Journal of Muscle Research & Cell Motility

, Volume 19, Issue 8, pp 825–837

A 7-amino-acid insert in the heavy chain nucleotide binding loop alters the kinetics of smooth muscle myosin in the laser trap

  • Anne-Marie Lauzon
  • Matthew J. Tyska
  • Arthur S. Rovner
  • Yelena Freyzon
  • David M. Warshaw
  • Kathleen M. Trybus


Two smooth muscle myosin heavy chain isoforms differ by a 7-amino- acid insert in a flexible surface loop located near the nucleotide binding site. The non-inserted isoform is predominantly found in tonic muscle, while the inserted isoform is mainly found in phasic muscle. The inserted isoform has twice the actin-activated ATPase activity and actin filament velocity in the in vitro motility assay as the non-inserted isoform. We used the laser trap to characterize the molecular mechanics and kinetics of the inserted isoform ((+)insert) and of a mutant lacking the insert ((−)insert), analogous to the isoform found in tonic muscle. The constructs were expressed as heavy meromyosin using the baculovirus/insect cell system. Unitary displacement (d) was similar for both constructs (∼10nm) but the attachment time (ton for the (−)insert was twice as long as for the (+)insert regardless of the [MgATP]. Both the relative average isometric force (Favg(−insert)/Favg(+insert))=1.1±0.2 (mean±se) using the in vitro motility mixture assay, and the unitary force (F∼1pN) using the laser trap, showed no difference between the two constructs. However, as under unloaded conditions, ton under loaded conditions was longer for the (−)insert compared with the (+)insert construct at limiting [MgATP]. These data suggest that the insert in this surface loop does not affect the mechanics but rather the kinetics of the cross-bridge cycle. Through comparisons of ton from d measurements at various [MgATP], we conclude that the insert affects two specific steps in the cross-bridge cycle, that is, MgADP release and MgATP binding.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. BABIJ, P. (1993) Tissue-specific and developmentally regulated alternative splicing of a visceral isoform of smooth muscle myosin heavy chain. Nucleic Acid Res. 21, 1467–71.Google Scholar
  2. BOBKOV, A. A., BOBKOVA, E. & REISLER, E. (1996) The role of surface loops (residues 204–216 and 627–646) in the motor function of the myosin head. Proc. Natl Acad. Sci. USA 93, 2285–9.Google Scholar
  3. COOKE, R. (1997) Actomysin interaction in striated muscle. Physiol. Rev. 77, 671–97.Google Scholar
  4. CREMO, C. R. & GEEVES, M. A. (1998) Interaction of actin and ADP with the head domain of smooth muscle myosin: implications for strain-dependent ADP release in smooth muscle. Biochem. 37, 1969–78.Google Scholar
  5. CUDA, G., PATE, E., COOKE, R. & SELLERS, J. R. (1997) In vitro actin filament sliding velocities produced by mixtures of different types of myosin. Biophys. J. 72, 1767–79.Google Scholar
  6. DUPUIS, D. E., GUILFORD, W. H., WU, J. & WARSHAW, D. M. (1997) Actin filament mechanics in the laser trap. J. Muscle Res. Cell Motil. 18, 17–30.Google Scholar
  7. EDDINGER, T. J. & MURPHY, R. A. (1988) Two smooth muscle myosin heavy chains differ in their light meromyosin fragment. Biochem. 27, 3807–11.Google Scholar
  8. FINER, J. T., SIMMONS, R. M. & SPUDICH, J. A. (1994) Single myosin molecule mechanics: piconewton forces and nanometer steps. Nature 386, 113–9.Google Scholar
  9. FUGLSANG, A., KHROMOV, A., TÖRÖK, K., SOMLYO, A. V. & SOMLYO, A. P. (1993) Flash photolysis studies of relaxation and cross-bridge detachment: higher sensitivity of tonic than phasic smooth muscle to MgADP. J. Muscle Res. Cell Motil. 14, 666–73.Google Scholar
  10. GOLDMAN, Y. E. (1987) Kinetics of the actomyosin ATPase in muscle fibers. Ann. Rev. Physiol. 49, 637–54.Google Scholar
  11. GUILFORDW. H., DUPUIS, D. E., KENNEDY, G., WU, J., PATLAK, J. B. & WARSHAW, D. M. (1997) Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap. Biophys. J. 72, 1006–21.Google Scholar
  12. HARRIS, D. E. & WARSHAW, D. M. (1993) Smooth and skeletal muscle actin are mechanically indistinguishable in the in vitro motility assay. Circ. Res. 72, 219–24.Google Scholar
  13. HARRIS, D. E., WORK, S. S., WRIGHT, R. K., ALPERT, N. R. & WARSHAW, D. M. (1994) Smooth, cardiac and skeletal muscle myosin force and motion generation assessed by cross-bridge mechanical interactions in vitro. J. muscle Res. Cell Motil 15, 11–19.Google Scholar
  14. HELLSTRAND, P. & PAULR. J. (1982) Vascular smooth muscle: relation between energy metabolism and mechanics. In Vascular Smooth Muscle: Metabolic, Ionic and Contractile Mechanism(edited by BARNES, C.D.) pp. 1–35. New York: Academic Press.Google Scholar
  15. HORIUTI, K., SOMLYO, A. V., GOLDMAN, Y. E. & SOMLYO, A. P. (1989) Kinetics of contraction initiated by flash photolysis of caged adenosine triphosphate in tonic and phasic smooth muscles. J. Gen. Physiol. 94, 769–81.Google Scholar
  16. HOROWITZ, A. & TRYBUS, K. M. (1992) Inhibition of smooth muscle myosin's activity and assembly by an anti-rod monoclonal antibody. J. Biol. Chem 267, 26091–6.Google Scholar
  17. HUXLEY, A. F. (1957) Muscle structure and theories of contraction. Prog. Biophys. 7, 255–317.Google Scholar
  18. KELLEY, C. A., TAKAHASHI, M., YU, J. H. & ADELSTEIN, R. S. (1993) An insert of seven amino acids confers functional differences between smooth muscle myosins from the intestines and vasculature. J. Biol. Chem. 268, 12848–54.Google Scholar
  19. KHROMOV, A. S., SOMLYO, A. V. & SOMLYO, A. P. (1996) Nucleotide binding by actomyosin as a determinant of relaxation kinetics of rabbit phasic and tonic smooth muscle. J. Physiol. 492, 669–73.Google Scholar
  20. MALMQVIST, U. & ARNER, A. (1991) Correlation between isoform composition of the 17 kDa myosin light chain and maximal shortening velocity in smooth muscle. Pflügers Arch. 418, 523–30.Google Scholar
  21. MARSTON, S. B., & TAYLOR, E. W. (1980) Comparison of the myosin and actomyosin ATPase mechanisms of the four types of vertebrate muscles. J. Mol. Biol. 139, 573–600.Google Scholar
  22. MEHTA, A. D., FINER, J. T. & SPUDICH, J. A. (1997) Detection of single-molecule interactions using correlated thermal diffusion. Proc. Natl Acad. Sci. USA 94, 7927–31.Google Scholar
  23. MOLLOY, J. E., BURNS, J. E., KENDRICK-JONES, J., TREGEAR, R. T. & WHITE, D. C. S. (1995) Movement and force produced by a single myosin head. Nature 378, 209–12.Google Scholar
  24. MURPHY, C. T. & SPUDICH, J. A. (1998) Dictyostelium myosin 25–50K loop substitutions specifically affect ADP release rates. Biochem. 37, 6738–44.Google Scholar
  25. MURPHY, R. A. (1976) Contractile system function in mammalian smooth muscle. Blood Vessels 13, 1–23.Google Scholar
  26. NAGAI, R., KURO-O, M., BABIJ, P. & PERIASAMY, M. (1989) Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis. J. Biol. Chem. 264, 9734–7.Google Scholar
  27. PARDEE, J. D. & SPUDICH, J. A. (1982) Purification of muscle actin. Methods Enzymol. 85, 164–81.Google Scholar
  28. PATLAK, J. B. (1993) Measuring kinetics of complex single ion channel data using mean-variance histograms. Biophys. J. 65, 29–42.Google Scholar
  29. PERREAULT-MICALE, C. L., KALABOKIS, V. N., NYITRAY, L. & SZENT-GYORGYI, A. G. (1996) Sequence variations in the surface loop near the nucleotide binding site modulate the ATP turnover rates of mulluscan myosins. J. Muscle Res. Cell Motil. 17, 543–53.Google Scholar
  30. RAYMENT, I., RYPNIEWSKI, W. R., SCHMIDT-BASE, K., SMITH, R., TOMCHICK, D. R., BENNING, M. M., WINKELMANN, D. A., WESENBERG, G. & HOLDEN, H. M. (1993) Three-dimensional structure of myosin subfragment-1; a molecular motor. Science 261, 50–65.Google Scholar
  31. ROVNER, A. S., FREYZON, Y. & TRYBUS, K. M. (1995) Chimeric substitutions of the actin-binding loop activate dephosphorylated but not phosphorylated smooth muscle heavy meromyosin. J. Biol. Chem. 270, 30260–3.Google Scholar
  32. ROVNER, A. S., FREYZON, Y. & TRYBUS, K. M. (1997) An insert in the motor domain determines the functional properties of expressed smooth muscle myosin isoforms. J. Muscle Res. Cell Motil. 18, 103–10.Google Scholar
  33. SOMLYO, A. P. (1993) Myosin isoforms in smooth muscle: how may they affect function and structure? J. Muscle Res. Cell Motil. 14, 557–63.Google Scholar
  34. SPUDICH, J. A. (1994) How molecular motors work? Nature 372, 515–8.Google Scholar
  35. SVOBODA, K., SCHMIDT, C. F., SCHNAPP, B. J. & BLOCK, S. M. (1993) Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–7.Google Scholar
  36. SWEENEY, H. L., ROSENFELD, S. S., BROWN, F., FAUST, L., SMITH, J, XING, J., STEIN, L. A. & SELLERS, J. R. (1998). Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket. J. Biol. Chem. 273, 6262–70.Google Scholar
  37. TRYBUS, K. M. & HENRY, L. (1989) Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin. J. Cell Biol. 109, 2879–86.Google Scholar
  38. UYEDA, T. Q. P., RUPPEL, K. M. & SPUDICH, J. A. (1994) Enzymatic activities correlate with chimaeric substitutions at the actin-binding face of myosin. Nature 368, 567–9.Google Scholar
  39. VANBUREN, P., HARRIS, D. E., ALPERT, N. R. & WARSHAW, D. M. (1995) Cardiac V1and V3myosins differ in their hydrolytic and mechanical activities in vitro. Circ. Res. 77, 439–44.Google Scholar
  40. VANBUREN, P., WORK, S. S. & WARSHAWD. M. (1994) Enhanced force generation by smooth muscle myosin in vitro. Proc. Natl Acad. Sci. USA 91, 202–5.Google Scholar
  41. WARSHAW, D. M., DESROSIERS, J. M., WORK, S. S. & TRYBUS, K. M. (1990) Smooth muscle myosin crossbridge interactions modulate actin filament sliding velocity in vitro J. Cell Biol. 111, 453–63.Google Scholar
  42. WARSHAW, D. M., HAYES, E., GAFFNEY, D., LAUZON, A.-M., WU., J., KENNEDY, G., TRYBUS, K. M., LOWEY, S. & BERGER, C. (1998) Myosin conformational states determined by single fluorophore polarization. Proc. Natl Acad. Sci. USA 95, 8034–9.Google Scholar
  43. WHITE, S., MARTIN, A. F. & PERIASAMY, M. (1993) Identi fication of a novel smooth muscle myosin heavy chain cDNA: isoform diversity in the S1 head region. Am. J. Physiol. 264, C1252–8.Google Scholar
  44. WORK, S. S. & WARSHAW, D. M. (1992) Computer-assisted tracking of actin filament motility. Analyt. Biochem. 202, 275–85.Google Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Anne-Marie Lauzon
  • Matthew J. Tyska
  • Arthur S. Rovner
  • Yelena Freyzon
  • David M. Warshaw
  • Kathleen M. Trybus

There are no affiliations available

Personalised recommendations