title: Purified, reconstituted cardiac Ca2+-ATPase is regulated by phospholamban but not by direct phosphorylation with Ca2+/calmodulin-dependent protein kinase.
abstract: Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to beta-adrenergic stimulation. This is due to phosphorylation of phospholamban by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca2+-ATPase). Recently, direct phosphorylation of Ca2+-ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca2+-ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban. The resulting proteoliposomes had high rates of calcium transport, which was tightly coupled to ATP hydrolysis (approximately 1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by approximately 50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosphorylation either with cAMP-dependent protein kinase or with calcium/calmodulin-dependent protein kinase. These effects were consistent with a change in the apparent calcium affinity of Ca2+-ATPase and not with a change in Vmax. Neither the purified, reconstituted cardiac Ca2+-ATPase nor the Ca2+-ATPase in longitudinal cardiac sarcoplasmic reticulum vesicles was a substrate for calcium/calmodulin-dependent protein kinase, and accordingly, we found no effect of calcium/calmodulin-dependent protein kinase phosphorylation on Vmax for calcium transport.
authors.affiliation: Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: L G
authors.initials: LG
authors.lastname: Reddy
authors.forename: L R
authors.initials: LR
authors.lastname: Jones
authors.forename: R C
authors.initials: RC
authors.lastname: Pace
authors.forename: D L
authors.initials: DL
authors.lastname: Stokes
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008081:Liposomes; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000099211:Phospholamban
chemical_list: D000255:Adenosine Triphosphate; D002118:Calcium; D002135:Calcium-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000252:Calcium-Transporting ATPases; D017868:Cyclic AMP-Dependent Protein Kinases; D008081:Liposomes; D011510:Proteolipids; D011994:Recombinant Proteins; D000099211:Phospholamban; C020485:proteoliposomes