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

5'-Nucleotidase, responsible for the conversion of adenosine-5'-monophospha te 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 Fe2+. The glutathione-mediated inactivation of 5'-nucleotidase was potentiated remarkably by Fe2+, but not Cu2+, in a concentration-dependent manner. Similarly, glutathione exhibite d a concentration-dependent enhancement of the Fe2+-mediated inactivation. In comparison, the glutathione/Fe2+ system was much more effective than the ascorbate/Fe2+ system in inactivating the enzyme. In support of an interme diary role of superoxide ions or H2O2 in the action of glutathione/Fe2+ sys tem, superoxide dismutase and catalase expressed a substantial protection a gainst the inactivation by the glutathione/Fe2+ system. Meanwhile, hydroxyl radical scavangers such as mannitol, benzoate or ethanol were incapable of preventing the inactivation, excluding the participation of extraneous hyd roxyl radicals. Whereas adenosine 5'-monophosphate as substrate exhibited a modest protection against the glutathione/Fe2+ action, a remarkable protec tion was expressed by divalent metal ions such as Zn2+ or Mn2+. Structure-a ctivity study with a variety of thiols indicates that the inactivating acti on of thiols in combination with Fe2+ resides in the free sulfhydyl group a nd amino group of thiols. Overall, thiols, expressing more inhibitory effec t on the activity of 5'-nucleotidase, were found to be more effective in po tentiating the Fe2+-mediated inactivation. Further, kinetic analyses indica te that Fe2+ and thiols inhibit the 5'-nucleotidase in a competitive or unc ompetitive manner, respectively. These results suggest that ecto-5'-nucleot idase from brain membrane is one of proteins susceptible to thiols/Fe2+-cat alyzed oxidation, and the oxidative inactivation may be related to the sele ctive association of Fe2+ and thiols to the enzyme molecule.