Studies on 5′-nucleotidases of rat liver

Abstract

Approximately 8% of the 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity (assayed with AMP as substrate at a pH of 7.4 in the presence of 10 mM tartrate) in a homogenate of rat liver behaved as a lysosomal enzyme when the homogenate was fractionated into its subcellular components by differential and density gradient centrifugation. The bulk of the activity sedimented with the nuclear and microsomal fractions. The 5′-nucleotidases in purified lysosomal and plasma membrane fractions exhibited nearly maximal activity at pH values between 7.0 and 9.5, and were similarly influenced by the addition of varying concentrations of Mg 2+ and Mn 2+ to the incubation mixture. When a lysosomal fraction solubilized by treatment with 1% Triton X-100 was passed through a Sephadex G-200 column, two peaks possessing 5′-nucleotidase activity appeared in the effluent. The first peak was identical chromatographically to the 5′-nucleotidase associated with plasma membranes and with a lysosomal membrane fraction. The second peak exhibiting 5′-nucleotidase activity contained a lower molecular weight enzyme which could be extracted from lysosomes by 1 mM NaHCO 3. Disc electrophoresis of these same fractions on polyacrylamide gel confirmed these chromatographic findings.