Calcium Binding to Troponin C and Troponin: Effects of Mg2+, Ionic Strength and pH1

Abstract

Calcium binding to troponin C and troponin was examined by a metallochromic indicator method under various conditions to obtain a further understanding of the regulatory roles of these proteins in muscle contraction. 1. Troponin C has four Ca binding sites, of which 2 sites have a high affinity of 4.5 × 10⁶ M⁻¹ for Ca²⁺ and the other 2 sites have a low affinity of 6.4 × 104 M⁻¹ in a reaction medium consisting of 100 mM KCl, 20 mM MOPS-KOH pH 6.80 and 0.13 mM tetramethylmurexide at 20°C. Magnesium also binds competitively to both the high and low affinity sites: the apparent binding constants are 1,000 M⁻² and 520 M⁻¹, respectively. Contrary to the claim by Potter and Gergely (J. Biol. Chem.250, 4628–4633, 1975), the low affinity sites are not specific only for Ca²⁺. 2. The high and low affinity sites of troponin C showed different dependence on the ionic strength: the high affinity sites were similar to GEDTA, while the low affinity sites were similar to calmodulin, which has a steeper ionic strength dependence than GEDTA. 3. Ca binding to troponin C was not affected by change of pH between 6.5 and 7.2. 4. Troponin I enhanced the apparent affinity of troponin C for Ca²⁺ to a value similar to that for troponin. Trifluoperazine also increased Ca binding to troponin C. 5. Troponin has four Ca binding sites as does troponin C, but the affinities are so high that the precise analysis was difficult by this method. The apparent binding constants for Ca²⁺ and Mg²⁺ were determined to be 3.5 × 10⁶ M−1 and 440 M−1, respectively, for low affinity sites under the same conditions as for troponin C, being independent of change in pH between 6.5 and 7.2. The competitive binding of Mg2+ to the low affinity sites of troponin is consistent with the results of Kohama (J. Biochem. 88, 591–599, 1980). The estimate for the high affinity sites is compatible with the reported results. 6. Stopped-flow analysis revealed that the association of troponin C with Ca²⁺ was so rapid that the process is diffusion-limited: the association rate constant was estimated to be 4 × 10⁷ M⁻¹.s⁻¹ or more at 20°C. This indicates that the apparent dissociation rate constant for the high affinity sites is as low as about 10 s⁻¹, while that for the low affinity sites is as high as a few hundred s⁻¹. In the presence of 10 mM Mg²⁺, the dissociation rate of Mg²⁺ was rate-limiting for Ca binding. These findings suggest that the rate of dissociation of Ca²⁺ from the high affinity sites may be too low to explain the rate of muscle relaxation.