Ca2+ binding to sarcoplasmic reticulum ATPase revisited. II. Equilibrium and kinetic evidence for a two-route mechanism.
The experiments reported in the present paper were designed to check the model proposed for Ca2+ binding in the preceding paper (Forge, V., Mintz, E., and Guillain, F. (1993) J. Biol. Chem. 268, 10953-10960). The pH dependence of the Mg(2+)-induced variation of the intrinsic fluorescence, as well as that of the phosphorylation by Pi, confirmed that there are several species of Ca(2+)-deprived ATPase. Kinetics of Ca2+ binding as a function of pH suggested that the deprotonated form of the ATPase binds Ca2+ rapidly (k > 50 s-1), whereas the protonated forms bind 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 intermediate pH values and Mg2+, which favors the deprotonated forms, result in biphasic kinetics. Mg2+ binds to all Ca(2+)-deprived species and to species having one bound Ca2+ but does not bind to ECa2. This is the reason why Mg2+ inhibits Ca2+ binding, and this inhibition is removed in the presence of adenosine-5'-O-(3-thiotriphosphate) which drives Mg2+ into the catalytic site.
Unité de Recherche Associée Centre National de la Recherche Scientifique 1290, Département de Biologie Cellulaire et Moléculaire, CE Saclay, Gif-sur-Yvette, France.
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Forge
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Mintz
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Guillain
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008433:Mathematics; D008954:Models, Biological; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium