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Fig. M10. Electron micrograph of myosin filament (From Huxley, H.E., reprinted with permission from the Structure and Function of Muscle, G. H. Bourne , Ed. , 1972, by Academic Press.).

A model for the arrangement of myosin molecules in the filaments is shown in Fig. M11. Since individual myosin molecules have a globular region (S1) at one end only, the filaments are formed probably by antiparallel association of myosin molecules. All the molecules in one half filament are oriented in one direction and all those in the other half of the filament are oriented in the opposite direction. Thus, in the middle of the filament the tails of antiparallel molecules overlap yielding a bare central shaft, and globular regions are projected at both ends of the filament. During contraction the S1 molecules (the cross bridges) are bending and this is illustrated by the dashed S1 molecules.

Fig. M11. Model of myosin filament (Courtesy of Dr. Helen Rarick).

Electron micrographs of thick filaments from muscle and synthetic thick filaments made from myosin are indistinguishable. However, synthetic thick filaments made from light meromyosin have no projections, as shown by electron microscopy.

To understand how myosin filaments function in contraction, it is necessary to know their native structure, composition, and biochemistry. This requires isolation of native myosin filaments directly from muscle and their purification free of thin filaments and contaminating proteins (Hidalgo et al., 2001).

Muscle fibers, myofibrils: Actomyosin threads produce much less tension than intact muscle and this initiated research on muscle fibers. In his classical experiments Szent-Gyorgyi divided rabbit psoas muscle in situ into fiber bundles about 1 mm in diameter. These were tied at resting length to a thin stick and placed in 50% glycerol at 0o C for 24 h. After exchanging the glycerol, the fiber bundles were stored at -20o C. Before use, the bundles were transferred to 20% glycerol, then washed with saline. The prolonged glycerol treatment destroys the muscle cell membrane, and the subsequent washing removes the inorganic and organic constituents of the muscle and over half of the sarcoplasmic proteins. Glycerol-treated psoas fibers no longer react to electrical stimulation, but upon addition of ATP produce powerful contraction.

H.H. Weber and Portzehl (1954) prepared single muscle fibers from glycerol treated psoas muscles that developed tension equal to the intact muscle and reproduced the entire contraction-relaxation cycle of the muscle (Fig. M12). Thus, it was proven without doubt that the interaction between actin, myosin and ATP is the basic mechanism for the contraction-relaxation cycle in skeletal muscle.

Fig. M12. Contraction-relaxation-contraction cycle of a single psoas fiber. The fiber was contracted by ATP and at the top of contraction washed with saline. The fiber was relaxed by adding pyrophosphate to the bath, arrow down, then recontracted by the addition of ATP, arrow up. (From Weber and Portzehl, 1954).

Myofibrils are tiny muscle fibers, prepared by homogenization of freshly dissected muscle in physiological salt solution. Their ATP-induced contraction can be followed under the microscope.

Both psoas fibers and myofibrils contain the contractile (myosin and actin) and the regulatory proteins (tropomyosin and troponin) of muscle. The individual component of these systems are well resolved by SDS-PAGE (Fig. M13).

Pearson Tropomyosin

Fig. M13. SDS-PAGE of purified skeletal muscle myofibrils.(Reprinted from Biochim. Biophys. Acta vol. 490, Porzio, M.A. and Pearson, A.M. " Improved resolution of myofibrillar proteins with sodium dodecyl sulfate gel electrophoresis," pp. 27-34, Copyright

1977, with permission from Elsevier Sciences).

Fig. M13. SDS-PAGE of purified skeletal muscle myofibrils.(Reprinted from Biochim. Biophys. Acta vol. 490, Porzio, M.A. and Pearson, A.M. " Improved resolution of myofibrillar proteins with sodium dodecyl sulfate gel electrophoresis," pp. 27-34, Copyright

1977, with permission from Elsevier Sciences).

Not shown in Fig. M13 are the thick filament proteins, titin, H protein, M band protein, and the thin filament protein nebulin.

Localization of myosin in the structure of muscle: Myofibrils were extracted under the microscope with a solution selective for myosin dissolution. The A-band disappeared as a result of the extraction; hence the conclusion was reached that myosin is localized in the A-band, the darkly staining part of the muscle. Antibodies, specific for myosin that were deposited in the A-band also confirmed the localization.

References,

Barany, M. (1967). ATPase activity of myosin correlated with speed of muscle shortening. J. Gen. Physiol. 50, 197-218.

Barany, M. and Barany, K. (1959). Studies on "active centers" of L-myosin. Biochim. Biophys. Acta, 35, 293-309.

Barany, M. and Close, R. I. (1971). The transformation of myosin in cross-innervated rat muscles. J. Physiol. 213, 455-474.

Engelhardt, V.A. and Lyubimova, M.N. (1939). Myosin and adenosinetriphosphatase. Nature, 144, 668.

Geeves, M.A. and Holmes, K.C. (1999). Structural mechanism of muscle contraction. Annu. Rev. Biiochem., 68, 687-728.

Hidalgo, C., Padron, R., Horowitz, R., Zhao, F-G., and Craig, R. (2001). Purification of native myosin filaments from muscle. Biophys. J. 81, 2817-2826.

Houdusse, A. and Cohen, C. (1996). Structure of the regulatory domain of scallop myosin at 2 angstrom resolution. Structure, 4, 21-32.

Huxley, H.E. (1972). Molecular basis for contraction in cross-striated muscles. ]n The structure and function of muscle, ( G.H. Bourne, Ed.) second edition, vol. I, Part 1, pp. 301-387.

Joel, P.B., Trybus, K.M., and Sweeney, H.L. (2001). Two conserved lysines at the 50/20-kDa junction of myosin are necessary for triggering actin activation. J. Biol. Chem. 276, 2998-3003.

Knetsch, M. L.W., Uyeda, T.Q. P., and Manstein, D.J. (1999). Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction. J. Biol. Chem. 274, 20133-20138.

Lowey, S., Slayter, H.S., Weeds, A.G., and Baker, H. (1969). Substructure of the myosin molecule I. Subfragments of myosin by enzymic degradation. J. Mol. Biol., 42, 1-20.

Lymn, R.W. and Taylor, E.W. (1970). Transient state phosphate production in the hydrolysis of nucleoside triphosphates by myosin. Biochemistry, 9, 2975-2983.

Nitao, L.K., and Reisler, E. (1998). Probing the conformational states of the SH1-SH2 helix in myosin: a cross-linking approach. Biochemistsry, 37, 16704-16710.

Perry, S.V. (1996). Molecular mechanisms in striated muscle. Cambridge University Press.

Porzio, M.A. and Pearson, A.M. (1977). Improved resolution of myofibrillar proteins with sodium dodecylsulfate polyacrylamide gel electrophoresis. Biochim. Biophys. Acta, 490, 27-34.

Rayment, I., Rypniewski, W.R., Schmidt-Base, K., Smith, R., Tomchick, D.R., Benning, M.M., Winkelman, D.A., Wesenberg, G., and Holden, H.M. (1993). Three-dimensional structure of myosin subfragment-1: a molecular motor. Science, 261, 50-58.

Reiser, P.J., Moss, R.L., Giulian, G.G., and Greaser, M.L. (1985). Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition. J. Biol. Chem. 260, 9077- 9080.

Sellers, J.R. and Goodson, H.V. (1995). Motor proteins 2: myosin. Protein Profile, 2, 1323-1423.

Taylor, E.W., Lymn, R.W., and Moll, G. (1970). Myosin-product complex and its effect on the steady-state rate of nucleoside triphosphate hydrolysis. Biochemistry, 9, 2984-2991.

Weber, H.H. and Portzehl, H. (1954). The transference of the muscle energy in the contraction cycle. Progress in Biophysics and Biophysical Chemistry, 4, 60-111.

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