Rapid purification of phospholamban by monoclonal antibody immunoaffinity chromatography

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Abstract
Monoclonal antibodies have been raised against canine phospholamban purified by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). Four of twenty-four antibodies were purified to close to homogeneity from mouse ascites. All four antibodies could react with isolated bovine cardiac sarcoplasmic reticulum (SR) to result in the stimulation of ATP-dependent Ca2+ pump activity and blocking of phospholamban phosphorylation by cAMP-dependent protein kinase. Relative efficiencies of antibodies in Ca2+ pump stimulation and on phospholamban phosphorylation were not correlated. An immunoabsorbent prepared by conjugating antibody Al to Affi-Gel 10 was used for the purification of phospholamban. Isolated bovine cardiac SR was solubilized in a buffer containing deoxycholate and the soluble fraction was applied to the immunoaffinity column. After washing the column with a series of detergent-containing buffer solutions, the column-bound protein which contained essentially pure phospholamban was eluted by a buffer containing 2.8 M MgCl2. The phospholamban recovery from the immunoaffinity column was close to 100%; the overall yield of purification from SR vesicles was about 70%. SDS–PAGE analysis showed that purified phospholamban consisted of a 25 and 5 kilodalton (kDa) protein species. Upon brief boiling (20 s) of the sample in SDS–PAGE sample buffer, five molecular species ranging from 5 to 25 kDa could be detected by immunotransblotting following SDS–PAGE. This observation supports the notion that phospholamban is composed of five 5-kDa polypeptides. The pure phospholamban could be phosphorylated maximally by cAMP-dependent protein kinase to 1–1.5 mol phosphate/mol phospholamban (25 000 g). This stoichiometry of phosphorylation could be increased to about 5 upon addition of the immunoaffinity column flow through fraction.

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