Binding of ADP to sarcoplasmic reticulum Ca(2+)-ATPase in the absence of Mg2+ is specifically inhibited by thapsigargin: observations on the ligand stoichiometry.
The conditions of nucleotide binding to native, though partly purified, Ca(2+)-ATPase from SR as well as the stoichiometry of nucleotide and strontium binding and the phosphorylation capacity was reevaluated. Binding of MgADP appeared to be aberrant whereas even high-affinity binding of [14C]-ADP took place in the absence of Mg2+. Also low-affinity ATP binding was possible in the absence of divalent cations. A heterogeneity in ADP binding compatible with a two-component model in the absence of thapsigargin was changed to an apparent homogeneity of low-affinity receptors following a mole:mole interaction of enzyme and thapsigargin. Since the affinity of both components was reduced by thapsigargin, high- as well as low-affinity ADP binding seem to be specific and probably to the substrate receptor proper. Analysis of ADP binding isotherms in the absence of Mg2+ according to a model of two independent populations of sites was compatible with a binding capacity of 8.49 +/- 0.43 nmoles/mg protein corresponding to a molecular mass of 118 +/- 6 kD per ADP site. The same total binding capacity was found for ATP. The phosphorylation capacity corresponded to more than one and less than two approximately P per two 110-kD peptides (formally one approximately P per 154 kD protein). Specific binding of Ca2+ and the congener Sr2+ to SR Ca(2+)-ATPase was compatible with their interaction with a single population of sites. The binding capacity was equal to one divalent cation per nucleotide binding peptide. The binding of one nucleotide and one divalent cation per approximately 110 kD peptide and the absence of cooperativity in divalent cation binding might imply that Ca(2+)-ATPase works as a monomer.
Hansen
O
O
Institute of Physiology, Aarhus University, Denmark.
Jensen
J
J
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology; D018656:Muscle Fibers, Fast-Twitch / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013326:Strontium Radioisotopes / Q000378:metabolism; D013379:Substrate Specificity; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin
D004791:Enzyme Inhibitors; D008024:Ligands; D013326:Strontium Radioisotopes; D013729:Terpenes; D000244:Adenosine Diphosphate; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium