Purification of Phospholamban from Canine Cardiac Sarcoplasmic Reticulum Vesicles by Use of Sulfhydryl Group Affinity Chromatography

Larry R. Jones, Adam D. Wegener, Heather K.B. Simmerman

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

This chapter focuses on the purification of phospholamban from canine cardiac sarcoplasmic reticulum vesicles by use of sulfhydryl group affinity chromatography. Phospholamban is an integral membrane protein localized to cardiac sarcoplasmic reticulum, which regulates the activity of the sarcoplasmic reticulum Ca-pump in response to phosphorylation. Phospholamban in isolated sarcoplasmic reticulum vesicles is a substrate for cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, and protein kinase C. Phospholamban is also readily phosphorylated in sarcoplasmic reticulum in intact hearts in response to β-adrenergic stimulation. A prerequisite for studying the protein in an isolated, reconstituted system is a method for preparing phospholamban from myocardium in reasonable yield and purity. Central to the isolation scheme is the use of sulfhydryl group affinity chromatography at the final stage of purification. The purpose of this chapter is to describe isolation method in detail. Hopefully, the availability of a highly purified phospholamban preparation will be useful in future studies directed to probe more deeply into the molecular structure and function of the protein.

Original languageEnglish (US)
Pages (from-to)360-369
Number of pages10
JournalMethods in Enzymology
Volume157
Issue numberC
DOIs
StatePublished - Jan 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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