Role of the High Affinity Ca Binding Sites of Cardiac and Fast Skeletal Troponins1

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Abstract
The mode of calcium binding to cardiac and fast skeletal troponins was studied at various proton and Mg ion concentrations, and the results thus obtained were compared with the superprecipitation of cardiac and fast skeletal myosin B under specified conditions. The high and low affinity sites of cardiac troponin showed many properties in common with those of fast skeletal troponin. 1.In low Mg ion concentrations the high affinity sites of both cardiac and fast skeletal troponins showed extraordinarily high affinity for Ca ions. They also exhibited very strong affinity for Mg ions in the virtual absence of Ca ions. These findings are discussed in relation to conformational changes of troponin C. 2.With increase in Mg ion concentration the extraordinarily high affinity for Ca ions gradually disappeared. The dissociation constant of the high affinity sites for Mg ions at milli-molar Mg ion concentrations of cardiac and skeletal troponins were 2.6×10−3M, and those of the low affinity sites were 1-2×10−2M. These observations are compatible with the Mg-induced shift of the relationship between pCa and contractile response (Ebashi & Endo (1968)Prog. Biophys. Mol. Biol. 18, 123–183). 3. In the presence of millimolar Mg ion concentrations, fast skeletal troponin showed different affinities for Ca ions at different pHs (6.2, 6.8, and 7.4), exhibiting higher affinity at higher pH. In the case of cardiac troponin, no difference in the affinity for Ca ions could be detected at different pHs in the presence of Mg ions, but in the absence of Mg ions a clear difference was seen at every pH. 4. Superprecipitation of cardiac and fast skeletal myosin B at low concentrations of Mg ions and ATP was regulated by Ca ions in the concentration range between 10−9-10−7M, where only the high affinity sites of troponin were involved in Ca binding. This observation further substantiates the view that the high affinity sites can directly regulate the contractile processes (Kohama, K. (1979) J. Biochem. 86, 811–820).

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