Protein kinase activity from vaccinia virions: solubilization and separation into heat-labile and heat-stable components.
A protein kinase was solubilized from whole vaccinia virions by using a solution containing deoxycholate, dithiothreitol, and sodium or potassium chloride. The released enzyme was completely dependent on Mg(2+) and was greatly stimulated by added basic proteins such as protamine or histones. Dithiothreitol was also stimulatory, whereas GTP, CTP, UTP, and P(i) at concentrations equimolar with ATP had little or no effect. Attempts to purify the protein kinase were initially unsuccessful, leading us to consider that either the enzyme was extremely labile or that two readily separable components were required for activity. The observation that the material extracted with NP-40 detergent during the preparation of viral cores stimulated the protein kinase activity of the intact cores supported the second possibility. As the protein kinase, now solubilized from viral cores, was passed through successive DEAE-cellulose columns, it became increasingly dependent for activity on addition of the NP-40 extract. A 30- to 40-fold stimulation of protein kinase activity, which afforded recovery of essentially all starting activity, could be effected by addition of the NP-40 extract to the partially purified enzyme. The NP-40 extract was shown to contain a heat stable, trypsin-sensitive protein, whose action could not be duplicated by cyclic nucleotides.
D002848:Chromatography, DEAE-Cellulose; D003840:Deoxycholic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D006367:HeLa Cells; D006358:Hot Temperature; D011494:Protein Kinases / Q000145:classification / Q000302:isolation & purification* / Q000494:pharmacology; D011506:Proteins / Q000494:pharmacology; D012995:Solubility; D014616:Vaccinia virus / Q000032:analysis / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification; D014764:Viral Proteins / Q000032:analysis
D011506:Proteins; D014764:Viral Proteins; D003840:Deoxycholic Acid; D011494:Protein Kinases