CA2-RETICULUM ATPASE REVISITED .2. EQUILIBRIUM AND KINETIC EVIDENCE FOR A 2-ROUTE MECHANISM( BINDING TO SARCOPLASMIC)

The experiments reported in the present paper were designed to check t he model proposed for Ca2+ binding in the preceding paper (Forge, V., Mintz, E., and Guillain, F. (1993) J. Biol. Chem. 268,10953-10960). Th e pH dependence of the Mg2+-induced variation of the intrinsic fluores cence, as well as that of the phosphorylation by P(i), confirmed that there are several species of Ca2+-deprived ATPase. Kinetics of Ca2+ bi nding as a function of pH suggested that the deprotonated form of the ATPase binds Ca2+ rapidly (k > 50 s-1), whereas the protonated forms b ind Ca2+ slowly (1.3-2.7 s-1). At variance with other models which are linear, slow and rapid Ca2+ binding take two different routes, and in termediate pH values and Mg2+, which favors the deprotonated forms, re sult in biphasic kinetics. Mg2+ binds to all Ca2+-deprived species and to species having one bound Ca2+ but does not bind to ECa2. This is t he reason why Mg2+ inhibits Ca2+ binding, and this inhibition is remov ed in the presence of adenosine-5'-O-(3-thiotriphosphate) which drives Mg2+ into the catalytic site.