Search tips
Search criteria 


Logo of jcinvestThe Journal of Clinical InvestigationCurrent IssueArchiveSubscriptionAbout the Journal
J Clin Invest. 1987 September; 80(3): 732–742.
PMCID: PMC442297

Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000.


A new method has been developed for purification of cytochrome b from stimulated human granulocytes offering the advantage of high yields from practical quantities of whole blood. Polymorphonuclear leukocytes were treated with diisopropylfluorophosphate, degranulated and disrupted by nitrogen cavitation. Membranes enriched in cytochrome b were prepared by differential centrifugation. Complete solubilization of the cytochrome from the membranes was achieved in octylglucoside after a 1-M salt wash. Wheat germ agglutinin-conjugated Sepharose 4B specifically bound the solubilized cytochrome b and afforded a threefold purification. Eluate from the immobilized wheat germ agglutinin was further enriched by chromatography on immobilized heparin. The final 260-fold purification of the b-type cytochrome with a 20-30% yield was achieved by velocity sedimentation in sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified preparation revealed two polypeptides of Mr 91,000 and Mr 22,000. Treatment of the 125I-labeled, purified preparation with peptide:N-glycosidase F, which removes N-linked sugars, decreased relative molecular weight of the larger species to approximately 50,000, whereas beta-elimination, which removes O-linked sugars, had little or no effect on the mobility of the Mr-91,000 polypeptide. Neither of the deglycosylation conditions had any effect on electrophoretic mobility of the Mr-22,000 polypeptide. Disuccinimidyl suberate cross-linked the two polypeptides to a new Mr of 120,000-135,000 by SDS-PAGE. Antibody raised to the purified preparation immunoprecipitated spectral activity and, on Western blots, bound to the Mr-22,000 polypeptide but not the Mr-91,000 polypeptide. Western blot analysis of granulocytes from patients with X-linked chronic granulomatous disease revealed a complete absence of the Mr-22,000 polypeptide. These results (a) suggest that the two polypeptides are in close association and are part of the cytochrome b, (b) provide explanation for the molecular weight discrepancies previously reported for the protein, and (c) further support the involvement of the cytochrome in superoxide production in human neutrophils.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.9M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • McRipley RJ, Sbarra AJ. Role of the phagocyte in host-parasite interactions. XI. Relationship between stimulated oxidative metabolism and hydrogen peroxide formation, and intracellular killing. J Bacteriol. 1967 Nov;94(5):1417–1424. [PMC free article] [PubMed]
  • Mandell GL. Bactericidal activity of aerobic and anaerobic polymorphonuclear neutrophils. Infect Immun. 1974 Feb;9(2):337–341. [PMC free article] [PubMed]
  • Lehrer RI, Cline MJ. Interaction of Candida albicans with human leukocytes and serum. J Bacteriol. 1969 Jun;98(3):996–1004. [PMC free article] [PubMed]
  • Babior BM, Kipnes RS, Curnutte JT. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest. 1973 Mar;52(3):741–744. [PMC free article] [PubMed]
  • Briggs RT, Drath DB, Karnovsky ML, Karnovsky MJ. Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method. J Cell Biol. 1975 Dec;67(3):566–586. [PMC free article] [PubMed]
  • Badwey JA, Karnovsky ML. Active oxygen species and the functions of phagocytic leukocytes. Annu Rev Biochem. 1980;49:695–726. [PubMed]
  • Karnovsky ML, Badwey JA. Determinants of the production of active oxygen species by granulocytes and macrophages. J Clin Chem Clin Biochem. 1983 Sep;21(9):545–553. [PubMed]
  • Segal AW, Jones OT. Novel cytochrome b system in phagocytic vacuoles of human granulocytes. Nature. 1978 Nov 30;276(5687):515–517. [PubMed]
  • Segal AW, Jones OT. Reduction and subsequent oxidation of a cytochrome b of human neutrophils after stimulation with phorbol myristate acetate. Biochem Biophys Res Commun. 1979 May 14;88(1):130–134. [PubMed]
  • Segal AW, Jones OT, Webster D, Allison AC. Absence of a newly described cytochrome b from neutrophils of patients with chronic granulomatous disease. Lancet. 1978 Aug 26;2(8087):446–449. [PubMed]
  • Hamers MN, de Boer M, Meerhof LJ, Weening RS, Roos D. Complementation in monocyte hybrids revealing genetic heterogeneity in chronic granulomatous disease. Nature. 1984 Feb 9;307(5951):553–555. [PubMed]
  • Gabig TG, Schervish EW, Santinga JT. 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]
  • Bellavite P, Cross AR, Serra MC, Davoli A, Jones OT, 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. [PubMed]
  • Cross AR, Higson FK, Jones OT, Harper AM, Segal AW. The enzymic reduction and kinetics of oxidation of cytochrome b-245 of neutrophils. Biochem J. 1982 May 15;204(2):479–485. [PubMed]
  • Cross AR, Jones OT, Harper AM, Segal AW. 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. [PubMed]
  • Pember SO, Heyl BL, Kinkade JM, Jr, Lambeth JD. Cytochrome b558 from (bovine) granulocytes. Partial purification from Triton X-114 extracts and properties of the isolated cytochrome. J Biol Chem. 1984 Aug 25;259(16):10590–10595. [PubMed]
  • Bellavite P, Papini E, Zeni L, Della Bianca V, Rossi F. Studies on the nature and activation of O2(-)-forming NADPH oxidase of leukocytes. Identification of a phosphorylated component of the active enzyme. Free Radic Res Commun. 1985;1(1):11–29. [PubMed]
  • Harper AM, Dunne MJ, Segal AW. Purification of cytochrome b-245 from human neutrophils. Biochem J. 1984 Apr 15;219(2):519–527. [PubMed]
  • Lutter R, van Schaik ML, van Zwieten R, Wever R, Roos D, Hamers MN. Purification and partial characterization of the b-type cytochrome from human polymorphonuclear leukocytes. J Biol Chem. 1985 Feb 25;260(4):2237–2244. [PubMed]
  • Henson PM, Oades ZG. Stimulation of human neutrophils by soluble and insoluble immunoglobulin aggregates. Secretion of granule constituents and increased oxidation of glucose. J Clin Invest. 1975 Oct;56(4):1053–1061. [PMC free article] [PubMed]
  • Harper AM, Chaplin MF, Segal AW. Cytochrome b-245 from human neutrophils is a glycoprotein. Biochem J. 1985 May 1;227(3):783–788. [PubMed]
  • Amrein PC, Stossel TP. Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate. Blood. 1980 Sep;56(3):442–447. [PubMed]
  • Borregaard N, Tauber AI. Subcellular localization of the human neutrophil NADPH oxidase. b-Cytochrome and associated flavoprotein. J Biol Chem. 1984 Jan 10;259(1):47–52. [PubMed]
  • Clarke S. The size and detergent binding of membrane proteins. J Biol Chem. 1975 Jul 25;250(14):5459–5469. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Wray W, Boulikas T, Wray VP, Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. [PubMed]
  • Merril CR, Goldman D, Sedman SA, Ebert MH. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. [PubMed]
  • Edge AS, Faltynek CR, Hof L, Reichert LE, Jr, Weber P. Deglycosylation of glycoproteins by trifluoromethanesulfonic acid. Anal Biochem. 1981 Nov 15;118(1):131–137. [PubMed]
  • Spiro RG, Bhoyroo VD. Structure of the O-glycosidically linked carbohydrate units of fetuin. J Biol Chem. 1974 Sep 25;249(18):5704–5717. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Udenfriend S, Stein S, Böhlen P, Dairman W, Leimgruber W, Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972 Nov 24;178(4063):871–872. [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PubMed]
  • Segal AW, Jones OT. 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. [PubMed]
  • Borregaard N, Heiple JM, Simons ER, Clark RA. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J Cell Biol. 1983 Jul;97(1):52–61. [PMC free article] [PubMed]
  • Parkos CA, Cochrane CG, Schmitt M, Jesaitis AJ. Regulation of the oxidative response of human granulocytes to chemoattractants. No evidence for stimulated traffic of redox enzymes between endo and plasma membranes. J Biol Chem. 1985 Jun 10;260(11):6541–6547. [PubMed]
  • Frank RN, 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. [PubMed]
  • Gabig TG, Lefker BA. Deficient flavoprotein component of the NADPH-dependent O2-.-generating oxidase in the neutrophils from three male patients with chronic granulomatous disease. J Clin Invest. 1984 Mar;73(3):701–705. [PMC free article] [PubMed]
  • Sheterline P, Hopkins CR. Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes. J Cell Biol. 1981 Sep;90(3):743–754. [PMC free article] [PubMed]
  • Apps DK, Pryde JG, Phillips JH. Cytochrome b561 is identical with chromomembrin B, a major polypeptide of chromaffin granule membranes. Neuroscience. 1980;5(12):2279–2287. [PubMed]
  • Duong LT, Fleming PJ. Isolation and properties of cytochrome b561 from bovine adrenal chromaffin granules. J Biol Chem. 1982 Aug 10;257(15):8561–8564. [PubMed]
  • Spatz L, Strittmatter P. A form of cytochrome b5 that contains an additional hydrophobic sequence of 40 amino acid residues. Proc Natl Acad Sci U S A. 1971 May;68(5):1042–1046. [PubMed]
  • Royer-Pokora B, Kunkel LM, Monaco AP, Goff SC, Newburger PE, Baehner RL, Cole FS, Curnutte JT, Orkin SH. Cloning the gene for an inherited human disorder--chronic granulomatous disease--on the basis of its chromosomal location. Nature. 1986 Jul 3;322(6074):32–38. [PubMed]
  • Dinauer MC, Orkin SH, Brown R, Jesaitis AJ, Parkos CA. The glycoprotein encoded by the X-linked chronic granulomatous disease locus is a component of the neutrophil cytochrome b complex. Nature. 327(6124):717–720. [PubMed]
  • Segal AW. Absence of both cytochrome b-245 subunits from neutrophils in X-linked chronic granulomatous disease. Nature. 1987 Mar 5;326(6108):88–91. [PubMed]
  • Teahan C, Rowe P, Parker P, Totty N, Segal AW. The X-linked chronic granulomatous disease gene codes for the beta-chain of cytochrome b-245. Nature. 327(6124):720–721. [PubMed]

Articles from The Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation