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.