Purification and characterization of bovine brain calmodulin-dependent protein kinase II. The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme |
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Authors: | Guang Yi Zhang Jerry H Wang Rajendra K Sharma |
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Institution: | (1) Cell Regulation Group, Department of Medical Biochemistry, Faculty of Medicine, The University of Calgary, T2N 4N1 Calgary, Alberta, Canada;(2) The Department of Pathology, College of Medicine, University of Saskatchewan, Royal University Hospital and Saskatoon Cancer Centre, S7N 0W0 Saskatoon, Saskatchewan, Canada |
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Abstract: | Bovine brain contains two calmodulin-dependent phosphodiesterase kinases which are separated on Sephacryl S-300 column. One of these kinases has been purified to homogeneity and shown to belong to the calmodulin-dependent protein kinase II family. Phosphorylation of the 63 kDa phosphodiesterase by this purified protein kinase results in the incorporation of 1.0 mol phosphate per mol subunit and an accompanying increase in Ca2+ concentrations required for the phosphodiesterase activation by calmodulin. The protein kinase undergoes autophosphorylation to incorporate 1.0 mol phosphate per mol of subunit of the enzyme and the autophosphorylated enzyme is active, independent of the presence of Ca2+. The autophosphorylation reaction as well as the protein kinase reaction are rendered Ca2+ independent in less than 15 seconds when approximately one mol phosphate per mol protein kinase is incorporated. The result suggests that activation of phosphodiesterase phosphorylation reaction may occur prior to the activation of phosphodiesterase and phosphatase during a cell Ca2+ flux via the protein kinase autophosphorylation mechanism.Abbreviations SDS
sodium dodecyl sulfate
- EGTA
ethylene glycol bis (-aminoethyl ether)
- N,N,N,N
tetra acetic acid
- EDTA
ethylenediamine-tetraacetic acid
- cAMP
cyclic adenosine 35 monophosphate
This work is supported by grants from the Medical Research Council of Canada (JHW), the Heart and Stroke Foundation of Alberta (JHW and RKS) and the Heart and Stroke Foundation of Saskatchewan (RKS) |
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Keywords: | Ca2+ calmodulin protein kinase phosphodiesterase |
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