Uric Acid Degrading Enzymes, Urate Oxidase and Allantoinase, Are Associated with Different Subcellular Organelles in Frog Liver and Kidney
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Journal of Cell Science 107, 1073-1081 (1994) 1073 Printed in Great Britain © The Company of Biologists Limited 1994 Uric acid degrading enzymes, urate oxidase and allantoinase, are associated with different subcellular organelles in frog liver and kidney Nobuteru Usuda1,*, Sueko Hayashi1,†, Satoko Fujiwara2, Tomoo Noguchi2, Tetsuji Nagata3, M. Sambasiva Rao1, Keith Alvares1, Janardan K. Reddy1 and Anjana V. Yeldandi1,‡ 1Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611, USA 2Department of Biochemistry, Kyushu Dental College, Kokura, Kitakyushu 803, Japan 3Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto 390, Japan *Present address: Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto 390, Japan †Present address: Kyushu Dental College, Kokura, Kitakyushu 803, Japan ‡Author for correspondence SUMMARY On the basis of differential and density gradient centrifu- found to be the exclusive site of urate oxidase localization. gation studies, the site of the uric acid degrading enzymes, Allantoinase was detected within mitochondria, but not in urate oxidase and allantoinase, in amphibia was previously peroxisomes of hepatocytes or proximal tubular epithe- assigned to the hepatic peroxisomes. Using specific anti- lium. No allantoinase was detected in the mitochondria of bodies against frog urate oxidase and allantoinase, we have nonhepatic parenchymal cells in liver and of the cells lining undertaken an immunocytochemical study of the localiza- the distal convoluted tubules of the kidney. These results tion of these two proteins in frog liver and kidney, and demonstrate that, unlike rat kidney peroxisomes which demonstrate that whereas urate oxidase is present in per- lack urate oxidase, peroxisomes of frog kidney contain this oxisomes, allantoinase is localized in mitochondria. Urate enzyme. Contrary to previous assumptions, these studies oxidase and allantoinase were detected by immunoblot also clearly establish that urate oxidase and allantoinase, analysis in both frog liver and kidney. The subcellular the first two enzymes involved in uric acid degradation, are localization of these two enzymes was ascertained by localized in different subcellular organelles in frog liver Protein A-gold immunocytochemical staining of Lowicryl and kidney. K4M-embedded tissue. Peroxisomes in frog liver parenchy- mal cells and kidney proximal tubular epithelium Key words: urate oxidase, allantoinase, purine metabolism, contained a semi-dense subcrystalloid core, which was peroxisome, mitochondrion INTRODUCTION The loss of urate oxidase activity in humans and hominoid primates, such as chimpanzee, gorilla and orangutan, due to Urate oxidase or uricase (urate: oxygen oxidoreductase, EC nonsense mutations in the urate oxidase gene, results in the 1.7.3.3), an enzyme that catalyzes the oxidation of uric acid to excretion of uric acid (Wu et al., 1989; Yeldandi et al., 1990; allantoin, occupies a pivotal position in the chain of enzymes 1991). In amphibia and fish liver, uric acid is degraded all the responsible for the metabolism of purines (Keilin, 1958). way to urea because all three enzymes, urate oxidase, allan- Degradation of purines to uric acid is common to all species toinase and allantoicase, are present (Takada and Noguchi, but the degradation of uric acid, however, varies from species 1983; Noguchi et al., 1986; Hayashi et al., 1989). to species. For example, bacteria and some marine inverte- In the rat, and in most other mammals that display urate brates degrade purines to uric acid, which is then oxidized to oxidase activity and metabolize uric acid to allantoin, this allantoin by urate oxidase (Keilin, 1958). Hydrolysis of enzyme is associated specifically with the crystalloid core allantoin by allantoinase (EC 3.5.2.5) results in the formation present within the peroxisomes in hepatic parenchymal cells of allantoic acid for further metabolism by allantoicase (EC (De Duve and Baudhuin, 1966; Shnitka, 1966; Tsukada et al., 3.5.3.4) to yield urea. The enzyme urease (EC 3.5.1.5) then 1966; Lata et al., 1977; Tolbert, 1981; Usuda et al., 1988a,b; converts urea to ammonia and carbon dioxide. Most mammals, Alvares et al., 1992). Allantoin generated within the peroxi- with the exception of human and hominoid primates, contain some in mammalian hepatocytes is excreted in urine, but the urate oxidase in their liver (Keilin, 1958; Freidman et al., 1985) precise mechanism of allantoin transport out of the peroxi- and excrete allantoin as the end product of purine metabolism, some remains unclear. In amphibia and fish liver, the as these animals do not contain allantoinase and allantoicase. discovery of the presence of all three enzymes of the uric acid 1074 N. Usuda and others degradation pathway led to studies on their subcellular distri- Immunocytochemical labeling bution (Scott et al., 1969; Visentin and Allen, 1969). For immunocytochemical localization, thin sections were incubated Following differential and density gradient centrifugation pro- on drops of 0.5 M Tris-HCl, pH 7.5, containing 0.05% Triton X-100, cedures, it was reported that urate oxidase, allantoinase and 5 mg/ml bovine serum albumin and 0.15 M NaCl for 1 hour. Subse- allantoicase are associated with the peroxisome (Visentin and quently, the sections were transferred onto drops of antibody solution Allen, 1969). The subcellular compartmentalization of these (1:1000 dilution of antiserum in 0.05 M Tris-HCl, pH 7.5, containing three enzymes within hepatic peroxisomes thus apparently 0.05% Triton X-100, 5 mg of bovine serum albumin and 0.15 M served to optimize the metabolic degradation of uric acid to NaCl) and incubated for 4 hours. They were then washed several urea (Hayashi et al., 1989). Recent subcellular fractionation times with 0.05 M Tris-HCl buffer containing 0.1% Triton X-100 and studies of liver demonstrated that marine fishes, such as 0.15 M NaCl. Protein A-gold labeling, using 15 nm size gold particles, (EY Laboratories) was carried out as previously described (Bendayan sardine and mackerel, contain allantoinase both in the perox- and Reddy, 1982; Bendayan et al., 1983). After counterstaining with isome and cytosol, whereas in fresh water fishes (e.g carp, uranyl acetate and lead citrate, the sections were examined in a JEOL bass) this enzyme is found only in the cytosol (Fujiwara et al., 100 CEX electron microscope. A minimum of 20 randomly selected 1989). electron micrographs were obtained from each animal. To demon- In view of the current interest in the evolutionary loss of per- strate the immunocytochemical specificity, control sections were oxisomal enzymes responsible for the degradation of uric acid reacted with nonimmune serum. (Wu et al., 1989; Yeldandi et al., 1990; 1991), the postulated role of uric acid as a potent biological antioxidant (Ames et al., Immunoperoxidase staining 1981), and the possible implications of the generation of the For the demonstration of allantoinase, 3- to 4-µm thick ethanol- or oxidant H2O2 as a result of uric acid metabolism by urate paraformaldehyde-fixed, paraffin-embedded sections were stained oxidase (De Duve and Baudhuin, 1966), it appeared necessary using the avidin-biotin-peroxidase procedure (Usuda et al., 1991). to confirm visually the peroxisomal localization of urate Briefly, the deparaffinized sections were incubated in normal goat oxidase, allantoinase and allantoicase in the frog liver by serum (1:50 dilution) for 20 minutes and then treated with anti-allan- immunocytochemical localization. Our objective in the present toinase antibodies (1:300 dilution) for 2 hours followed by bio- tinylated goat anti-rabbit IgG (1:100 dilution) for 1 hour, and rabbit study was to localize urate oxidase and allantoinase, the first peroxidase anti-peroxidase complex (1:100 dilution) for 1 hour. two enzymes responsible for uric acid degradation in the liver Antibody solutions were made with 50 mM Tris-HCl, pH 7.5, con- and kidney, using Protein A-gold immunocytochemical local- taining 150 mM NaCl and normal goat serum. Sections were then ization. The results show that urate oxidase is localized exclu- incubated in the medium for peroxidase which contained 0.05% sively to the subcrystalloid core present within the peroxisomes 3,3′-diaminobenzidine tetrahydrochloride and 0.01% H2O2 in 0.05 M of both parenchymal cells of liver and the proximal tubular Tris-HCl, pH 7.2, for 10 minutes, and counterstained with 1% epithelium of kidney. Allantoinase, however, was not methyl green. detectable within the peroxisomes in these cells, but was localized to the mitochondrion. Subcellular fractionation Frogs were fasted overnight and their liver perfused in situ with 0.25 M sucrose under light ether anesthesia. The liver and kidneys MATERIALS AND METHODS were removed, finely chopped and homogenized in 5 vols of 0.25 M sucrose/10 mM Tris-HCl, pH 7.5, using a Potter-Elvehjem tissue Antibodies grinder. The homogenate was centrifuged at 960 g for 10 minutes to remove nuclei and unbroken cells. The supernatant was carefully Urate oxidase and allantoinase were purified from the liver of adult bull decanted and centrifuged at 15,000 g for 20 minutes to separate frogs (Rana catesbeiana) and used to raise polyclonal monospecific anti- crude mitochondrial fraction (De Duve et al., 1955). The crude bodies in rabbits, as previously described (Fujiwara et al., 1987; Noguchi mitochondrial fraction was suspended in 20 ml of 0.25 M et al., 1986). Antibodies against