Ca²+ and Mg²+ binding to troponin and the regulation of muscle contraction

Troponin (Tn) isolated as the complex of TnC, TnI and TnT binds 4 moles of Ca²⁺ per mole in the presence of 2 mM MgCl₂, 150 mM KCl, pH 7.0 with a single affinity constant of 5 x 10'M^-1. In the absence of Mg²⁺ Tn contains 2 classes of sites with n₁ = 2, K₁ = 5 x 10⁸-^-1; and n₂ = 2, K₂ = 5 x 10⁶-^-1. Purified TnC, the calcium binding component of Tn, contains two classes of binding sites with n₁ = 2, K₁ = 2 x 10⁶M^-1; and n₂ = 2, K₂ = 2 x 10⁵-^-1 (100 mM KCl, 2 mM MgCl₂, pH 7.0). In the absence of Mg²⁺ the higher affinity constant increases to 2x10⁷M^-1 with no effect on the sites of lower affinity. The complex of TnC and the inhibitory component TnI has the same binding characteristics as Tn. Mg²⁺ binding studies are consistent with there being competition between Mg²⁺(K=10³M^-1) and Ca²⁺. Studies of myofibrillar ATPase activity as a function of pCa at several pMg levels show that full activation occurs only when all 4 sites of TnC are occupied.