The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase

The cardiac troponin (Tn) complex, consisting of a Ca ²⁺-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca ²⁺ binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca ²⁺ concentration. Cardiac Tn binds 3 mol Ca ²⁺/mol and contains two Ca ²⁺-binding sites with a binding constant of 3 x 10 ⁸ M ^-1 and one binding site with a binding constant of 2 x 10 ⁶ M ^-1. In the presence of 4 mM MgCl ₂, the binding constant of the sites of higher affinity is reduced to 2 x 10 ⁷ M ^-1, while Ca ²⁺ binding to the site at the lower affinity is unaffected. The two high affinity Ca ²⁺ binding sites of cardiac Tn are analogous to the two Ca ⁺-Mg ²⁺ sites of skeletal Tn, while the single low affinity site is similar to the two Ca ²⁺ specific sites of skeletal Tn. The Ca ²⁺ binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca ²⁺/mol and contains two sites with a binding constant of 1 x 10 ⁷ M ^-1 and a single site with a binding constant of 2 x 10 ⁵ M ^-1. Assuming competition between Mg ²⁺ and Ca ²⁺ for the high affinity sites of TnC and Tn, the binding constants for Mg ²⁺ were 0.7 and 3.0 x 10 ³ M ^-1, respectively. The Ca ²⁺ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison of the Ca ²⁺ binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca ²⁺] and at millimolar [Mg ²⁺] suggests that activation of the ATPase occurs over the same range of [Ca ²⁺] where the Ca ²⁺-specific site of cardiac Tn binds Ca ²⁺.