Purification and characterization of a 2′-phosphodiesterase from bovine spleen

• Published in: The Journal of biological chemistry Vol. 262; no. 17; pp. 8377 - 8382
• Main Authors: Johnston, M I, Hearl, W G
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15.06.1987; American Society for Biochemistry and Molecular Biology
• Subjects: 2'-phosphodiesterase; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cattle; Chromatography - methods; Chromatography, Ion Exchange - methods; Durapatite; Enzymes and enzyme inhibitors; Exoribonucleases - isolation & purification; Exoribonucleases - metabolism; Fundamental and applied biological sciences. Psychology; Hydrolases; Hydroxyapatites; Kinetics; spleen; Spleen - enzymology; Substrate Specificity; Ultrafiltration - methods; Characterization; Spleen; Vertebrata; Mammalia; Purification; Bovine; Phosphodiesterase II; Animal; Artiodactyla; Ungulata; Comparative study
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)47574-6

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.