Calmodulin-dependent protein kinase II from bovine cardiac muscle: purification and differential activation by calcium.
Calmodulin-dependent protein kinase II was purified to apparent homogeneity with a high yield from the total calmodulin-binding protein fraction of bovine cardiac muscle in a single step by gel filtration column chromatography. This procedure is simple and suitable for adaptation to large scale preparations. The purified calmodulin-dependent protein kinase has a specific enzymic activity of 2.4 mumol/min/mg when mixed histone was used as a substrate. The preparation of enzyme appears to be homogeneous when examined by SDS-PAGE. The molecular weight of the enzyme was determined to be 570 kDa by gel filtration. SDS-PAGE of the enzyme subunit showed a single protein band with an apparent molecular weight of 56 kDa. These results suggest that the calmodulin-dependent protein kinase II from bovine heart is composed of 10 identical subunits. Anti-peptide antibody raised against multifunctional calmodulin-dependent protein kinase II from rat brain showed a single immunoreactive band of 56 kDa on Western blot. These results suggested that bovine cardiac muscle calmodulin-dependent protein kinase could resemble the brain isozyme. Calmodulin-dependent protein kinase II undergoes autophosphorylation with a maximal incorporation of 1 mol of phosphate per mol of the subunit of the enzyme and the autophosphorylated enzyme remains active in the absence of Ca2+ and calmodulin. The concentration of Ca2+ required for the activation of calmodulin-dependent protein kinase II depends on the level of calmodulin in the reaction.
Kakkar
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R
Department of Pathology, Saskatoon Cancer Centre, College of Medicine, University of Saskatchewan, Canada.
Raju
R V
RV
Sharma
R K
RK
D000180:Acrylic Resins; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002845:Chromatography; D004789:Enzyme Activation; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation
D000180:Acrylic Resins; D002364:Caseins; D007527:Isoenzymes; C037239:Sephacryl Superfine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium