PURIFICATION AND CHARACTERIZATION OF CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE-IV KINASE FROM RAT-BRAIN

被引:76
作者
OKUNO, S
KITANI, T
FUJISAWA, H
机构
[1] Department of Biochemistry, Asahikawa Medical College, Asahikawa
关键词
BRAIN; CA2+; CALMODULIN; PROTEIN KINASE; PURIFICATION;
D O I
10.1093/oxfordjournals.jbchem.a124617
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Calmodulin-dependent protein kinase IV (CaM-kinase IV) kinase was recently discovered in the rat brain by its activity to activate the inactive recombinant CaM-kinase IV expressed in Escherichia coli [Okuno, S. and Fujisawa, H. (1993) J. Biochem. 114, 167-170]. In the present study, CaM-kinase IV kinase was purified approximately 2,000-fold from rat cerebral cortex by purification procedures including calmodulin affinity chromatography, and its properties were examined. The highly purified CaM-kinase IV kinase gave one major protein band corresponding to a molecular weight of about 66,000 upon SDS-PAGE. The purified CaM-kinase IV kinase phosphorylated and concomitantly activated CaM-kinase IV purified from rat brain as well as the recombinant kinase expressed in Escherichia coli in a Ca2+/calmodulin-dependent manner. The phosphorylation of CaM-kinase IV by CaM-kinase IV kinase occurred on only serine residue(s). Among a number of proteins, including several known to be phosphorylated by the various protein kinases tested, CaM-kinase IV was the best substrate for CaM-kinase IV kinase. Since syntide-2, a synthetic peptide known to be a good peptide substrate for calmodulin-dependent protein kinase II (CaM-kinase II), was a fairly good substrate for CaM-kinase IV kinase, some kinetic properties of CaM-kinase IV kinase were examined using syntide-2 as a substrate. The K-m value for the peptide substrate in the presence of Ca2+/calmodulin was almost two orders of magnitude lower than that in its absence, although the V-max value was almost the same in the presence and absence of Ca2+/calmodulin.
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页码:923 / 930
页数:8
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