Phospholamban stoichiometry in canine cardiac muscle sarcoplasmic reticulum

Abstract

Treatment of cardiac sarcoplasmic reticulum with the crosslinking reagent dithiobis (succinimidyl propionate) in the presence of 125I-calmodulin, resulted in the formation of a 40,000-dalton affinity labeled component, consisting of a 1:1, phospholamban: 125I-calmodulin complex. In parallel experiments, sarcoplasmic reticulum was phosphorylated in the presence of calmodulin and [gamma-32P]ATP, and then treated with the crosslinking reagent to produce an affinity labeled component consisting of a 1:1, calmodulin: 32P-phospholamban complex. These experiments permitted determination of the amount of 125I and 32P incorporated into the 40,000-dalton complexes, as well as the amount of 32P incorporated into the 23,000-dalton form of phospholamban. If 1 mol of Ca2+-dependent ATPase phosphoprotein represents 1 mol of 100,000-dalton Ca2+-dependent ATPase monomer, then there are 4.88 +/- 1.33 mol Ca2+-dependent ATPase/mol of phospholamban. If there are 2 mol of Ca2+-dependent ATPase phosphoprotein/mol of 100,000-dalton Ca2+-dependent ATPase monomer, then there are 9.76 +/- 2.66 mol Ca2+-dependent ATPase/mol phospholamban.