Key Points
- To identify and characterize the specific endogenous calmodulin-binding proteins present in canine cardiac sarcoplasmic reticulum membranes.
- Cross-linked canine cardiac sarcoplasmic reticulum vesicles to membrane-bound 125I-calmodulin using the homobifunctional cross-linker dithiobis(succinimidyl propionate).
- Analyzed cross-linked complexes and [gamma-32P]ATP-phosphorylated products via sodium dodecyl sulfate-polyacrylamide gel electrophoresis under varying heat and cold-storage conditions.
- Detected a major 125I-labeled product at Mr = 40,000 and a minor product at Mr = 120,000, corresponding to 1:1 complexes of calmodulin with phospholamban and the 100,000-dalton Ca2+-ATPase, respectively.
- Demonstrated that boiling dissociated the 40,000-dalton complex into 26,000- and 28,000-dalton components, representing calmodulin bound to 8,000- and 11,000-dalton phospholamban subunits, which reassociated upon storage at -70 °C.
- Confirmed the 40,000-dalton species as a 1:1 phospholamban-calmodulin complex using phosphorylation with [gamma-32P]ATP in the presence of 3 microM calmodulin.
Structured PICO
PPopulationCanine cardiac sarcoplasmic reticulum vesicles
IInterventionAffinity labeling by covalent cross-linking to membrane-bound 125I-calmodulin with dithiobis(succinimidyl propionate)
OOutcomeIdentification of calmodulin-binding componentssurrogate
This preclinical study identifies phospholamban as the likely endogenous receptor for calmodulin in canine cardiac sarcoplasmic reticulum membranes.