Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Sep 1;286(Pt 2):549–554. doi: 10.1042/bj2860549

Isolation and characterization of grancalcin, a novel 28 kDa EF-hand calcium-binding protein from human neutrophils.

C G Teahan 1, N F Totty 1, A W Segal 1
PMCID: PMC1132932  PMID: 1530588

Abstract

A novel 28 kDa protein, which we have named 'grancalcin', has been identified in human neutrophils. The protein was isolated from the cytosol and found to be a homodimer, with an apparent molecular mass of 55 kDa on gel filtration. Polyclonal antibodies were raised to the native protein. N-Terminal sequence analysis and tryptic-peptide sequence analysis was performed. The protein exhibits sequence similarity to sorcin, a 24 kDa calcium-binding protein over-expressed in certain multi-drug-resistant cell lines. It appears to be a member of the EF-hand family of calcium-binding proteins. The association of a high proportion of this protein with the membranes and granules in the presence of physiological concentrations of calcium may indicate a role in granule-membrane fusion and degranulation.

Full text

PDF
549

Images in this article

550Fig. 1.
on p.550
551Fig. 2.
on p.551
551Fig. 3.
on p.551
552Fig. 6.
on p.552
552Fig. 7.
on p.552

Selected References

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

  1. Borregaard N., Heiple J. M., Simons E. R., Clark R. A. 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. doi: 10.1083/jcb.97.1.52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyhan A., Casimir C. M., French J. K., Teahan C. G., Segal A. W. Molecular cloning and characterization of grancalcin, a novel EF-hand calcium-binding protein abundant in neutrophils and monocytes. J Biol Chem. 1992 Feb 15;267(5):2928–2933. [PubMed] [Google Scholar]
  3. Burgess W. H., Jemiolo D. K., Kretsinger R. H. Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate. Biochim Biophys Acta. 1980 Jun 26;623(2):257–270. doi: 10.1016/0005-2795(80)90254-8. [DOI] [PubMed] [Google Scholar]
  4. Charuk J. H., Pirraglia C. A., Reithmeier R. A. Interaction of ruthenium red with Ca2(+)-binding proteins. Anal Biochem. 1990 Jul;188(1):123–131. doi: 10.1016/0003-2697(90)90539-l. [DOI] [PubMed] [Google Scholar]
  5. Clark R. A., Malech H. L., Gallin J. I., Nunoi H., Volpp B. D., Pearson D. W., Nauseef W. M., Curnutte J. T. Genetic variants of chronic granulomatous disease: prevalence of deficiencies of two cytosolic components of the NADPH oxidase system. N Engl J Med. 1989 Sep 7;321(10):647–652. doi: 10.1056/NEJM198909073211005. [DOI] [PubMed] [Google Scholar]
  6. Dinauer M. C., Orkin S. H., Brown R., Jesaitis A. J., Parkos C. A. The glycoprotein encoded by the X-linked chronic granulomatous disease locus is a component of the neutrophil cytochrome b complex. 1987 Jun 25-Jul 1Nature. 327(6124):717–720. doi: 10.1038/327717a0. [DOI] [PubMed] [Google Scholar]
  7. Garrigos M., Deschamps S., Viel A., Lund S., Champeil P., Møller J. V., le Maire M. Detection of Ca(2+)-binding proteins by electrophoretic migration in the presence of Ca2+ combined with 45Ca2+ overlay of protein blots. Anal Biochem. 1991 Apr;194(1):82–88. doi: 10.1016/0003-2697(91)90154-l. [DOI] [PubMed] [Google Scholar]
  8. HUMMEL J. P., DREYER W. J. Measurement of protein-binding phenomena by gel filtration. Biochim Biophys Acta. 1962 Oct 8;63:530–532. doi: 10.1016/0006-3002(62)90124-5. [DOI] [PubMed] [Google Scholar]
  9. Hamada H., Okochi E., Oh-hara T., Tsuruo T. Purification of the Mr 22,000 calcium-binding protein (sorcin) associated with multidrug resistance and its detection with monoclonal antibodies. Cancer Res. 1988 Jun 1;48(11):3173–3178. [PubMed] [Google Scholar]
  10. Heyworth P. G., Segal A. W. Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils. Biochem J. 1986 Nov 1;239(3):723–731. doi: 10.1042/bj2390723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heyworth P. G., Shrimpton C. F., Segal A. W. Localization of the 47 kDa phosphoprotein involved in the respiratory-burst NADPH oxidase of phagocytic cells. Biochem J. 1989 May 15;260(1):243–248. doi: 10.1042/bj2600243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Klee C. B., Crouch T. H., Krinks M. H. Calcineurin: a calcium- and calmodulin-binding protein of the nervous system. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6270–6273. doi: 10.1073/pnas.76.12.6270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Koch G., Smith M., Twentyman P., Wright K. Identification of a novel calcium-binding protein (CP22) in multidrug-resistant murine and hamster cells. FEBS Lett. 1986 Jan 20;195(1-2):275–279. doi: 10.1016/0014-5793(86)80176-4. [DOI] [PubMed] [Google Scholar]
  14. Lomax K. J., Leto T. L., Nunoi H., Gallin J. I., Malech H. L. Recombinant 47-kilodalton cytosol factor restores NADPH oxidase in chronic granulomatous disease. Science. 1989 Jul 28;245(4916):409–412. doi: 10.1126/science.2547247. [DOI] [PubMed] [Google Scholar]
  15. Maruyama K., Mikawa T., Ebashi S. Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J Biochem. 1984 Feb;95(2):511–519. doi: 10.1093/oxfordjournals.jbchem.a134633. [DOI] [PubMed] [Google Scholar]
  16. Maruyama K., Nonomura Y. High molecular weight calcium binding protein in the microsome of scallop striated muscle. J Biochem. 1984 Sep;96(3):859–870. doi: 10.1093/oxfordjournals.jbchem.a134904. [DOI] [PubMed] [Google Scholar]
  17. Meyers M. B., Schneider K. A., Spengler B. A., Chang T. D., Biedler J. L. Sorcin (V19), a soluble acidic calcium-binding protein overproduced in multidrug-resistant cells. Identification of the protein by anti-sorcin antibody. Biochem Pharmacol. 1987 Jul 15;36(14):2373–2380. doi: 10.1016/0006-2952(87)90606-x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Persechini A., Moncrief N. D., Kretsinger R. H. The EF-hand family of calcium-modulated proteins. Trends Neurosci. 1989 Nov;12(11):462–467. doi: 10.1016/0166-2236(89)90097-0. [DOI] [PubMed] [Google Scholar]
  21. Segal A. W., Harper A. M., Cross A. R., Jones O. T. Cytochrome b-245. Methods Enzymol. 1986;132:378–394. doi: 10.1016/s0076-6879(86)32023-8. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Segal A. W., Jones O. T. 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. doi: 10.1016/0014-5793(80)80035-4. [DOI] [PubMed] [Google Scholar]
  24. Segal A. W. The electron transport chain of the microbicidal oxidase of phagocytic cells and its involvement in the molecular pathology of chronic granulomatous disease. J Clin Invest. 1989 Jun;83(6):1785–1793. doi: 10.1172/JCI114083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Teahan C. G., Totty N., Casimir C. M., Segal A. W. Purification of the 47 kDa phosphoprotein associated with the NADPH oxidase of human neutrophils. Biochem J. 1990 Apr 15;267(2):485–489. doi: 10.1042/bj2670485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Van der Bliek A. M., Meyers M. B., Biedler J. L., Hes E., Borst P. A 22-kd protein (sorcin/V19) encoded by an amplified gene in multidrug-resistant cells, is homologous to the calcium-binding light chain of calpain. EMBO J. 1986 Dec 1;5(12):3201–3208. doi: 10.1002/j.1460-2075.1986.tb04630.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Volpp B. D., Nauseef W. M., Donelson J. E., Moser D. R., Clark R. A. Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7195–7199. doi: 10.1073/pnas.86.18.7195. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES