Modulation of Calcium Binding in Sarcoplasmic Reticulum Adenosinetriphosphatase

High-affinity calcium binding to sarcoplasmic reticulum (SR) ATPase occurs with a stoichiometric ratio of 2 with respect to sites phosphorylated with ATP [Yamamoto, T., & Tonomura, Y. (1967) J. Biochem. (Tokyo) 62, 558; Yamamoto, T., & Tonomura, Y. (1968) J. Biochem. (Tokyo) 64, 137; Makinose, M. (1969) Eur. J. Biochem. 10, 74] in steady-state conditions, but with a stoichiometric ratio of 1 with respect to the total number of sites available for phosphorylation with P in the absence of Ca2+ [Masuda, H., & de Meis, L. (1973) Biochemistry 12, 4581] in equilibrium conditions. Additional cation binding sites of intermediate and low affinity for Ca2+ are also present in the enzyme. The cooperative character of calcium binding noted with SR vesicles [Inesi, G., Kurzmack, M., Coan, C., & Lewis, D. (1980) J. Biol. Chem. 255, 3025] is observed with purified ATPase as well. A cooperative behavior is also displayed by the Ca2+ concentration dependence of ATPase enzymatic activity. This cooperativity is abolished, and the apparent affinity for Ca2+ is reduced by a detergent known to dissociate ATPase into enzymatically active single chains [Dean, W., & Tanford, C. (1978) Biochemistry 17,1683]. Stoichiometry and cooperative behavior demonstrate that within one enzyme unit two interacting sites are occupied by calcium, while only one out of two phosphorylation sites is phosphorylated by ATP in steady-state conditions. The effect of detergent solubilization indicates that single polypeptide chains retain enzymatic activity which is dependent on the occupancy of noninteracting calcium site(s). Therefore, the cooperative behavior observed with native enzyme is due to chain-chain interactions, or reversible segmental interactions within one chain. The affinity of calcium binding to ATPase is increased by a rise in pH but is not significantly affected by changes in temperature or by ATP analogues which do not phosphorylate the enzyme. Analysis of the pH effect indicates that one group, dissociating with pKapp = 7.3, participates as a ligand for calcium site modulation during the transport cycle. Ca2+ exchange with H+ is an integral part of the active transport mechanism [Chiesi, M., & Inesi, G. (1980) Biochemistry 19, 2912]. Enzyme phosphorylation with ATP in native vesicles is followed by transfer of bound calcium into a location that is not accessible to the medium [Inesi, G., Kurzmack, M., & Verjovski- Almeida, S. (1978) Ann. N.Y. Acad. Sci. 307, 224]. On the other hand, when leaky vesicles are used, a stoichiometric release of two calcium ions per enzyme unit is observed following phosphorylation with ATP, consistent with reduction of site affinity for the divalent cation [Ikemoto, N. (1975) J. Biol. Chem. 250, 7219]. Observation of the release phenomenon requires addition of dimethyl sulfoxide to the medium, in order to inhibit rapid hydrolytic cleavage of the phosphoenzyme following a slow calcium release [Takakuwa, Y., & Kanazawa, T. (1979) Biochem. Biophys. Res. Commun. 88, 1209; Dupont, Y. (1980) Eur. J. Biochem. 109, 231]. Therefore, changes in site orientation and reduction in affinity are identified with the mechanistic step utilizing free energy for vectorial transport of Ca2+ against a concentration gradient. In the normal operation of the pump, conversion of the phosphoenzyme and inward calcium release from the nonaccessible (“occluded”) sites is a slow step which is rapidly followed by hydrolytic cleavage of the phosphoenzyme.