PURIFIED, RECONSTITUTED CARDIAC CA2-ATPASE IS REGULATED BY PHOSPHOLAMBAN BUT NOT BY DIRECT PHOSPHORYLATION WITH CA2+()CALMODULIN-DEPENDENT PROTEIN-KINASE/

Regulation of calcium transport by sarcoplasmic reticulum provides inc reased cardiac contractility in response to beta-adrenergic stimulatio n. This is due to phosphorylation of phospholamban by cAMP-dependent p rotein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca2+-ATPase), Recently, direct phosphoryl ation of Ca2+-ATPase by calcium/calmodulin-dependent protein kinase ha s been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca2+-ATPase from cardiac sarcopla smic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban, The resulting proteoliposomes ha d high rates of calcium transport, which was tightly coupled to ATP hy drolysis (similar to 1.7 calcium ions transported per ATP molecule hyd ro lyzed). Co-reconstitution with phospholamban suppressed both calciu m uptake and ATPase activities by similar to 50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosp horylation either with cAMP-dependent protein kinase or with calcium/c almodulin-dependent protein kinase, These effects were consistent with a change in the apparent calcium affinity of Ca2+-ATPase and not with a change in V-max. Neither the purified, reconstituted cardiac Ca2+-A TPase nor the Ca2+-ATPase in longitudinal cardiac sarcoplasmic reticul um vesicles was a substrate for calcium/calmodulin-dependent protein k inase, and accordingly, we found no effect of calcium/calmodulin-depen dent protein kinase phosphorylation on V-max for calcium transport.