Purification and characterization of a multifunctional calmodulin-dependent protein kinase from canine myocardial cytosol

• Published in: Archives of Biochemistry and Biophysics Vol. 248; no. 1; pp. 21 - 29
• Main Authors: Iwasa, Takafumi, Inoue, Nobuhiro, Fukunaga, Kohji, Isobe, Toshiaki, Okuyama, Tsuneo, Miyamoto, Eishichi
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.07.1986; Elsevier BV; Elsevier
• Subjects: Animals; Applied sciences; Calcium; Calcium - pharmacology; Calcium-Binding Proteins; Calcium-Binding Proteins - metabolism; Calmodulin; Calmodulin - metabolism; Calmodulin - pharmacology; Cattle; Centrifugation, Density Gradient; Chickens; Cytosol; Cytosol - enzymology; Dogs; Electrophoresis, Polyacrylamide Gel; Exact sciences and technology; Glycogen Synthase; Glycogen Synthase - metabolism; Molecular Weight; Myocardium; Myocardium - enzymology; Other techniques and industries; Protein Kinases; Protein Kinases - isolation & purification; Protein Kinases - metabolism; Rabbits; Substrate Specificity; Troponin; Troponin - metabolism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/0003-9861(86)90396-6

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 M r of 550,000 with a sedimentation coefficient of 16.6 S, and showed a single protein band with a M r 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 K a values of 67 nM and 1.1 μM for calmodulin and Ca 2+, 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 Ca 2+, calmodulin-dependent cellular functions of the canine myocardium, and that the enzyme resembles enzymes detected in the brain, liver, and skeletal muscle.