Phospholamban, the regulator of the cardiac sarcoplasmic reticulum calcium pump, does not copurify with the Ca2+-ATPase enzyme.
Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3',5'-monophosphate (cAMP)-dependent and by Ca2+-calmodulin-dependent protein kinases on an Mr 22 000 protein called phospholamban. Both types of phosphorylation are associated with an increase in the initial rate of Ca2+ 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 Ca2+-ATPase with its regulator, phospholamban. In this study, we report that phospholamban does not copurify with a Ca2+-ATPase preparation of high specific activity. Although 32P-labeled phospholamban is solubilized in the same fraction as the Ca2+-ATPase from cardiac sarcoplasmic reticulum, it dissociates from the Ca2+ pump during subsequent purification steps. Our isolation procedure results in an increase of over 4-fold in the specific activity of the Ca2+-ATPase, but a decrease of 2.5-fold in the specific activity of 32Pi-phosphoester bonds (pmol Pi/mg). Furthermore, the purified Ca2+-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 Ca2+-ATPase from cardiac sarcoplasmic reticulum. Isolation of a Ca2+-ATPase preparation essentially free of phospholamban will aid in future kinetic studies designed to elucidate similarities and differences in the Ca2+-ATPase parameters from cardiac and skeletal muscle (which is known not to contain phospholamban).
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification; D000242:Cyclic AMP / Q000378:metabolism; D004285:Dogs; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D017830:Octoxynol; D010710:Phosphates / Q000378:metabolism; D011092:Polyethylene Glycols; D011494:Protein Kinases / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D000242:Cyclic AMP; D017830:Octoxynol; D010710:Phosphates; D011092:Polyethylene Glycols; D011494:Protein Kinases; D000099211:Phospholamban