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 sarcoplasmic reticulum (SR) labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin has been shown to decrease on phosphorylation of the ATPase with P(i), this providing a convenient measure of the level of phosphorylation. 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<==>E2P(i)Mg at pH 6.0 at about 2. Studies of the pH-dependence of phosphorylation show that H2PO4- and HPO4(2)- 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 1.3 x 10(3) and 1.7 x 10(3) 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.
Khan
Y M
YM
Department of Biochemistry and Institute for Biomolecular Sciences, University of Southampton, U.K.
East
J M
JM
Lee
A G
AG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008956:Models, Chemical; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014638:Vanadates / Q000494:pharmacology
D003374:Coumarins; D005456:Fluorescent Dyes; D010710:Phosphates; D014638:Vanadates; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium