Catalytic site of calmodulin-dependent protein phosphatase from bovine brain resides in subunit A.
The calmodulin-dependent protein phosphatase from bovine brain is composed of two subunits: subunit A, Mr 60,000, and subunit B, Mr 16,500. Using in vitro immunization techniques, we have produced a monoclonal antibody specific for the phosphatase. The antibody was immobilized to Sepharose 4B to prepare an immunoabsorbent column, which was used to purify the enzyme. Phosphatase isolated from the column showed a polypeptide with Mr 60,000, equivalent to subunit A, which showed calmodulin-dependent phosphatase activity. Subunit B was not obtained from the column. Limited trypsin digestion stimulated phosphatase activity, yielding polypeptides of Mr 59,000, 43,000, and 16,000; the phosphatase activity after trypsin digestion was calmodulin independent. Chromatography of trypsin-treated phosphatase on an immunoaffinity column yielded two proteins, Mr 59,000 and 43,000, that were catalytically active and calmodulin independent. In a separate experiment, the two subunits of the phosphatase were separated by gel filtration in 6 M urea. Subunit A isolated from the filtration column showed little or no activity in the presence of Ca2+ and calmodulin, but it showed calmodulin-dependent phosphatase activity in the presence of 0.8 mM Mn2+. Subunit B was catalytically inactive. Collectively, these results indicate that subunit A and its proteolytic fragment contain the catalytic site and the antigenic determinant for the monoclonal antibody.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002148:Calmodulin-Binding Proteins; D002417:Cattle; D002846:Chromatography, Affinity; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002148:Calmodulin-Binding Proteins; D046911:Macromolecular Substances; D010749:Phosphoprotein Phosphatases