Liquid chromatography purification and study of uridilylpolynucleotide-(5′P→O)-tyrosine phosphodiesterase

Summary LC has been used as a tool for studying uridilylpolynucleotide-(5′P→O)-phosphodiesterase—an enzyme which hydrolyses specifically the phosphodiester bond between picarnaviral RNA and viral protein VPg. According to various chromatographic data, the enzyme forms two types of complex with nucleic acids: weak ones which dissociate in 200 mM KCl, and others which are stable at concentrations up to 900 mM KCl. 2.5-3-fold (preparative) or 6-fold (normal scale) purification of the enzyme was obtained by size-exclusion chromatography (SEC). Cation-exchange separation (4-fold purification) was found to be more suitable as the second enzyme purification step than the earlier anionexchange method used. Three forms of enzyme activity were discovered by hydrophobic-interaction chromatography on the enzyme preparation obtained by SEC. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996