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

The Ca(2+)-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 Ca(2+)-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.