V. Forge et al., CA2-RETICULUM ATPASE REVISITED .2. EQUILIBRIUM AND KINETIC EVIDENCE FOR A 2-ROUTE MECHANISM( BINDING TO SARCOPLASMIC), The Journal of biological chemistry, 268(15), 1993, pp. 961-968
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.