Activation of bovine brain calmodulin-dependent protein phosphatase by limited trypsinization

A calmodulin-dependent protein phosphatase isolated from bovine brain [Tallant, E.A., & Cheung, W.Y. (1983) Biochemistry 22, 3630-3635] is stimulated by limited trypsinization to the same activity level as that by calmodulin. Prolonged trypsinization caused gradual loss of phosphatase activity, a process retarded in the presence of Ca2+, and even more in the presence of calmodulin. Trypsinized phosphatase, when fully activated, had a molecular weight of 60 000 and was composed of two protein species of 43 000 and 16 000 daltons. Trypsinization decreased the Km of phosphatase for casein from 10.8 to 1.2 microM and increased the Vmax from 4.9 to 30.9 nmol (mg of protein)-1 min-1. The proteolyzed enzyme was insensitive to calmodulin and did not bind to a calmodulin-Sepharose affinity column. It was, however, stimulated by Ca2+, requiring 0.4 microM Ca2+ for half-maximal activation. Both native and trypsinized phosphatase were stimulated by Mn2+ to a level considerably higher than that by Ca2+.