Nuclear poly(A) polymerase, the enzyme involved in the posttranscriptional addition of poly(A) to the 3'-termini of eukaryotic mRNA, was purified from the transplanted rat tumor Morris hepatoma 3924A, from primary hepatomas induced by an azo dye [N,N-dimethyl-p(m-tolylazo)aniline], and from normal rat liver. Phosphocellulose column chromatography separated two species of liver poly(A) polymerase, a predominant enzyme of Mr 38,000 (38K) and a minor enzyme of Mr 48,000 (48K), which constituted approximately 1% of the total enzyme prior to extensive purification. In contrast, poly(A) polymerase from the transplanted or primary hepatoma consisted of a single 48K species that was identical to the minor liver enzyme with respect to chromatographic characteristics. The tumor enzyme and the 48K liver enzyme exhibited very similar CNBr cleavage maps, which were distinct from that of the major liver enzyme (38K). Antibodies raised against the purified Morris hepatoma enzyme reacted with poly(A) polymerase from the primary tumor and the 48K liver enzyme but not with the 38K liver species. These data indicate that rat liver contains different genes that code for two structurally and immunologically distinct nuclear poly(A) polymerases, one of which is present in very limited quantities in normal liver but is the only detectable species in either the primary or transplanted hepatoma. The cyclic nucleotide-independent protein kinase activity that is separated from poly(a) polymerase during its purification from nuclei of Morris hepatoma 3924A was purified essentially to homogeneity. The protein kinase copurified with the hepatoma poly(A) polymerase on the phosphocellulose column and was separated (greater than 95%) following further chromatography on a hydroxylapatite column. Removal of the protein kinase by gel filtration on Sephacryl S-200 removed the kinase completely from poly(A) polymerase. This resulted in complete inactivation of poly(A) polymerase, which could be dramatically restored to normal levels by addition of the purified kinase. These data indicate that: (1) phosphorylation of poly(a) polymerase is obligatory to its basal activity; (2) the close association of poly(A) polymerase and the protein kinase might permit continuous phosphorylation of the polymerase in vivo; and (3) activation of poly(A) polymerase by phosphorylation might have a regulatory role in mRNA polyadenylation. (G)
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