FIGURE 4.

Dose dependence of ATP and ADP on the rate of cross-bridge detachment. Panel A shows the effect of increasing ATP on the apparent rate (fast phase) of cross-bridge detachment from Ca²⁺-free TnC_IANBD^T53C rabbit myofibrils. TnC_IANBD^T53C rabbit myofibrils in Buffer A + 5 mm EGTA were mixed against equal volumes of Buffer A + 5 (0.51 ± 0.02/s), 10 (1.1 ± 0.2/s), 50 (5.5 ± 0.6/s), or 100 (16 ± 2/s) μm ATP (traces shown top to bottom, respectively). Panel B shows the triphasic fluorescence change that occurs upon ATP addition to the Ca²⁺-free TnC_IANBD^T53C rabbit myofibrils with increasing ATP from 5 to 100 μm over an extended period of time. The Ca²⁺-free base line over these long times displayed a linear decrease in fluorescence that was due to either the bleaching of the signal or myofibril settling, which was subtracted from the experimental traces. Buffer conditions were identical to those described for panel A. Panel C shows the dose-dependent effect that increasing ATP had on the apparent fast rate of cross-bridge detachment from Ca²⁺-free TnC_IANBD^T53C rabbit myofibrils. The inset shows the transmitted light microscopy image of the myofibrils after being mixed with 50 μm ATP in the stopped flow. Panel D shows the effect that increasing ADP had on the apparent fast rate of cross-bridge detachment of Ca²⁺-free TnC_IANBD^T53C rabbit myofibrils. Increasing concentrations of ADP (0–2000 μm) were added to the Ca²⁺-free TnC_IANBD^T53C myofibrils in Buffer A + 5 mm EGTA and rapidly mixed against Buffer A + 5 mm EGTA + 2 mm ATP at 15 °C. Both the concentrations of ATP and ADP shown in panels C and D were the final concentration after mixing in the stopped-flow. The top inset shows the transmitted light microscopy image of the myofibrils after being mixed with 2 mm ATP in the stopped flow. The bottom inset shows an enlarged myofibril from this population.