Stimulation of the ATP-dependent interaction between actin and myosin by a myosin-binding fragment of smooth muscle caldesmon.

We reported previously that smooth muscle caldesmon stimulates the ATP-dependent interaction between actin and phosphorylated smooth muscle myosin, as monitored by ATPase measurement and in vitro motility assay. Furthermore, this effect changes from stimulatory to inhibitory with increasing concentrations of caldesmon [Ishikawa et al., 1991: J. Biol. Chem. 266:21784-21790]. The N-terminal (myosin-binding) fragment and the C-terminal (actin-binding) fragment were purified from digests of caldesmon. The effects of the myosin-binding fragment and the actin-binding fragment on the interaction were stimulatory and inhibitory, respectively, indicating that stimulatory and inhibitory domains are localized in the myosin-binding domain and actin-binding domain of caldesmon, respectively. The effect of the myosin-binding fragment on the interaction was exclusively stimulatory when the interaction was challenged by caldesmon, both at lower and higher concentrations. However, the actin-binding fragment had no effect on the interaction at lower concentrations and inhibited the interaction at higher concentrations. Thus, the stimulatory effect of caldesmon that is observed at lower concentrations can be explained by the hypothesis that the stimulatory effect of the myosin-binding domain predominates over the inhibitory effect of the actin-binding domain when the concentration of caldesmon is low. With uncleaved caldesmon, we also emphasized the role of the myosin-binding domain in the stimulation as follows; the stimulatory effect of caldesmon became obscured when binding of caldesmon to myosin was competed by the exogenous caldesmon-binding fragment of myosin.