[29] Purification of phospholamban from canine cardiac sarcoplasmic reticulum vesicles by use of sulfhydryl group affinity chromatography

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 Ca2÷-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.