Thermodynamic studies on the binding of adenosine diphosphate and calcium to beef cardiac myosin.

Thermodynamic quantities for the binding of MgADP, CaADP and Ca2+ to purified beef cardiac myosin have been determined by flow calorimetry at 25 degrees C and by equilibrium dialysis at 4 degrees C in 0.5 M KCl, 20 mM tris-HCl (pH 7.5). About 1.65 +/- 0.15 mol MgADP and 1.9 +/- 0.1 mol CaADP were bound per mol myosin. Free energies of binding of MgADP and CaADP were -6.7 and -5.7 kcal/mol, respectively. Enthalpies for binding of MgADP and CaADP were about -12.5 and -19.0 kcal/mol, respectively. Furthermore, there were 1.8 +/- 0.2 mol high affinity Ca2+ binding sites per mol myosin with an affinity constant of about 10(5) M-1. The enthalpy of Ca2+ binding was about zero. It is concluded that CaADP binds to cardiac myosin with a much greater negative enthalpy than MgADP. Also, the free energy of MgADP binding to cardiac myosin is similar to values reported for skeletal myosin. However, the enthalpy of binding is much less negative than the value obtained for skeletal myosin by Kodama and Woledge (J. Biol. Chem. (1976) 251, 7499--7503). The latter results suggest a subtle difference in the nucleotide binding sites of these myosins.