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. 1987 Feb;79(2):643–648. doi: 10.1172/JCI112861

Altered binding of 125I-labeled calmodulin to a 46.5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis.

E A Tallant, R W Wallace
PMCID: PMC424155  PMID: 3805287

Abstract

The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca2+/calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblasts or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of 125I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis.

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Selected References

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  1. Bolton J. E., Field M. Ca ionophore-stimulated ion secretion in rabbit ileal mucosa: relation to actions of cyclic 3',5'-AMP and carbamylcholine. J Membr Biol. 1977 Jun 30;35(2):159–173. doi: 10.1007/BF01869947. [DOI] [PubMed] [Google Scholar]
  2. Brady R. C., Karnaky K. J., Jr, Dedman J. R. Colonic glycoprotein secretion and calmodulin-acceptor proteins in the reserpine-treated rat. Am J Physiol. 1985 Jan;248(1 Pt 1):G54–G60. doi: 10.1152/ajpgi.1985.248.1.G54. [DOI] [PubMed] [Google Scholar]
  3. Cheung W. Y. Calmodulin plays a pivotal role in cellular regulation. Science. 1980 Jan 4;207(4426):19–27. doi: 10.1126/science.6243188. [DOI] [PubMed] [Google Scholar]
  4. Conti M. A., Adelstein R. S. The relationship between calmodulin binding and phosphorylation of smooth muscle myosin kinase by the catalytic subunit of 3':5' cAMP-dependent protein kinase. J Biol Chem. 1981 Apr 10;256(7):3178–3181. [PubMed] [Google Scholar]
  5. Donowitz M., Asarkof N. Calcium dependence of basal electrolyte transport in rabbit ileum. Am J Physiol. 1982 Jul;243(1):G28–G35. doi: 10.1152/ajpgi.1982.243.1.G28. [DOI] [PubMed] [Google Scholar]
  6. Frizzell R. A. Active chloride secretion by rabbit colon: calcium-dependent stimulation by ionophore A23187. J Membr Biol. 1977 Jun 30;35(2):175–187. doi: 10.1007/BF01869948. [DOI] [PubMed] [Google Scholar]
  7. Frizzell R. A., Koch M. J., Schultz S. G. Ion transport by rabbit colon. I. Active and passive components. J Membr Biol. 1976;27(3):297–316. doi: 10.1007/BF01869142. [DOI] [PubMed] [Google Scholar]
  8. Frizzell R. A., Rechkemmer G., Shoemaker R. L. Altered regulation of airway epithelial cell chloride channels in cystic fibrosis. Science. 1986 Aug 1;233(4763):558–560. doi: 10.1126/science.2425436. [DOI] [PubMed] [Google Scholar]
  9. Gnegy M. E., Erickson R. P., Markovac J. Increased calmodulin in cultured skin fibroblasts from patients with cystic fibrosis. Biochem Med. 1981 Dec;26(3):294–298. doi: 10.1016/0006-2944(81)90004-1. [DOI] [PubMed] [Google Scholar]
  10. Gopalakrishna R., Anderson W. B. Ca2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-Sepharose affinity chromatography. Biochem Biophys Res Commun. 1982 Jan 29;104(2):830–836. doi: 10.1016/0006-291x(82)90712-4. [DOI] [PubMed] [Google Scholar]
  11. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  12. Ilundain A., Naftalin R. J. Role of Ca(2+)-dependent regulator protein in intestinal secretion. Nature. 1979 May 31;279(5712):446–448. doi: 10.1038/279446a0. [DOI] [PubMed] [Google Scholar]
  13. Knowles M. R., Stutts M. J., Spock A., Fischer N., Gatzy J. T., Boucher R. C. Abnormal ion permeation through cystic fibrosis respiratory epithelium. Science. 1983 Sep 9;221(4615):1067–1070. doi: 10.1126/science.6308769. [DOI] [PubMed] [Google Scholar]
  14. Knowles M., Gatzy J., Boucher R. Relative ion permeability of normal and cystic fibrosis nasal epithelium. J Clin Invest. 1983 May;71(5):1410–1417. doi: 10.1172/JCI110894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Knowlton R. G., Cohen-Haguenauer O., Van Cong N., Frézal J., Brown V. A., Barker D., Braman J. C., Schumm J. W., Tsui L. C., Buchwald M. A polymorphic DNA marker linked to cystic fibrosis is located on chromosome 7. 1985 Nov 28-Dec 4Nature. 318(6044):380–382. doi: 10.1038/318380a0. [DOI] [PubMed] [Google Scholar]
  16. Knowlton R. G., Cohen-Haguenauer O., Van Cong N., Frézal J., Brown V. A., Barker D., Braman J. C., Schumm J. W., Tsui L. C., Buchwald M. A polymorphic DNA marker linked to cystic fibrosis is located on chromosome 7. 1985 Nov 28-Dec 4Nature. 318(6044):380–382. doi: 10.1038/318380a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. LaPorte D. C., Storm D. R. Detection of calcium-dependent regulatory protein binding components using 125I-labeled calcium-dependent regulatory protein. J Biol Chem. 1978 May 25;253(10):3374–3377. [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Mandel K. G., Dharmsathaphorn K., McRoberts J. A. Characterization of a cyclic AMP-activated Cl-transport pathway in the apical membrane of a human colonic epithelial cell line. J Biol Chem. 1986 Jan 15;261(2):704–712. [PubMed] [Google Scholar]
  21. Pallen C. J., Wang J. H. A multifunctional calmodulin-stimulated phosphatase. Arch Biochem Biophys. 1985 Mar;237(2):281–291. doi: 10.1016/0003-9861(85)90279-6. [DOI] [PubMed] [Google Scholar]
  22. Quinton P. M., Bijman J. Higher bioelectric potentials due to decreased chloride absorption in the sweat glands of patients with cystic fibrosis. N Engl J Med. 1983 May 19;308(20):1185–1189. doi: 10.1056/NEJM198305193082002. [DOI] [PubMed] [Google Scholar]
  23. Quinton P. M. Chloride impermeability in cystic fibrosis. Nature. 1983 Feb 3;301(5899):421–422. doi: 10.1038/301421a0. [DOI] [PubMed] [Google Scholar]
  24. Rugolo M., Romeo G., Lenaz G. Kinetic analysis of chloride efflux from normal and cystic fibrosis fibroblasts. Biochem Biophys Res Commun. 1986 Jan 14;134(1):233–239. doi: 10.1016/0006-291x(86)90552-8. [DOI] [PubMed] [Google Scholar]
  25. Sato K., Sato F. Defective beta adrenergic response of cystic fibrosis sweat glands in vivo and in vitro. J Clin Invest. 1984 Jun;73(6):1763–1771. doi: 10.1172/JCI111385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sharma R. K., Wang J. H. Differential regulation of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isoenzymes by cyclic AMP-dependent protein kinase and calmodulin-dependent phosphatase. Proc Natl Acad Sci U S A. 1985 May;82(9):2603–2607. doi: 10.1073/pnas.82.9.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Smith P. L., Field M. In vitro antisecretory effects of trifluoperazine and other neuroleptics in rabbit and human small intestine. Gastroenterology. 1980 Jun;78(6):1545–1553. [PubMed] [Google Scholar]
  28. Smith P. L., Welsh M. J., Stoff J. S., Frizzell R. A. Chloride secretion by canine tracheal epithelium: I. Role of intracellular c AMP levels. J Membr Biol. 1982;70(3):217–226. doi: 10.1007/BF01870564. [DOI] [PubMed] [Google Scholar]
  29. Tallant E. A., Wallace R. W. Characterization of a calmodulin-dependent protein phosphatase from human platelets. J Biol Chem. 1985 Jun 25;260(12):7744–7751. [PubMed] [Google Scholar]
  30. Tallant E. A., Wallace R. W., Cheung W. Y. Purification and radioimmunoassay of calmodulin-dependent protein phosphatase from bovine brain. Methods Enzymol. 1983;102:244–256. doi: 10.1016/s0076-6879(83)02025-x. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Wainwright B. J., Scambler P. J., Schmidtke J., Watson E. A., Law H. Y., Farrall M., Cooke H. J., Eiberg H., Williamson R. Localization of cystic fibrosis locus to human chromosome 7cen-q22. 1985 Nov 28-Dec 4Nature. 318(6044):384–385. doi: 10.1038/318384a0. [DOI] [PubMed] [Google Scholar]
  33. Wallace R. W., Cheung W. Y. Calmodulin. Production of an antibody in rabbit and development of a radioimmunoassay. J Biol Chem. 1979 Jul 25;254(14):6564–6571. [PubMed] [Google Scholar]
  34. Wallace R. W., Lynch T. J., Tallant E. A., Cheung W. Y. Purification and characterization of an inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. J Biol Chem. 1979 Jan 25;254(2):377–382. [PubMed] [Google Scholar]
  35. Wallace R. W., Tallant E. A., Cheung W. Y. Assay of calmodulin by Ca2+-dependent phosphodiesterase. Methods Enzymol. 1983;102:39–47. doi: 10.1016/s0076-6879(83)02006-6. [DOI] [PubMed] [Google Scholar]
  36. White R., Woodward S., Leppert M., O'Connell P., Hoff M., Herbst J., Lalouel J. M., Dean M., Vande Woude G. A closely linked genetic marker for cystic fibrosis. 1985 Nov 28-Dec 4Nature. 318(6044):382–384. doi: 10.1038/318382a0. [DOI] [PubMed] [Google Scholar]
  37. Widdicombe J. H., Welsh M. J., Finkbeiner W. E. Cystic fibrosis decreases the apical membrane chloride permeability of monolayers cultured from cells of tracheal epithelium. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6167–6171. doi: 10.1073/pnas.82.18.6167. [DOI] [PMC free article] [PubMed] [Google Scholar]

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