Purification and characterization of Novikoff ascites tumor protein kinase.
A protein kinase, designed KII, has been purified 5000-fold from Novikoff ascites tumor cells. The purification procedure also allows for the purification of a second 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 A, suggesting a molecular weight of about 122000. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate suggests the enzyme is composed of subunits of molecular weights 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. Protein kinase II 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 micron for ATP and 6.5 micronM for GTP were determined in the presence of 4mM Mg2+. The enzyme is neither stimulated by cyclic adenosine 3',5'-monophosphate or cyclic guanosine 3', 5'-monophosphate 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.
D000242:Cyclic AMP / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000201:enzymology*; D010750:Phosphoproteins / Q000096:biosynthesis; D011189:Potassium Chloride / Q000494:pharmacology; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology
D010750:Phosphoproteins; D011189:Potassium Chloride; D000242:Cyclic AMP; D011494:Protein Kinases; D012321:DNA-Directed RNA Polymerases