Journal of Muscle Research & Cell Motility

, Volume 5, Issue 1, pp 81–96 | Cite as

Modification of crossbridge states by ethylene glycol in insect flight muscle

  • M. L. Clarke
  • C. D. Rodger
  • R. T. Tregear


Substitution of ethylene glycol for part of the solvent water changes the mechanical properties, structure and nucleotide binding of glycerol-extracted flight muscle fibres from the waterbugLethocerus. On addition of ethylene glycol the rigor tension falls, rapidly and reversibly. With increasing glycol concentration the effect saturates at a non-zero tension. The isotonic stiffness is unchanged on adding ethylene glycol. Adding MgAMPPNP (adenylylimidodiphosphate) to a muscle fibre in 50% ethylene glycol causes a further rapid tension fall; above 100 µm AMPPNP the tension reaches zero. The isotonic stiffness of restretched muscle is then close to that of a relaxed fibre. Removal of MgAMPPNP from the bathing medium has no immediate mechanical effect. After several hours the isotonic stiffness rises to some extent; on removal of the glycol both tension and stiffness rise to rigor values within one minute.3H-Labelled AMPPNP binds to muscle fibres in 50% ethylene glycol in a similar amount to the number of myosin heads present. The binding is tighter than that in aqueous solution and the nucleotide is only released very slowly. Upon removal of the ethylene glycol nucleotide is rapidly released. X-ray diffraction of muscle in 50% ethylene glycol reveals a highly ordered structure, in which both the 14 nm and the 38 nm layer lines are sharply sampled and are of intermediate values between rigor and relaxation. The two inner equatorial peaks are also of intermediate values. On adding MgAMPPNP the pattern resembles that of relaxed muscle. Upon removal of the nucleotide the pattern does not revert towards rigor but on removal of glycol it does. These results are interpreted in terms of changes within the myosin heads and their array within the filament lattice.


Myosin Head Flight Muscle Layer Line Rigor Solution Heavy Meromyosin 
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  1. ANDO, T. & ASAI, H. (1977) The effects of solvent viscosity on the kinetic parameters of myosin and heavy meromyosin ATPase.J. Bioenerget. Biomemb. 9, 283–88.CrossRefGoogle Scholar
  2. ARATA, T. & PODOLSKY, R. J. (1982) Crossbridge flexibility derived from the influence of lattice spacing on mechanical properties of muscle fibres in rigor.Biophys. J. 37, 362a.Google Scholar
  3. ARMITAGE, P. M., TREGEAR, R. T. & MILLER, A. (1975) Effect of activation by calcium on the X-ray diffraction pattern from insect flight muscle.J. molec. Biol. 92, 39–53.PubMedCrossRefGoogle Scholar
  4. BARRINGTON-LEIGH, J., HOLMES, K. C., MANNHERZ, H. G., ROSENBAUM, G., ECKSTEIN, F. & GOODY, R. S. (1972) Effects of ATP analogues on the low angle x-ray diffraction pattern of insect flight muscle.Cold Spring Harb. Symp. quant. Biol. 37, 443–8.Google Scholar
  5. BECHET, J. J., BREDA, C., GUINAND, S., HILL, M. D'ALBIS, A. (1979) Magnesium-ion dependent ATPase activity of HMM as a function of temperature between +20 and −15°C.Biochemistry 18, 4080–9.PubMedCrossRefGoogle Scholar
  6. CLARKE, M. L., RODGER, C. D., TREGEAR, R. T., BORDAS, J. & KOCH, M. (1980) The effect of ethylene glycol and low temperature on the structure and function of insect flight muscle.J. Musc. Res. Cell. Motility 1, 195–6.Google Scholar
  7. CLARKE, M. L. (1982)The attachment of myosin heads to actin in the presence and absence of an unhydrolysable analogue of ATP. D.Phil. thesis, University of Oxford.Google Scholar
  8. DOUZOU, P. (1977)Cryobiochemistry. New York: Academic Press.Google Scholar
  9. FARUQI, A. R. (1975) High spatial resolution position-sensitive counter for use in muscle diffraction.J. Phys. E. Sci. Instrum. 8, 633–5.CrossRefGoogle Scholar
  10. FORD, L. E., HUXLEY, A. F. & SIMMONS, R. M. (1981) The relation between stiffness and filament overlap in stimulated frog muscle fibres.J. Physiol. 311, 219–49.PubMedGoogle Scholar
  11. GABRIEL, A. (1977) Position sensitive x-ray detector.Rev. Sci. Instrum. 48, 1303–5.CrossRefGoogle Scholar
  12. GEKKO, R. & TIMASHEFF, S. N. (1981) Mechanism of protein stabilisation by glycerol: preferential hydration in glycerol-water mixtures.Biochemistry 20, 4667–76.PubMedCrossRefGoogle Scholar
  13. GOODNO, C. C. (1979) Inhibition of myosin ATPase by vanadate ion.Proc. natn. Acad. Sci. U.S.A. 76, 2620–4.CrossRefGoogle Scholar
  14. GOODNO, C. C. & TAYLOR, E. W. (1982) Inhibition of actomyosin ATPase by vanadate.Proc. natn. Acad. Sci. U.S.A. 79, 21–5.CrossRefGoogle Scholar
  15. GOODY, R. S., BARRINGTON-LEIGH, J., MANNHERZ, H. G. TREGEAR, R. T. & ROSENBAUM, G. (1976) X-ray titration of binding of β, γ, imidoATP to myosin in insect flight muscle.Nature 262, 613–5.PubMedCrossRefGoogle Scholar
  16. GOODY, R. S. & HOFMANN, W. (1980) Stereochemical aspects of the interaction of myosin and actomyosin with nucleotides.J. Musc. Res. Cell Motility 1, 101–16.CrossRefGoogle Scholar
  17. GOODY, R. S., HOFMANN, W., REEDY, M. K., MAGID, A. & GOODNO, C. C. (1980) Relaxation of glycerinated insect flight muscle by vanadate.J. Musc. Res. Cell Motility 1, 198–9.Google Scholar
  18. GOODY, R. S., HOLMES, K. C., MANNHERZ, H. G., BARRINGTON-LEIGH, J. & ROSENBAUM, G. (1975) X-ray studies of insect flight muscle with ATP analogues.Biophys. J. 15, 687–705.PubMedCrossRefGoogle Scholar
  19. HOLMES, K. C., TREGEAR, R. T., & BARRINGTON-LEIGH, J. (1980) Interpretation of the low angle x-ray diffraction pattern from insect flight muscle in rigor.Proc. R. Soc. 207, 13–33.CrossRefGoogle Scholar
  20. KAY, C. M. & BRAHMS, J. (1963) The influence of ethylene glycol on the enzymatic ATPase activity and molecular conformation of fibrous muscle proteins.J. biol. Chem. 238, 2945–9.PubMedGoogle Scholar
  21. KODAMA, T. (1981) Temperature-modulated binding of ADP and AMPPNP to myosin S-1 studied by calorimetric titration.J. biol. Chem. 256, 11503–8.PubMedGoogle Scholar
  22. KUHN, H. J. (1973) Transformation of chemical energy into mechanical energy by glycerol-extracted fibres of insect flight muscle in the absence of nucleosidetriphosphate hydrolysis.Experientia 29, 1086–8.PubMedCrossRefGoogle Scholar
  23. 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 by TREGEAR, R. T.), pp. 307–315. Amsterdam: North-Holland.Google Scholar
  24. LOXDALE, H. D. (1980)Molecular parameters of diverse muscle systems. D.Phil. thesis, University of Oxford.Google Scholar
  25. MARSTON, S. B. (1973)Kinetic studies of the contractile mechanism of muscle. D.Phil. thesis, University of Oxford.Google Scholar
  26. MARSTON, S. B. (1980) Evidence for an altered structure of actin-S1 complexes when magnesium adenylylimidodiphosphate binds.J. Musc. Res. Cell Motility 1, 305–20.CrossRefGoogle Scholar
  27. MARSTON, S. B. (1982) The rates of formation and dissociation of actin-myosin complexes: effect of solvent, temperature, nucleotide binding and head-head interactions.Biochem. J. 203, 453–60.PubMedGoogle Scholar
  28. 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.PubMedCrossRefGoogle Scholar
  29. MARSTON, S. B., TREGEAR, R. T., RODGER, C. D. & CLARKE, M. L. (1979) Coupling between the enzymatic site of myosin and the mechanical output of muscle.J. molec. Biol. 128, 111–26.PubMedCrossRefGoogle Scholar
  30. MAUREL, P. (1978) Relevance of dielectric constant and solvent hydrophobicity to the organic solvent effect in enzymology.J. biol. Chem. 253, 1677–83.PubMedGoogle Scholar
  31. PERRIN, D. D. & SAYCE, I. G. (1967) Computer calculation of equilibrium concentrations of metal ions and complexing species.Talanta 14, 833–42.CrossRefPubMedGoogle Scholar
  32. PETTIT, L. D. & SIDDIQUI, K. F. (1976) The protein and metal complexes of adenyl-5-yl imidodiphosphate.Biochem. J. 159, 169–71.PubMedGoogle Scholar
  33. PODOLSKY, R. J., NAYLOR, G. R. S. & ARATA, T. (1982) Crossbridge properties in the rigor state. InBasic Biology of Muscles (edited by TWAROG, B., LEVINE, R. and DEWEY, M.), pp. 79–90. New York: Raven Press.Google Scholar
  34. REEDY, M. C., REEDY, M. K. & GOODY, R. S. (1983) Co-ordinated electron microscope and x-ray studies of glycerinated insect flight muscle. II. Electron microscopy and image reconstruction of muscle fibres fixed in rigor, in ATP and in AMPPNP.J. Musc. Res. Cell Motility 4, 55–81.CrossRefGoogle Scholar
  35. SHRIVER, J. W. & SYKES, B. D. (1981) Phosphorus-31 nuclear magnetic resonance evidence for two conformations of myosin subfragment-one nucleotide complexes.Biochemistry 20, 2004–12.PubMedCrossRefGoogle Scholar
  36. SILLEN, L. G. & MARTELL, A. E. (1971)Stability Constants of Metal Ion Complexes. Suppl. No. 1. London: The Chemical Society.Google Scholar
  37. TRAVERS, F. & HILLAIRE, D. (1979) Cryoenzymological studies on myosin subfragment-1.Eur. J. Biochem. 98, 293–9.PubMedCrossRefGoogle Scholar
  38. TREGEAR, R. T. (1981) Activation and action in insect flight muscle. InDevelopment and Specialisation of Skeletal Muscle (edited by GOLDSPRINK, D.), pp. 107–122. Cambridge: Cambridge University Press.Google Scholar
  39. TREGEAR, R. T., CLARKE, M. L., MARSTON, S. B., RODGER, C. D., BORDAS, J. & KOCH, M. (1982) A study of demembranated muscle fibres under equilibrium conditions. InBasic Biology of Muscles: A Comparative Approach (edited by TWAROG, B., LEVINE, R. and DEWEY, M.), pp. 131–141. New York: Raven Press.Google Scholar
  40. WELLS, J. A., SHELDON, M. & YOUNT, R. G. (1980) Magnesium nucleotide is stoichiometrically trapped at the active site of myosin and its active proteolytic fragments by thiol cross-linking reagents.J. biol. Chem. 255, 1598–602.PubMedGoogle Scholar
  41. WILKINSON, C. N. (1961) Statistical estimations in enzyme kinetics.Biochem. J. 80, 324–32.PubMedGoogle Scholar

Copyright information

© Chapman and Hall Ltd 1984

Authors and Affiliations

  • M. L. Clarke
    • 1
  • C. D. Rodger
    • 1
  • R. T. Tregear
    • 1
  1. 1.Biophysics UnitARC Institute of Animal PhysiologyCambridgeUK

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