Summary
The change in orientation of myosin crossbridges in contracting muscle during sudden length changes was examined by fluorescence polarization. This study used a fluorescent ATP analogue, 1,N 6-etheno-2-aza-ATP(ε-2-aza-ATP) as a probe. Its fluorescence is considerably enhanced upon binding with myosin and is dependent on the chemical state of the myosin-nucleotide complex in muscle. The results showed that nucleotides bound to crossbridges in the intermediate attached state (presumably AM-ε-2-aza-ADP-Pi) during isometric contraction are highly oriented at the same angle as that of AM in rigor with bound ε-2-aza-ADP. Furthermore the orientation of nucleotides bound to crossbridges in the attached state is not altered during sudden changes in length of isometrically contracting muscle. The results of this time-resolved measurement support the conclusion obtained from a previous steady-state experiment that change in axial orientation of the active site of the myosin head is not involved in force generation.
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References
ARATA, T., MUKOHATA, Y. & TONOMURA, Y. (1977) Structure and function of the two heads of the myosin molecule VI. ATP hydrolysis, shortening and tension development of myofibrils.J. Biochem. 82, 801–12.
ARATA, T. & SHIMIZU, H. (1980) Spin-label study of actin-myosin-nucleotide interactions glycerinated muscle fibers.J. molec. Biol. 151, 411–37.
COOKE, R., CROWDER, M. S. & THOMAS, D. D. (1982) Orientation of spin-labels attached to crossbridges in contracting muscle fiber.Nature 300, 776–8.
HUXLEY, A. F. (1974) Review lecture on muscle contraction.J. Physiol. 243, 1–43.
HUXLEY, A. F. & SIMMONS, R. M. (1971) Proposed mechanism of force generation in striated muscle.Nature 233, 533–8.
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.
HUXLEY, H. E., SIMMONS, R. M., FARUQI, A. R., KRESS, M., BORDAS, J. & KOCH, M. H. J. (1981)Proc. natn. Acad. Sci. U.S.A. 78, 2297–301.
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.
MARSTON, S. B. & TREGEAR, R. T. (1972) Evidence for a complex between myosin and ADP in relaxed muscle fibers.Nature 235, 23–5.
MIYATA, H. & ASAI, H. (1982) 1,N 6-etheno-2-aza-adenosine triphosphate: Enormous increase in fluorescence intensity induced by its binding to heavy meromyosin and estimation of the kinetic parameters.Biochem. biophys. Res. Commun. 105, 269–302.
NAGANO, H. & YANAGIDA, T. (1984) Predominant attached state of myosin cross-bridges during contraction and relaxation at low ionic strength.J. molec. Biol. 177, 769–85.
PRÓCHNIEWICZ-NAKAYAMA, E. & YANAGIDA, T. (1984) Oriented polymerization of actin in muscle ghost fibre. InAbstracts of the 22nd Japan Biophysical Conference, p. 577.
THOMAS, D. D. & COOKE, R. (1980) Orientation of spin-labeled myosin heads in glycerinated muscle fibers.Biophys. J. 32, 891–906.
TREGEAR, R. T. & SQUIRE, J. M. (1973) Myosin content and filament structure in smooth and striated muscle.J. molec. Biol. 77, 279–90.
TUSO, K. C., MILLER, E. E. & LO, K. W. (1974) Synthesis of 1,N 6-etheno-2-aza-adenosine(2-aza-ε-adenosine): a new cytotaxic fluorescent nucleotide.Nucleic Acid Res. 1, 531–47.
WILSON, M. G. A. & MENDELSON, R. A. (1983) A comparison of order and orientation of crossbridges in rigor and relaxed muscle fibers using fluorescence polarization.J. Musc. Res. Cell Motility 4, 671–93.
YANAGIDA, T. (1980) The angles of cross-bridges bound to nucleotide at various concentrations of Mg-(ε)-ATP. InMuscle Contraction: Its Regulatory Mechanisms (edited by EBASHI, S., MARUYAMA, K. and ENDO, M.), pp. 173–80. Tokyo: Japanese Scientific Press/Berlin: Springer-Verlag.
YANAGIDA, T. (1981) Angles of nucleotides bound to cross-bridges in glycerinated muscle fiber at various concentrations of ε-ATP, ε-ADP and ε-AMPPNP detected by polarized fluorescence.J. molec. Biol. 146, 539–60.
YANAGIDA, T. (1984a) Neo-sliding mechanism of muscle contraction and cell motility. InAbstracts of the 10th Yamada International Conference on Cell Motility II: Mechanism and Regulation (edited by ISHIKAWA, H.). Tokyo: University of Tokyo Press. (In press).
YANAGIDA, T. (1984b) Angles of fluorescently labelled myosin heads and actin monomers in contracting and rigor stained muscle fiber. InCross-bridge Mechanisms in Muscle Contraction (edited by POLLACK, G. H. and SUGI, H.), pp. 397–411. New York: Plenum Press.
YANAGIDA, T., KURANAGA, I. & INOUE, A. (1982) Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength.J. Biochem. 92, 407–12.
YANAGIDA T. & OOSAWA, F. (1978) Polarized fluorescence from ε-ADP incorporated into F-actin in a myosin-free single fiber: conformation of F-actin and changes induced in it by heavy meromyosin.J. molec. Biol. 126, 507–24.
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Yanagida, T. Angle of active site of myosin heads in contracting muscle during sudden length changes. J Muscle Res Cell Motil 6, 43–52 (1985). https://doi.org/10.1007/BF00712310
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DOI: https://doi.org/10.1007/BF00712310