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.
Kitani
T
T
Department of Biochemistry, Asahikawa Medical College, Hokkaido.
Okuno
S
S
Fujisawa
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D011994:Recombinant Proteins; D004492:Edetic Acid; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517686:Camk4 protein, rat; D008274:Magnesium