Abstract
Poly (A) polymerase [EC 2. 7. 7. 19] was highly purified from beef liver nuclei by the use of column chromatographies on heparin-Sepharose 4 B and Blue Dextran-Sepharose 4 B. The purified enzyme showed one major protein band of the molecular weight of 57, 000 in SDS polyacrylamide gel electrophoresis, which agreed with the molecular weight estimated from glycerol gradient centrifugation. The enzyme required the presence of Mn2+ for its activity but was almost completely inactive with Mg2+. It incorporated specifically ATP into polynucleotide as a sole substrate. The enzyme activity depended entirely on the addition of exogenous polynucleotide primer. It showed certain selectivity for the primers. The most effective among the tested polynucleotides was a short poly (A), for which the Km of the enzyme was shown to be 7μM. Poly (G, U) and short poly (U) also primed the reaction, but tRNA, phage RNA, poly (G), and poly (C) were inactive. Based on observed specificity for the primer, the role of this enzyme in the cell nuclei was discussed. Digestion of the reaction product of this enzyme by two specific exonucleases, snake venom and spleen phospho-diesterases, suggested that this enzyme catalyzed the covalent bonding of the substrate to the 3' terminus of the primer as in the manner expected for in vivo polyadenylation.
