Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

X-ray diffraction studies of the structural state of crossbridges in skinned frog sartorius muscle at low ionic strength

  • 39 Accesses

  • 31 Citations

Summary

Low-angle X-ray diffraction diagrams were obtained from chemically skinned frog sartorius muscles under low ionic strength relaxing conditions. Experiments on single muscle fibres from rabbit muscle and on muscle proteins in solution have suggested the presence of a ‘low ionic strength attached state’ of the myosin crossbridges to actin, in which the overall ATP splitting and force-generating cycle is still blocked. This opened up the possibility that structural information about one of the intermediate states in the crossbridge cycle might be obtained under these conditions. Using synchrotron radiation as a high intensity X-ray source we were able to record the appropriate diffraction diagrams with short exposure times and were able to compare the same muscles at normal and at low ionic strength. Changes in the intensities of the equatorial reflections are consistent with the existence of some actin-attached crossbridges at low ionic strength in a relaxing medium and an increase in the 143 Å meridional intensity can be interpreted in a similar way. However, these attached bridges do not give rise to changes in the actin-based layer line reflections, nor is their presence associated with a weakening of the myosin layer line pattern. These results provide further evidence for the existence of bound states of crossbridges, in which their orientation relative to actin is not sharply defined.

This is a preview of subscription content, log in to check access.

References

  1. Arndt, U. W., Barrington-Leigh, J., Mallet, J. F. W. &Twinn, K. E. (1969) A mechanical microdensitometer.J. Sci. Inst. 2, 385–7.

  2. Barrington-Leigh, J., Holmes, K. C., Mannherz, H. G., Rosenbaum, A., Eckstein, F. &Goody, R. S. (1972) Effects of ATP analogs on the low-angle X-ray diffraction pattern of insect flight muscle.Cold Spring Harbor Symp. quant. Biol. 37, 443–7.

  3. Brenner, B., Schoenberg, M., Chalovich, J. M., Greene, L. E. &Eisenberg, E. (1982) Evidence for cross-bridge attachment in relaxed muscle at low ionic strength.Proc. natn. Acad. Sci. U.S.A. 79, 7288–91.

  4. Brenner, B., Yu, L. C. &Podolsky, R. J. (1984) X-ray diffraction evidence for cross-bridge formation in relaxed muscle fibers at various ionic strengths.Biophys. J. 46, 299–306.

  5. Chalovich, J. M., Chock, P. B. &Eisenberg, E. (1981) Mechanism of action of troponin-tropomyosin.J. biol. Chem. 256, 575–8.

  6. Chalovich, J. M. &Eisenberg, E. (1982) Inhibition of actomyosin ATPase activity by troponin-tropomyosin without blocking the binding of myosin to actin.J. biol. Chem. 257, 2432–7.

  7. Chalovich, J. M., Stein, L. A., Greene, L. E. &Eisenberg, E. (1984) Interactions of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide.Biochemistry 23, 4885–9.

  8. Clarke, F. M. &Masters, C. J. (1975) On the association of glycolytic enzymes with structural proteins of skeletal muscle.Biochim. biophys. Acta 381, 37–46.

  9. Egelman, E. H. &Derosier, D. J. (1983) Helical disorder and the filament structure of F-actin are elucidated by the angle-layered aggregate.J. molec. Biol. 166, 623–9.

  10. Goody, R. S., Holmes, K. C., Mannherz, H. G., Barrington-Leigh, J. &Rosenbaum, G. (1975) X-ray studies of insect flight muscle with ADP analogues.Biophys. J. 15, 687–705.

  11. Haselgrove, J. C. (1980) A model of myosin crossbridge structure consistent with the low angle X-ray diffraction pattern of vertebrate muscle.J. Muse. Res. Cell Motility 2, 177–91.

  12. Hendrix, J., Fuerst, H., Hartfiel, B. &Dainton, D. (1982) A wire per wire detector system for high counting rate X-ray experiments.Nucl. Instrum. Meth. 201, 139–44.

  13. Hendrix, J., Koch, M. H. J. &Bordas, J. (1979) A double focusing X-ray camera for use with synchrotron radiation.J. appl. Crystallogr. 12, 467–72.

  14. Huxley, H. E. (1968) Structural differences between resting and rigor muscle; evidence from intensity changes in the low angle X-ray diagram.J. molec. Biol. 37, 507–20.

  15. Huxley, H. E. &Brown, W. (1967) The low angle X-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor.J. molec. Biol. 30, 383–434.

  16. Huxley, H. E., Faruqi, A. R., Kress, M., Bordas, J. &Koch, M. H. J. (1982) Time resolved X-ray diffraction studies of the myosin layer-line reflections during muscle contraction.J. molec. Biol. 158, 637–84.

  17. Huxley, H. E. &Kress, M. (1985) Crossbridge behaviour during muscle contraction.J. Musc. Res. Cell Motility 6, 153–62.

  18. Huxley, H. E., Simmons, R. M., Faruqi, A. R., Kress, M., Bordas, J. &Koch, M. H. J. (1983) Changes in the X-ray reflections from contracting muscle during rapid mechanical transients and their structural implications.J. molec. Biol. 169, 469–506.

  19. Kensler, R. W. &Stewart, M. (1983) Frog skeletal muscle thick filaments are three-stranded.J. Cell Biol. 96, 1797–1802.

  20. Kuhn, H. J. (1977) Reversible transformation of mechanical work into chemical free energy by stretch dependent binding of AMPPNP in glycerinated fibrillar muscle fibres. InInsect Flight Muscle (edited byTregear, R. T.), pp. 307–15. Amsterdam: North Holland Publishing Company.

  21. Lymn, R. W. (1975) X-ray diagrams from skeletal muscle.J. molec. Biol. 99, 567–82.

  22. Lymn, R. W. &Huxley, H. E. (1972) X-ray diagrams from skeletal muscle in the presence of ATP analogs.Cold Spring Harbor Symp. quant. Biol. 37, 449–53.

  23. Magid, A. &Reedy, M. (1980) X-ray diffraction observations of chemically skinned frog skeletal muscle processed by an improved method.Biophys. J. 30, 27–40.

  24. Marston, S. B., Rodger, C. D. &Tregear, R. T. (1976) Changes in muscle crossbridges when β-γ-imido-ATP binds to myosin.J. molec. Biol. 104, 263–76.

  25. Matsuda, T. &Podolsky, R. J. (1984) X-ray evidence for two structural states of the actomyosin cross-bridge in muscle fibers.Proc. natn. Acad. Sci. U.S.A. 81, 2364–8.

  26. Poulsen, F. R. &Lowy, J. (1983) Small-angle X-ray scattering from myosin heads in relaxed and rigor frog skeletal muscles.Nature, Lond. 303, 146–52.

  27. Padron, R. &Huxley, H. E. (1984) The effect of the ATP analogue AMPPNP on the structure of crossbridges in vertebrate skeletal muscles: X-ray diffraction and mechanical studies.J. Musc. Res. Cell Motility 5, 613–55.

  28. Schoenberg, M., Brenner, B. &Chalovich, J. M. (1984) Cross-bridge attachment in relaxed muscle. InContractile Mechanisms in Muscle (edited byPollack, G. H. &Sugi, H.), pp. 269–84. New York, London: Plenum Press.

  29. Squire, J. M. (1973) General model of myosin filament structure. III. Molecular packing arrangements in myosin filaments.J. molec. Biol. 77, 291–323.

  30. Squire, J. M., Harford, J. J., Edman, A. C. &Sjöstrom, M. (1982) Fine structure of the A-band in cryo-sections. III. Crossbridge distribution and the axial structure of the human C-zone.J. molec. Biol. 155, 467–94.

  31. Stewart, M., Morton, D. J. &Clarke, F. M. (1979) Changes associated with glycolytic-enzyme binding in the equatorial X-ray diffraction pattern of glycerinated rabbit psoas muscle.Biochem. J. 183, 663–7.

  32. Stewart, M., Kensler, R. W. &Levine, R. J. C. (1985) Three dimensional reconstruction of thick filaments fromLimulus and scorpion muscle.J. Cell Biol. 101, 402–11.

  33. Thomas, D. D. &Cooke, R. (1980) Orientation of spin-labeled myosin heads in glycerinated muscle fibers.Biophys. J. 32, 891–906.

  34. Wagner, P. D. &Giniger, E. (1981) Calcium sensitive binding of heavy meromyosin to regulated actin in the presence of ATP.J. biol. Chem. 256, 12647–50.

  35. Wray, J. W. (1984) Cross-bridge states in invertebrate muscles. InContractile Mechanisms in Muscle (edited byPollack, G. H. &Sugi, H.), pp. 185–92. New York, London: Plenum Press.

  36. Yagi, N., O'Brien, E. J. &Matsubara, I. (1981) Changes in thick filament structure during contraction of frog striated muscle.Biophys. J. 33, 121–38.

  37. Yoshimura, H., Nishio, T. &Scmihashi, K. (1984) Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range.J. molec. Biol. 179, 453–67.

Download references

Author information

Additional information

Seceased December 1985.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Xu, S., Kress, M. & Huxley, H.E. X-ray diffraction studies of the structural state of crossbridges in skinned frog sartorius muscle at low ionic strength. J Muscle Res Cell Motil 8, 39–54 (1987). https://doi.org/10.1007/BF01767263

Download citation

Keywords

  • Short Exposure Time
  • Layer Line
  • Skinned Frog
  • Rabbit Muscle
  • Attached State