Journal of Cell Science 107, 1073-1081 (1994) 1073 Printed in Great Britain © The Company of Biologists Limited 1994

Uric acid degrading , urate 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 localization. gation studies, the site of the degrading enzymes, Allantoinase was detected within mitochondria, but not in urate oxidase and allantoinase, in amphibia was previously 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 . 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, , contained a semi-dense subcrystalloid core, which was , mitochondrion

INTRODUCTION The loss of urate oxidase activity in humans and hominoid , such as chimpanzee, gorilla and orangutan, due to Urate oxidase or uricase (urate: oxygen , 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; , 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 (Keilin, 1958). way to 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, and some marine inverte- In the rat, and in most other 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 (EC 3.5.1.5) then 1966; Lata et al., 1977; Tolbert, 1981; Usuda et al., 1988a,b; converts urea to 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 , 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 of , 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 (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 - 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 rat liver were generated and characterized as described elsewhere (Reddy and Kumar, 1979). sucrose/10 mM Tris-HCl and washed twice to remove adsorbed cytosol. The homogenates, and the mitochondrial and 100,000 g Preparation of tissue for Lowicryl K4M embedding supernatant fractions, were processed for immunoblotting as Adult bull frogs and tadpoles (stages 51 to 60) were used in this study. described below. Samples of the crude mitochondrial fractions were The developmental stages were based on the criteria outlined by Taylor also fixed in 4% paraformaldehyde/0.1% glutaraldehyde and and Kollros (1946). For electron microscopic immunohistochemistry, embedded in Lowicryl K4M for immunocytochemical use, as pieces of liver and kidney from four frogs and three tadpoles were fixed decribed above. by immersion in 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M Gel electrophoresis and immunoblotting sodium phosphate, pH 7.4, for 16 hours (Usuda et al., 1988b). After rinsing in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.15 M Homogenates of liver and kidney were prepared from three adult frogs NaCl and 0.1 M lysine for 1 hour, tissues were dehydrated in a graded and six tadpoles, and the proteins separated by SDS-PAGE using 10% series of ethanol and embedded in Lowicryl K4M at −20°C. Post-osmi- polyacrylamide gels (Laemmli (1970). In addition, crude mitochon- fication of the tissues was omitted. Ultrathin sections (0.1 µm) were drial and 100,000 g supernatant fractions were also subjected to SDS- cut on a Dupont-Sorvall MT2B ultramicrotome and mounted on nickel PAGE. Immunoblotting with frog anti-urate oxidase, frog anti-allan- grids with Formvar membrane. For light microscopic immunohisto- toinase, rat anti-catalase and rat anti-urate oxidase antibodies was then chemistry, tissues from three adult frogs and five tadpoles were fixed performed (Towbin et al., 1979). The antigen and antibody complexes in 70% ethanol or paraformaldehyde at 4°C, and embedded in paraffin were identified by immunoperoxidase staining using 4-chloro-1- (Usuda et al., 1991). Sections, 5 µm thick, were subjected to staining naphthol as reagent. The immunoblotting of homogenates was after deparaffinization and rehydration. repeated at least four times. Urate oxidase and allantoinase localization 1075

RESULTS the present study (Fig. 4). The immunogold labeling depicting urate oxidase localization is clearly restricted to the Localization of urate oxidase and catalase in the core-like densities in the peroxisomal matrix (Fig. 4A and C). liver and kidney Catalase is present in all peroxisomes in both liver and kidney In earlier studies, using immunoblotting and immunocyto- cells. chemical approaches, we showed that polyclonal antibodies generated against rat liver urate oxidase recognized urate Localization of allantoinase in the liver and kidney oxidase present in bovine, murine, canine and feline liver Immunoblot analysis revealed the presence of allantoinase in (Usuda et al., 1988a). In the present study, immunoblot kidney and liver homogenates obtained from adult frogs and analyses of frog liver and kidney homogenates with anti-rat tadpoles (Fig. 5). The molecular mass of allantoinase subunit urate oxidase antibodies failed to show cross-reactivity with is approximately Mr 54,000 (Noguchi et al., 1986). The anti- the frog enzyme (data not shown). Likewise, antibodies bodies used in this study are highly specific for allantoinase; generated against frog liver urate oxidase did not cross-react these antibodies inhibited allantoinase activity on immunoti- with rat hepatic urate oxidase (data not shown), but these frog tration and precipitated an Mr 54,000 subunit protein (Noguchi anti-urate oxidase antibodies recognized a single polypeptide et al., 1986). The cellular distribution of allantoinase in frog in the liver and kidney of adult frogs and tadpoles (Fig. 1). The liver and kidney was investigated by immunoperoxidase subunit molecular mass of urate oxidase in the liver and kidney staining (Fig. 6). Hepatocytes showed a strongly positive of frogs and tadpoles is approximately Mr 32,500, which is reaction in the cytoplasm (Fig. 6A); no staining was seen in smaller than that of the rat urate oxidase subunit (Mr 35,000) the sinusoidal cells and bile duct epithelium. In the frog kidney (Alvares et al., 1992). Polyclonal antibodies raised against rat (Fig. 6B), allantoinase appeared to be specifically localized to liver catalase reacted with frog liver and kidney catalase when the cytoplasm of cells lining the proximal convoluted tubules. the homogenates were analyzed by immunoblotting (Fig. 2), No immunostaining was detected in the glomeruli and distal indicating that catalase is highly conserved. The molecular renal tubules. mass of catalase subunit in the rat and the frog is the same (Mr At the electron microscopic level, allantoinase was localized 60,000). predominantly over the mitochondria of liver parenchymal Peroxisomes are few in number in the hepatic parenchy- cells (Fig. 7A and C). Labeling over peroxisomes was not mal cells and kidney proximal tubular epithelium of frog and detected, indicating that allantoinase is not associated with this tadpole. A small proportion of peroxisomes from these two organelle in the frog or tadpole liver. All mitochondria in the cell types in the adult frog have a dense subcrystalloid core hepatic parenchymal cells showed immunolabeling for allan- in the matrix; this core is not as prominent as the crystalloid toinase. We evaluated over 300 hepatocyte mitochondria from core of mammalian liver peroxisomes (Shnitka, 1966, Lata et several different electron micrographs and found labeling over al., 1977; Usuda et al., 1988a). In the adult frog hepatic and all of them. Few gold particles were detected over the endo- renal peroxisomes, urate oxidase is localized to this subcrys- plasmic reticulum which, in some electron micrographs, talloid core (Fig. 3A and C), whereas catalase is distributed appeared slightly more prominent than the overall background. throughout the matrix of all peroxisomes (Fig. 3B and D). In In the adult and tadpole kidney, allantoinase was found also in the tadpole, urate oxidase-containing peroxisomes with the the mitochondria of cells lining the proximal tubules, but not subcrystalloid core are detected in the liver and kidney during in the peroxisome (Fig. 7B and D). In the kidney, the mito- developmental stages 51 through 60 that were examined in chondria present in distal tubules contained no allantoinase. It

Fig. 2. Immunoblot analysis of homogenates obtained from adult frog kidney (lane 1) and frog liver (lane 2) for catalase, using anti-rat Fig. 1. Immunoblot analysis of adult frog liver (lane 1) and kidney catalase antibodies. Rat liver homogenate is used in lane 3 for (lane 2), and tadpole liver (lane 3) and kidney (lane 4) homogenates comparison. The antibodies raised against rat catalase cross-react (100 µg protein per lane) for urate oxidase, using antibodies with the frog catalase. The catalase subunit molecular mass (Mr generated against frog urate oxidase. Lane M, molecular mass 60,000) is similar in the rat and the frog. Lane M, molecular mass −3 −3 standards (×10 ). Urate oxidase is approximately Mr 32,500. standards (Mr ×10 ). 1076 N. Usuda and others

Fig. 3. Immunocytochemical localization of urate oxidase and catalase in Lowicryl-embedded adult frog liver and kidney by the Protein A- gold method. Urate oxidase was localized using frog anti-urate oxidase antibodies (A and C), and catalase by rat anti-catalase antibodies (B and D) in the adult frog liver (A and B) and kidney (C and D). Note that urate oxidase is localized within the subcrystalloid nucleoid in the peroxisome (A and C), whereas catalase is localized diffusely over the peroxisomal matrix (B and D). P, peroxisome; M, mitochondrion. Bars, 20 µm. Urate oxidase and allantoinase localization 1077

Fig. 4. Immunocytochemical localization of urate oxidase and catalase in Lowicryl-embedded tadpole liver and kidney by the Protein A-gold method. Urate oxidase was localized using antibodies raised against frog liver urate oxidase and catalase by antibodies generated against rat liver catalase. (A) Tadpole liver urate oxidase and (B) tadpole liver catalase. (C) Tadpole kidney urate oxidase and (D) tadpole kidney catalase. Urate oxidase labeling is restricted to the subcrystalloid core of the peroxisome (P). M, mitochondrion. Bars, 20 µm. 1078 N. Usuda and others

To confirm further the distribution of allantoinase in the mitochondria, crude mitochondrial fractions from liver and kidney were processed for immunocytochemical localization. Allantoinase was found by Protein-A gold staining to be localized in mitochondria isolated from rat liver (Fig. 8A and B), as was observed in tissue sections stained in situ. In mito- chondrial fractions isolated from the kidney, allantoinase was detected only in some mitochondria and not in others (Fig. 8C). This was expected, since allantoinase distribution is restricted to the mitochondria in specific segments of the renal tubules and not to those in other segments. Immunoblot analysis of the crude mitochondrial fractions that were washed free of contaminating cytosolic proteins also provided strong evidence for the presence of allantoinase in Fig. 5. Immunoblot analysis of adult frog liver (lane 1) and kidney this particulate fraction (data not shown). It should be noted (lane 2), and tadpole liver (lane 3) and kidney (lane 4) homogenates µ that the crude mitochondrial fractions were invariably con- (100 g protein per lane) for allantoinase, using antibodies against taminated with a few peroxisomes and accordingly the frog allantoinase. Lane M, molecular mass standards (×10−3). immunoblot results of these subcellular fractionations do not unequivocally prove that allantoinase is associated with mito- chondria. Nevertheless, the immunocytochemical localization appears that urate oxidase and allantoinase are co-localized in studies on these crude mitochondrial fractions convincingly the same renal epithelial cells, albeit in different subcellular demonstrate that allantoinase is in the mitochondria. organelles. A trace amount of immunostaining was detected in the

Fig. 6. Immunoperoxidase localization of allantoinase in liver (A) and kidney (B) using antibodies against frog liver allantoinase. In liver, allantoinase is localized in the cytoplasm of hepatocytes and the proximal tubular epithelium. No staining is observed in the distal tubules and the glomerulus in kidney. (A) ×240 and (B) ×400 Urate oxidase and allantoinase localization 1079

100,000 g supernatant of liver, possibly due to leakage during pool, reflecting that a fraction of the newly synthesized protein homogenization. Nuclear-encoded mitochondrial proteins are is in the process of translocating from the site of synthesis to sometimes detected outside the mitochondria in the cytosolic the target organelle (Grivell, 1988). Abundant mitochondrial

Fig. 7. Immunocytochemical localization of allantoinase in Lowicryl-embedded tissues by the Protein A-gold method, using frog liver anti- allantoinase antibodies. Adult frog liver (A) and tadpole liver (C), and adult frog kidney (B) and tadpole kidney (D). Allantoinase is localized within the mitochondria (M), adjacent to the cristae. No allantoinase labeling is seen in peroxisomes (P) in liver and kidney. Subcrystalloid urate oxidase core is seen in peroxisomes in (B) and (D). Bars, 20 µm. 1080 N. Usuda and others

Fig. 8. Immunocytochemical localization of allantoinase in isolated mitochondria obtained from subcellular fractions of liver (A and B) and kidney (C) from adult frogs embedded in Lowicryl. In the liver mitochondrial fractions, allantoinase is distinctly visualized in the mitochondrial cristae (A,B). Not all mitochondria in the kidney fractions (C) show allantoinase because of heterogeneous distribution of this enzyme in different segments of the renal tubule. Bars, 20 µm. proteins are known to be detected in the cytosolic fractions and frogs and tadpoles. Urate oxidase is exclusively localized to the pool varies under different physiological conditions the subcrystalloid core of peroxisomes in the liver and kidney (Grivell, 1988). of the adult frog and tadpole, whereas allantoinase is present predominantly in proximity to mitochondrial cristae. The localization of urate oxidase to the hepatocyte peroxi- DISCUSSION somes of liver is consistent with the distribution pattern of this enzyme in most mammals. However, unlike in most mammals, The degradation of adenine and guanine, the purine moieties we show that urate oxidase is expressed in the peroxisomes of of nucleic acid, results in the formation of uric acid in all proximal convoluted tubules of the frog and tadpole kidney. animals with the exception of leeches, fresh water mussels and This distribution pattern of urate oxidase is similar to that noted spiders, where the degradation of these purines does not go in the bovine liver and kidney (Zaar et al., 1986; Usuda et al., beyond the stage of hypoxanthine or xanthine (Keilin, 1958). 1988b). The reasons for this particular hepatic and renal dis- Uric acid is an important biological molecule which exerts an tribution of urate oxidase remain to be elucidated. Neverthe- antioxidant action and is postulated to protect against cancer less, it could be speculated that this pattern is probably related and other disorders, including ageing, caused by oxygen free to increased production of uric acid from and other radicals (Ames et al., 1981). The peroxisomal localization of dietary sources in the frog, which may not be efficiently urate oxidase, the first of the three enzymes responsible for the degraded by the liver urate oxidase. Alternatively, hepatic urate degradation of uric acid to urea (urate oxidase, allantoinase and oxidase may not be readily accessible to uric acid for allantoicase), and the observation that the urate oxidase gene oxidation; as a result renal urate oxidase could participate in in humans and hominoid primates is mutated, have generated the further degradation of uric acid prior to excretion. Hence, considerable interest in the biological and evolutionary impli- it is important to determine the relative proportions of urate cations of uric acid and the enzymes responsible for its metab- oxidase in amphibian liver and kidney. It is interesting to note olism (Wu et al., 1989; Yeldandi et al., 1990; 1991). Informa- that in the dalmatian dog, abnormalities of uric acid handling tion about genes encoding urate oxidase, allantoinase and result in hyperuriciemia (Yu et al., 1971). Similar abnormali- allantoicase from several species, as well as the precise sub- ties of uric acid handling by the liver may be responsible for cellular localization of these enzymes and their regulation at the dual organ expression of this gene in the amphibia. the molecular level, will be essential to an understanding of the The localization of allantoinase to the mitochondria of the biological role of uric acid in development and differentiation. liver of frog is inconsistent with its previously reported local- In the present study, we have demonstrated by immunoblot- ization in hepatic peroxisomes (Visentin and Allen, 1969). This ting the presence of urate oxidase and allantoinase in both liver inconsistency can be explained by the fact that previous studies and kidney. The presence of these two enzymes in the frog on the localization of allantoinase consisted of density gradient kidney is demonstrated for the first time. In addition, using the and differential centrifugations, which cannot fully separate highly specific Protein A-gold immunocytochemical peroxisomes from mitochondria (Scott et al., 1969; Visentin technique, we have localized the two uric acid degrading and Allen, 1969). The present study provides direct visual enzymes, urate oxidase and allantoinase, to peroxisomes and evidence for the presence of allantoinase within the mitochon- mitochondria respectively, both in the liver and kidney of adult dria, and not in the peroxisomes as was previously thought. Urate oxidase and allantoinase localization 1081

Protein A-gold immunocytochemistry is a highly specific and purification of its peroxisomal membrane-bound form. J. Biol. Chem. technique at the ultrastructural level and this method clearly 264, 3211-3215. establishes the unequivocal presence of allantoinase in the Keilin, J. (1959). The biological significance of uric acid and guanine excretion. Biol. Rev. 34, 265-296. mitochondrial cristae. Laemmli U. K. (1970). Cleavage of structural proteins during the assembly of Our data herein raise several interesting questions as to the the head of bacteriophage T4. Nature 227, 680-685. role of peroxisomes and mitochondria in uric acid degradation Lata, G. F., Mamrak, F., Block, P. and Baker. B. (1977). An electron in amphibia. One issue of interest is to explore how the microscopic and enzymic study of rat liver peroxisomal nuceloid core and its allantoin generated in the peroxisome moves to the mitochon- association with urate oxidase. J. Supramol. Struc. 7, 419-434. 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