Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum

Larry Jones, H. K B Simmerman, W. W. Wilson, F. R. Gurd, A. D. Wegener

Research output: Contribution to journalArticle

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Abstract

Phospholamban, the putative protein regulator of the Ca2+ 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 (~2 mg of phospholamban/g of sarcoplasmic reticulum protein). Nondissociated phospholamban exhibited an apparent M(r) = 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 P(i)/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. Phospholamban was localized by immunochemical methods to cardiac membranes enriched in sarcoplasmic reticulum, but was absent from sarcoplasmic reticulum membranes prepared from fast skeletal muscle. The method described for isolation of cardiac phospholamban is highly reproducible and relatively simple, and should be useful for further detailed studies designed to probe the molecular structure of the protein.

Original languageEnglish
Pages (from-to)7721-7730
Number of pages10
JournalJournal of Biological Chemistry
Volume260
Issue number12
StatePublished - 1985

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Sarcoplasmic Reticulum
Purification
Canidae
Sodium Dodecyl Sulfate
Proteins
zwittergent 3-14
phospholamban
Molecular Weight
Molecular weight
Sodium Cholate
Proteolipids
Membranes
Calcium Oxalate
Phosphorylation
Chloroform
Cyclic AMP-Dependent Protein Kinases
Molecular Structure
Stoichiometry
Sepharose
Boiling liquids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Jones, L., Simmerman, H. K. B., Wilson, W. W., Gurd, F. R., & Wegener, A. D. (1985). Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum. Journal of Biological Chemistry, 260(12), 7721-7730.

Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum. / Jones, Larry; Simmerman, H. K B; Wilson, W. W.; Gurd, F. R.; Wegener, A. D.

In: Journal of Biological Chemistry, Vol. 260, No. 12, 1985, p. 7721-7730.

Research output: Contribution to journalArticle

Jones, L, Simmerman, HKB, Wilson, WW, Gurd, FR & Wegener, AD 1985, 'Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum', Journal of Biological Chemistry, vol. 260, no. 12, pp. 7721-7730.
Jones, Larry ; Simmerman, H. K B ; Wilson, W. W. ; Gurd, F. R. ; Wegener, A. D. / Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum. In: Journal of Biological Chemistry. 1985 ; Vol. 260, No. 12. pp. 7721-7730.
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