Purification and characterization of a brain-specific multifunctional calmodulin-dependent protein kinase from rat cerebellum.
A brain-specific multifunctional calmodulin-dependent protein kinase, calmodulin-dependent protein kinase IV, which exhibited characteristic properties quite different from those of calmodulin-dependent protein kinase II, was purified approximately 230-fold from rat cerebellum. The purified preparation gave two protein bands with molecular weights of 63,000 (alpha) and 66,000 (beta) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both of which showed protein kinase activity as examined by the activity gel method. The molecular weight of the enzyme was estimated as about 67,000 from sedimentation coefficient (3.2 S) and Stokes radius (50 A), indicating a monomeric structure of the enzyme. The enzyme phosphorylated smooth muscle myosin light chain, synapsin I, microtubule-associated protein 2, tau protein, myelin basic protein, histone H1, and tyrosine hydroxylase in a Ca2+/calmodulin dependent manner, suggesting that the enzyme is a multifunctional calmodulin-dependent protein kinase capable of phosphorylating a large number of substrates. A synthetic peptide, Lys-Ser-Asp-Gly-Gly-Val-Lys-Lys-Arg-Lys-Ser-Ser-Ser-Ser, was found to be a specific substrate for this kinase and, using this peptide as substrate, the distribution of the enzyme activity in various rat tissues was examined. The activity was found in cerebral cortex, brain stem, and cerebellum, most abundantly in cerebellum, but other tissues tested, including liver, spleen, kidney, lung, heart, skeletal muscle, and adrenal gland showed very little activity.
Miyano
O
O
Department of Biochemistry, Asahikawa Medical College, Hokkaido, Japan.
Kameshita
I
I
Fujisawa
H
H
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002531:Cerebellum / Q000201:enzymology*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D036341:Intercellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014018:Tissue Distribution
D036341:Intercellular Signaling Peptides and Proteins; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D010455:Peptides; C063391:syntide-2; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases