Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum
Phospholamban, the putative protein regulator of the Ca/sup 2 +/ pump of cardiac sarcoplasmic reticulum, was purified to apparent homogeneity from canine cardiac sarcoplasmic reticulum vesicles by selective extraction with sodium cholate, followed by adsorption to calcium oxalate, solubilization in Zwittergent 3-14, and specific elution from p-hydroxymercuribenzoate-agarose. Phospholamban, isolated in the dephosphorylated state, was purified 80-fold in 15% yield (approximately 2 mg of phospholamban/g of sarcoplasmic reticulum protein). Nondissociated phospholamban exhibited an apparent Mr = 25,000 in sodium dodecyl sulfate-polyacrylamide gels. Partially dissociated phospholamban, induced by boiling in sodium dodecyl sulfate, exhibited five distinct mobility forms in sodium dodecyl sulfate-polyacrylamide gels, of apparent molecular weights between 5,000-6,000 and 25,000. Phospholamban was phosphorylated to a level of 190 nmol of Pi/mg of protein by cAMP-dependent protein kinase, consistent by minimum stoichiometry with a subunit molecular weight of approximately 5,000. Phospholamban prepared by the present method was different in several respects from the proteins that have been isolated in other laboratories. Pure phospholamban was cysteine rich, containing 6 residues/100 amino acid residues. Dephosphorylated phospholamban was strongly basic with a pI = 10; phosphorylation decreased the pI to approximately 6.7. Pure phospholamban (and phospholamban present in sarcoplasmic reticulum vesicles) was not readily extracted into acidified chloroform/methanol, suggesting that the protein does not behave as an acidic proteolipid. The purified protein was highly antigenic.
- OSTI ID:
- 6528860
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 12; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
AMINO ACIDS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CALCIUM COMPOUNDS
CARBOXYLIC ACIDS
CARDIOVASCULAR SYSTEM
CATIONS
CELL CONSTITUENTS
CHARGED PARTICLES
CHEMICAL COMPOSITION
CHEMICAL REACTIONS
DOGS
HEART
IONS
ISOTOPES
KINETICS
MAMMALS
MEMBRANE TRANSPORT
MOLECULAR WEIGHT
MUSCLES
MYOCARDIUM
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PHOSPHORUS ISOTOPES
PHOSPHORYLATION
PROTEINS
PURIFICATION
REACTION KINETICS
SARCOPLASMIC RETICULUM
VERTEBRATES