Purification and characterization of a 2'-phosphodiesterase from bovine spleen.
Interferon-induced 2',5'-oligoadenylates are transiently produced during viral infection and are believed to play a role in the interferon-mediated inhibition of replication of at least some viruses. 2',5'-Oligoadenylates must be catabolized but are resistant to degradation by most known ribonucleases. A 2'-phosphodiesterase that degrades 2',5'-oligoadenylates was purified 1500-fold from a low speed homogenate of bovine spleen by precipitation at pH 5.2, ammonium sulfate fractionation, differential ultrafiltration, and successive chromatography on DEAE-Sephacel, hydroxylapatite, and a fast protein liquid chromatography Mono P column. No other 2-5A-degrading activity was observed during the purification procedure. The molecular mass of the enzyme estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 65,000. The enzyme is distinct from bovine spleen phosphodiesterase II. The 2'-phosphodiesterase cleaves 2',5'- and 3',5'-linked oligonucleotides, as well as branched oligoadenylate, A(2'pA)(3'pA), but appears to be most active on 3',5'-oligoribonucleotides. The enzyme cleaves 5'-AMP from the 2' terminus of 2',5'-oligoadenylates and appears to require a free 2' terminus and a 3'-oxygen on the penultimate nucleotide. Substrate length, 5'-phosphorylation, and base composition do not appear to be critical factors in determining enzyme activity. The effects of pH, Mg2+, Mn2+, EDTA, phosphate, 2-mercaptoethanol, and N-ethylmaleimide are also described. This enzyme may be involved in the catabolism of the interferon-induced 2',5'-oligoadenylates and other 2',5'-linked RNAs in the cell.
D000818:Animals; D002417:Cattle; D002845:Chromatography / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D017886:Durapatite; D005095:Exoribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D006882:Hydroxyapatites; D007700:Kinetics; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity; D014462:Ultrafiltration / Q000379:methods
D006882:Hydroxyapatites; D017886:Durapatite; D005095:Exoribonucleases; C029364:2'-phosphodiesterase