Characterization of Cyclic Nucleotide-Independent Protein Kinase Produced Enzymatically from Its Proenzyme by Calcium-Dependent Neutral Protease from Rat Liver

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The protein kinase which is produced from its proenzyme partially purified from rat liverthrough limited proteolysis by a Ca⁺²-dependent protease (Takai, Y., Yamamoto, M., Inoue, M., Kishimoto, A., & Nishizuka, Y. (1977) Biochein. Biophys. Res. Commun. 77, 542-550) is characterized. The protein kinase is entirely independent of cyclic nucleotides. The enzyme phosphorylates histone and protamine but not casein or phosvitin. The active protein kinase shows an optimum pH of 8.5 to 9.0, and requires high concentrations of Mg²⁺ (50 to 100mM) and 2-mercaptoethanol (K_a 6mM). The enzyme shows a Stokes radius of about 38Å, and an S value of about 3.9 which corresponds to a molecular weight of 5.1×10⁴. Analysis on the phosphorylated sites in histone fractions indicates that the enzyme phosphorylates Ser-38 in HI histone and Ser-32 and Ser-36 in H2B histone, which have all been identified as the sites for cyclic AMP-dependent as well as for cyclic GMP-dependent protein kinases. However, the enzyme is not inhibited by the regulatory subunit nor by the protein inhibitor described for cyclic AMP-dependent protein kinase. The enzyme also differs from cyclic GMP-de-pendent protein kinase in physical and kinetic properties. The exact relationship of this new species of protein kinase to cyclic nucleotide-dependent enzymes has remained unexplored.