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 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.
Mochizuki
H
H
Department of Pharmacology, Nagoya University School of Medicine, Japan.
Ito
T
T
Hidaka
H
H
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity
D007546:Isoquinolines; D010455:Peptides; D010879:Piperazines; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C088320:calmodulin-dependent protein kinase V