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.
(Received for publication, May 10, 1983). This investigation was supported in part by National Institutes of Health Grants NS17660 and 1 T32 GM07616, and by a Gordon Ross fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We would like to thank Dr. Jeremy Brockes for use of his tissue culture facilities, Dr. Robert Adelstein for a gift of smooth muscle myosin light chain, Dr. Claude Klee for a gift of rabbit anti-calcineurin, and C. Hochenedel and C. Oto for help in preparing the manuscript.
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