Thermal unfolding of monomeric Ca(II),Mg(II)‐ATPase from sarcoplasmic reticulum of rabbit skeletal muscle

Abstract

The thermal unfolding of monomeric and delipidated Ca²⁺-ATPase, solubilized in C₁₂E₈, can be appropriately described as a non-two-state irreversible denaturation, with only one endothermic peak. In the Ca²⁺ concentration range (0–0.5 mM) which stimulates the ATPase activity of solubilized monomeric ATPase, Ca²⁺ shifts the critical temperature midpoint of the denaturation process (T _m) from 42 to 50°C without segregation of the endothermic peak into two separate components. Because 20 mM Mg²⁺ only shifts the T _m from 42 to 44°C, we conclude that the effect of Ca²⁺ upon the T _m is likely to be due to binding to the high affinity Ca²⁺ sites in the ATPase. The effect of Ca²⁺ upon the enthalpy of denaturation is biphasic, suggesting the presence of low affinity Ca²⁺ sites (K _0.5 in the millimolar range) in monomeric and solubilized ATPase.