Catalytic site of calmodulin-dependent protein phosphatase from bovine brain resides in subunit A.

Abstract

The calmodulin-dependent protein phosphatase from bovine brain is composed of 2 subunits: subunit A, MW 60,000, and subunit B, MW 16,500. Using in vitro immunization techniques, a monoclonal antibody specific for the phosphatase was produced. 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 MW 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 MW 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 2 proteins, MW 59,000 and 43,000, that were catalytically active and calmodulin independent. In a separate experiment, the 2 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, subunit A and its proteolytic fragment contain the catalytic site and the antigenic determinant for the monoclonal antibody.