Rapid purification of phospholamban by monoclonal antibody immunoaffinity chromatography.
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.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008861:Microsomes / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D011494:Protein Kinases; D000099211:Phospholamban