The fluorescence polarization from rhodamine labels specifically attached to the fast-reacting thiol of the myosin cross-bridge in glycerinated muscle fibers has been measured to determine the angular distribution of the cross-bridges in different physiological states of the fibers as a function of temperature. To investigate the fibers at temperatures below 0 °C, we have added glycerol to the bathing solution as an antifreezing agent. We find that the fluorescence polarization from the rhodamine probe detects distinct angular distributions of the cross-bridges in isometric-active, rigor, MgADP, and low ionic strength relaxed fibers at 4 °C. We also find that the rigor cross-bridges in the presence of glycerol can maintain at least two distinct orientations relative to the actin filament, one dominant at temperatures T>2 °C and another dominant at T <-10 °C. MgADP cross-bridges in the presence of glycerol maintain approximately the same orientation for all temperatures investigated. The rigor cross-bridge orientation at T <-10 °C is similar to both the MgADP cross-bridge orientation in the presence of glycerol and the active muscle cross-bridge orientation at 4 °C. These findings show that the rigor cross-bridge in the presence of glycerol has at least two distinct orientations while attached to actin: one of them dominant at high temperature, the other dominant at low temperature or when MgADP is present. The latter orientation resembles that present in isometric-active fibers. These findings suggest that force generation in the activated cross-bridge cycle may occur as a result of an actin-attached cross-bridge transition between these two orientations.
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