Open AccessCa 2+ gradient and drugs reveal different binding sites for P i and Mg 2+ in phosphorylation of the sarcoplasmic reticulum ATPase
Author(s) -
MEIS Leopoldo,
SUZANO Valdecir A.,
CALDEIRA Teresa,
MINTZ Elisabeth,
GUILLAIN Florent
Publication year - 1991
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1991.tb21069.x
Subject(s) - ruthenium red , chemistry , trifluoperazine , endoplasmic reticulum , phosphorylation , atpase , calmodulin , enzyme , spermidine , pi , stereochemistry , calcium , biochemistry , organic chemistry
The first step towards ATP synthesis by the Ca 2 ‐ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by P i . Phosphoenzyme formation requires both P i and Mg 2+ . At 35°C, the presence of a Ca 2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for P i more than 10‐fold, whereas it had no effect on the apparent affinity for Mg 2+ . In the absence of a Ca 2+ gradient, the phosphorylation reaction is inhibited by both K + and Na + at all Mg 2+ concentrations used. However, in the presence of 1 mM Mg 2+ and of a transmembrane Ca 2+ gradient, the reaction is still inhibited by Na + , but the inhibition promoted by K + is greatly decreased. When the Mg 2+ 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 Ca 2+ gradient, trifluoperazine competes with the binding to the enzyme of both P i and Mg 2+ , whereas spermidine and ruthenium red were found to compete only with Mg 2+ . The data presented suggest that the enzyme has different binding sites for Mg 2+ and for P i .