Ca²⁺ Binding to Cardiac Troponin C in the Myofilament Lattice and Its Relation to the Myofibrillar ATPase Activity

The Ca²⁺‐binding properties of troponin C in the intact myofilament lattice and their relation to the activation of ATPase were investigated with isolated porcine cardiac myofibrils. Ca²⁺ binding, which is composed of two classes of binding sites with different affinities (classes 1 and 2), was clearly detected by a novel method for subtracting the large background activity of myofibrillar Ca²⁺ binding. The classes 1 and 2 were equivalent stoichiometrically to the two high‐affinity sites (sites III and IV) and a single low‐affinity site (site II) of troponin C. In the presence of ATP, positive cooperativity was observed in the Ca²⁺ binding of class‐2 sites and the Hill equation parameters were in excellent agreement with those for the Ca²⁺‐activated myofibrillar ATPase activity, which indicated that the activation of ATPase is a linear function of the Ca²⁺ occupancy of site II. In the absence of ATP, a marked increase in the affinity of only class‐2 sites was observed while the cooperativity was lost. These results provide direct evidence that some feedback mechanism exists between myosin crossbridge attachment and the Ca²⁺ binding to site II of troponin C, which may thus confer positive cooperativity on the Ca²⁺ activation of myofibrillar ATPase activity.