Key Points
- Purify and biochemically characterize phospholamban and (Ca2+ + Mg2+)-ATPase from canine cardiac sarcoplasmic reticulum using low detergent concentrations.
- Extracted proteins from canine cardiac sarcoplasmic reticulum using sequentially low concentrations of the detergent deoxycholate.
- Purified the 22,000-dalton phospholamban using Sephadex G-75 gel filtration chromatography and verified purity with SDS-polyacrylamide gel electrophoresis.
- Assessed cyclic AMP-dependent protein kinase phosphorylation stoichiometry, phospholipid binding, amino acid composition, and ATP hydrolytic activity.
- Purified phospholamban yielded a single band on SDS-PAGE, specifically phosphorylated by cyclic AMP-dependent protein kinase at 0.15 mol phosphate/mol protein.
- Phospholamban bound tightly to phospholipids at a ratio of 5 to 8 mol phospholipid/mol protein, containing an excess of acidic residues and approximately 40% hydrophobic residues.
- The (Ca2+ + Mg2+)-ATPase achieved at least 95% purity with an initial hydrolytic activity of ~1.25 pmol Pi/mg/min, which increased to ~2.5 pmol Pi/mg/min upon additional deoxycholate.
Structured PICO
PPopulationCanine cardiac sarcoplasmic reticulum
IInterventionPurification using very low concentrations of deoxycholate and column chromatography
OOutcomeIsolation and biochemical characterization of phospholamban and (Ca2+ + Mg2+)-ATPasesurrogate
The successful purification of phospholamban and (Ca2+ + Mg2+)-ATPase from canine cardiac sarcoplasmic reticulum enables further study of calcium transport regulation in the heart.