Purification and characterization of Ca2+/calmodulin-dependent protein kinase V from rat cerebrum.

Abstract

A novel Ca2+/calmodulin-dependent protein kinase (CaM kinase V) from rat cerebrum was purified. This kinase phosphorylates the synthetic peptide substrate syntide-2. The purified enzyme showed a single protein band with a molecular mass of 41 kDa on SDS-polyacrylamide gel electrophoresis. The Stokes radius and the sedimentation coefficient were 31.8 A and 2.83 S, respectively. An approximate molecular mass of 37 kDa was calculated for the native enzyme, and a monomeric structure of the enzyme was suggested. Expression of the enzymatic activity required the presence of both Ca2+ and calmodulin (apparent Ka = 24 +/- 7 nM). The CaM kinase V had an apparent Km for ATP of 75 +/- 11 microM and for syntide-2 of 20 +/- 4 microM. CaM kinase V undergoes autophosphorylation in response to Ca2+ and calmodulin. CaM kinase V was digested with lysyl endopeptidase, and the partial amino acid sequence was determined. A computer homology search revealed no identical protein. KN-62, a selective inhibitor of Ca2+/calmodulin-dependent protein kinase II, inhibited CaM kinase V, with a Ki of 0.8 microM. CaM kinase V phosphorylates a number of endogenous proteins.