Purification and Characterization of a Ca2+- and Calmodulin-Dependent Protein Kinase from Rat Brain |
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Authors: | Kohji Fukunaga Hideyuki Yamamoto Kazuo Matsui Kenji Higashi Eishichi Miyamoto |
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Institution: | Department of Pharmacology, Kumamoto University Medical School, Kumamoto, Japan |
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Abstract: | Abstract: A Ca2+- and calmodulin-dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800-fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S-300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the native enzyme has a large molecular weight and consists of 11 to 14 identical subunits. The purified enzyme exhibited K m values of 109 and 30 μM for ATP and chicken gizzard myosin light chain, respectively, and K a values of 12 n M and 1.9 μM for brain calmodulin and Ca2+, respectively. In addition to myosin light chain, myelin basic protein, casein, arginine-rich histone, microtubule protein, and synaptosomal proteins were phosphorylated by the enzyme in a Ca2+- and calmodulin-dependent manner. The purified enzyme was phosphorylated without the addition of the catalytic subunit of cyclic AMP-dependent protein kinase. Our findings indicate that there is a multifunctional Ca2+- and calmodulin-dependent protein kinase in the brain and that this enzyme may regulate the reactions of various endogenous proteins. |
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Keywords: | Brain cytosol fraction Ca2+- and calmodulin-dependent protein kinase Microtubule Synaptosomal fraction |
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