Abstract
We have used electron paramagnetic resonance (EPR) spectroscopy to monitor the orientation of spin labels attached specifically to a reactive sulfhydryl on the myosin heads in glycerinated rabbit psoas skeletal muscle. Previous work has shown that the paramagnetic probes are highly ordered in rigor muscle and display a random angular distribution in relaxed muscle (Thomas and Cooke, 1980). Addition of ADP to rigor fibers caused no spectral changes, while addition of AMPPNP or PPi increased the fraction of disordered probes. We show here that the application of stress to fibers in the presence of ADP, AMPPNP or PH causes no change in their spectra. During the generation of isometric tension approximately 80% of the probes display a random angular distribution as in relaxed muscle while the remaining 20% are highly oriented at the same angle as found in rigor muscle. In each of the above cases the spectrum consists of two components, one highly ordered as in rigor and one highly disordered. Saturation transfer EPR has shown that the ordered component is rigid while the disordered component is mobile on the microsecond time scale (Thomas, Ishiwata, Seidel and Gergely, 1980). These data lead to the conclusion that the disordered spectral component arises from myosin heads that are detached from actin while the ordered component comes from heads that are attached to actin. The observation that the ordered component displays an identical angular distribution under all conditions indicates that its orientation is not linked to force generation.
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References
Aronson, J.F., and Morales, M.F. (1989). Polarization of tryptophan fluorescence in muscle. Biochemistry 8: 4517–4522.
Barden, J., and Mason, P. (1978). Muscle cross-bridge stroke and activity revealed by optical diffraction. Science ( Wash. D.C. ). 199: 1212–1213.
Barnett, V.A., and Thomas, D.D. (1982). STEPR of spin labeled fibers; dependence on sarcomere length. J. Mol. Biol., submitted.
Borejdo, J. and Putnam, S. (1977). Polarization of fluorescence from single skinned glyceri- nated rabbit psoas fibers in rigor and relaxation. Biochem. Biophys. Acta 459: 578.
Borejdo, J., Putnam, S., Morales, M.F. (1979). Fluctuations in polarized fluorescence: Evidence that muscle cross-bridges rotate repetitively during contraction. Proc. Natl. Acad. Sci. USA 76: 8346.
Cooke, R. (1981). Stress does not alter the conformation of a domain of the myosin cross-bridge in rigor muscle fibers. Nature 294: 570–571.
Cooke, R. and Franks, K. (1978). Generation of force by single-headed myosin. J. Mol. Biol. 120: 361.
Curtin, N.A., Gilbert, C., Kretzschmar, K.M. and Wilkie, D.R. (1974). The effect of the performance of work on total energy output and metabolism during muscular contraction. J. Physiol. 238: 455.
Chalovich, J.M. and Eisenberg, E. (1982). Inhibition of actomyosin ATPase activity by troponin without blocking the binding of myosin to actin. J. Biol. Chem. 257: 2432.
Dos Remedios, C.G., Millikan, R.G.C., Morales, M.F., (1972). Polarization of tryptophan fluorescence from single striated muscle fibers. J. Gen. Physiol. 59: 103–120.
Eisenberg, E., Hill, T.L. and Chen, Y. (1980). Cross-bridge model of contraction: quantitative analysis. Biophys. J. 29: 195–227.
Goldman, Y.E. and Simmons, R.M. (1977). Active and rigor muscle stiffness. J. Physiol. 269: 55P - 57 P.
Haselgrove, J. and Huxley, H.E. (1973). X-ray evidence for radial cross-bridge movement and for the sliding filament model in actively contracting skeletal muscle. J. Mol. Biol. 77: 549.
Huxley, H.E. (1969). The mechanism of muscular contraction. Science ( Wash. D.C. ) 114: 1356–1366.
Huxley, A.F. and Simmons, R.M. (1971). Proposed mechanism of force generation in striated muscle. Nature 233: 533–538.
Huxley, H.E., Simmons, R.M., Faruqi, A.R., Kress, M., Bordas, J. and Koch, M.H. (1981). Millisecond time-resolved changes in X-ray reflections from contracting muscle during rapid mechanical transients, recorded using synchrotron radiation. Proc. Natl. Acad. Sei. USA 78: 2297.
Ishiwata, S., Seidel, J. and Gergely, J. (1979). Regulation by Ca ions of cross-bridge attachment in myofibrils studied by STEPR. Biophys. J. 25: 19a
Mendelson, R.A., Morales, M.F. and Botts, J. (1973). Segmental flexibility of the S-1 moiety of myosin. Biochemistry 12: 2250–2255.
Mendelson, R.A. and Wilson, M.G.A. (1982). Three dimensional disorder of dipolar probes in a helical array: application to muscle crossbridges. Biophys J. (in press).
Podolsky, R.J. and Nolan, A.C. (1972). Muscle contraction transients, cross-bridge kinetics, and the Fenn Effect. Cold Spring Harbor Symp. Quant. Biol. 37: 661.
Reedy, M.K., Holmes, K.C. Tregear, R.T. (1965). Induced changes in orientation of the crossbridges of glycerinated insect flight muscle. Nature (tond) 207: 1276–1280.
Thomas, D.D., Seidel, J.C., Gergely, J. and Hyde, J.S. (1975). Motion of subfragment-1 in myosin and its supramolecular complexes: saturation transfer electron paramagnetic resonance. Proc. Natl. Acad. Sci. USA 72: 1729–1733.
Thomas, D.D. and Cooke, R. (1980). Orientation of spin-labeled myosin heads in glycerinated muscle fibers. Biophys. J. 32: 891–906.
Thomas, D.D., Ishiwata, S., Seidel, J.C. and Gergely, J. (1980). Submillisecond rotational dynamics of spin-labeled crossbridges in myofibrils. Biophys. J. 32: 873–890.
Thomas, D.D., Barnett, B. an Cooke, R. (1982). Spectra of spin labels attached to insect flight muscle. J. Muscle Research and Cell Motility, (in press).
Yanagida, T. (1981). Angles of nucleotides bound to cross-bridges in glycerinated muscle fibers at various concentrations of a-ATP, a-ADP and s-AMPPNP detected by polarized fluorescence. J. Mol. Biol. 148: 539.
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© 1984 Plenum Press, New York
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Cooke, R., Crowder, M.S., Wendt, C.H., Barnett, V.A., Thomas, D.D. (1984). Muscle Cross-Bridges: Do They Rotate?. In: Pollack, G.H., Sugi, H. (eds) Contractile Mechanisms in Muscle. Advances in Experimental Medicine and Biology, vol 37. Springer, Boston, MA. https://doi.org/10.1007/978-1-4684-4703-3_37
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DOI: https://doi.org/10.1007/978-1-4684-4703-3_37
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