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. 1990 Aug 1;111(2):453–463. doi: 10.1083/jcb.111.2.453

Smooth muscle myosin cross-bridge interactions modulate actin filament sliding velocity in vitro

PMCID: PMC2116193  PMID: 2143195

Abstract

Although it is generally believed that phosphorylation of the regulatory light chain of myosin is required before smooth muscle can develop force, it is not known if the overall degree of phosphorylation can also modulate the rate at which cross-bridges cycle. To address this question, an in vitro motility assay was used to observe the motion of single actin filaments interacting with smooth muscle myosin copolymers composed of varying ratios of phosphorylated and unphosphorylated myosin. The results suggest that unphosphorylated myosin acts as a load to slow down the rate at which actin is moved by the faster cycling phosphorylated cross-bridges. Myosin that was chemically modified to generate a noncycling analogue of the "weakly" bound conformation was similarly able to slow down phosphorylated myosin. The observed modulation of actin velocity as a function of copolymer composition can be accounted for by a model based on mechanical interactions between cross-bridges.

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Selected References

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  1. Brenner B., Schoenberg M., Chalovich J. M., Greene L. E., Eisenberg E. Evidence for cross-bridge attachment in relaxed muscle at low ionic strength. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7288–7291. doi: 10.1073/pnas.79.23.7288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Butler T. M., Siegman M. J., Davies R. E. Rigor and resistance to stretch in vertebrate smooth muscle. Am J Physiol. 1976 Nov;231(5 Pt 1):1509–1514. doi: 10.1152/ajplegacy.1976.231.5.1509. [DOI] [PubMed] [Google Scholar]
  3. Butler T. M., Siegman M. J. High-energy phosphate metabolism in vascular smooth muscle. Annu Rev Physiol. 1985;47:629–643. doi: 10.1146/annurev.ph.47.030185.003213. [DOI] [PubMed] [Google Scholar]
  4. Butler T. M., Siegman M. J., Mooers S. U. Slowing of cross-bridge cycling in smooth muscle without evidence of an internal load. Am J Physiol. 1986 Dec;251(6 Pt 1):C945–C950. doi: 10.1152/ajpcell.1986.251.6.C945. [DOI] [PubMed] [Google Scholar]
  5. Chalovich J. M., Greene L. E., Eisenberg E. Crosslinked myosin subfragment 1: a stable analogue of the subfragment-1.ATP complex. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4909–4913. doi: 10.1073/pnas.80.16.4909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clark T., Ngai P. K., Sutherland C., Gröschel-Stewart U., Walsh M. P. Vascular smooth muscle caldesmon. J Biol Chem. 1986 Jun 15;261(17):8028–8035. [PubMed] [Google Scholar]
  7. Cohen D. M., Murphy R. A. Cellular thin filament protein contents and force generation in porcine arteries and veins. Circ Res. 1979 Nov;45(5):661–665. doi: 10.1161/01.res.45.5.661. [DOI] [PubMed] [Google Scholar]
  8. Dillon P. F., Aksoy M. O., Driska S. P., Murphy R. A. Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science. 1981 Jan 30;211(4481):495–497. doi: 10.1126/science.6893872. [DOI] [PubMed] [Google Scholar]
  9. Edman K. A. The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibres. J Physiol. 1979 Jun;291:143–159. doi: 10.1113/jphysiol.1979.sp012804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eisenberg E., Hill T. L., Chen Y. Cross-bridge model of muscle contraction. Quantitative analysis. Biophys J. 1980 Feb;29(2):195–227. doi: 10.1016/S0006-3495(80)85126-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Greene L. E., Sellers J. R., Eisenberg E., Adelstein R. S. Binding of gizzard smooth muscle myosin subfragment 1 to actin in the presence and absence of adenosine 5'-triphosphate. Biochemistry. 1983 Feb 1;22(3):530–535. doi: 10.1021/bi00272a002. [DOI] [PubMed] [Google Scholar]
  12. Hai C. M., Murphy R. A. Regulation of shortening velocity by cross-bridge phosphorylation in smooth muscle. Am J Physiol. 1988 Jul;255(1 Pt 1):C86–C94. doi: 10.1152/ajpcell.1988.255.1.C86. [DOI] [PubMed] [Google Scholar]
  13. Harada Y., Noguchi A., Kishino A., Yanagida T. Sliding movement of single actin filaments on one-headed myosin filaments. Nature. 1987 Apr 23;326(6115):805–808. doi: 10.1038/326805a0. [DOI] [PubMed] [Google Scholar]
  14. Kamm K. E., Stull J. T. The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol. 1985;25:593–620. doi: 10.1146/annurev.pa.25.040185.003113. [DOI] [PubMed] [Google Scholar]
  15. Kishino A., Yanagida T. Force measurements by micromanipulation of a single actin filament by glass needles. Nature. 1988 Jul 7;334(6177):74–76. doi: 10.1038/334074a0. [DOI] [PubMed] [Google Scholar]
  16. Kron S. J., Spudich J. A. Fluorescent actin filaments move on myosin fixed to a glass surface. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6272–6276. doi: 10.1073/pnas.83.17.6272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Margossian S. S., Lowey S. Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol. 1982;85(Pt B):55–71. doi: 10.1016/0076-6879(82)85009-x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Paul R. J., Glück E., Rüegg J. C. Cross bridge ATP utilization in arterial smooth muscle. Pflugers Arch. 1976 Feb 24;361(3):297–299. doi: 10.1007/BF00587295. [DOI] [PubMed] [Google Scholar]
  20. Pemrick S., Weber A. Mechanism of inhibition of relaxation by N-ethylmaleimide treatment of myosin. Biochemistry. 1976 Nov 16;15(23):5193–5198. doi: 10.1021/bi00668a038. [DOI] [PubMed] [Google Scholar]
  21. Perrie W. T., Perry S. V. An electrophoretic study of the low-molecular-weight components of myosin. Biochem J. 1970 Aug;119(1):31–38. doi: 10.1042/bj1190031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reiser P. J., Kasper C. E., Greaser M. L., Moss R. L. Functional significance of myosin transitions in single fibers of developing soleus muscle. Am J Physiol. 1988 May;254(5 Pt 1):C605–C613. doi: 10.1152/ajpcell.1988.254.5.C605. [DOI] [PubMed] [Google Scholar]
  23. Sellers J. R. Mechanism of the phosphorylation-dependent regulation of smooth muscle heavy meromyosin. J Biol Chem. 1985 Dec 15;260(29):15815–15819. [PubMed] [Google Scholar]
  24. Sellers J. R., Pato M. D., Adelstein R. S. Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin. J Biol Chem. 1981 Dec 25;256(24):13137–13142. [PubMed] [Google Scholar]
  25. Sellers J. R., Spudich J. A., Sheetz M. P. Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments. J Cell Biol. 1985 Nov;101(5 Pt 1):1897–1902. doi: 10.1083/jcb.101.5.1897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sheetz M. P., Spudich J. A. Movement of myosin-coated fluorescent beads on actin cables in vitro. Nature. 1983 May 5;303(5912):31–35. doi: 10.1038/303031a0. [DOI] [PubMed] [Google Scholar]
  27. Siegman M. J., Butler T. M., Mooers S. U., Davies R. E. Chemical energetics of force development, force maintenance, and relaxation in mammalian smooth muscle. J Gen Physiol. 1980 Nov;76(5):609–629. doi: 10.1085/jgp.76.5.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Somlyo A. V., Goldman Y. E., Fujimori T., Bond M., Trentham D. R., Somlyo A. P. Cross-bridge kinetics, cooperativity, and negatively strained cross-bridges in vertebrate smooth muscle. A laser-flash photolysis study. J Gen Physiol. 1988 Feb;91(2):165–192. doi: 10.1085/jgp.91.2.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stone D. B., Prevost S. C. Characterization of modified myosin at low ionic strength. Enzymatic and spin-label studies. Biochemistry. 1973 Oct 9;12(21):4206–4211. doi: 10.1021/bi00745a026. [DOI] [PubMed] [Google Scholar]
  30. TAUSSKY H. H., SHORR E. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem. 1953 Jun;202(2):675–685. [PubMed] [Google Scholar]
  31. Toyoshima Y. Y., Kron S. J., McNally E. M., Niebling K. R., Toyoshima C., Spudich J. A. Myosin subfragment-1 is sufficient to move actin filaments in vitro. Nature. 1987 Aug 6;328(6130):536–539. doi: 10.1038/328536a0. [DOI] [PubMed] [Google Scholar]
  32. Trybus K. M. Filamentous smooth muscle myosin is regulated by phosphorylation. J Cell Biol. 1989 Dec;109(6 Pt 1):2887–2894. doi: 10.1083/jcb.109.6.2887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Trybus K. M., Henry L. Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin. J Cell Biol. 1989 Dec;109(6 Pt 1):2879–2886. doi: 10.1083/jcb.109.6.2879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Trybus K. M., Lowey S. Conformational states of smooth muscle myosin. Effects of light chain phosphorylation and ionic strength. J Biol Chem. 1984 Jul 10;259(13):8564–8571. [PubMed] [Google Scholar]
  35. Warshaw D. M., Fay F. S. Cross-bridge elasticity in single smooth muscle cells. J Gen Physiol. 1983 Aug;82(2):157–199. doi: 10.1085/jgp.82.2.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Warshaw D. M. Force: velocity relationship in single isolated toad stomach smooth muscle cells. J Gen Physiol. 1987 May;89(5):771–789. doi: 10.1085/jgp.89.5.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. White H. D. Special instrumentation and techniques for kinetic studies of contractile systems. Methods Enzymol. 1982;85(Pt B):698–708. doi: 10.1016/0076-6879(82)85057-x. [DOI] [PubMed] [Google Scholar]
  38. Work S. S., Warshaw D. M. Detection of surface movements on single smooth muscle cells: digital video microscopy. Comput Biol Med. 1988;18(6):385–393. doi: 10.1016/0010-4825(88)90056-x. [DOI] [PubMed] [Google Scholar]
  39. Yamakawa M., Harris D. E., Fay F. S., Warshaw D. M. Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium. J Gen Physiol. 1990 Apr;95(4):697–715. doi: 10.1085/jgp.95.4.697. [DOI] [PMC free article] [PubMed] [Google Scholar]

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