Purification and Cahracyterization of te Brain Calmodulin‐Dependent Protein Kinase (Kinase II), Which Is involved in the Activtion of Tryptophan 5‐Monooxygnase

Abstract

Calmodulin‐dependent protein kinase (kinase II) [Yamauchi, T. and Fujisawa, H. (1980) FEBS Lett. 116 141–144] which is involed in the activation of tryptophan 5‐monoosygenase was purified 720‐fold wiht a 36% yield from rat cerebvral cortex using ammonium sulfate and chromatography on Sepharose CL‐4B, Calmodulin‐Sepharose 4B and phosphocellulose. The purified enzyme showed one major protein band corresponding in to a molecular weight of 55000 and a faint band upo0n sodium dodecyl sufate/polyacrylamide disc gel electophoresis, wherase it gave a single protein band upon polyacrylamide gel electroporesis whithout soudium dodecyl sulfate. The molecualar weight ad th sedimentation coeffieient of the kinase were dtermined to be 540000 by sedimentation equilibrium and 16.5 S by sucrose density gradient cntrifugation. The Kinase required absolutely calmodulin and Ca 2+ for its activity and the apparent K a values for calmodulin and Ca+2 were 10 nM and 1.6 μM respectively. The K m values for ATP and Mg 2+ wer calculated to be 0.06 mM and 1 mM, respectively. The concentration of tryptophan 5‐monooxygenase required to produece half‐maximal effects on ist activation by the kinase was estimated to be as low as 0.3 nM, on the basis of th finding that the molecular weight and the specific activity of trytophan 5‐monooxygenas were 245000 and 374 nmol min−1 mg oprotein−1 respectively [Nakata, H. and Fuisawa, H. (1982) Eur. J. Biochem. 122, 41–47]. The kinase phosphorylated casein, smooth muscle myosin light chain as well some endogenous protiens of brain cytosol. The enzyme did not phoisphorylte significantly histone, protamine, and phsphorlyase. b. Some other proterties of he Kinase were exiamined.