Purified, reconstituted cardiac Ca2+-ATPase is regulated by phospholamban but not by direct phosphorylation with Ca2+/calmodulin-dependent protein kinase

Laxma G. Ready, Larry R. Jones, Ronald C. Pace, David L. Stokes

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Abstract

Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to β-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 (∼1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by ∼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.

Original languageEnglish (US)
Pages (from-to)14964-14970
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number25
StatePublished - Dec 1 1996

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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