Purification and characterization of Ca2+/calmodulin-dependent protein kinase I from bovine brain.
Ca2+/calmodulin-dependent protein kinase (Ca2+/CaM kinase I), which phosphorylates site I of synapsin I, has been highly purified from bovine brain. The physical properties and substrate specificity of Ca2+/CaM kinase I were distinct from those of all other known Ca2+/CaM kinases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified enzyme preparation consisted of two major polypeptides of Mr 37,000 and 39,000 and a minor polypeptide of Mr 42,000. In the presence of Ca2+ and calmodulin (CaM), all three polypeptides bound CaM, were autophosphorylated on threonine residues, and were labeled by the photoaffinity label 8-azido-ATP. Peptide maps of the three autophosphorylated polypeptides were very similar. The Stokes radius and the sedimentation coefficient of the enzyme were, respectively, 31.8 A and 3.25 s. A molecular weight of 42,400 and a frictional ratio of 1.38 were calculated from the above values, suggesting that Ca2+/CaM kinase I is a monomer. It is possible that the polypeptides of lower molecular weight are derived from the polypeptide of Mr 42,000 by proteolysis; alternatively, the polypeptides may represent isozymes of Ca2+/CaM kinase I. Synapsin I (site I) was the best substrate tested (Km, 2-4 microM) for Ca2+/CaM kinase I. Of many additional proteins tested, only protein III (a phosphoprotein related to synapsin I) and smooth muscle myosin light chain were phosphorylated. Ca2+/CaM kinase I was found in highest concentration in brain, where it showed widespread regional and subcellular distributions. In addition, the enzyme had a widespread and predominantly cytosolic tissue distribution. The widespread neuronal and tissue distribution of Ca2+/CaM kinase I suggests that other substrates might exist for this enzyme in both neuronal and non-neuronal tissues.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity; D016704:Synapsins; D014018:Tissue Distribution
D000345:Affinity Labels; D001386:Azides; D002147:Calmodulin; D009419:Nerve Tissue Proteins; D016704:Synapsins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D002118:Calcium