A Monoclonal Antibody against Brain Calmodulin-Dependent Protein Kinase Type II Detects Putative Conformational Changes Induced by Ca²⁺-Calmodulin

A mouse monoclonal IgG l antibody has been generated against the soluble form of the calmodulin-dependent protein kinase type II. This antibody recognizes both the soluble and cytoskeletal forms of the enzyme, requiring Ca²⁺(EC₅₀= 20 μM) for the interaction. Other divalent cations such as Zn²⁺, Mn²⁺, Cd²⁺, Co²⁺, and Ni²⁺will substitute for Ca²⁺, while Mg²⁺and Ba²⁺will not. The antibody reacts with both the a- and β-subunits on Western blots in a similar Ca²⁺-dependent fashion but with a lower sensitivity. The affinity of the antibody for the kinase is 0.13 nM determined by displacement of¹²⁵I Bolton-Hunter-labeled kinase with unlabeled enzyme. A variety of other proteins including tubulin do not compete for antibody binding. The M_r30 000 catalytic fragment obtained by proteolysis of either the soluble or the cytoskeletal form of the kinase fails to react with the antibody. Calmodulin and antibody reciprocally potentiate each other's interaction with the enzyme. This is illustrated both by direct binding studies and by a decrease of the k_mappfor calmodulin and an increase in the V_maxfor the autophosphorylation reaction of the enzyme. The antibody thus appears to recognize and stabilize a conformation of the kinase which favors calmodulin binding although it does not itself activate the kinase in the absence of calmodulin. Since themr 30 000 catalytic fragment of the kinase is not immunoreactive, either the antibody combining site of the kinase must be present in the noncatalytic portion of the protein along with the calmodulin binding site or proteolysis interferes with the putative Ca²⁺-dependent conformational change. Thus, monoclonal antibodies can be useful tools in elucidating the mechanism by which Ca²⁺and calmodulin act on the kinase molecule.