FUNCTIONAL-CHARACTERIZATION OF LANTHANIDE BINDING-SITES IN THE SARCOPLASMIC-RETICULUM CA2+-ATPASE - DO LANTHANIDE IONS BIND TO THE CALCIUM-TRANSPORT SITE

Gd3+ binding sites on the purified Ca2+-ATPase of sarcoplasmic reticulum were characterized at 2 and 6-degrees-C and pH 7.0 under conditions in which Ca-45(2+) and Mn-54(2+) specifically labeled the calcium transport site and the catalytic site of the enzyme, respectively. We detected several classes of Gd3+ binding sites that affected enzyme function: (a) Gd3+ exchanged with Mn-54(2+) of the (MnATP)-Mn-54 complex bound at the catalytic site. This permitted slow phosphorylation of the enzyme when two Ca2+ ions were bound at the transport site. The Gd3+ ion bound at the catalytic site inhibited decomposition of the ADP-sensitive phosphoenzyme. (b) High-affinity binding of Gd3+ to site(s) distinct from both the transport site and the catalytic site inhibited the decomposition of the ADP-sensitive phosphoenzyme. (c) Gd3+ enhanced 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence in NBD-modified enzyme by probably binding to the Mg2+ site that is distinct from both the transport site and the catalytic site. (d) Gd3+ inhibited high-affinity binding of Ca-45(2+) to the transport site not by directly competing with Ca2+ for the transport site but by occupying site(s) other than the transport site. This conclusion was based mainly on the result of kinetic analysis of displacement of the enzyme-bound Ca-45(2+) ions by Gd3+ and vice versa, and the inability of Gd3+ to phosphorylate the enzyme under conditions in which GdATP served as a substrate. These results strongly suggest that Ln3+ ions cannot be used as probes to structurally and functionally characterize the calcium transport site on the Ca2+-ATPase.