Ca2+ Bindings of Pig Cardiac Myosin, Subfragment-1, and g2 Light Chain1

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Abstract
Ca2+ bindings to pig cardiac myosin, subfragment-1 (S-1), and g2 light chain were investigated by the equilibrium dialysis method. Two different S-1s, one of which can bind Ca2+ and another which cannot, were prepared. In order to calculate the free Ca2+ concentrations adequately, the amounts of Ca2+ included in various chemicals and proteins were measured by atomic absorption spectroscopy. Ca2+ contamination was greatest in KCl among the chemicals tested. In addition, the (Ca2+ strongly bound to myosth and S-1 was released in the presence of Mg2+ When Mg2+ was not added, the Ca2+-binding constant of myosin was 4×105 M−1 and the maximum binding number was 1.8 mol per mol of myosin. Cooperativity between the 2 Ca2+ bindings could not be demonstrated. Mg2+ strongly inhibited the Ca2+ binding: at a free Ca2+ concentration of 1×10−5M, 1.3 mol Ca2+ was bound to myosin in the absence of Mg2+ but 0.6 and 0.2 mol were bound in the presence of 0.3 and 4.5 mM Mg2+ respectively. The Ca2+-binding constant of S-1, which contained a 15,000 dalton component, was 8.6×105 M−1 and the maximum binding number was 0.7 mol per mol of S-1. The 15,000 dalton component could be exchanged with extraneous g2 S-1 which lacked the 15,000 component could not bind Ca2+ at free Ca2+ concentrations less than 0.1 mM. The Ca2+ binding to free g2 light chain was about 100 times weaker than the binding to myosin, as indicated previously for skeletal myosin (Okamoto, Y. & Yagi, K. (1976) J. Biochem. 80, 111–120). The Ca2+ constant was obtained as 4.1×103M−1 in the absence of added Mg2+ Phosphorylation of g2, light chain did not affect the Ca2+ binding to the free g2, light chain or to myosin. Ca2+ binding to cardiac native tropomyosin was also measured.

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