Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1991 Apr;87(4):1345–1351. doi: 10.1172/JCI115138

Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils.

R C Woodman 1, J M Ruedi 1, A J Jesaitis 1, N Okamura 1, M T Quinn 1, R M Smith 1, J T Curnutte 1, B M Babior 1
PMCID: PMC295171  PMID: 1849148

Abstract

Resting and phorbol-activated human neutrophils were separated by treatment with Triton X-100 into detergent-extractable and cytoskeleton fractions. Respiratory burst oxidase activity was restricted entirely to the cytoskeleton. The cytoskeleton also contained approximately 15% of the neutrophil cytochrome b558, an oxidase-associated heme protein, as well as most of the oxidase-related cytosolic polypeptide p67phox. In contrast, the components of the oxidase-associated phosphoprotein family p47phox were found almost exclusively in the detergent extract, suggesting that p47phox is needed for oxidase activation but not for O2- production by the activated oxidase. Activation of the oxidase had no apparent effect on the distribution of any of these species between the cytoskeleton and the detergent extract. Our results support earlier studies implying that the cytoskeleton participates in an important way in regulating the activity of the O2(-)-forming respiratory burst oxidase of neutrophils.

Full text

PDF
1345

Images in this article

1347Image
on p.1347
1348Image
on p.1348
1348Image
on p.1348
1349Image
on p.1349
1349Image
on p.1349
1349Image
on p.1349
1349Image
on p.1349
1350Image
on p.1350
1350Image
on p.1350
1350Image
on p.1350

Selected References

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

  1. Al-Mohanna F. A., Ohishi I., Hallett M. B. Botulinum C2 toxin potentiates activation of the neutrophil oxidase. Further evidence of a role for actin polymerization. FEBS Lett. 1987 Jul 13;219(1):40–44. doi: 10.1016/0014-5793(87)81187-0. [DOI] [PubMed] [Google Scholar]
  2. Babior B. M., Woodman R. C. Chronic granulomatous disease. Semin Hematol. 1990 Jul;27(3):247–259. [PubMed] [Google Scholar]
  3. Bradford M. M. 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. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Curnutte J. T. Classification of chronic granulomatous disease. Hematol Oncol Clin North Am. 1988 Jun;2(2):241–252. [PubMed] [Google Scholar]
  6. Curnutte J. T., Kuver R., Scott P. J. Activation of neutrophil NADPH oxidase in a cell-free system. Partial purification of components and characterization of the activation process. J Biol Chem. 1987 Apr 25;262(12):5563–5569. [PubMed] [Google Scholar]
  7. Fox J. E. Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes. J Biol Chem. 1985 Oct 5;260(22):11970–11977. [PubMed] [Google Scholar]
  8. Glass G. A., DeLisle D. M., DeTogni P., Gabig T. G., Magee B. H., Markert M., Babior B. M. The respiratory burst oxidase of human neutrophils. Further studies of the purified enzyme. J Biol Chem. 1986 Oct 5;261(28):13247–13251. [PubMed] [Google Scholar]
  9. Hayakawa T., Suzuki K., Suzuki S., Andrews P. C., Babior B. M. A possible role for protein phosphorylation in the activation of the respiratory burst in human neutrophils. Evidence from studies with cells from patients with chronic granulomatous disease. J Biol Chem. 1986 Jul 15;261(20):9109–9115. [PubMed] [Google Scholar]
  10. Hornbeck P., Paul W. E. Anti-immunoglobulin and phorbol ester induce phosphorylation of proteins associated with the plasma membrane and cytoskeleton in murine B lymphocytes. J Biol Chem. 1986 Nov 5;261(31):14817–14824. [PubMed] [Google Scholar]
  11. Jesaitis A. J., Tolley J. O., Allen R. A. Receptor-cytoskeleton interactions and membrane traffic may regulate chemoattractant-induced superoxide production in human granulocytes. J Biol Chem. 1986 Oct 15;261(29):13662–13669. [PubMed] [Google Scholar]
  12. Kramer I. M., Verhoeven A. J., van der Bend R. L., Weening R. S., Roos D. Purified protein kinase C phosphorylates a 47-kDa protein in control neutrophil cytoplasts but not in neutrophil cytoplasts from patients with the autosomal form of chronic granulomatous disease. J Biol Chem. 1988 Feb 15;263(5):2352–2357. [PubMed] [Google Scholar]
  13. Leto T. L., Lomax K. J., Volpp B. D., Nunoi H., Sechler J. M., Nauseef W. M., Clark R. A., Gallin J. I., Malech H. L. Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src. Science. 1990 May 11;248(4956):727–730. doi: 10.1126/science.1692159. [DOI] [PubMed] [Google Scholar]
  14. Maly F. E., Nakamura M., Gauchat J. F., Urwyler A., Walker C., Dahinden C. A., Cross A. R., Jones O. T., de Weck A. L. Superoxide-dependent nitroblue tetrazolium reduction and expression of cytochrome b-245 components by human tonsillar B lymphocytes and B cell lines. J Immunol. 1989 Feb 15;142(4):1260–1267. [PubMed] [Google Scholar]
  15. Markert M., Andrews P. C., Babior B. M. Measurement of O2- production by human neutrophils. The preparation and assay of NADPH oxidase-containing particles from human neutrophils. Methods Enzymol. 1984;105:358–365. doi: 10.1016/s0076-6879(84)05048-5. [DOI] [PubMed] [Google Scholar]
  16. Nathan C. F. Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J Clin Invest. 1987 Dec;80(6):1550–1560. doi: 10.1172/JCI113241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nunoi H., Rotrosen D., Gallin J. I., Malech H. L. Two forms of autosomal chronic granulomatous disease lack distinct neutrophil cytosol factors. Science. 1988 Dec 2;242(4883):1298–1301. doi: 10.1126/science.2848319. [DOI] [PubMed] [Google Scholar]
  18. Ohno Y., Hirai K., Kanoh T., Uchino H., Ogawa K. Subcellular localization of H2O2 production in human neutrophils stimulated with particles and an effect of cytochalasin-B on the cells. Blood. 1982 Jul;60(1):253–260. [PubMed] [Google Scholar]
  19. Okamura N., Curnutte J. T., Roberts R. L., Babior B. M. Relationship of protein phosphorylation to the activation of the respiratory burst in human neutrophils. Defects in the phosphorylation of a group of closely related 48-kDa proteins in two forms of chronic granulomatous disease. J Biol Chem. 1988 May 15;263(14):6777–6782. [PubMed] [Google Scholar]
  20. Okamura N., Malawista S. E., Roberts R. L., Rosen H., Ochs H. D., Babior B. M., Curnutte J. T. Phosphorylation of the oxidase-related 48K phosphoprotein family in the unusual autosomal cytochrome-negative and X-linked cytochrome-positive types of chronic granulomatous disease. Blood. 1988 Aug;72(2):811–816. [PubMed] [Google Scholar]
  21. Osborn M., Weber K. The detertent-resistant cytoskeleton of tissue culture cells includes the nucleus and the microfilament bundles. Exp Cell Res. 1977 May;106(2):339–349. doi: 10.1016/0014-4827(77)90179-3. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Pontremoli S., Melloni E., Michetti M., Sparatore B., Salamino F., Sacco O., Horecker B. L. Phosphorylation and proteolytic modification of specific cytoskeletal proteins in human neutrophils stimulated by phorbol 12-myristate 13-acetate. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3604–3608. doi: 10.1073/pnas.84.11.3604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pontremoli S., Melloni E., Michetti M., Sparatore B., Salamino F., Sacco O., Horecker B. L. Phosphorylation by protein kinase C of a 20-kDa cytoskeletal polypeptide enhances its susceptibility to digestion by calpain. Proc Natl Acad Sci U S A. 1987 Jan;84(2):398–401. doi: 10.1073/pnas.84.2.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Quinn M. T., Parkos C. A., Jesaitis A. J. The lateral organization of components of the membrane skeleton and superoxide generation in the plasma membrane of stimulated human neutrophils. Biochim Biophys Acta. 1989 Dec 11;987(1):83–94. doi: 10.1016/0005-2736(89)90458-6. [DOI] [PubMed] [Google Scholar]
  26. Schwarz H. P., Heeb M. J., Wencel-Drake J. D., Griffin J. H. Identification and quantitation of protein S in human platelets. Blood. 1985 Dec;66(6):1452–1455. [PubMed] [Google Scholar]
  27. Segal A. W., Heyworth P. G., Cockcroft S., Barrowman M. M. Stimulated neutrophils from patients with autosomal recessive chronic granulomatous disease fail to phosphorylate a Mr-44,000 protein. Nature. 1985 Aug 8;316(6028):547–549. doi: 10.1038/316547a0. [DOI] [PubMed] [Google Scholar]
  28. 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. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Volpp B. D., Nauseef W. M., Clark R. A. Two cytosolic neutrophil oxidase components absent in autosomal chronic granulomatous disease. Science. 1988 Dec 2;242(4883):1295–1297. doi: 10.1126/science.2848318. [DOI] [PubMed] [Google Scholar]
  30. Wessel D., Flügge U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. doi: 10.1016/0003-2697(84)90782-6. [DOI] [PubMed] [Google Scholar]
  31. Yassin R., Shefcyk J., White J. R., Tao W., Volpi M., Molski T. F., Naccache P. H., Feinstein M. B., Sha'afi R. I. Effects of chemotactic factors and other agents on the amounts of actin and a 65,000-mol-wt protein associated with the cytoskeleton of rabbit and human neutrophils. J Cell Biol. 1985 Jul;101(1):182–188. doi: 10.1083/jcb.101.1.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yea C. M., Cross A. R., Jones O. T. Purification and some properties of the 45 kDa diphenylene iodonium-binding flavoprotein of neutrophil NADPH oxidase. Biochem J. 1990 Jan 1;265(1):95–100. doi: 10.1042/bj2650095. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES