Purification and characterization of Novikoff ascites tumor protein kinase

Abstract

A protein kinase, designated KII, was purified 5000-fold from Novikoff ascites [rat hepatoma] cells. The purification procedure also allows for the purification of a 2nd major protein kinase, designated KI, as well as RNA polymerase I and II. Purified KII has a sedimentation constant of 7.6 S and a Stokes radius of 39 .ANG., suggesting a molecular weight of .apprx. 122,000. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate suggests the enzyme is composed of subunits of MW 44,000, 40,000 and 26,000 present in a molar ratio of 1:1:2. Incubation of the enzyme alone in the presence of [.gamma.-32P]ATP results in the phosphorylation of the 26,000 dalton subunit. KII actively phosphorylates phosvitin, casein and nonhistone chromosomal proteins but does not phosphorylate basic proteins such as histones or protamine to an appreciable extent. Km values of 3.6 .mu.M for ATP and 6.5 .mu.M for GTP were determined in the presence of 4 mM Mg2+. The enzyme is neither stimulated by cyclic[c]AMP or cGMP, nor inhibited by the regulatory subunit of rabbit muscle protein kinase. Its activity is stimulated by KCl at concentrations below 0.2 M and inhibited by higher concentrations.