Isolation from Ascites Carcinoma Krebs II Cells of an Unlinking Enzyme Hydrolyzing a Covalent Bond between Picornavirus RNA and VPg

R. A. Yusupova^*, A. Yu. Gulevich, and Yu. F. Drygin

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-3181; E-mail: yusup@genebee.msu.su

^* To whom correspondence should be addressed.

Received March 3, 2000; Revision received April 6, 2000

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A new purification procedure for the isolation of the “unlinking” enzyme, which hydrolyzes the phosphodiester bond between 5´-terminal uridylic acid of the encephalomyocarditis viral RNA and protein VPg has been developed. The enzyme (tyrosine-(5´P-->O)-uridylylpolynucleotide phosphodiesterase, Y-pUpN PDE) was purified from frozen mouse carcinoma Krebs II cells. The purification procedure included ammonium sulfate fractionation of the cell extract, pH fractionation by acidification of the protein solution to pH 4.0, cation-exchange chromatography on CM-52-cellulose, chromatofocusing, and size-exclusion HPLC on a TSK 2000 SW column. The enzyme was shown to exist as several forms characterized by different isoelectric points (ranging from 4.0 to 5.2) and molecular masses. The pH fractionation and ion-exchange chromatography on CM-cellulose influenced the pI and molecular mass values for each form (pI increased, whereas molecular mass decreased from 30 to 26 kD). The employment of these two stages removed (almost completely) an accompanying proteolytic activity, which co-purified with Y-pUpN PDE and digested free VPg. The molecular mass of 26 kD determined by HPLC for the native form coincided with the molecular mass of the major protein band determined by SDS-PAGE for the denatured form of the enzyme.

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KEY WORDS: “unlinking enzyme”, size-exclusion HPLC, picornavirus, RNA-VPg, chromatofocusing, ascites carcinoma Krebs II

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