Oxidative inactivation of brain ecto-5′-nucleotidase by thiols/Fe2+ system

5'-Nucleotidase, responsible for the conversion of adenosine-5'-monophosphate into adenosine, was purified from bovine brain membranes, and subjected to oxidative inactivation. The 5'-nucleotidase activity decreased slightly after the exposure to either glutathione or Fe(2+). The glutathione-mediated inactivation of 5'-nucleotidase was potentiated remarkably by Fe(2+), but not Cu(2+), in a concentration-dependent manner. Similarly, glutathione exhibited a concentration-dependent enhancement of the Fe(2+)-mediated inactivation. In comparison, the glutathione/Fe(2+) system was much more effective than the ascorbate/Fe(2+) system in inactivating the enzyme. In support of an intermediary role of superoxide ions or H(2)O(2) in the action of glutathione/Fe(2+) system, superoxide dismutase and catalase expressed a substantial protection against the inactivation by the glutathione/Fe(2+) system. Meanwhile, hydroxyl radical scavangers such as mannitol, benzoate or ethanol were incapable of preventing the inactivation, excluding the participation of extraneous hydroxyl radicals. Whereas adenosine 5'-monophosphate as substrate exhibited a modest protection against the glutathione/Fe(2+) action, a remarkable protection was expressed by divalent metal ions such as Zn(2+) or Mn(2+). Structure-activity study with a variety of thiols indicates that the inactivating action of thiols in combination with Fe(2+) resides in the free sulfhydyl group and amino group of thiols. Overall, thiols, expressing more inhibitory effect on the activity of 5'-nucleotidase, were found to be more effective in potentiating the Fe(2+)-mediated inactivation. Further, kinetic analyses indicate that Fe(2+) and thiols inhibit the 5'-nucleotidase in a competitive or uncompetitive manner, respectively. These results suggest that ecto-5'-nucleotidase from brain membrane is one of proteins susceptible to thiols/Fe(2+)-catalyzed oxidation, and the oxidative inactivation may be related to the selective association of Fe(2+) and thiols to the enzyme molecule.