Purification and properties of a multifunctional calcium/calmodulin-dependent protein kinase from rat pancreas.
A calcium/calmodulin-dependent protein kinase (Ca/calmodulin protein kinase) was purified from rat pancreas using hydrophobic chromatography followed by gel filtration and affinity chromatography. Ca/calmodulin protein kinase from pancreas resembled previously described multifunctional Ca/calmodulin protein kinases from other tissues with respect to substrate specificity, autophosphorylation on serine and threonine residues, and catalytic and hydrodynamic properties. While Ca/calmodulin protein kinase from other tissues contains subunits of 53-60 kDa with variable proportions of a smaller 50-52 kDa subunit, pancreatic Ca/calmodulin protein kinase was found to contain a single component of 51 kDa. Experiments mixing brain Ca/calmodulin protein kinase with pancreatic homogenate suggest that the absence of a larger subunit in the pancreatic Ca/calmodulin protein kinase is not due to proteolytic degradation during enzyme preparation. Ca/calmodulin protein kinase binding to 125I-labeled calmodulin in solution was demonstrated using the photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azidobenzoate. 125I-labeled calmodulin binding to Ca/calmodulin protein kinase was also demonstrated using filters containing Ca/calmodulin protein kinase transferred from polyacrylamide gels after two-dimensional gel electrophoresis. Finally, the ribosomal substrate for Ca/calmodulin protein kinase was identified as the ribosomal protein, S6. The purification procedure presented in this study promises to be useful in characterizing Ca/calmodulin protein kinase in other tissues and in clarifying the role of these enzymes in cellular function.
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology*; D010766:Phosphorylation; D010777:Photochemistry; D011487:Protein Conformation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D016704:Synapsins
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D016704:Synapsins; D011494:Protein Kinases; D002118:Calcium