Stimulation of bovine cardiac sarcoplasmic reticulum Ca2+ pump and blocking of phospholamban phosphorylation and dephosphorylation by a phospholamban monoclonal antibody.
A monoclonal antibody, A1, was produced against sodium dodecyl sulfate-polyacrylamide gel electrophoresis purified canine phospholamban and isolated from mouse ascites by chromatography on a hydroxylapatite column. Western immunoblotting experiments showed that the antibody was specific for phospholamban and cross-reacted with the protein from a bovine source. Incubation of bovine cardiac sarcoplasmic reticulum (SR) vesicles with the antibody resulted in a marked increase in the ATP-dependent Ca2+ pump activity which was slightly higher than that brought about by cyclic AMP-dependent protein kinase. This observation provides direct proof for the involvement of phospholamban as a SR Ca2+ pump regulatory protein. In addition to stimulating the Ca2+ pump activity, antibody A1 was capable of blocking the phosphorylation of phospholamban by cyclic AMP-dependent protein kinase and by an endogenous SR Ca2+/calmodulin-dependent protein kinase. It was also capable of blocking the dephosphorylation of phosphorylated phospholamban by an endogenous SR protein phosphatase. From these observations, it may be suggested that the antigenic site of A1 antibody is proximal to the phosphorylation sites of phospholamban.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002417:Cattle; D004285:Dogs; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D000099211:Phospholamban