Purification and Properties of 5'-Nucleotidase from Snapper Muscle.

Abstract

The IMP-degrading enzyme from ordinary muscle of the snapper Pagrus auratus was purified by ammonium sulfate fractionation and two steps of affinity chromatography on Con A-Sepharose and 5'-AMP Sepharose 4B. The purified enzyme was shown to be homogeneous by disc polyacrylamide gel electrophoresis. The apparent molecular weight of the purified enzyme was estimated to be about 8.9×104 by SDS-polyacrylamide gel electrophoresis and about 3.6×105 by gel filtration with Sephacryl S-300HR, thus this enzyme was shown to be a tetramer. The purified enzyme was identified as 5'-nucleotidase (EC 3.1.3.5) by substrate specificity, it has maximal activity at pH 8.5 in Tris-acetate buffer in the presence of MgC12, and it hydrolyzed 5'-AMP most effectively. Of several metal ions, Mn2+ was the most effective activator and Co2+ also activated this enzyme. The enzyme activity was inhibited by Zn2+, Cu2+, Bat2+, and EDTA and complete inhibition of the enzyme occurred in the presence of 1 mM 5'-ATP and 5'-ADP. The Km value for 5'-IMP was 3.0 × 10-5 M. The enzyme was competitively inhibited by ATP and ADP, and the K1 values for ATP and ADP were 0.4 and 0.13μM, respectively.