Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1981 Mar 1;193(3):875–885. doi: 10.1042/bj1930875

Isolation and characterization of calmodulin from an insulin-secreting tumour.

J C Hutton, E J Penn, P Jackson, C N Hales
PMCID: PMC1162680  PMID: 6272721

Abstract

A major protein constituent of a rat islet cell tumour that exhibited Ca2+-dependent changes in electrophoretic mobility has been purified to homogeneity and compared in its physicochemical and biological properties with bovine brain and rat brain calmodulin (synonymous with phosphodiesterase activator protein, calcium-dependent regulator, troponin C-like protein and modulator protein). The protein, like these calmodulins, contained trimethyl-lysine, exhibited a blocked N-terminus and had an identical amino-acid composition and molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Peptide "maps' prepared after digestion of the three proteins with trypsin, papain or Staphylococcus V-8 proteinase were virtually superimposable. Ca2+ altered the electrophoretic mobilities the enhanced the native protein fluorescence in an equivalent manner with all three proteins. Equilibrium dialysis experiments demonstrated in each case the binding of 4g-atoms of calcium/mol of protein; the binding sites were equivalent and showed Kd 0.8 microM. Tumour and brain proteins were equipotent as Ca2+-dependent activators of partially purified rat brain cyclic nucleotide phosphodiesterase, and in this action were inhibited in an identical manner by trifluoperazine. The proteins also exhibited the common property of Ca2+-dependent binding to troponin I, histone H2B and myelin basic protein. The estimated tumour content of calmodulin was 450 mg/kg fresh wt., a value similar to that reported in islets of Langerhans. These results further document the validity of the islet cell tumour as an experimental model of Ca2+-mediated molecular events associated with insulin secretion. They also suggest that brain calmodulin may be substituted for endogenous calmodulin in experimental investigations into the mechanism of insulin secretion.

Full text

PDF
875

Images in this article

879Fig. 1.
on p.879

Selected References

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

  1. Brostrom C. O., Huang Y. C., Breckenridge B. M., Wolff D. J. Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Proc Natl Acad Sci U S A. 1975 Jan;72(1):64–68. doi: 10.1073/pnas.72.1.64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Charles M. A., Fanska R., Schmid F. G., Forsham P. H., Grodsky G. M. Adenosine 3',5'-monophosphate in pancreatic islets: glucose-induced insulin release. Science. 1973 Feb 9;179(4073):569–571. doi: 10.1126/science.179.4073.569. [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. Chick W. L., Warren S., Chute R. N., Like A. A., Lauris V., Kitchen K. C. A transplantable insulinoma in the rat. Proc Natl Acad Sci U S A. 1977 Feb;74(2):628–632. doi: 10.1073/pnas.74.2.628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Cohen P., Burchell A., Foulkes J. G., Cohen P. T., Vanaman T. C., Nairn C. Identification of the Ca2+-dependent modulator protein as the fourth subunit of rabbit skeletal muscle phosphorylase kinase. FEBS Lett. 1978 Aug 15;92(2):287–293. doi: 10.1016/0014-5793(78)80772-8. [DOI] [PubMed] [Google Scholar]
  7. Collins J. H., Potter J. D., Horn M. J., Wilshire G., Jackman N. The amino acid sequence of rabbit skeletal muscle troponin C: gene replication and homology with calcium-binding proteins from carp and hake muscle. FEBS Lett. 1973 Nov 1;36(3):268–272. doi: 10.1016/0014-5793(73)80388-6. [DOI] [PubMed] [Google Scholar]
  8. Dabrowska R., Sherry J. M., Aromatorio D. K., Hartshorne D. J. Modulator protein as a component of the myosin light chain kinase from chicken gizzard. Biochemistry. 1978 Jan 24;17(2):253–258. doi: 10.1021/bi00595a010. [DOI] [PubMed] [Google Scholar]
  9. Dedman J. R., Jackson R. L., Schreiber W. E., Means A. R. Sequence homology of the Ca2+-dependent regulator of cyclic nucleotide phosphodiesterase from rat testis with other Ca2+-binding proteins. J Biol Chem. 1978 Jan 25;253(2):343–346. [PubMed] [Google Scholar]
  10. Dedman J. R., Potter J. D., Jackson R. L., Johnson J. D., Means A. R. Physicochemical properties of rat testis Ca2+-dependent regulator protein of cyclic nucleotide phosphodiesterase. Relationship of Ca2+-binding, conformational changes, and phosphodiesterase activity. J Biol Chem. 1977 Dec 10;252(23):8415–8422. [PubMed] [Google Scholar]
  11. Deibler G. E., Martenson R. E., Kies M. W. Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Prep Biochem. 1972;2(2):139–165. doi: 10.1080/00327487208061467. [DOI] [PubMed] [Google Scholar]
  12. Grand R. J., Perry S. V. The binding of calmodulin to myelin basic protein and histone H2B. Biochem J. 1980 Aug 1;189(2):227–240. doi: 10.1042/bj1890227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grand R. J., Perry S. V., Weeks R. A. Troponin C-like proteins (calmodulins) from mammalian smooth muscle and other tissues. Biochem J. 1979 Feb 1;177(2):521–529. doi: 10.1042/bj1770521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grodsky G. M., Bennett L. L. Cation requirements for insulin secretion in the isolated perfused pancreas. Diabetes. 1966 Dec;15(12):910–913. doi: 10.2337/diab.15.12.910. [DOI] [PubMed] [Google Scholar]
  15. HOFSTEE B. H. Non-inverted versus inverted plots in enzyme kinetics. Nature. 1959 Oct 24;184:1296–1298. doi: 10.1038/1841296b0. [DOI] [PubMed] [Google Scholar]
  16. Hales C. N., Milner R. D. Cations and the secretion of insulin from rabbit pancreas in vitro. J Physiol. 1968 Nov;199(1):177–187. doi: 10.1113/jphysiol.1968.sp008647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hartley B. S. Strategy and tactics in protein chemistry. Biochem J. 1970 Oct;119(5):805–822. doi: 10.1042/bj1190805f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hedeskov C. J. Mechanism of glucose-induced insulin secretion. Physiol Rev. 1980 Apr;60(2):442–509. doi: 10.1152/physrev.1980.60.2.442. [DOI] [PubMed] [Google Scholar]
  19. Jackson P., Amphlett G. W., Perry S. V. The primary structure of troponin T and the interaction with tropomyosin. Biochem J. 1975 Oct;151(1):85–97. doi: 10.1042/bj1510085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jarrett H. W., Penniston J. T. Purification of the Ca2+-stimulated ATPase activator from human erythrocytes. Its membership in the class of Ca2+-binding modulator proteins. J Biol Chem. 1978 Jul 10;253(13):4676–4682. [PubMed] [Google Scholar]
  21. Kakiuchi S., Yamazaki R. Calcium dependent phosphodiesterase activity and its activating factor (PAF) from brain studies on cyclic 3',5'-nucleotide phosphodiesterase (3). Biochem Biophys Res Commun. 1970 Dec 9;41(5):1104–1110. doi: 10.1016/0006-291x(70)90199-3. [DOI] [PubMed] [Google Scholar]
  22. Klee C. B. Conformational transition accompanying the binding of Ca2+ to the protein activator of 3',5'-cyclic adenosine monophosphate phosphodiesterase. Biochemistry. 1977 Mar 8;16(5):1017–1024. doi: 10.1021/bi00624a033. [DOI] [PubMed] [Google Scholar]
  23. Klee C. B., Crouch T. H., Richman P. G. Calmodulin. Annu Rev Biochem. 1980;49:489–515. doi: 10.1146/annurev.bi.49.070180.002421. [DOI] [PubMed] [Google Scholar]
  24. Klee C. B., Krinks M. H. Purification of cyclic 3',5'-nucleotide phosphodiesterase inhibitory protein by affinity chromatography on activator protein coupled to Sepharose. Biochemistry. 1978 Jan 10;17(1):120–126. doi: 10.1021/bi00594a017. [DOI] [PubMed] [Google Scholar]
  25. Kohnert K. D., Hahn H. J., Gylfe E., Borg H., Hellman B. Calcium and pancreatic beta-cell function. 6. Glucose and intracellular 45Ca distribution. Mol Cell Endocrinol. 1979 Dec;16(3):205–220. doi: 10.1016/0303-7207(79)90027-3. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Lacy P. E., Howell S. L., Young D. A., Fink C. J. New hypothesis of insulin secretion. Nature. 1968 Sep 14;219(5159):1177–1179. doi: 10.1038/2191177a0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Malaisse-Lagae F., Malaisse W. J. The stimulus-secretion coupling of glucose-induced insulin release. 3. Uptake of 45 calcium by isolated islets of Langerhans. Endocrinology. 1971 Jan;88(1):72–80. doi: 10.1210/endo-88-1-72. [DOI] [PubMed] [Google Scholar]
  30. Malaisse W. J., Herchuelz A., Levy J., Sener A. Calcium antagonists and islet function--III. The possible site of action of verapamil. Biochem Pharmacol. 1977 Apr 15;26(8):735–740. doi: 10.1016/0006-2952(77)90217-9. [DOI] [PubMed] [Google Scholar]
  31. Means A. R., Dedman J. R. Calmodulin--an intracellular calcium receptor. Nature. 1980 May 8;285(5760):73–77. doi: 10.1038/285073a0. [DOI] [PubMed] [Google Scholar]
  32. Milner R. D., Hales C. N. The role of calcium and magnesium in insulin secretion from rabbit pancreas studied in vitro. Diabetologia. 1967 Mar;3(1):47–49. doi: 10.1007/BF01269910. [DOI] [PubMed] [Google Scholar]
  33. PORTZEHL H., CALDWELL P. C., RUEEGG J. C. THE DEPENDENCE OF CONTRACTION AND RELAXATION OF MUSCLE FIBRES FROM THE CRAB MAIA SQUINADO ON THE INTERNAL CONCENTRATION OF FREE CALCIUM IONS. Biochim Biophys Acta. 1964 May 25;79:581–591. doi: 10.1016/0926-6577(64)90224-4. [DOI] [PubMed] [Google Scholar]
  34. Rüegg U. T., Rudinger J. Alkylation of cysteine thiols with 1,3-propane sultone. Methods Enzymol. 1977;47:116–122. doi: 10.1016/0076-6879(77)47013-7. [DOI] [PubMed] [Google Scholar]
  35. SANGER F., THOMPSON E. O. Halogenation of tyrosine during acid hydrolysis. Biochim Biophys Acta. 1963 May 14;71:468–471. doi: 10.1016/0006-3002(63)91108-9. [DOI] [PubMed] [Google Scholar]
  36. Schubart U. K., Erlichman J., Fleischer N. The role of calmodulin in the regulation of protein phosphorylation and insulin release in hamster insulinoma cells. J Biol Chem. 1980 May 10;255(9):4120–4124. [PubMed] [Google Scholar]
  37. Schulman H., Greengard P. Ca2+-dependent protein phosphorylation system in membranes from various tissues, and its activation by "calcium-dependent regulator". Proc Natl Acad Sci U S A. 1978 Nov;75(11):5432–5436. doi: 10.1073/pnas.75.11.5432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simpson R. J., Neuberger M. R., Liu T. Y. Complete amino acid analysis of proteins from a single hydrolysate. J Biol Chem. 1976 Apr 10;251(7):1936–1940. [PubMed] [Google Scholar]
  39. Sugden M. C., Ashcroft S. J., Sugden P. H. Protein kinase activities in rat pancreatic islets of Langerhans. Biochem J. 1979 Apr 15;180(1):219–229. doi: 10.1042/bj1800219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sugden M. C., Christie M. R., Ashcroft S. J. Presence and possible role of calcium-dependent regulator (calmodulin) in rat islets of Langerhans. FEBS Lett. 1979 Sep 1;105(1):95–100. doi: 10.1016/0014-5793(79)80894-7. [DOI] [PubMed] [Google Scholar]
  41. 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. doi: 10.1126/science.178.4063.871. [DOI] [PubMed] [Google Scholar]
  42. Valverde I., Vandermeers A., Anjaneyulu R., Malaisse W. J. Calmodulin activation of adenylate cyclase in pancreatic islets. Science. 1979 Oct 12;206(4415):225–227. doi: 10.1126/science.225798. [DOI] [PubMed] [Google Scholar]
  43. Waisman D. M., Singh T. J., Wang J. H. The modulator-dependent protein kinase. A multifunctional protein kinase activatable by the Ca2+-dependent modulator protein of the cyclic nucleotide system. J Biol Chem. 1978 May 25;253(10):3387–3390. [PubMed] [Google Scholar]
  44. Watterson D. M., Harrelson W. G., Jr, Keller P. M., Sharief F., Vanaman T. C. Structural similarities between the Ca2+-dependent regulatory proteins of 3':5'-cyclic nucleotide phosphodiesterase and actomyosin ATPase. J Biol Chem. 1976 Aug 10;251(15):4501–4513. [PubMed] [Google Scholar]
  45. Watterson D. M., Sharief F., Vanaman T. C. The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J Biol Chem. 1980 Feb 10;255(3):962–975. [PubMed] [Google Scholar]
  46. Weiss B., Levin R. M. Mechanism for selectively inhibiting the activation of cyclic nucleotide phosphodiesterase and adenylate cyclase by antipsychotic agents. Adv Cyclic Nucleotide Res. 1978;9:285–303. [PubMed] [Google Scholar]
  47. Welsh M. J., Dedman J. R., Brinkley B. R., Means A. R. Calcium-dependent regulator protein: localization in mitotic apparatus of eukaryotic cells. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1867–1871. doi: 10.1073/pnas.75.4.1867. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES