Effects of pH on phosphorylation of the Ca2+-ATPase of sarcoplasmic reticulum by inorganic phosphate

Abstract

The fluorescence intensity of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin has been shown to decrease on phosphorylation of the ATPase with Pi, this providing a convenient measure of the level of phosphorylation. Comparison of the fluorescence decrease observed with ATP and with high concentrations of Pi fix the value of the equilibrium constant for the phosphorylation reaction E2PMg ⇌ E2PiMg at pH 6.0 at about 2. Studies of the pH-dependence of phosphorylation show that H2PO4- and HPO42- bind to the ATPase with equal affinity, but that only binding of H2PO4- leads to phosphorylation, described by an equilibrium constant of 2.3. Luminal Ca2+ can bind to a pair of sites on the ATPase, with affinities of 1.3ȕ103 and 1.7ȕ103 M-1 for the unphosphorylated and phosphorylated forms of the ATPase respectively, with stronger binding of Ca2+ to the phosphorylated form resulting in an increase in the effective equilibrium constant for phosphorylation.