Isolation and characterization of 5'-nucleotidase inhibitor from rat liver

5'-Nucleotidase (EC 3.1.3.5) is widely distributed in nature. However, it could not be detected in rat liver, because of the presence of specific inhibitors. Such inhibitors were also found in other tissues of rat, but at lower concentrations than that in the liver. The inhibitor activity was enriched in the membrane fraction and was also present in the cytosol fraction. It was sensitive to treatment with 6M urea and trypsin, while heating in a boiling water bath for 10 min or dialysis reduced the activity only slightly. Gel filtration or Sephadex G-50 yielded two types of inhibitors. Inhibitor I inhibited brain 5'-nucleotidase while inhibitor II inhibited both the brain and liver enzymes. Inhibitor II on further purification on CM Sephadex C-25 yielded five fractions with inhibitor activity of which inhibitor IIC was electrophoretically homogeneous. It had a molecular weight of 8500 by SDS gel electrophoresis, was rich in basic amino acids and had a high proportion of beta structure. Interaction of the inhibitor with 5'-nucleotidase brought about modifications in the secondary structure of the inhibitor as seen from the circular dichroism spectrum.