Skip to main content
NCBI home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1968 May 1;51(5):655–676. doi: 10.1085/jgp.51.5.655

The Site of Calcium Binding in Relation to the Activation of Myofibrillar Contraction

Franklin Fuchs 1, F Norman Briggs 1
PMCID: PMC2201203  PMID: 5654405

Abstract

Skeletal muscle myofibrils, in the presence of 2 mM MgCl2 at pH 7.0, were found to have two classes of calcium-binding sites with apparent affinity constants of 2.1 x 106 M -1 (class 1) and ∼3 x 104 M -1 (class 2), respectively. At free calcium concentrations essential for the activation of myofibrillar contraction (∼10-6 M) there would be significant calcium binding only to the class 1 sites. These sites could bind about 1.3 µmoles of calcium per g protein. Extraction of myosin from the myofibrils did not alter their calcium-binding parameters. Myosin A, under identical experimental conditions, had little affinity for calcium. The class 1 sites are, therefore, presumed to be located in the I filaments. The class 1 sites could only be detected in F actin and myosin B preparations which were contaminated with the tropomyosin-troponin complex. Tropomyosin bound very little calcium. Troponin, which in conjunction with tropomyosin confers calcium sensitivity on actomyosin systems, could bind 22 µmoles of calcium per g protein with an apparent affinity constant of 2.4 x 106 M -1. In view of the identical affinity constants of the myofibrils and troponin and the much greater number of calcium-binding sites on troponin it is suggested that calcium activates myofibrillar contraction by binding to the troponin molecule.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Azuma N., Watanabe S. The major component of metin from rabbit skeletal and bovine cardiac muscle. J Biol Chem. 1965 Oct;240(10):3847–3851. [PubMed] [Google Scholar]
  2. Azuma N., Watanabe S. The minor component of metin from rabbit skeletal muscle. J Biol Chem. 1965 Oct;240(10):3852–3857. [PubMed] [Google Scholar]
  3. Borle A. B., Briggs F. N. Microdetermination of calcium in biological material by automatic fluorometric titration. Anal Chem. 1968 Feb;40(2):339–344. doi: 10.1021/ac60258a056. [DOI] [PubMed] [Google Scholar]
  4. CARSTEN M. E., MOMMAERTS W. F. A study of actin by means of starch gel electrophoresis. Biochemistry. 1963 Jan-Feb;2:28–32. doi: 10.1021/bi00901a006. [DOI] [PubMed] [Google Scholar]
  5. Cohen C., Longley W. Tropomyosin paracrystals formed by divalent cations. Science. 1966 May 6;152(3723):794–796. doi: 10.1126/science.152.3723.794. [DOI] [PubMed] [Google Scholar]
  6. Corsi A., Ronchetti I., Cigognetti C. Observations on the actin content of the rabbit myofibril. Biochem J. 1966 Jul;100(1):110–113. doi: 10.1042/bj1000110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DAVIES R. E. A MOLECULAR THEORY OF MUSCLE CONTRACTION: CALCIUM-DEPENDENT CONTRACTIONS WITH HYDROGEN BOND FORMATION PLUS ATP-DEPENDENT EXTENSIONS OF PART OF THE MYOSIN-ACTIN CROSS-BRIDGES. Nature. 1963 Sep 14;199:1068–1074. doi: 10.1038/1991068a0. [DOI] [PubMed] [Google Scholar]
  8. DRAPER M. H., HODGE A. J. Electron-induced microincineration with the electron microscope. I. Distribution of residual mineral content in vertebrate striated muscle. Aust J Exp Biol Med Sci. 1950 Sep;28(5):549–557. doi: 10.1038/icb.1950.54. [DOI] [PubMed] [Google Scholar]
  9. EBASHI S., EBASHI F. A NEW PROTEIN COMPONENT PARTICIPATING IN THE SUPERPRECIPITATION OF MYOSIN B. J Biochem. 1964 Jun;55:604–613. doi: 10.1093/oxfordjournals.jbchem.a127933. [DOI] [PubMed] [Google Scholar]
  10. Ebashi S., Ebashi F., Kodama A. Troponin as the Ca++-receptive protein in the contractile system. J Biochem. 1967 Jul;62(1):137–138. doi: 10.1093/oxfordjournals.jbchem.a128628. [DOI] [PubMed] [Google Scholar]
  11. Ebashi S., Kodama A. A new protein factor promoting aggregation of tropomyosin. J Biochem. 1965 Jul;58(1):107–108. doi: 10.1093/oxfordjournals.jbchem.a128157. [DOI] [PubMed] [Google Scholar]
  12. HUXLEY H. E. ELECTRON MICROSCOPE STUDIES ON THE STRUCTURE OF NATURAL AND SYNTHETIC PROTEIN FILAMENTS FROM STRIATED MUSCLE. J Mol Biol. 1963 Sep;7:281–308. doi: 10.1016/s0022-2836(63)80008-x. [DOI] [PubMed] [Google Scholar]
  13. Katz A. M. Purification and properties of a tropomyosin-containing protein fraction that sensitizes reconstituted actomyosin to calcium-binding agents. J Biol Chem. 1966 Apr 10;241(7):1522–1529. [PubMed] [Google Scholar]
  14. LAKI K., MARUYAMA K., KOMINZ D. R. Evidence for the interaction between tropomyosin and actin. Arch Biochem Biophys. 1962 Aug;98:323–330. doi: 10.1016/0003-9861(62)90190-x. [DOI] [PubMed] [Google Scholar]
  15. LEVY H. M., RYAN E. M. EVIDENCE THAT CALCIUM ACTIVATES THE CONTRACTION OF ACTOMYOSIN BY OVERCOMING SUBSTRATE INHIBITION. Nature. 1965 Feb 13;205:703–705. doi: 10.1038/205703b0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Levy H. M., Ryan E. M. Heat inactivation of the relaxing site of actomyosin: prevention and reversal with dithiothreitol. Science. 1967 Apr 7;156(3771):73–74. doi: 10.1126/science.156.3771.73. [DOI] [PubMed] [Google Scholar]
  18. MARTONOSI A. Studies on actin. VII. Ultracentrifugal analysis of partially polymerized actin solutions. J Biol Chem. 1962 Sep;237:2795–2803. [PubMed] [Google Scholar]
  19. MARTONOSII A., MOLINO C. M., GERGELY J. THE BINDING OF DIVALENT CATIONS TO ACTIN. J Biol Chem. 1964 Apr;239:1057–1064. [PubMed] [Google Scholar]
  20. MARUYAMA K., ISHIKAWA Y. EFFECTS OF SOME SULFHYDRYL COMPOUNDS ON THE MAGNESIUM-ENHANCED ADENOSINETRIPHOSPHATASE ACTIVITY OF MYOSIN B. J Biochem. 1964 Oct;56:372–374. doi: 10.1093/oxfordjournals.jbchem.a128004. [DOI] [PubMed] [Google Scholar]
  21. NANNINGA L. B. The binding of magnesium, calcium, and chlorine ions to heavy and light meromyosin. Arch Biochem Biophys. 1957 Aug;70(2):346–366. doi: 10.1016/0003-9861(57)90122-4. [DOI] [PubMed] [Google Scholar]
  22. Otsuki I., Masaki T., Nonomura Y., Ebashi S. Periodic distribution of troponin along the thin filament. J Biochem. 1967 Jun;61(6):817–819. doi: 10.1093/oxfordjournals.jbchem.a128619. [DOI] [PubMed] [Google Scholar]
  23. PARKER C. J., Jr, GERGELY J. The role of calcium in the adensine triphosphatase activity of myofibrils and in the mechanism of the relaxing factor system of muscle. J Biol Chem. 1961 Feb;236:411–415. [PubMed] [Google Scholar]
  24. Pepe F. A. Some aspects of the structural organization of the myofibril as revealed by antibody--staining methods. J Cell Biol. 1966 Mar;28(3):505–525. doi: 10.1083/jcb.28.3.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. SZENT-GYOERGYI A., KAMINER B. METIN AND METACTOMYOSIN. Proc Natl Acad Sci U S A. 1963 Dec;50:1033–1036. doi: 10.1073/pnas.50.6.1033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sandow A. Excitation-contraction coupling in skeletal muscle. Pharmacol Rev. 1965 Sep;17(3):265–320. [PubMed] [Google Scholar]
  27. TONOMURA Y., YOSHIMURA J. Inhibition of myosin B-adenosinetriphosphatase by excess substrate. Arch Biochem Biophys. 1960 Sep;90:73–81. doi: 10.1016/0003-9861(60)90614-7. [DOI] [PubMed] [Google Scholar]
  28. WATANABE S., YASUI T. EFFECTS OF MAGNESIUM AND CALCIUM ON THE SUPERPRECIPITATION OF MYOSIN B. J Biol Chem. 1965 Jan;240:105–111. [PubMed] [Google Scholar]
  29. WEBER A., HERZ R., REISS I. On the mechanism of the relaxing effect of fragmented sarcoplasmic reticulum. J Gen Physiol. 1963 Mar;46:679–702. doi: 10.1085/jgp.46.4.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. WEBER A., HERZ R. Requirement for calcium in the synaeresis of myofibrils. Biochem Biophys Res Commun. 1961 Dec 20;6:364–368. doi: 10.1016/0006-291x(61)90146-2. [DOI] [PubMed] [Google Scholar]
  31. WEBER A., HERZ R. The binding of calcium to actomyosin systems in relation to their biological activity. J Biol Chem. 1963 Feb;238:599–605. [PubMed] [Google Scholar]
  32. WEBER A., WINICUR S. The role of calcium in the superprecipitation of actomyosin. J Biol Chem. 1961 Dec;236:3198–3202. [PubMed] [Google Scholar]
  33. WINEGRAD S. AUTORADIOGRAPHIC STUDIES OF INTRACELLULAR CALCIUM IN FROG SKELETAL MUSCLE. J Gen Physiol. 1965 Jan;48:455–479. doi: 10.1085/jgp.48.3.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Watanabe S., Staprans I. Purification of the relaxing protein of rabbit skeletal muscle. Proc Natl Acad Sci U S A. 1966 Aug;56(2):572–577. doi: 10.1073/pnas.56.2.572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Woods E. F. The dissociation of tropomyosin by urea. J Mol Biol. 1966 Apr;16(2):581–584. doi: 10.1016/s0022-2836(66)80199-7. [DOI] [PubMed] [Google Scholar]
  36. Yasui B., Fuchs F., Briggs F. N. The role of the sulfhydryl groups of tropomyosin and troponin in the calcium control of actomyosin contractility. J Biol Chem. 1968 Feb 25;243(4):735–742. [PubMed] [Google Scholar]

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press

RESOURCES