Characterization of CrATP-induced calcium occlusion in membrane-bound and soluble monomeric sarcoplasmic reticulum Ca2+-ATPase.

Occlusion of Ca2+ induced by beta, gamma-bidentate CrATP in membrane bound and in soluble monomeric sarcoplasmic reticulum Ca2+-ATPase was studied by previously developed filtration and HPLC techniques (Vilsen and Andersen (1986) Biochim. Biophys. Acta 855, 429-431). Activation of Ca2+ occlusion occurred at micromolar free Ca2+ and depended on the concentration of Ca2+, H+ and Mg2+ in a similar way as activation of Ca2+ transport and equilibrium Ca2+ binding to high-affinity Ca2+ transport sites. The slopes of the Ca2+ titration curves indicated that Ca2+ binding is a cooperative process both in membraneous and in soluble monomeric enzyme. At alkaline pH and absence of Mg2+, occlusion of Ca2+ was inhibited by 1 mM Ca2+ in membrane-bound, but not in soluble monomeric Ca2+-ATPase. Parallel studies of phosphorylation from [gamma-32P]CrATP indicated a stoichiometry of 2 mol Ca2+ occluded per mol Ca2+-dependent EP formed, at saturating as well as at desaturating Ca2+ concentrations. Tryptic digestion of the CrATP induced Ca2+ occluded complex indicated that it belongs to the E1 conformational class (E1P). In the absence of Ca2+ and Mg2+, but presence of CrATP the conformational state was E2. When Mg2+ was added together with CrATP at alkaline pH the conformation was shifted in direction of E1.