CA2-C IN THE MYOFILAMENT LATTICE AND ITS RELATION TO THE MYOFIBRILLARATPASE ACTIVITY( BINDING TO CARDIAC TROPONIN)

The Ca2+-binding properties of troponin C in the intact myofilament la ttice and their relation to the activation of ATPase were investigated with isolated porcine cardiac myofibrils. Ca2+ binding, which is comp osed of two classes of binding sites with different affinities (classe s 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 sit es (sites III and TV) and a single low-affinity site (site II) of trop onin C. In the presence of ATP, positive cooperativity was observed in the Ca2+ binding of class-2 sites and the Hill equation parameters we re in excellent agreement with those for the Ca2+-activated myofibrill ar ATPase activity, which indicated that the activation of ATPase is a linear function of the Ca2+ occupancy of site II. In the absence of A TP, a marked increase in the affinity of only class-2 sites was observ ed while the cooperativity was lost. These results provide direct evid ence that some feedback mechanism exists between myosin crossbridge at tachment and the Ca2+ binding to site II of troponin C, which may thus confer positive cooperativity on the Ca2+ activation of myofibrillar ATPase activity.