AN INVESTIGATION OF THE MECHANISM OF INHIBITION OF THE CA2-ATPASE BY PHOSPHOLAMBAN()

The Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum has been rec onstituted with peptides corresponding to the hydrophobic domain of ph ospholamban (PLB) with or without the three Cys residues replaced by A la, and with PLB with the three Cys residues replaced by Ala [PLB(Cys- )(1-52)]. Reconstitution with the hydrophobic domain of PLB [PLB(25-52 )] was found to decrease the apparent affinity of the ATPase for Ca2with no effect on the maximal rate of ATP hydrolysis observed at satur ating concentrations of Ca2+. Reconstitution with PLB(Cys-)(1-52) decr eased both the apparent affinity for Ca2+ and the maximal activity; th e effect on maximal activity followed from a decrease in the rate of t he Ca2+ transport step (E1PCa(2) --> E2P) as observed with the hydroph ilic domain PLB(1-25). The concentration dependences of the effects of the hydrophobic domain and of the whole PLB molecule were very simila r, suggesting that the hydrophilic domain made little contribution to the affinity of the ATPase for PLB. The effect of PLB on the ATPase wa s dependent on the molar ratio of phospholipid to ATPase, suggesting p artition of the PLB between its binding site on the ATPase and the bul k lipid phase in the membrane. Neither PLB nor its hydrophobic domain affected the rates of phosphorylation or dephosphorylation of the ATPa se. Despite their effects on the apparent affinity of the ATPase for C a2+ neither PLB nor its hydrophobic domain had any effect on the true affinity of the ATPase for Ca2+, as measured from changes in the trypt ophan fluorescence of the ATPase. The effects of PLB on the activity o f the ATPase are the sum of the effects of its hydrophilic and hydroph obic domains.