Ca²⁺ binding to sarcoplasmic reticulum Ca²⁺-ATPase in the absence of chelators

Abstract

The Ca²⁺ affinity of the sarcoplasmic reticulum Ca²⁺-ATPase is in the submicromolar range. Since buffer solutions will contain Ca²⁺ above this level, Ca²⁺ concentrations in binding studies have to be controlled by chelators such as EGTA. Obviously the accuracy of the calculated free concentrations of Ca²⁺ is crucial for determination of the characteristic features of Ca²⁺ binding to the enzyme. The relationship between calculated Ca²⁺ concentrations and binding in e.g. Hill plots is usually interpreted as positive cooperativity in binding. This interpretation seems compatible with 2 Ca²⁺ sites per phosphorylation unit of Ca²⁺-ATPase encompassing only half of the 110-kDa peptides present in purified Ca²⁺-ATPase preparations. Apparently no role is assigned to half of the protein (for refs., see 1). Artefactually high Hill numbers have been noticed even in soluble monomers (1) and recently it was emphasized that EGTA should be used as Ca²⁺ chelator with caution (2).