Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum.
A protein kinase which phosphorylated histone and protamine was partially purified from bovine cerebellum. Casein and phosvitin were inert as substrates. The enzyme did not require any cyclic nucleotide. A sulfhydryl compound such as 2-mercaptoethanol, glutathione, or cysteine was necessary for the reaction. The optimum pH was 8.5 to 9.0 Km values for ATP and whole histone were 3.3 X 10(-6) M and 150 microgram/ml, respectively. The optimum concentration of Mg2+ varied with histone fractions employed; with H2B histone as substrate the enzyme was most active at 50 to 100 nM Mg2", whereas with H1 and H2A histones the maximum activity was observed at 5 to 10 mM Mg2+ and with H3 and H4 histones the enzyme was active over a range of 5 to 75 mM Mg2+. The enzyme phosphorylated Ser-32 and Ser-36 in H2B histone and Ser-38 in H1 histone, although the reaction with Ser-36 in H2B histone was very slow. The molecular weight was 6.4 X 10(4). The sedimentation coefficient and Stokes radium were about 4.5 and 29 A, respectively. The enzyme showed heterogeneity upon isoelectrofocusing electrophoresis with isoelectric points of 5.6, 6.0, and 6.6. The enzyme was not inhibited by protein inhibitor nor by the regulatory subunit of cyclic AMP-dependent protein kinase. Preliminary analysis suggested that the enzyme was produced from its precursor protein by a limited proteolytic reaction.
D000818:Animals; D002417:Cattle; D002531:Cerebellum / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D006656:Protamine Kinase / Q000302:isolation & purification / Q000378:metabolism; D011479:Protamines; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D046911:Macromolecular Substances; D011479:Protamines; D008623:Mercaptoethanol; D011494:Protein Kinases; D006656:Protamine Kinase; D008274:Magnesium