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J Cell Biol. 1983 Jul 1; 97(1): 52–61.
PMCID: PMC2112494

Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation


We describe a new method for subcellular fractionation of human neutrophils. Neutrophils were disrupted by nitrogen cavitation and the nuclei removed by centrifugation. The postnuclear supernatant was applied on top of a discontinuous Percoll density gradient. Centrifugation for 15 min at 48,000 g resulted in complete separation of plasma membranes, azurophil granules, and specific granules. As determined by ultrastructure and the distribution of biochemical markers of these organelles, approximately 90% of the b-cytochrome in unstimulated cells was recovered from the band containing the specific granules and was shown to be in or tightly associated with the membrane. During stimulation of intact neutrophils with phorbol myristate acetate or the ionophore A23187, we observed translocation of 40-75% of the b-cytochrome to the plasma membrane. The extent of this translocation closely paralleled release of the specific granule marker, vitamin B12-binding protein. These data indicate that the b- cytochrome is in the membrane of the specific granules of unstimulated neutrophils and that stimulus-induced fusion of these granules with the plasma membrane results in a translocation of the cytochrome. Our observations provide a basis for the assembly of the microbicidal oxidase of the human neutrophil.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • 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]
  • Root RK, Metcalf J, Oshino N, Chance B. H2O2 release from human granulocytes during phagocytosis. I. Documentation, quantitation, and some regulating factors. J Clin Invest. 1975 May;55(5):945–955. [PMC free article] [PubMed]
  • Green MR, Hill HA, Okolow-Zubkowska MJ, Segal AW. The production of hydroxyl and superoxide radicals by stimulated human neutrophils- measurements by EPR spectroscopy. FEBS Lett. 1979 Apr 1;100(1):23–26. [PubMed]
  • SBARRA AJ, KARNOVSKY ML. The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. J Biol Chem. 1959 Jun;234(6):1355–1362. [PubMed]
  • Holmes B, Page AR, Good RA. Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function. J Clin Invest. 1967 Sep;46(9):1422–1432. [PMC free article] [PubMed]
  • 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]
  • 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]
  • Mandell GL, Hook EW. Leukocyte bactericidal activity in chronic granulomatous disease: correlation of bacterial hydrogen peroxide production and susceptibility to intracellular killing. J Bacteriol. 1969 Oct;100(1):531–532. [PMC free article] [PubMed]
  • Clark RA, Klebanoff SJ. Studies on the mechanism of antibody-dependent polymorphonuclear leukocyte-mediated cytotoxicity. J Immunol. 1977 Oct;119(4):1413–1418. [PubMed]
  • Hafeman DG, Lucas ZJ. Polymorphonuclear leukocyte-mediated, antibody-dependent, cellular cytotoxicity against tumor cells: dependence on oxygen and the respiratory burst. J Immunol. 1979 Jul;123(1):55–62. [PubMed]
  • Borregaard N, Kragballe K. Role of oxygen in antibody-dependent cytotoxicity mediated by monocytes and neutrophils. J Clin Invest. 1980 Oct;66(4):676–683. [PMC free article] [PubMed]
  • Clark RA, Szot S. Chemotactic factor inactivation by stimulated human neutrophils mediated by myeloperoxidase-catalyzed methionine oxidation. J Immunol. 1982 Apr;128(4):1507–1513. [PubMed]
  • Carp H, Janoff A. Potential mediator of inflammation. Phagocyte-derived oxidants suppress the elastase-inhibitory capacity of alpha 1-proteinase inhibitor in vitro. J Clin Invest. 1980 Nov;66(5):987–995. [PMC free article] [PubMed]
  • Clark RA, Stone PJ, El Hag A, Calore JD, Franzblau C. Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils. J Biol Chem. 1981 Apr 10;256(7):3348–3353. [PubMed]
  • Rossi F, Zatti M. Biochemical aspects of phagocytosis in polymorphonuclear leucocytes. NADH and NADPH oxidation by the granules of resting and phagocytizing cells. Experientia. 1964 Jan 15;20(1):21–23. [PubMed]
  • Babior BM, Curnutte JT, Kipnes BS. Pyridine nucleotide-dependent superoxide production by a cell-free system from human granulocytes. J Clin Invest. 1975 Oct;56(4):1035–1042. [PMC free article] [PubMed]
  • Hohn DC, Lehrer RI. NADPH oxidase deficiency in X-linked chronic granulomatous disease. J Clin Invest. 1975 Apr;55(4):707–713. [PMC free article] [PubMed]
  • Babior BM. Oxygen-dependent microbial killing by phagocytes (first of two parts). N Engl J Med. 1978 Mar 23;298(12):659–668. [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]
  • Shinagawa Y, Tanaka C, Teraoka A, Shinagawa Y. A new cytochrome in neurophilic granules of rabbit leucocyte. J Biochem. 1966 Jun;59(6):622–624. [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]
  • Borregaard N, Johansen KS, Esmann V. Quantitation of superoxide production in human polymorphonuclear leukocytes from normals and 3 types of chronic granulomatous disease. Biochem Biophys Res Commun. 1979 Sep 12;90(1):214–219. [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. Absence of cytochrome b reduction in stimulated neutrophils from both female and male patients with chronic granulomatous disease. FEBS Lett. 1980 Jan 28;110(1):111–114. [PubMed]
  • Borregaard N, Simons ER, Clark RA. Involvement of cytochrome b-245 in the respiratory burst of human neutrophils. Infect Immun. 1982 Dec;38(3):1301–1303. [PMC free article] [PubMed]
  • Segal AW, Cross AR, Garcia RC, Borregaard N, Valerius NH, Soothill JF, Jones OT. 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. [PubMed]
  • Sloan EP, Crawford DR, Schneider DL. Isolation of plasma membrane from human neutrophils and determination of cytochrome b and quinone content. J Exp Med. 1981 May 1;153(5):1316–1328. [PMC free article] [PubMed]
  • Segal AW, Jones OT. Rapid incorporation of the human neutrophil plasma membrane cytochrome b into phagocytic vacuoles. Biochem Biophys Res Commun. 1980 Jan 29;92(2):710–715. [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. [PMC free article] [PubMed]
  • Millard JA, Gerard KW, Schneider DL. The isolation from rat peritoneal leukocytes of plasma membrane enriched in alkaline phosphatase and a B-type cytochrome. Biochem Biophys Res Commun. 1979 Sep 12;90(1):312–319. [PubMed]
  • Klempner MS, Mikkelsen RB, Corfman DH, André-Schwartz J. Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation. J Cell Biol. 1980 Jul;86(1):21–28. [PMC free article] [PubMed]
  • Nachman R, Hirsch JG, Baggiolini M. Studies on isolated membranes of azurophil and specific granules from rabbit polymorphonuclear leukocytes. J Cell Biol. 1972 Jul;54(1):133–140. [PMC free article] [PubMed]
  • Luna EJ, Fowler VM, Swanson J, Branton D, Taylor DL. A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity. J Cell Biol. 1981 Feb;88(2):396–409. [PMC free article] [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. [PMC free article] [PubMed]
  • Bos A, Wever R, Roos D. Characterization and quantification of the peroxidase in human monocytes. Biochim Biophys Acta. 1978 Jul 7;525(1):37–44. [PubMed]
  • DeChatelet LR, Cooper MR. A modified procedure for the determination of leukocyte alkaline phosphatase. Biochem Med. 1970 Aug;4(1):61–68. [PubMed]
  • Brittinger G, Hirschhorn R, Douglas SD, Weissmann G. Studies on lysosomes. XI. Characterization of a hydrolase-rich fraction from human lymphocytes. J Cell Biol. 1968 May;37(2):394–411. [PMC free article] [PubMed]
  • COOPERSTEIN SJ, LAZAROW A. A microspectrophotometric method for the determination of cytochrome oxidase. J Biol Chem. 1951 Apr;189(2):665–670. [PubMed]
  • Klass HJ, Hopkins J, Neale G, Peters TJ. The estimation of serum lysozyme: a comparison of four assay methods. Biochem Med. 1977 Aug;18(1):52–57. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Spitznagel JK, Dalldorf FG, Leffell MS, Folds JD, Welsh IR, Cooney MH, Martin LE. Character of azurophil and specific granules purified from human polymorphonuclear leukocytes. Lab Invest. 1974 Jun;30(6):774–785. [PubMed]
  • Bretz U, Baggiolini M. Biochemical and morphological characterization of azurophil and specific granules of human neutrophilic polymorphonuclear leukocytes. J Cell Biol. 1974 Oct;63(1):251–269. [PMC free article] [PubMed]
  • West BC, Rosenthal AS, Gelb NA, Kimball HR. Separation and characterization of human neutrophil granules. Am J Pathol. 1974 Oct;77(1):41–66. [PMC free article] [PubMed]
  • Kane SP, Peters TJ. Analytical subcellular fractionation of human granulocytes with reference to the localization of vitamin B12-binding proteins. Clin Sci Mol Med. 1975 Aug;49(2):171–182. [PubMed]
  • Dewald B, Bretz U, Baggiolini M. Release of gelatinase from a novel secretory compartment of human neutrophils. J Clin Invest. 1982 Sep;70(3):518–525. [PMC free article] [PubMed]
  • Shirley PS, Wang P, DeChatelet LR, Waite M. Absence of the membrane marker enzyme 5'-nucleotidase in human polymorphonuclear leukocytes. Biochem Med. 1976 Jun;15(3):289–295. [PubMed]
  • Strauss RG, Burrows SE. Letter: Activity of 5'nucleotidase in polymorphonuclear leukocytes. Blood. 1975 Oct;46(4):655–656. [PubMed]
  • Lichtman MA, Weed RI. The monovalent cation content and adenosine triphosphatase activity of human normal and leukemic granulocytes and lymphocytes: relationship to cell volume and morphologic age. Blood. 1969 Nov;34(5):645–660. [PubMed]
  • Taylor DL, Condeelis JS, Moore PL, Allen RD. The contractile basis of amoeboid movement. I. The chemical control of motility in isolated cytoplasm. J Cell Biol. 1973 Nov;59(2 Pt 1):378–394. [PMC free article] [PubMed]
  • Bainton DF, Ullyot JL, Farquhar MG. The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. J Exp Med. 1971 Oct 1;134(4):907–934. [PMC free article] [PubMed]
  • Estensen RD, White JG, Holmes B. Specific degranulation of human polymorphonuclear leukocytes. Nature. 1974 Mar 22;248(446):347–348. [PubMed]
  • Goldstein IM, Hoffstein ST, Weissmann G. Mechanisms of lysosomal enzyme release from human polymorphonuclear leukocytes. Effects of phorbol myristate acetate. J Cell Biol. 1975 Sep;66(3):647–652. [PMC free article] [PubMed]
  • Simon JD, Houck WS, Albala MM. Release of unsaturated vitamin B12 binding capacity from human granulocytes by the calcium ionophore A23187. Biochem Biophys Res Commun. 1976 Nov 22;73(2):444–450. [PubMed]
  • Voetman AA, Weening RS, Hamers MN, Meerhof LJ, Bot AA, Roos D. Phagocytosing human neutrophils inactivate their own granular enzymes. J Clin Invest. 1981 May;67(5):1541–1549. [PMC free article] [PubMed]
  • Olsson I. Isolation of human leukocyte granules using colloidal silica-polysaccharide density gradients. Exp Cell Res. 1969 Mar;54(3):325–330. [PubMed]
  • HIRSCH JG. Cinemicrophotographic observations on granule lysis in polymorphonuclear leucocytes during phagocytosis. J Exp Med. 1962 Dec 1;116:827–834. [PMC free article] [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, Dorling J, Coade S. Kinetics of fusion of the cytoplasmic granules with phagocytic vacuoles in human polymorphonuclear leukocytes. Biochemical and morphological studies. J Cell Biol. 1980 Apr;85(1):42–59. [PMC free article] [PubMed]
  • Crawford DR, Schneider DL. Identification of ubiquinone-50 in human neutrophils and its role in microbicidal events. J Biol Chem. 1982 Jun 25;257(12):6662–6668. [PubMed]

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