Distinction of the roles of the two high‐affinity calcium sites in the functional activities of the Ca2+‐ATPase of sarcoplasmic reticulum

Abstract

The effects of trypsin digestion and low temperature on Ca²⁺ binding and on Ca²⁺ activation of ATP hydrolysis by the high‐affinity transport sites of the Ca²⁺,Mg²⁺‐ATPase of sarcoplasmic reticulum were examined. Sarcoplasmic reticulum vesicles contain 0.7–1.1 high‐affinity Ca²⁺ sites per 10⁵ g sarcoplasmic reticulum with K= 3–5 × 10⁵ M⁻¹, as well as sites of lower affinity. The first cleavage of the ATPase with trypsin (TD1) has no effect on the binding properties of the high affinity sites. The second tryptic cleavage (TD2) decreases the affinity of the high sites to K= 3 × 10⁴ M⁻¹ with conservation of the total number of sites. The purified ATPase contains 1.6 – 2.0 high affinity Ca²⁺ sites per 10⁵ g protein when measured at 23°C, while at 0–4°C there is ∼ 1 high‐affinity (K= 5 – 10 × 10⁵ M⁻¹) affinity site and ∼ 1 intermediate‐affinity (K= 3 × 10⁴ M⁻¹) site per 10⁵ g. Trypsin digestion to the point of TD1 has no effect on either the number or the binding constants of the high‐affinity sites. Upon TD2 cleavage, one of the sites is converted to the intermediate‐affinity state, while the other remains at high affinity. After TD2 modification of the enzyme both of the sites are in the intermediate affinity state at 4°C. On the basis of the binding data, several models for the roles of the Ca²⁺ sites in the activation of ATP hydrolysis are derived. The results are summarized by a scheme in which the two high‐affinity Ca²⁺ sites are heterogeneous with respect to sensitivity to temperature and to TD2 modification. The results of this and a previous study (Scott, T. L. and Shamoo, A. E. (1982) J. Membr. Biol. 64, 137–144] indicate that while occupation of either of the two Ca²⁺ sites can stimulate ATP hydrolysis, the site which is sensitive to TD2 is essential for the coupling of hydrolysis to Ca²⁺ transport.