Purification of phospholamban, a 22,000-dalton protein from cardiac sarcoplasmic reticulum that is specifically phosphorylated by cyclic AMP-dependent protein kinase.

Abstract

Very low concentrations deoxycholate (DOC) were used to isolate two proteins from canine cardiac sarcoplasmic reticulum. These two proteins are phospholamban, a 22,000 dalton protein, and the Ca/sup 2 +/ + Mg/sup 2 +/-ATPase, the major protein of the sarcoplasmic reticulum, responsible for the active transport of calcium. The 22,000 dalton protein is first solubilized in a very low concentration of DOC and then subjected to column chromatography. After molecular weight sieving on a Sephadex G-75 column, the 22,000 dalton protein appears as a purified protein on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The purified protein is specifically phosphorylated by cyclic AMP-dependent protein kinase. Phospholipids are still bound to the isolated protein. The Ca/sup 2 +/ + Mg/sup 2 +/-ATPase is purified by first solubilizing all the extrinsic proteins with a low concentration of DOC. An increasing amount of DOC is then added to yield the purified Ca/sup 2 +/ + Mg/sup 2 +/-ATPase. This protein is at least 95% pure. Adding additional DOC to the purified enzyme enhances the enzyme's ability to hydrolyze ATP. (ERB)