Inactivation of Ca²⁺/Calmodulin-Dependent Protein Kinase IV by Ca²⁺/Calmodulin and Restoration of the Activity by Mg²⁺/EGTA

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The activity of calmodulin-dependent protein kinase IV (CaM-kinase IV) was progressively decreased by incubation at 30°C with calmodulin in the presence of Ca²⁺, becoming one-half to one-fifth of the original activity within several minutes. The amount of calmodulin necessary to produce the maximal inactivation was approximately 1 mol for 1 mol of the enzyme. The inactivation of CaM-kinase IV by Ca²⁺/calmodulin was prevented by ATP in the presence of Mg²⁺, but such protection was not observed with either of the two alone or with a peptide substrate such as syntide-2. The activity of the calmodulininactivated enzyme was increased by incubation at 30°C with Mg²⁺ in the presence of EGTA, being completely restored to the original level within several minutes, indicating that the Ca²⁺/calmodulin-induced inactivation of the enzyme was not due to irreversible denaturation of the enzyme. Both the inactivation of CaM-kinase IV by Ca²⁺/calmodulin and the restoration of its activity by Mg²⁺/EGTA were time- and temperature-dependent reactions. Kinetic analysis revealed that the alterations of the enzyme activity were due mainly to changes in V_max of the enzyme.