Calcium-activated, phospholipid-dependent protein kinase from rat brain. Subcellular distribution, purification, and properties.

The subcellular distribution of Ca2+-activated, phospholipid-dependent protein kinase (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishia k a , Y. (1979) J. Biol. Chem 254, 3692-3695) in rat brain was investigated. Approximately one-third of the protein kinase was recovered in the soluble cytosol fraction, another one-third was in the crude mitochondrial fraction, and the rest was in nuclear and microsomal fractions. Upon further analysis of the crude mitochondrial fraction, most of the enzyme was found to be associated with synaptosomal membranes. The cytosol protein kinase was purified approximately 800fold to apparent homogeneity by DEAE-cellulose and Sephadex G-150 column chromatographies, followed by isoelectrofocusing electrophoresis, blue-Sepharose CL-GB, and phenyl-Sepharose CG4B column chromatographies. The molecular weight of the protein kinase was about 77,000 as estimated by sucrose density gradient ultracentrifugation. The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M, 82,000, indicating that the enzyme is composed of one polypeptide chain. The enzyme was free of calmodulin. The protein kinase associated with membrane was solubilized with Triton X100, and partially purified by DEAE-cellulose and Sephadex G-150 column chromatographies. The membraneassociated protein kinase thus obtained was indistinguishable from the cytosol protein kinase in physical, kinetic, and catalytic properties. Both enzymes were fully activated by diacylglycerol in the presence of phospholipid and less than micromolar concentrations of Ca2+.