Purification and characterization of a multifunctional calmodulin-dependent protein kinase from canine myocardial cytosol |
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| Institution: | 1. Department of Biology, University of Copenhagen, 13 Universitetsparken, Copenhagen Ø DK-2100, Denmark;2. Facultad de Biología, Departamento de Microbiología, Universidad de Sevilla, Avenida Reina Mercedes 6, Sevilla E-41012, Spain;3. Cancer and Human Molecular Genetics Area – Oto-Neurosurgery Research Group, University Hospital La Paz Research Institute (IdiPAZ), Paseo de la Castellana 261, Madrid E-28046, Spain;1. Department of Chemistry and Bioscience, Aalborg University, Frederik Bajers vej 7H, 9220 Aalborg, Denmark;2. Institute of Biotechnology, University of Helsinki, PO Box 65, Helsinki, FIN-00014, Finland |
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| Abstract: | A calmodulin-dependent protein kinase from canine myocardial cytosol was purified 1150-fold to apparent homogeneity with a 1.5% yield. The purified enzyme had a Mr of 550,000 with a sedimentation coefficient of 16.6 S, and showed a single protein band with a Mr of 55,000 (55K protein), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 1.6 μmol/mg protein/min, and Ka values of 67 nM and 1.1 μM for calmodulin and Ca2+, respectively, using chicken gizzard myosin light chain as substrate. Calmodulin bound to the 55K protein. The purified enzyme had a broad substrate specificity. Endogenous proteins including glycogen synthase, phospholamban, and troponin I from the canine heart were phosphorylated by the enzyme. These results suggest that the purified enzyme works as a multifunctional protein kinase in the Ca2+, calmodulin-dependent cellular functions of the canine myocardium, and that the enzyme resembles enzymes detected in the brain, liver, and skeletal muscle. |
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