Localization of xanthine oxidoreductase activity using the tissue protectant polyvinyl alcohol and final electron acceptor Tetranitro BT.
We have detected xanthine oxidoreductase activity in unfixed cryostat sections of rat and chicken liver, rat duodenum, and bovine mammary gland using the tissue protectant polyvinyl alcohol, the electron carrier 1-methoxyphenazine methosulfate, the final electron acceptor Tetranitro BT, and hypoxanthine as a substrate. Enzyme activity was localized in rat duodenum at lateral membranes and brush borders of enterocytes and in goblet cells and mucus. Hepatocytes in pericentral areas and especially sinusoidal cells showed high activity in rat liver. Xanthine oxidoreductase was also detected in epithelial cells and milk lipid globules of lactating bovine mammary gland, which is known to contain large quantities of the oxidase form of the enzyme. Chicken liver, which contains an inconvertible dehydrogenase form, also showed high activity in sinusoidal cells. Therefore, we conclude that the tetrazolium reaction demonstrates both the dehydrogenase and the oxidase form of xanthine oxidoreductase. Control activity, in the absence of hypoxanthine or in the presence of the competitive inhibitor allopurinol, was low in all tissues studied. Addition of O2 or NAD to the incubation medium did not change the specific reaction in bovine mammary gland or chicken liver, implying that the dehydrogenase and the oxidase form are not dependent on their natural electron acceptors in this tetrazolium salt reaction. We conclude that the present light microscopic method gives specific and precise localization of xanthine oxidoreductase activity in situ.
Kooij
A
A
Laboratory of Cell Biology and Histology, University of Amsterdam, The Netherlands.
Frederiks
W M
WM
Gossrau
R
R
Van Noorden
C J
CJ
D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D004386:Duodenum / Q000201:enzymology / Q000648:ultrastructure; D004848:Epithelium / Q000201:enzymology; D005260:Female; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000494:pharmacology; D007774:Lactation; D008099:Liver / Q000201:enzymology; D008297:Male; D008321:Mammary Glands, Animal / Q000201:enzymology; D008773:Methylphenazonium Methosulfate / Q000031:analogs & derivatives; D008871:Microvilli / Q000201:enzymology; D009243:NAD / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology; D011142:Polyvinyl Alcohol; D011247:Pregnancy; D051381:Rats; D011919:Rats, Inbred Strains; D013778:Tetrazolium Salts; D014968:Xanthine Dehydrogenase / Q000032:analysis*; D014969:Xanthine Oxidase / Q000032:analysis*
D007042:Hypoxanthines; D013778:Tetrazolium Salts; D009243:NAD; C028130:tetranitrotetrazolium blue; C033411:1-methoxyphenazine; D008773:Methylphenazonium Methosulfate; D019271:Hypoxanthine; D000493:Allopurinol; D011142:Polyvinyl Alcohol; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase; D010100:Oxygen