title: Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase.
abstract: The first step towards ATP synthesis by the Ca2-ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35 degrees C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10-fold, whereas it had no effect on the apparent affinity for Mg2+. In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations. Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+. The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.
authors.affiliation: Departamento Bioquímica, Universidade Federal do Rio de Janeiro, Brazil.
authors.forename: L
authors.initials: L
authors.lastname: De Meis
authors.forename: V A
authors.initials: VA
authors.lastname: Suzano
authors.forename: T
authors.initials: T
authors.lastname: Caldeira
authors.forename: E
authors.initials: E
authors.lastname: Mintz
authors.forename: F
authors.initials: F
authors.lastname: Guillain
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D010758:Phosphorus / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
chemical_list: D010758:Phosphorus; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium