Ultrastructural cytochemical localization of uricase in peroxisomes of rat liver.
Ultrastructural localization of uricase (urate: oxygen oxidoreductase, E.C.1.7.3.3.) in rat liver parenchymal cells has been studied with the cerium technique. The cerous ions react with H2O2 generated by the activity of the enzyme in the presence of urate, forming the electron-dense reaction product of cerous perhydroxide. Tissue fixation is carried out by perfusion for 5 min with a low concentration (0.25%) of glutaraldehyde. Since in a biochemical assay it was found that the activity of uricase determined in Trismaleate buffer is substantially weaker than in the Pipes buffer, the classical medium of Briggs et al. (6) was modified, and the latter buffer was substituted for the Trismaleate. Vibratome sectons are incubated at 37 degrees C for 60 min in 0.1 M Pipes buffer, pH 7.8, containing 3 mM cerium chloride and 0.1 mM sodium urate. Under these conditions, the reaction product is localized in the crystalline cores of hepatic peroxisomes. The intensity of the staining is dependent on the concentration of the substrate and the incubation time. In control preparations incubated without urate or with 2,6,8-trichloropurine, a specific inhibitor of uricase, staining is almost completely abolished. In sections incubated with 5 mM cerium and 0.1 mM sodium urate, fine granules with a distribution corresponding to peroxisomes are also visible at the light microscopic level. This latter observation is invaluable for correlative light and electron microscopic studies.
Angermüller
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Fahimi
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D000818:Animals; D002021:Buffers; D002563:Cerium / Q000494:pharmacology; D005976:Glutaral / Q000494:pharmacology; D006651:Histocytochemistry; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008830:Microbodies / Q000201:enzymology* / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors; D014503:Urate Oxidase / Q000032:analysis*
D002021:Buffers; D002563:Cerium; D014503:Urate Oxidase; D005976:Glutaral