CA2+ BINDING TO OCCLUDED SITES IN THE CRATP-ATPASE COMPLEX OF SARCOPLASMIC-RETICULUM - EVIDENCE FOR 2 INDEPENDENT HIGH-AFFINITY SITES

Occluded Ca2+ sites in the CrATP-ATPase complex are studied by first forming the complex in the presence of EGTA so that the sites can be occluded while vacant. Ca-45(2+) binding to the occluded sites is then studied under equilibrium conditions. Binding curves are produced for two independent Ca2+ sites with K(d)(1) = 0.2 muM and K(d)(2) = 1.6 muM. When both sites are saturated, only the Ca2+ bound to the lower affinity site can exchange with free Ca2+. On addition of EGTA (15 vs 0.5 mM Ca2+) all bound Ca2+ dissociates, the net dissociation rate of one-half of the Ca2+ being approximately 10-fold greater than that of the other one-half (at 37-degrees-C). When Ca2+ is bound only to the higher affinity site, this Ca2+ will exchange slowly if the concentration of free Ca2+ is below the saturation level of the lower affinity site. An ionophore dependency of the rates of binding and dissociation indicates that the access to the sites is through the interior of the vesicle. Solubilization in C-12E9 releases the Ca2+ in the higher affinity site. Our observations are consistent with a model of the ATPase where the lower affinity of two transport sites is associated with the interior position (closest to the lumen) in a transmembrane channel. It is further evident that when in the occluded state, the higher affinity site is available without Ca2+ first being bound to the lower affinity site, eliminating cooperativity from the binding mechanism. In turn, this implies a connection between the integrety of the high-affinity binding site and the linking of sections of the catalytic site by CrATP.