Comparison of Ca2+/calmodulin-dependent protein kinase IV from rat brain, expressed in insect cells, and expressed in Escherichia coli.

Calmodulin-dependent protein kinase IV (CaM-kinase IV) is thought to play crucial roles in the functioning of Ca2+ in the central nervous system and immune system, and the regulation of its activity is therefore very important. Recombinant CaM-kinase IV is invaluable for studies of its regulatory mechanism, because of its large-amount availability and ready site-specific mutagenesis. In the present study, rat CaM-kinase IV was expressed in Sf9 cells and Escherichia coli, and the kinetic properties were examined with syntide-2 and peptide-gamma as substrates. The recombinant enzymes were produced highly efficiently, comprising as much as about 15% of the total protein in Sf9 cells and 9% in E. coli. The brain enzyme shows two Km values for syntide-2 in the presence of Ca2+/calmodulin, but the recombinant enzymes showed normal kinetic behavior. The brain enzyme and Sf9 enzyme showed Km values for peptide-gamma of 53 and 82 microM, respectively, but the Km of the E. coli enzyme was as high as 1.7 mM, in the presence of Ca2+/calmodulin. Thus, the three enzymes differed in their kinetic properties, but all the three were markedly activated upon incubation with CaM-kinase IV kinase under the Ca2+/calmodulin-dependent protein phosphorylation conditions.