Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain.
A calcium and calmodulin-dependent protein kinase has been purified from rat brain. It was monitored during the purification by its ability to phosphorylate the synaptic vesicle-associated protein, synapsin I. A 300-fold purification was sufficient to produce kinase that is 90-95% pure as determined by scans of stained sodium dodecyl sulfate-polyacrylamide gels and has a specific activity of 2.9 mumol of 32P transferred per min/mg of protein. Thus, the kinase is a relatively abundant brain enzyme, perhaps comprising as much as 0.3% of the total brain protein. The Stokes radius (95 A) and sedimentation coefficient (16.4 S) of the kinase indicate a holoenzyme molecular weight of approximately 650,000. The holoenzyme is composed of three subunits as judged by their co-migration with kinase activity during the purification steps and co-precipitation with kinase activity by a specific anti-kinase monoclonal antibody. The three subunits have molecular weights of 50,000, 58,000, and 60,000, and have been termed alpha, beta', and beta, respectively. The alpha- and beta-subunits are distinct peptides, however, beta' may have been generated from beta by proteolysis. All three of these subunits bind calmodulin in the presence of calcium and are autophosphorylated under conditions in which the kinase is active. The subunits are present in a ratio of about 3 alpha-subunits to 1 beta/beta'-subunit. We therefore postulate that the 650,000-Da holoenzyme consists of approximately 9 alpha-subunits and 3 beta/beta'-subunits. The abundance of this calmodulin-dependent protein kinase indicates that its activation is likely to be an important biochemical response to increases in calcium ion concentration in neuronal tissue.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D011485:Protein Binding; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D002147:Calmodulin; D046911:Macromolecular Substances; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases