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

Abstract

A calmodulin-dependent protein phosphatase isolated from bovine brain 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 MW of 60,000 and was composed of 2 protein species of 43,000 and 16,000 daltons. Trypsinization decreased the Km of phosphatase for casein from 10.8-1.2 .mu.M and increased the Vmax from 4.9-30.9 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 .mu.M Ca2+ for half-maximal activation. Both native and trypsinized phosphatase were stimulated by Mn2+ to a level considerably higher than that by Ca2+.