Abstract
Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%. Inhibitory caldesmon concentrations had little effect upon the rate of S1 binding to actin, actin-S1 dissociation by ATP, and dissociation of ADP from actin-S1 x ADP; however the rate of phosphate release from the actin-S1 x ADP x P(i) complex was decreased by more than 80%. In addition the transient of phosphate release displayed a lag of up to 200 ms. The presence of a lag phase indicates that a step on the pathway prior to phosphate release has become rate-limiting. Premixing the actin-tropomyosin filaments with myosin heads resulted in the disappearance of the lag phase. We conclude that caldesmon inhibition of the rate of phosphate release is caused by the thin filament being switched by caldesmon to an inactive state. The active and inactive states correspond to the open and closed states observed in skeletal muscle thin filaments with no evidence for the existence of a third, blocked state. Taken together these data suggest that at physiological concentrations, caldesmon controls the isomerization of the weak binding complex to the strong binding complex, and this causes the inhibition of the rate of phosphate release. This inhibition is sufficient to account for the inhibition of the steady state actomyosin ATPase by caldesmon and tropomyosin.
Highlights
The smooth muscle thin filament is Ca2ϩ-regulated and is made up of actin, tropomyosin, caldesmon, and a Ca2ϩbinding protein in molar ratios ϳ14:2:1:1 [7]
We have recently demonstrated a direct correlation between caldesmon inhibition of the actin-S1 ATPase and the conformational change accompanying the switch of the actin-tropomyosin complex to the OFF state, measured by the excimer fluorescence of pyrene iodoacetamide-labeled tropomyosin [18]
With the recent development of probes to monitor most of the elementary steps of the actomyosin ATPase, it is possible to attempt a detailed investigation of the effect of smooth muscle regulatory proteins on these steps
Summary
The smooth muscle thin filament is Ca2ϩ-regulated and is made up of actin, tropomyosin, caldesmon, and a Ca2ϩbinding protein in molar ratios ϳ14:2:1:1 [7]. A competition model proposes that ATPase inhibition by caldesmon is caused by a reduction in the formation of the weakly bound actomyosin complex as a result of caldesmon binding to the same site on actin as S11⁄7ADP1⁄7Pi [13] This is supported by the inability of caldesmon to inhibit the crosslinked actin-S1 complex, which does not dissociate on ATP binding [14] and the reduction of the Km of the steady state ATPase [8]. Transient kinetic studies of the effect of Ca2ϩ on thin filament activation of the S1 ATPase in skeletal muscle has been successful in probing the effect of the skeletal muscle regulatory protein complex, troponin-tropomyosin on the rate of the elementary steps of the actin-S1 ATPase (19 –24) They led to the proposition of a cooperative-allosteric model as a mechanism of regulation of skeletal muscle actin-S1 ATPase. The effect of caldesmon on subsequent steps of the actomyosin ATPase has not been investigated
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