Local melting in the subfragment-2 region of myosin in activated muscle and its correlation with contractile force

Local melting within the subfragment-2 region of activated rabbit skeletal glycerinated muscle fibers has been investigated over the temperature range 5 to 37 °C, using an enzyme (chymotrypsin)-probe method. The cleavage rates were determined from the time-course of formation of digestion products by electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. We found the cleavage sites to be localized in a restricted region M r = 64,000 to 90,000/polypeptide chain, measured from the C terminus of the myosin rod (the subfragment-2 hinge domain). The cleavage rate constant for activated muscle fibers in the presence of an ATP-regenerating system was about 100 times larger at each temperature than that for rigor or for relaxed muscle fibers and showed a marked increase in magnitude with increasing temperature. Comparative plots of the apparent rate-constant for cleavage within the subfragment-2 hinge domain and the isometric force generated by active fibers versus MgATP concentration gave closely similar profiles suggesting a strong positive correlation. Thus, there appears to be a close coupling between the conformational transition within the subfragment-2 hinge domain and contractile force when the cross-bridges undergo cycling.