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(2+) 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(2+), S-1·ATP and S-1·ADP·P(i) bind to actin-troponin-tropomyosin (regulated actin) with almost the same affinity as in the presence of Ca(2+). 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(2+), the rate of ATP hydrolysis is more than 20 times greater than in the absence of Ca(2+). In contrast, the association constant of S-1·ATP and S-1·ADP·P(i) with regulated actin is virtually the same in the absence of Ca(2+) (1.4 × 10(4) m(−1)) as in the presence of Ca(2+) (1.5 × 10(4) m(−1)). Similarly, at 50 mm ionic strength, the ATPase rate is inhibited about 98% in the absence of Ca(2+) although the association constant is not significantly changed compared to that in the presence of Ca(2+). Finally, it has been shown that, at 18 mm ionic strength, the inhibition of the actin-activated ATPase rate in the absence of Ca(2+) is due to a large decrease in the maximum ATPase rate (to 4% of the Ca(2+) 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(2+), troponin-tropomyosin inhibits a kinetic step, perhaps P(i) release, in the cycle of ATP hydrolysis.