PURIFICATION AND CHARACTERIZATION OF CA2+ CALMODULIN-DEPENDENT PROTEIN KINASE-V FROM RAT CEREBRUM

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 angstrom 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 K(a) = 24 +/- 7 nm). The CaM kinase V had an apparent K(m) for ATP of 75 +/- 11 muM and for syntide-2 of 20 +/- 4 muM. 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 K(i) of 0.8 muM. CaM kinase V phosphorylates a number of endogenous proteins.