Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain.
A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D004355:Drug Stability; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D010446:Peptide Fragments; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D002147:Calmodulin; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011494:Protein Kinases; D002118:Calcium