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 Fe2+. The glutathione-mediated inactivation of 5'-nucleotidase was potentiated remarkably by Fe2+, but not Cu2+, in a concentration-dependent manner. Similarly, glutathione exhibited 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 intermediary role of superoxide ions or H2O2 in the action of glutathione/Fe2+ system, superoxide dismutase and catalase expressed a substantial protection against the inactivation by the glutathione/Fe2+ system. Meanwhile, hydroxyl radical scavengers 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/Fe2+ action, a remarkable protection was expressed by divalent metal ions such as Zn2+ or Mn2+. Structure-activity study with a variety of thiols indicates that the inactivating action of thiols in combination with Fe2+ resides in the free sulfhydryl 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 Fe2+-mediated inactivation. Further, kinetic analyses indicate that Fe2+ 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/Fe2+-catalyzed oxidation, and the oxidative inactivation may be related to the selective association of Fe2+ and thiols to the enzyme molecule.
Liu
X W
XW
College of Pharmacy, Chungnam National University, Taejon, Korea.
Sok
D E
DE
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D002417:Cattle; D005296:Ferrous Compounds / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D005296:Ferrous Compounds; D016877:Oxidants; D013438:Sulfhydryl Compounds; D015720:5'-Nucleotidase