Plausible stoichiometry of the interacting nucleotide-binding sites in theCa2+-ATPase from sarcoplasmic reticulum membranes

The Ca2+,Mg2+-ATPase from sarcoplasmic reticulum couples ATP hydrolysis to Ca2+ transport toward the lumen of the muscular vesicular system. Combined structural and functional studies suggest that the Ca2+ binding sites are f ormed by six amino acids of the same polypeptide and that cation translocat ion may take place through a channel inside a monomer of the ATPase, Howeve r, calorimetric, fluorescent, and kinetic studies suggest that the ATPase m ay assemble into functional oligomers of as yet unknown stoichiometry, We h ave addressed this question and attempted to determine the ATPase stoichiom etry using a biophysical approach based on the analysis of the ATPase inhib ition by fluorescein 5'-isothiocyanate in the presence of increasing ATP co ncentrations. For native SR membranes, our inhibition data are well describ ed by a model consisting of two interacting nucleotide-binding sites per ol igomer. This stoichiometry was disrupted in detergent C12E8-solubilized ATP ase, Thus, these findings suggest that interacting nucleotide binding sites of the ATPase may appear as dimers, and imply that interactions of the glo bular cytoplasmic domains would play a modulatory role of the protein enzym atic activity. (C) 1999 Academic Press.