Because of controversial data in the literature, we studied the localization of uric acid oxidase (UAOX) activity in rat liver by light miaompy (LM) and electron microscopy (EM). UAOX is partially inactivated by aldehyde fmtion and therefore we developed a technique that permits the use of unfmed cryostat sections for both LM and EM studies. Sections of rat liver were mounted on a semipermeable membrane stretched over a gelled incubation medium containing urate as specific substrate for UAOX and cerium ions to capture H20z produced by oxidase activity. The specificity of the reaction was checked by comparing incubations in the presence of substrate with incubations either in the absence of substrate or in the presence of substrate and 2,6,8-Uichloropurine, a competitive inhibitor of UAOX. After incubation the sections were either fmed immediately for EM or visual- IntroductionUric acid oxidase (urate: 0 2 oxidoreductase, E.C. 1.7.3.3.; urate oxidase, uricase, UAOX) is a cuproprotein involved in the purine catabolism. It catalyzes the breakdown of uric acid with the use of molecular oxygen, yielding H202, C02, and allantoin (Mahler et al., 1955;Keilin and Hartree, 1936; Battelli and Stern, 1909). Since urate is assumed to be a potent antioxidant, UAOX is directly involved in the oxygen radical scavenger system of the cell and the circulation.UAOX is found in all vertebrates except primates, birds, and some reptiles, and in most invertebrates except insects and spiders (Shnitka, 1966;Keilin, 1959). UAOX has also been demonstrated in higher plants (Vaughn et al., 1982; Thomas and Trelease, 1981). In the animal body, UAOX is predominantly found in peroxisomes ized for LM with a second-step incubation. At the LM level, final reaction product was found in a granular form, homogeneously distributed throughout the hepatocytes. EM revealed excellent subcellular morphology and electron-dense reaction product in both the core and the matrix of peroxisomes, but not in other organelles or the cytoplasmic matrix. After incubations without substrate or with substrate and inhibitor, hardly any reaction product was found. We conclude that, because of the use of unfiied tissue, UAOX is not inactivated, which results in localization of UAOX activity not only in the COR of peroxisomes but also in the perox- KEY WORDS: Uric acid oxidase; Urate oxidase; Uricase; Enzyme histochemistry; Electron microscopy; Cerium salt capture method; Unfixed cryostat section; Semipermeable membrane. of liver and kidney (De Duve, 1960). However, the exact localization of UAOX activity in peroxisomes is still a matter of debate.Biochemical studies have demonstrated UAOX activity within the crystalline core of peroxisomes (Hayashi et al., 1971; ?Sukada et al., 1971; De Duve and Baudhuin, 1966; Baudhuin et al., 1965; Hruban and Swift, 1964), but it is still not clear whether a quantitative correlation exists between UAOX activity and core volume. De Duve and Baudhuin (1966) calculated that 10% of the protein content of the core consists of UAOX, wherea...
Data concerning the substrate specificity and the exact intracellular localization of the polyamine-catabolizing enzyme polyamine oxidase are conflicting. Biochemical studies have shown that N1-acetylation of spermine and spermidine dramatically increases the specificity of these compounds for peroxisomal polyamine oxidase to produce spermidine and putrescine, respectively. On the other hand, polyamine oxidase activity was demonstrated histochemically both in peroxisomes and in cytoplasm of several tissues, using spermidine and/or spermine as substrate. To elucidate the in situ substrate specificity of polyamine oxidase and the localization of its activity, enzyme activity was detected in rat liver, kidney, and duodenum at the light and electron microscopic levels. For this purpose, unfixed cryostat sections were applied to avoid changes in enzyme activity owing to chemical fixation. Spermine, spermidine, their N1-acetylated forms, and putrescine were used as substrates, and cerium ions as capturing agent for H2O2. Control reactions were performed in the absence of substrate or in the presence of substrate and specific oxidase inhibitors. At the light microscopic level, final reaction product specifically generated by polyamine oxidase activity was found exclusively in a granular form in hepatocytes, epithelial cells of proximal tubules of the kidney, and epithelial cells of duodenal villi with N1-acetylspermidine or N1-acetylspermine as substrates. Final reaction product was not observed in any of the tissues after incubation in the presence of putrescine, spermidine, or spermine. Formation of specific final reaction product was prevented by incubation in the presence of a specific polyamine oxidase inhibitor, but it was not affected by a diamine oxidase inhibitor. Ultrastructural studies revealed that polyamine oxidase activity is localized exclusively to the matrix of peroxisomes of kidney and liver and to microperoxisomes of the duodenum. The localization patterns obtained with unfixed tissues are in agreement with biochemical data. Strong intraperoxisomal, interperoxisomal, and intercellular heterogeneity in polyamine oxidase activity was found in all tissues investigated.
(3A3155).A tecently developed histochemical technique to demonstrate xanthine oxidase activity in milk globules of bovine mammary gland and in epithelial cells of rat small intestine using cerium ions and a semipermeable membrane was slightly modified. The semipermeable membrane method was replaced by the addition of 10% (wh) polyvinyl alcohol to the incubation medium. This technically more simple procedure enabled detection of xanthine oxidase activity in unfmed cryostat sections of rat liver. Both methods gave qualitatively and quantitatively similar results. Activity was found in sinusoidal cells and in liver parenchymal cells, with 50% higher activity in pericentral than in periportal areas. The specificip of the reaaion was proven by the generation of only small amounts of fmal reaction product on incubation either in the absence of the substrates hypoxanthine or oxygen or in the presence of hypoxanthine and allopurinol. Allopurinol is a specific inhibitor of xanthine oxidase activity. The amount of final reaction product, as measured cytophotometrically in rat liver, increased linearly with incubation time (15-90 min) and with section thickness (up to 12 pm). By varying the hypoxanthine concentrations, a Km value of 0.05 mM was found. Addition of dithiothreitol to the incubation medium reduced the amount of final reaction product by 85%, which was caused by conversion of reversible xanthine oxidase into xanthine dehydrogenase. This histochemical method can be used for quantitative analysis of in situ xanthine oxidase activity. (]Hisrochem Cytochem 42:1091-1097, 1994)
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