title: Plausible stoichiometry of the interacting nucleotide-binding sites in the Ca(2+)-ATPase from sarcoplasmic reticulum membranes.
abstract: The Ca(2+),Mg(2+)-ATPase from sarcoplasmic reticulum couples ATP hydrolysis to Ca(2+) transport toward the lumen of the muscular vesicular system. Combined structural and functional studies suggest that the Ca(2+) binding sites are formed by six amino acids of the same polypeptide and that cation translocation may take place through a channel inside a monomer of the ATPase. However, calorimetric, fluorescent, and kinetic studies suggest that the ATPase may assemble into functional oligomers of as yet unknown stoichiometry. We have addressed this question and attempted to determine the ATPase stoichiometry using a biophysical approach based on the analysis of the ATPase inhibition by fluorescein 5'-isothiocyanate in the presence of increasing ATP concentrations. For native SR membranes, our inhibition data are well described by a model consisting of two interacting nucleotide-binding sites per oligomer. This stoichiometry was disrupted in detergent C(12)E(8)-solubilized ATPase. Thus, these findings suggest that interacting nucleotide binding sites of the ATPase may appear as dimers, and imply that interactions of the globular cytoplasmic domains would play a modulatory role of the protein enzymatic activity.
authors.affiliation: Facultad de Ciencias, Universidad de Extremadura, Badajoz, 06080, Spain. jmmerino@unex.es
authors.forename: J M
authors.initials: JM
authors.lastname: Merino
authors.forename: C
authors.initials: C
authors.lastname: Gutiérrez-Merino
authors.forename: F
authors.initials: F
authors.lastname: Henao
mesh_terms: D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D007425:Intracellular Membranes / Q000737:chemistry / Q000201:enzymology; D018482:Muscle, Skeletal / Q000378:metabolism / Q000648:ultrastructure; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology*
chemical_list: D009711:Nucleotides; D000252:Calcium-Transporting ATPases