Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1996 Oct;71(4):1891–1904. doi: 10.1016/S0006-3495(96)79388-8

Calcium alone does not fully activate the thin filament for S1 binding to rigor myofibrils.

D R Swartz 1, R L Moss 1, M L Greaser 1
PMCID: PMC1233656  PMID: 8889164

Abstract

Skeletal muscle contraction is regulated by calcium via troponin and tropomyosin and appears to involve cooperative activation of cross-bridge binding to actin. We studied the regulation of fluorescent myosin subfragment 1 (fS1) binding to rigor myofibrils over a wide range of fS1 and calcium levels using highly sensitive imaging techniques. At low calcium and low fS1, the fluorescence was restricted to the actin-myosin overlap region. At high calcium and very low fS1, the fluorescence was still predominantly in the overlap region. The ratio of nonoverlap to overlap fluorescence intensity showed that increases in the fS1 level resulted in a shift in maximum fluorescence from the overlap to the nonoverlap region at both low and high calcium; this transition occurred at lower fS1 levels in myofibrils with high calcium. At a fixed fS1 level, increases in calcium also resulted in a shift in maximum fluorescence from the overlap region to the nonoverlap region. These results suggest that calcium alone does not fully activate the thin filament for rigor S1 binding and that, even at high calcium, the thin filament is not activated along its entire length.

Full text

PDF
1891

Images in this article

1895FIGURE 2
on p.1895
1895FIGURE 3
on p.1895
1896FIGURE 4
on p.1896
1896FIGURE 5
on p.1896
1897FIGURE 7
on p.1897
1899FIGURE 9
on p.1899
1900FIGURE 11
on p.1900

Selected References

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

  1. Balazs A. C., Epstein I. R. Kinetic model for the interaction of myosin subfragment 1 with regulated actin. Biophys J. 1983 Nov;44(2):145–151. doi: 10.1016/S0006-3495(83)84286-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brandt P. W., Diamond M. S., Rutchik J. S., Schachat F. H. Co-operative interactions between troponin-tropomyosin units extend the length of the thin filament in skeletal muscle. J Mol Biol. 1987 Jun 20;195(4):885–896. doi: 10.1016/0022-2836(87)90492-x. [DOI] [PubMed] [Google Scholar]
  3. Bremel R. D., Weber A. Cooperation within actin filament in vertebrate skeletal muscle. Nat New Biol. 1972 Jul 26;238(82):97–101. doi: 10.1038/newbio238097a0. [DOI] [PubMed] [Google Scholar]
  4. Brenner B. Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction. Proc Natl Acad Sci U S A. 1988 May;85(9):3265–3269. doi: 10.1073/pnas.85.9.3265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cantino M. E., Allen T. S., Gordon A. M. Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle. Biophys J. 1993 Jan;64(1):211–222. doi: 10.1016/S0006-3495(93)81358-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carter S. G., Karl D. W. Inorganic phosphate assay with malachite green: an improvement and evaluation. J Biochem Biophys Methods. 1982 Dec;7(1):7–13. doi: 10.1016/0165-022x(82)90031-8. [DOI] [PubMed] [Google Scholar]
  7. Chalovich J. M. Actin mediated regulation of muscle contraction. Pharmacol Ther. 1992;55(2):95–148. doi: 10.1016/0163-7258(92)90013-p. [DOI] [PubMed] [Google Scholar]
  8. Chalovich J. M., Chock P. B., Eisenberg E. Mechanism of action of troponin . tropomyosin. Inhibition of actomyosin ATPase activity without inhibition of myosin binding to actin. J Biol Chem. 1981 Jan 25;256(2):575–578. [PMC free article] [PubMed] [Google Scholar]
  9. Chalovich J. M., Eisenberg E. Inhibition of actomyosin ATPase activity by troponin-tropomyosin without blocking the binding of myosin to actin. J Biol Chem. 1982 Mar 10;257(5):2432–2437. [PMC free article] [PubMed] [Google Scholar]
  10. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 1988;157:378–417. doi: 10.1016/0076-6879(88)57093-3. [DOI] [PubMed] [Google Scholar]
  11. Fritz J. D., Swartz D. R., Greaser M. L. Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins. Anal Biochem. 1989 Aug 1;180(2):205–210. doi: 10.1016/0003-2697(89)90116-4. [DOI] [PubMed] [Google Scholar]
  12. GORNALL A. G., BARDAWILL C. J., DAVID M. M. Determination of serum proteins by means of the biuret reaction. J Biol Chem. 1949 Feb;177(2):751–766. [PubMed] [Google Scholar]
  13. Geeves M. A., Lehrer S. S. Dynamics of the muscle thin filament regulatory switch: the size of the cooperative unit. Biophys J. 1994 Jul;67(1):273–282. doi: 10.1016/S0006-3495(94)80478-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geeves M. A. The dynamics of actin and myosin association and the crossbridge model of muscle contraction. Biochem J. 1991 Feb 15;274(Pt 1):1–14. doi: 10.1042/bj2740001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Godt R. E., Lindley B. D. Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog. J Gen Physiol. 1982 Aug;80(2):279–297. doi: 10.1085/jgp.80.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grabarek Z., Tao T., Gergely J. Molecular mechanism of troponin-C function. J Muscle Res Cell Motil. 1992 Aug;13(4):383–393. doi: 10.1007/BF01738034. [DOI] [PubMed] [Google Scholar]
  17. Greene L. E., Eisenberg E. Cooperative binding of myosin subfragment-1 to the actin-troponin-tropomyosin complex. Proc Natl Acad Sci U S A. 1980 May;77(5):2616–2620. doi: 10.1073/pnas.77.5.2616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Greene L. E., Williams D. L., Jr, Eisenberg E. Regulation of actomyosin ATPase activity by troponin-tropomyosin: effect of the binding of the myosin subfragment 1 (S-1).ATP complex. Proc Natl Acad Sci U S A. 1987 May;84(10):3102–3106. doi: 10.1073/pnas.84.10.3102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Güth K., Potter J. D. Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers. J Biol Chem. 1987 Oct 5;262(28):13627–13635. [PubMed] [Google Scholar]
  20. Head J. G., Ritchie M. D., Geeves M. A. Characterization of the equilibrium between blocked and closed states of muscle thin filaments. Eur J Biochem. 1995 Feb 1;227(3):694–699. doi: 10.1111/j.1432-1033.1995.tb20190.x. [DOI] [PubMed] [Google Scholar]
  21. Hill T. L., Eisenberg E., Greene L. E. Alternate model for the cooperative equilibrium binding of myosin subfragment-1-nucleotide complex to actin-troponin-tropomyosin. Proc Natl Acad Sci U S A. 1983 Jan;80(1):60–64. doi: 10.1073/pnas.80.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hill T. L., Eisenberg E., Greene L. Theoretical model for the cooperative equilibrium binding of myosin subfragment 1 to the actin-troponin-tropomyosin complex. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3186–3190. doi: 10.1073/pnas.77.6.3186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ishii Y., Lehrer S. S. Fluorescence probe studies of the state of tropomyosin in reconstituted muscle thin filaments. Biochemistry. 1987 Aug 11;26(16):4922–4925. doi: 10.1021/bi00390a005. [DOI] [PubMed] [Google Scholar]
  24. Kress M., Huxley H. E., Faruqi A. R., Hendrix J. Structural changes during activation of frog muscle studied by time-resolved X-ray diffraction. J Mol Biol. 1986 Apr 5;188(3):325–342. doi: 10.1016/0022-2836(86)90158-0. [DOI] [PubMed] [Google Scholar]
  25. Leavis P. C., Gergely J. Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction. CRC Crit Rev Biochem. 1984;16(3):235–305. doi: 10.3109/10409238409108717. [DOI] [PubMed] [Google Scholar]
  26. Lehman W., Craig R., Vibert P. Ca(2+)-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature. 1994 Mar 3;368(6466):65–67. doi: 10.1038/368065a0. [DOI] [PubMed] [Google Scholar]
  27. Lehrer S. S., Morris E. P. Dual effects of tropomyosin and troponin-tropomyosin on actomyosin subfragment 1 ATPase. J Biol Chem. 1982 Jul 25;257(14):8073–8080. [PubMed] [Google Scholar]
  28. Lehrer S. S. The regulatory switch of the muscle thin filament: Ca2+ or myosin heads? J Muscle Res Cell Motil. 1994 Jun;15(3):232–236. doi: 10.1007/BF00123476. [DOI] [PubMed] [Google Scholar]
  29. Lorenz M., Poole K. J., Popp D., Rosenbaum G., Holmes K. C. An atomic model of the unregulated thin filament obtained by X-ray fiber diffraction on oriented actin-tropomyosin gels. J Mol Biol. 1995 Feb 10;246(1):108–119. doi: 10.1006/jmbi.1994.0070. [DOI] [PubMed] [Google Scholar]
  30. McKillop D. F., Geeves M. A. Regulation of the acto.myosin subfragment 1 interaction by troponin/tropomyosin. Evidence for control of a specific isomerization between two acto.myosin subfragment 1 states. Biochem J. 1991 Nov 1;279(Pt 3):711–718. doi: 10.1042/bj2790711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McKillop D. F., Geeves M. A. Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament. Biophys J. 1993 Aug;65(2):693–701. doi: 10.1016/S0006-3495(93)81110-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moss R. L. Effects on shortening velocity of rabbit skeletal muscle due to variations in the level of thin-filament activation. J Physiol. 1986 Aug;377:487–505. doi: 10.1113/jphysiol.1986.sp016199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Murray J. M., Weber A. Cooperativity of the calcium switch of regulated rabbit actomyosin system. Mol Cell Biochem. 1981 Feb 26;35(1):11–15. doi: 10.1007/BF02358183. [DOI] [PubMed] [Google Scholar]
  34. Murray J. M., Weber A., Knox M. K. Myosin subfragment 1 binding to relaxed actin filaments and steric model of relaxation. Biochemistry. 1981 Feb 3;20(3):641–649. doi: 10.1021/bi00506a030. [DOI] [PubMed] [Google Scholar]
  35. Nagashima H., Asakura S. Studies on co-operative properties of tropomyosin-actin and tropomyosin-troponin-actin complexes by the use of N-ethylmaleimide-treated and untreated species of myosin subfragment 1. J Mol Biol. 1982 Mar 15;155(4):409–428. doi: 10.1016/0022-2836(82)90479-x. [DOI] [PubMed] [Google Scholar]
  36. Pan B. S., Gordon A. M., Luo Z. X. Removal of tropomyosin overlap modifies cooperative binding of myosin S-1 to reconstituted thin filaments of rabbit striated muscle. J Biol Chem. 1989 May 25;264(15):8495–8498. [PubMed] [Google Scholar]
  37. Pardee J. D., Spudich J. A. Purification of muscle actin. Methods Enzymol. 1982;85(Pt B):164–181. doi: 10.1016/0076-6879(82)85020-9. [DOI] [PubMed] [Google Scholar]
  38. Parry D. A., Squire J. M. Structural role of tropomyosin in muscle regulation: analysis of the x-ray diffraction patterns from relaxed and contracting muscles. J Mol Biol. 1973 Mar 25;75(1):33–55. doi: 10.1016/0022-2836(73)90527-5. [DOI] [PubMed] [Google Scholar]
  39. Popp D., Maéda Y. Calcium ions and the structure of muscle actin filament. An X-ray diffraction study. J Mol Biol. 1993 Jan 20;229(2):279–285. doi: 10.1006/jmbi.1993.1032. [DOI] [PubMed] [Google Scholar]
  40. Rayment I., Holden H. M. The three-dimensional structure of a molecular motor. Trends Biochem Sci. 1994 Mar;19(3):129–134. doi: 10.1016/0968-0004(94)90206-2. [DOI] [PubMed] [Google Scholar]
  41. Rosenfeld S. S., Taylor E. W. The mechanism of regulation of actomyosin subfragment 1 ATPase. J Biol Chem. 1987 Jul 25;262(21):9984–9993. [PubMed] [Google Scholar]
  42. Squire J. M. The actomyosin interaction--shedding light on structural events: 'Plus ça change, plus c'est la même chose'. J Muscle Res Cell Motil. 1994 Jun;15(3):227–231. doi: 10.1007/BF00123475. [DOI] [PubMed] [Google Scholar]
  43. Swartz D. R., Greaser M. L., Marsh B. B. Regulation of binding of subfragment 1 in isolated rigor myofibrils. J Cell Biol. 1990 Dec;111(6 Pt 2):2989–3001. doi: 10.1083/jcb.111.6.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Swartz D. R., Moss R. L. Influence of a strong-binding myosin analogue on calcium-sensitive mechanical properties of skinned skeletal muscle fibers. J Biol Chem. 1992 Oct 5;267(28):20497–20506. [PubMed] [Google Scholar]
  45. Trybus K. M., Taylor E. W. Kinetic studies of the cooperative binding of subfragment 1 to regulated actin. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7209–7213. doi: 10.1073/pnas.77.12.7209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Weeds A. G., Pope B. Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility. J Mol Biol. 1977 Apr;111(2):129–157. doi: 10.1016/s0022-2836(77)80119-8. [DOI] [PubMed] [Google Scholar]
  47. Williams D. L., Jr, Greene L. E., Eisenberg E. Cooperative turning on of myosin subfragment 1 adenosinetriphosphatase activity by the troponin-tropomyosin-actin complex. Biochemistry. 1988 Sep 6;27(18):6987–6993. doi: 10.1021/bi00418a048. [DOI] [PubMed] [Google Scholar]
  48. Yates L. D., Greaser M. L. Quantitative determination of myosin and actin in rabbit skeletal muscle. J Mol Biol. 1983 Jul 25;168(1):123–141. doi: 10.1016/s0022-2836(83)80326-x. [DOI] [PubMed] [Google Scholar]
  49. Young I. T. Image fidelity: characterizing the imaging transfer function. Methods Cell Biol. 1989;30:1–45. doi: 10.1016/s0091-679x(08)60974-7. [DOI] [PubMed] [Google Scholar]
  50. Zot A. S., Potter J. D. Reciprocal coupling between troponin C and myosin crossbridge attachment. Biochemistry. 1989 Aug 8;28(16):6751–6756. doi: 10.1021/bi00442a031. [DOI] [PubMed] [Google Scholar]
  51. Zot A. S., Potter J. D. The effect of [Mg2+] on the Ca2+ dependence of ATPase and tension development of fast skeletal muscle. The role of the Ca2+-specific sites of troponin C. J Biol Chem. 1987 Feb 15;262(5):1966–1969. [PubMed] [Google Scholar]

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

RESOURCES