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Published April 1983 | Published
Journal Article Open

A calcium/calmodulin-dependent protein kinase from mammalian brain that phosphorylates Synapsin I: partial purification and characterization


A calcium/calmodulin-dependent protein kinase, which phosphorylates a synaptic vesicle-associated protein designated Synapsin I, has been shown to be present in both soluble and particulate fractions of rat brain homogenates. In the present study, the particulate activity was solubilized by washing with a low ionic strength solution, and the enzymes from the two fractions were partially purified by ion exchange chromatography and calmodulin-Sepharose affinity chromatography. By each of several criteria, the partially purified enzymes from the two sources were indistinguishable. These criteria included specificity for various substrate proteins, concentration dependence of activation by calcium and calmodulin, pH dependence, and apparent affinities for the substrates Synapsin I and ATP. The mild conditions that released the particulate enzyme indicated that it was not tightly bound to the membrane and suggested that it may exist in a dynamic equilibrium between soluble and particulate-bound states. The partially purified enzyme preparations from both the soluble and particulate fractions contained three proteins that were phosphorylated in the presence of calcium and calmodulin, a 50-kilodalton (Kd) protein and two proteins in the 60-Kd region. When compared by phosphopeptide mapping and two-dimensional gel electrophoresis, the proteins were indistinguishable from three proteins of corresponding molecular weights that were shown by Schulman and Greengard (Schulman, H., and P. Greengard (1978) Nature 271: 478-479) to be prominent substrates for calcium/calmodulin-dependent protein kinase in a crude particulate preparation from rat brain. The 50-Kd substrate was the major Coomassie blue staining protein in both partially purified enzyme preparations. The peak of this protein coincided with that of enzyme activity during DEAE-cellulose and calmodulin-Sepharose chromatography. These results suggest that the 50-Kd phosphoprotein may be an autophosphorylatable subunit of the Synapsin I Kinase or may exist in a complex with it.

Additional Information

© 1983 by Society for Neuroscience. Received August 11, 1982; Revised October 28, 1982; Accepted November 10, 1982. This work was supported by National Institutes of Health grants MH-17387 and NS-08440, and a grant from the McKnight Foundation (P. G.); National Institutes of Health Grant NS-17660, the Church Fund, the Muscular Dystrophy Association, the Pew Fund, and the Sloan Fund (M. K.). We wish to thank Dr. Claude Klee for her gift of cahnodulin-Sepharose, Barbara Moore for technical assistance, and Candace Hochenedel and Annette Gwardyak for help with preparation of the manuscript.

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August 19, 2023
August 19, 2023