Posttranslational inactivation of human xanthine oxidoreductase by oxygen under standard cell culture conditions.
Xanthine oxidoreductase (XOR) catalyzes the final reactions of purine catabolism and may account for cell damage by producing reactive oxygen metabolites in cells reoxygenated after hypoxia. We found a three- to eightfold higher XOR activity in cultured human bronchial epithelial cells exposed to hypoxia (0.5-3% O2) compared with cells grown in normoxia (21% O2) but no difference in XOR protein or mRNA. XOR promoter constructs failed to respond to hypoxia. The cellular XOR activity at 3% O2 returned to basal levels when the cells were returned to 21% O2, and hyperoxia (95% O2) abolished enzyme activity with no change in XOR protein. Our data suggest reversible enzyme inactivation by oxygen or its metabolites. NADH was normally oxidized by the oxygen-inactivated enzyme, which rules out damage to the flavin adenine dinucleotide cofactor. Hydrogen peroxide partially inactivated the molybdenum center of XOR, as shown by a parallel decrease in XOR-catalyzed xanthine oxidation and dichlorophenolindophenol reduction. We conclude that the transcription or translation of XOR is not influenced by hypoxia or hyperoxia. Instead, the molybdenum center of XOR is posttranslationally inactivated by oxygen metabolites in "normal" (21% O2) cell culture atmosphere. This inactivation is reversed in hypoxia and accounts for the apparent induction.
Linder
Nina
N
Research Program for Developmental and Reproductive Biology, University of Helsinki, Biomedicum Helsinki, 00290, Helsinki, Finland. nina.linder@hus.fi
Martelin
Eeva
E
Lapatto
Risto
R
Raivio
Kari O
KO
D015687:Cell Hypoxia / Q000502:physiology; D002461:Cell Line, Transformed; D002470:Cell Survival; D003035:Cobalt / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D006801:Humans; D018496:Hyperoxia / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism* / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D015427:Reperfusion Injury / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000201:enzymology*; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000502:physiology; D014969:Xanthine Oxidase / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D003035:Cobalt; D014969:Xanthine Oxidase; D010100:Oxygen