Two types of proton-modulated calcium binding in the sarcoplasmic reticulum Ca(2+)-ATPase. I. A model of two different conformations of chemically equivalent ATPase molecules.

Two pools of calcium binding sites of the sarcoplasmic reticulum Ca(2+)-ATPase have been found at 0 degrees C: Half of the calcium sites are in a slow (t1/2 > or = 2 s)/rapid (t1/2 < 2 s) binding site dependent on pH, and the other half are in a slow binding state independent of pH (Nakamura, J. (1989) J. Biol. Chem. 264, 17029-17031). Herein, to clarify the molecular basis of the two calcium sites, the relation between calcium binding to the enzyme and phosphorylation of the enzyme was examined at pH 7.40, where the calcium sites are split into rapid and slow binding states. The enzyme was slowly or rapidly phosphorylated with ATP, accompanied by slow or rapid calcium binding with a stoichiometry of about 1:2. Analysis of the amino acid sequence of lysyl endopeptidase peptides of the ATPase preparation confirmed the homogeneity of the preparation, which was of fast twitch muscle type. These results suggest that each of the two pools of calcium sites belongs to one of the two different conformations of chemically equivalent ATPase molecules, which are in pH-dependent equilibrium between E1 (high affinity state for calcium) and E2 (low affinity state for calcium) and predominantly in E2 independent of pH, respectively, before calcium binding.