Purification and partial characterization of a protein kinase from the Prague-C strain of rous sarcoma virus

A protein kinase has been purified from Rous sarcoma virus strain Prague-C by chromatography on DEAE-cellulose and phosphocellulose, followed by glycerol density gradient centrifugation. The purified enzyme had a sedimentation coefficient of about 2.5 S, corresponding to an M_Γ of about 25,000. Electrophoresis in SDS-polyacrylamide gels revealed a polypeptide with an M_Γ of about 11,000. The sedimentation coefficient of the enzyme in virus lysates was 3.9 S, corresponding to an M_Γ of about 45,000. Comparison of its chymotrypic and CNBr cleavage products with those of p15, p12, and p10 showed that it was not identical with any of these polypeptides. The enzyme required divalent metal ions for activity, Mgt²⁺ being preferred to Mn²⁺, It did not depend on cyclic nucleotides for activity. Its K_m for ATP and GTP were 40 and 870 μM respectively, but the V_max at saturating triphosphate concentrations was five times higher with GTP than with ATP. The most efficient exogenous phosphate acceptor was casein, followed by phosvitin and arginine-rich histone. The most efficient phosphate acceptors in virus lysates were, in order, two small polypeptides that were probably the enzyme subunit itself and p12, followed by p19 and unidentified proteins with molecular weights larger than 50,000 that were probably traces of host cell polypeptides in the virus envelope. In virus lysates that had been heated for 2 min at 90° and supplemented with purified protein kinase, the most. active phosphate acceptor was an unidentified protein with an M_Γ of about 38,000.