The (2′‐5′)Oligoadenylate (pppA2′‐5′A2′‐5′A) Synthetase and Protein Kinase(s) from Interferon‐Treated Cells

Two enzyme activities have been partially purified from interferon‐treated mouse L cells: the (2′‐5′)A n synthetase responsible for the synthesis of pppA2′‐5′A2′‐5′A (and related oligomers) and one or more protein kinases responsible for the phosphorylation of (a) a polypeptide of molecular weight 67000 (67000‐ M r polypeptide kinase), (b) the 35000‐ M r subunit of the protein synthesis initiation factor eIF‐2 and (c) added histones. Chromatography on columns of poly(I) · poly(C)‐Sepharose provided a more than 1000‐fold purification of these enzymes in a single step and in high yield. The partially‐purified poly(I) · poly(C)‐Sepharose‐bound (2′‐5′)A n synthetase is stable and on incubation with ATP is active for several days. This provides a convenient means for the synthesis and accumulation of pppA2′‐5′A2′‐5′A. A simple two‐step elution of the bound enzymes from the poly(I) · poly(C)‐Sepharose yielded a kinase fraction free of detectable (2′‐5′)A n synthetase. The (2′‐5′)A n synthetase from these columns, on the other hand, always contained detectable kinase activity. This latter could be removed on DEAE‐cellulose, but the resultant ‘kinase‐free′ (2′‐5′)A n synthetase required, in addition to double‐stranded RNA (dsRNA), a second component for optimum activity. This second component chromatographed with the kinase but no evidence for phosphorylation of the synthetase by it was obtained. It may be, therefore, that the association of the second component with the kinase(s) is fortuitous. The most purified kinase fractions still contained the 67000 ‐ M r polypeptide kinase, eIF‐2 kinase and histone kinase activities. A partial separation of the histone kinase activity was obtained, however, emphasising the possible heterogeneity of the kinase fraction. In contrast to the purified (2′‐5′)A n synthetase, the purified kinase activities were no longer dependent upon dsRNA.