The effect of Mg²⁺ on the Ca²⁺ binding to troponin C in rabbit fast skeletal myofibrils

The effect of Mg²⁺ on the Ca²⁺ binding to rabbit fast skeletal troponin C and the Ca²⁺ dependence of myofibrillar ATPase activity was studied in the physiological state where troponin C was incorporated into myofibrils. The Ca²⁺ binding to troponin C in myofibrils was measured directly by ⁴⁵Ca using the CDTA-treated myofibrils as previously reported (Morimoto, S. and Ohtsuki, I. (1989) J. Biochem. 105, 435-439). It was found that the Ca²⁺ binding to the low and high affinity sites of troponin C in myofibrils was affected by Mg²⁺ competitively and the Ca²⁺- and Mg²⁺-binding constants were 6.20 · 10⁶ and 1.94 · 10² M^-1, respectively, for the low affinity sites, and 1.58 · 10⁸ and 1.33 · 10³ M^-1, respectively, for the high affinity sites. The Ca²⁺ dependence of myofibrillar ATPase was also affected by Mg²⁺, with the apparent Ca²⁺- and Mg²⁺-binding constants of 1.46 · 10⁶ and 2.76 · 10² M^-1, respectively, suggesting that the myofibrillar ATPase was modulated through a competitive action of Mg²⁺ on Ca²⁺ binding to the low affinity sites, though the Ca²⁺ binding to the low affinity sites was not simply related to the myofibrillar ATPase.