Binding of Calcium and Magnesium to Myosin in Skeletal Muscle Myofibrils

Binding profiles for divalent cation to myosin have been obtained in myofibrils where myosin is assembled in arrays typical of the in vivo organization. Protection by Ca²⁺ and Mg²⁺ ions of the regions of myosin susceptible to chymotryptic attack provided the means to monitor metal ion binding. The effect of various concentrations of divalent cations on the chymotryptic digestion patterns was assessed by densitometry of Coomassie Blue stained gels obtained by Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by the rate of myofibrillar solubilization. The results indicate the presence of two classes of binding sites differing in affinity by 4 orders of magnitude. The fractional saturation of the high-affinity site associated with the 5,5′-dithiobis(2-nitrobenzoic acid)-dissociable light chains of myosin regulated the production of subfragment 1 of myosin. From the digestion profiles as a function of metal ion concentration, binding constants for Mg²⁺ and Ca²⁺ were obtained. The value for Mg²⁺ was 5.7 × 10⁶ M⁻¹, which is about 1 order of magnitude higher than the most recently determined values for free myosin in solution; the value for Ga²⁺ was 6.3 × 10⁶ M⁻¹. Binding to the low-affinity site regulated the production of the heavy meromyosin fragment and yielded association constants for Ca²⁺ and Mg²⁺ of 0.9 × 10³ and 0.7 × 10³ M⁻¹, respectively.