Inhibition of actomyosin ATPase activity by troponin-tropomyosin without blocking the binding of myosin to actin.

Relaxation of vertebrate skeletal muscle is thought to occur in the absence of Ca as a result of tropomyosin physically blocking the binding of myosin to actin. This steric blocking model of muscle relaxation predicts that myosin subfragment 1 (S-1) will not bind to actin under conditions where the acto-S-1 ATPase rate is inhibited. Using stopped flow absorbance as a measure of binding, we have previously shown that when the rate of ATP hydrolysis is only 4% of the rate in the presence of Ca, S-1·ATP and S-1·ADP·P bind to actin-troponin-tropomyosin (regulated actin) with almost the same affinity as in the presence of Ca. This result has now been confirmed using sedimentation in an air-driven ultracentrifuge to directly measure the binding at pH 7.0, 25 °C, and μ = 18 mm. In the presence of Ca, the rate of ATP hydrolysis is more than 20 times greater than in the absence of Ca. In contrast, the association constant of S-1·ATP and S-1·ADP·P with regulated actin is virtually the same in the absence of Ca (1.4 × 10 m) as in the presence of Ca (1.5 × 10 m). Similarly, at 50 mm ionic strength, the ATPase rate is inhibited about 98% in the absence of Ca although the association constant is not significantly changed compared to that in the presence of Ca. Finally, it has been shown that, at 18 mm ionic strength, the inhibition of the actin-activated ATPase rate in the absence of Ca is due to a large decrease in the maximum ATPase rate (to 4% of the Ca value) with only a small change in the apparent binding constant of S-1 to actin. These data do not support a simple steric blocking model of muscle relaxation. Rather they suggest that, in the absence of Ca, troponin-tropomyosin inhibits a kinetic step, perhaps P release, in the cycle of ATP hydrolysis.