Phospholamban, the regulator of the cardiac sarcoplasmic reticulum calcium pump, does not copurify with the Ca 2+-ATPase enzyme

Abstract

Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3′,5′-monophosphate (cAMP)-dependent and by Ca 2+-calmodulin-dependent protein kinases on an M r 22000 protein called phospholamban. Both types of phosphorylation are associated with an increase in the initial rate of Ca 2+ transport. Thus, phospholamban appears to be a regulator for the calcium pump in cardiac sarcoplasmic reticulum. However, there is conflicting evidence as to the degree of association of the Ca 2+-ATPase with its regulator, phospholamban. In this study, we report that phospholamban does not copurify with a Ca 2+-ATPase preparation of high specific activity. Although 32P-labeled phospholamban is solubilized in the same fraction as the Ca 2+-ATPase from cardiac sarcoplasmic reticulum, it dissociates from the Ca 2+ pump during subsequent purification steps. Our isolation procedure results in an increase of over 4-fold in the specific activity of the Ca 2+-ATPase, but a decrease of 2.5-fold in the specific activity of 32P i-phosphoester bonds (pmol P i/mg). Furthermore, the purified Ca 2+-ATPase enzyme preparation is not a substrate for protein kinase in vitro to any significant extent. These data indicate that phospholamban does not copurify with the Ca 2+-ATPase from cardiac sarcoplasmic reticulum. Isolation of a Ca 2+-ATPase preparation essentially free of phospholamban will aid in future kinetic studies designed to elucidate similarities and differences in the Ca 2+-ATPase parameters from cardiac and skeletal muscle (which is known not to contain phospholamban).