Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase.
Calmodulin-dependent protein kinase (kinase II) [Yamauchi, T. and Fujisawa, H. (1980) FEBS Lett. 116, 141-144], which is involved in the activation of tryptophan 5-monooxygenase, was purified 720-fold with a 36% yield from rat cerebral cortex using ammonium sulfate fractionation and chromatography on Sepharose CL-4B, calmodulin-Sepharose 4B and phosphocellulose. The purified enzyme showed one major protein band corresponding in mobility to a molecular weight of 55000 and a faint band upon sodium dodecyl sulfate/polyacrylamide disc gel electrophoresis, whereas it gave a single protein band upon polyacrylamide gel electrophoresis without sodium dodecyl sulfate. The molecular weight and the sedimentation coefficient of the kinase were determined to be 540000 by sedimentation equilibrium and 16.5 S by sucrose density gradient centrifugation. The kinase required absolutely calmodulin and Ca2+ for its activity and the apparent Ka values for calmodulin and Ca2+ were 10 nM and 1.6 microM respectively. The Km values for ATP and Mg2+ were calculated to be 0.06 mM and 1 mM, respectively. The concentration of tryptophan 5-monooxygenase required to produce half-maximal effects on its activation by the kinase was estimated to be as low as 0.3 nM, on the basis of the finding that the molecular weight and the specific activity of tryptophan 5-monooxygenase were 245000 and 374 nmol min-1 mg protein-1 respectively [Nakata, H. and Fujisawa, H. (1982) Eur. J. Biochem. 122, 41-47]. The kinase phosphorylated casein, smooth muscle myosin light chain as well as some endogenous proteins of brain cytosol. The enzyme did not phosphorylate significantly histone, protamine, and phosphorylase b. Some other properties of the kinase were examined.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000502:physiology; D051381:Rats; D014365:Tryptophan Hydroxylase / Q000378:metabolism*
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D014365:Tryptophan Hydroxylase; D011494:Protein Kinases