. T., Fleming P. J. Isolation and properties of cytochrome b561 from bovine adrenal chromaffin granules. J Biol Chem. 1982 Aug 10;257(15):8561–8564. [PubMed] [Google Scholar]
  11. Eckhardt A. E., Hayes C. E., Goldstein I. J. A sensitive fluorescent method for the detection of glycoproteins in polyacrylamide gels. Anal Biochem. 1976 May 21;73(1):192–197. doi: 10.1016/0003-2697(76)90154-8. [DOI] [PubMed] [Google Scholar]
  12. Faeder E. J., Siegel L. M. A rapid micromethod for determination of FMN and FAD in mixtures. Anal Biochem. 1973 May;53(1):332–336. doi: 10.1016/0003-2697(73)90442-9. [DOI] [PubMed] [Google Scholar]
  13. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  14. Frank R. N., Rodbard D. Precision of sodium dodecyl sulfate-polyacrylamide-gel electrophoresis for the molecular weight estimation of a membrane glycoprotein: studies on bovine rhodopsin. Arch Biochem Biophys. 1975 Nov;171(1):1–13. doi: 10.1016/0003-9861(75)90001-6. [DOI] [PubMed] [Google Scholar]
  15. Gabig T. G., Schervish E. W., Santinga J. T. Functional relationship of the cytochrome b to the superoxide-generating oxidase of human neutrophils. J Biol Chem. 1982 Apr 25;257(8):4114–4119. [PubMed] [Google Scholar]
  16. Gabig T. G. The NADPH-dependent O-.2-generating oxidase from human neutrophils. J Biol Chem. 1983 May 25;258(10):6352–6356. [PubMed] [Google Scholar]
  17. Glossmann H., Neville D. M., Jr Glycoproteins of cell surfaces. A comparative study of three different cell surfaces of the rat. J Biol Chem. 1971 Oct 25;246(20):6339–6346. [PubMed] [Google Scholar]
  18. Guengerich F. P., Martin M. V. Purification of cytochrome P-450, NADPH-cytochrome P-450 reductase, and epoxide hydratase from a single preparation of rat liver microsomes. Arch Biochem Biophys. 1980 Dec;205(2):365–379. doi: 10.1016/0003-9861(80)90119-8. [DOI] [PubMed] [Google Scholar]
  19. Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Light D. R., Walsh C., O'Callaghan A. M., Goetzl E. J., Tauber A. I. Characteristics of the cofactor requirements for the superoxide-generating NADPH oxidase of human polymorphonuclear leukocytes. Biochemistry. 1981 Mar 17;20(6):1468–1476. doi: 10.1021/bi00509a010. [DOI] [PubMed] [Google Scholar]
  22. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  23. Marres C. M., Slater E. C. Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria. Biochim Biophys Acta. 1977 Dec 23;462(3):531–548. doi: 10.1016/0005-2728(77)90099-8. [DOI] [PubMed] [Google Scholar]
  24. Roberts P. J., Cross A. R., Jones O. T., Segal A. W. Development of cytochrome b and an active oxidase system in association with maturation of a human promyelocytic (HL-60) cell line. J Cell Biol. 1982 Dec;95(3):720–726. doi: 10.1083/jcb.95.3.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Root R. K., Cohen M. S. The microbicidal mechanisms of human neutrophils and eosinophils. Rev Infect Dis. 1981 May-Jun;3(3):565–598. doi: 10.1093/clinids/3.3.565. [DOI] [PubMed] [Google Scholar]
  26. Segal A. W., Cross A. R., Garcia R. C., Borregaard N., Valerius N. H., Soothill J. F., Jones O. T. Absence of cytochrome b-245 in chronic granulomatous disease. A multicenter European evaluation of its incidence and relevance. N Engl J Med. 1983 Feb 3;308(5):245–251. doi: 10.1056/NEJM198302033080503. [DOI] [PubMed] [Google Scholar]
  27. Segal A. W., Garcia R. C., Harper A. M., Banga J. P. Iodination by stimulated human neutrophils. Studies on its stoichiometry, subcellular localization and relevance to microbial killing. Biochem J. 1983 Jan 15;210(1):215–225. doi: 10.1042/bj2100215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Segal A. W., Garcia R., Goldstone H., Cross A. R., Jones O. T. Cytochrome b-245 of neutrophils is also present in human monocytes, macrophages and eosinophils. Biochem J. 1981 Apr 15;196(1):363–367. doi: 10.1042/bj1960363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Segal A. W., Jones O. T. Novel cytochrome b system in phagocytic vacuoles of human granulocytes. Nature. 1978 Nov 30;276(5687):515–517. doi: 10.1038/276515a0. [DOI] [PubMed] [Google Scholar]
  30. Segal A. W., Jones O. T. The subcellular distribution and some properties of the cytochrome b component of the microbicidal oxidase system of human neutrophils. Biochem J. 1979 Jul 15;182(1):181–188. doi: 10.1042/bj1820181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Segal A. W., Peters T. J. Analytical subcellular fractionation of human granulocytes with special reference to the localization of enzymes involved in microbicidal mechanisms. Clin Sci Mol Med. 1977 Apr;52(4):429–442. doi: 10.1042/cs0520429. [DOI] [PubMed] [Google Scholar]
  32. Tauber A. I., Goetzl E. J. Structural and catalytic properties of the solubilized superoxide-generating activity of human polymorphonuclear leukocytes. Solubilization, stabilization in solution, and partial characterization. Biochemistry. 1979 Dec 11;18(25):5576–5584. doi: 10.1021/bi00592a009. [DOI] [PubMed] [Google Scholar]
  33. Thomas P. E., Ryan D., Levin W. An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal Biochem. 1976 Sep;75(1):168–176. doi: 10.1016/0003-2697(76)90067-1. [DOI] [PubMed] [Google Scholar]
  34. Wakeyama H., Takeshige K., Takayanagi R., Minakami S. Superoxide-forming NADPH oxidase preparation of pig polymorphonuclear leucocyte. Biochem J. 1982 Sep 1;205(3):593–601. doi: 10.1042/bj2050593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. van GELDER B., SLATER E. C. The extinction coefficient of cytochrome c. Biochim Biophys Acta. 1962 Apr 23;58:593–595. doi: 10.1016/0006-3002(62)90073-2. [DOI] [PubMed] [Google Scholar]
  36. van der Hoeven T. A., Coon M. J. Preparation and properties of partially purified cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from rabbit liver microsomes. J Biol Chem. 1974 Oct 10;249(19):6302–6310. [PubMed] [Google Scholar]

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