EFFECTS OF PH ON PHOSPHORYLATION OF THE CA2-ATPASE OF SARCOPLASMIC-RETICULUM BY INORGANIC-PHOSPHATE()

The fluorescence intensity of the Ca2+-ATPase of skeletal muscle sarco plasmic reticulum (SR) labelled with 4-(bromomethyl)-6,7-dimethoxycoum arin has been shown to decrease on phosphorylation of the ATPase with P-i, this providing a convenient measure of the level of phosphorylati on. Comparison of the fluorescence decrease observed with ATP and with high concentrations of P-i fix the value of the equilibrium constant for the phosphorylation reaction E2PMg reversible arrow E2P(i)Mg 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 tha t only binding of H2PO4- leads to phosphorylation, described by an equ ilibrium constant of 2.3. Luminal Ca2+ can bind to a pair of sites on the ATPase, with affinities of 1.3 x 10(3) and 1.7 x 10(3) M(-1) for t he unphosphorylated and phosphorylated forms of the ATPase respectivel y, with stronger binding of Ca2+ to the phosphorylated form resulting in an increase in the effective equilibrium constant for phosphorylati on.