PURIFICATION AND CHARACTERIZATION OF A PROTEIN-KINASE FROM DWARF PEA EPICOTYLS

A protein kinase was purified about 330-fold from dwarf pea epicotyls, using purified dwarf pea RNA polymerase II as a substrate protein. After casein-Sepharose affinity chromatography, the preparation showed a single protein stained band on native acrylamide gel electrophoresis. Serial scanning of the parallel unstained gel also showed a single activity peak of protein kinase coinciding with the protein stained band. Fractionation of purified protein kinase on SDS-PAGE showed that the M(r) of the protein subunit was 63 000. Molecular sieve chromatography of the casein-Sepharose fraction on Sephacryl S-200 revealed that the M(r) of the native protein kinase was 76 000. Thus, it appears that the pea protein kinase is a monomer. The pH optimum of protein kinase activity was 8.0, and the optimum concentration of Mg2+ was 15 mM. Purified pea protein kinase was considerably more efficient in phosphorylating purified RNA polymerase II and casein in comparison with histones and phosvitin. The in vitro phosphorylation of extensively purified RNA polymerase II brought about a two-fold stimulation of enzyme activity over that of the controls. The in vitro phosphorylated RNA polymerase II showed radioactivity associated with authentic phosphoserine.