Characterization of bovine brain calmodulin-dependent protein phosphatase.
Calmodulin-dependent protein phosphatase of bovine brain exhibited a pH optimum of 7 and appeared to require sulfhydryl groups for activity. Phosphatase activity was inhibited by both NaF and ZnCl2, but was stimulated approximately 2-fold by MnCl2. The enzyme exhibited broad substrate specificity, dephosphorylating casein, troponin I, protamine, histone, and phosvitin, and was not phosphorylated by cAMP-dependent protein kinase. With 32P-labeled casein as a substrate, phosphatase was activated 15-fold by calmodulin; the dissociation constant of phosphatase for calmodulin was 30 nM. Activation of the enzyme by calmodulin as a function of Ca2+ was highly cooperative; the Hill coefficient was 4.9. At a saturating concentration of calmodulin, half-maximal activation of phosphatase was obtained at 0.3 microM Ca2+. Calmodulin increased the Vmax from 1.7 to 41 nmol mg protein-1 min-1 with no significant change in its Km. Formation of a Ca2+-dependent complex between calmodulin and the phosphatase was demonstrated by a calmodulin-Sepharose affinity column, gel-filtration chromatography, and sedimentation on a sucrose density gradient. The rate of formation and dissociation of the calmodulin X phosphatase complex was rapid and readily reversible in response to changes in Ca2+ concentration. The calmodulin X phosphatase complex consists of 1 mol of calmodulin and 1 mol of phosphatase.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology; D002148:Calmodulin-Binding Proteins; D002417:Cattle; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D013379:Substrate Specificity
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010749:Phosphoprotein Phosphatases; D002118:Calcium