Degradation of Uric Acid in Fish Liver Peroxisomes INTRAPEROXISOMAL LOCALIZATION OF HEPATIC ALLANTOICASE AND PURIFICATION OF ITS PEROXISOMAL MEMBRANE-BOUND FORM*

Urate-degrading enzymes such as uricase, allantoinase, and allantoicase are located in the peroxisomes of marine fish liver (Noguchi, T., Takada, Y., and Fujiwara, S. (1979) J. Biol. Chem. 254, 5272-5275). On the basis of intraperoxisomal localization of hepatic allantoicase, 13 different fishes were classified into two groups: mackerel group and sardine group. Allantoicase is located on the outer surface of the peroxisomal membrane in the mackerel group and in the peroxisomal soluble matrix in the sardine group. The peroxisomal membrane enzyme and the peroxisomal matrix enzyme are not distinguishable on the basis of the number and molecular weight of the subunits, but differ in isoelectric point and electrophoretic mobility. The molecular weight of the fish allantoicase subunit is identical with that of the small subunit (allantoicase subunit) of amphibian allantoinase-allantoicase complex, suggesting that the subunit of fish allantoicase changed to the small subunit of the amphibian complex during evolution: allantoinase and allantoicase are present as a complex in amphibian liver (Noguchi, T., Fujiwara, S., and Hayashi, S. (1986) J. Biol. Chem. 261, 4221-4223).

The end product of purine degradation varies from species to species (1). The degradation of purines to urate is common to all animal species thus far studied, whereas the degradation of urate is much less complete in higher animals. In some marine fishes, enzymes that convert purines to urate are located in the cytosol and those that convert urate to urea (uricase (EC 1.7.3.31, allantoinase (EC 3.5.2.5) and allantoicase (EC 3.5.3.4)) are present in the peroxisomes, suggesting that in purine degradation, peroxisomal enzymes have been lost during animal evolution (2). We have reported that uricase and allantoinase are located in the peroxisomal matrix and allantoicase in the peroxisomal membrane in some marine fish liver (2, 3). In the present study, fishes were found to be classified into two groups on the basis of intraperoxisomal localization of hepatic allantoicase: one with allantoicase located on the outer surface of liver peroxisomal membranes and the other with allantoicase located in the liver peroxisomal soluble matrix. Peroxisomal membrane allantoicase was * This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and by the Cancer Research Fund from Fukuoka Cancer Research Association, Fukuoka, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ T o whom correspondence and reprint requests should be addressed. purified from mackerel liver and compared with peroxisomal soluble matrix allantoicases from other fish livers.

Intracellular
Localization of Fish Liver Allantoicase-The intracellular distribution of allantoicase was examined by sucrose density gradient centrifugation with the postnuclear fractions of the livers of 13 different fishes (Figs. 1 and 2). In each case, the peroxisomes and mitochondria were separated; the peroxisomes, identified by their catalase activity (EC 1.11.1.6), were at a density of about 1.25 g/ml, and the mitochondria, identified by their glutamate dehydrogenase activity, at a density of about 1.18 g/ml. Acid phosphatase (used as a lysosomal marker) was distributed over a broad density range with a peak of about 1.19 g/ml, and in the soluble top fraction formed presumably from broken peroxisomes (not shown). On the basis of sedimentation profiles of allantoicase, 13 different fishes were classified into two groups: sardine group and mackerel group. In the sardine group (sardine, bonito, young yellowtail, flying fish, yellow mackerel, gray mullet, carp, and pale chub), allantoicase was recovered both in the peroxisomes and in the soluble fraction ( Fig. 1). In each case, the activity ratio of the peroxisomal fraction to the soluble fraction of allantoicase was nearly identical with that of catalase as the soluble matrix enzyme of the peroxisomes. These results suggest that hepatic allantoicase is located only in the peroxisomal matrix in the sardine group, because catalase activity in the soluble top fraction is from broken peroxisomes. In the mackerel group (mackerel, grunt, crucian carp, sunfish and large mouth bass), hepatic allantoicase was recovered only in the peroxisomal fraction, whereas catalase was recovered both in the peroxisomal fraction and in the soluble fraction from broken peroxisomes (Fig.  2). The results suggest that hepatic allantoicase is located in the peroxisomal membrane or core in the mackerel group.
Intraperoxisomal localization of allantoicase was examined in the livers of the two prototypes i.e. sardine and mackerel. Peroxisomal suspensions of fish livers from the sucrose density gradients were separately diluted with the same volume of 0.01 M pyrophosphate buffer, pH 9.0, which is known to break peroxisomes (15). After storage overnight at 4 "C, each suspension was subjected to sucrose density gradient centrifugation. Under these conditions, in rat liver, all of the catalase Portions of this paper (including "Materials and Methods," Figs. 3-5, and 7, and Table I)

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Degradation of Uric Acid in Fish Liver Peroxisomes allantoicase is located in the peroxisomal membrane or core in mackerel liver. Fig. 5, in the Miniprint, shows inactivation profiles of allantoicase by the antibody against sardine liver allantoicase using the peroxisomes and the peroxisomal extracts from sardine or mackerel liver. In each case, allantoicase activities of the peroxisomal extract were inactivated by the antibody. On the other hand, allantoicase activity of the peroxisomes from mackerel liver was inactivated by the antibody, whereas the activity of the peroxisomes from sardine liver was not inactivated. These findings show that hepatic allantoicase is located on the outer surface of the peroxisomes in mackerel liver and in the soluble matrix of peroxisomes in sardine liver.
All fishes of the sardine group were found to be identical with sardine and all of the mackerel group with mackerel with respect to the intraperoxisomal localization of hepatic allantoicase.
Purification of Peroxisomal Membrane Allantoicase from Mackerel Liver-We have purified and characterized the peroxisomal soluble matrix allantoicase from sardine liver (12). In the present study, the purification of the peroxisomal membrane allantoicase from mackerel liver was carried out and compared with the peroxisomal soluble matrix allantoicases of other fishes. The peroxisomal membranes were prepared from the peroxisomal fraction of mackerel liver as described under "Materials and Methods." Allantoicase could be solubilized from the peroxisomal membrane by treatment with n-octyl-0-D-thioglucoside. An approximately 1,500-fold purification was achieved from liver homogenates with a yield of about 2.1% (Table I, Miniprint). The enzyme preparation may be stored at -20 "C for at least 5 weeks without loss of activity, and there was little loss with storage at 0-4 "C for at least 2 weeks. The enzyme preparation showed a single protein-staining band upon polyacrylamide gel electrophoresis a t pH 9.0 (Fig. 6). The molecular weight of the enzyme was estimated to be approximately 100,000 by sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave a single protein in the soluble matrix is recovered in the soluble top fraction, uricase in the core is located at a density of 1.23 g/ml, and NADH-cytochrome-c reductase in the membrane is distributed over a broad density range with a peak of about 1.17 g/ ml (8, 16). Different sedimentation profiles of allantoicase activity were observed between sardine liver and mackerel liver. In the sardine, allantoicase as well as catalase were completely solubilized and recovered only in the soluble top fraction, showing that allantoicase is located only in the peroxisomal soluble matrix (Fig. 3, Miniprint). In contrast, in the mackerel, allantoicase was not solubilized and was distributed over a broad density range with a peak of about 1.17 g/ml, whereas catalase is completely solubilized and recovered only in the soluble top fraction (Fig. 4, Miniprint). In fish liver, uricase is not a marker enzyme of the peroxisomal core and NADH-cytochrome-c reductase is not a marker enzyme of the peroxisomal membrane, because uricase is located in the peroxisomal soluble matrix, and NADH-cytochrome-c reductase has not been demonstrated in the peroxisomal membrane (2, 3). However, the present results suggest that   component that had an estimated molecular weight of about 48,000, showing that the enzyme consists of two identical subunits each with a M, = 48,000 (Fig. 6).
Some physical, enzymatic, and immunological properties of peroxisomal membrane-bound allantoicase purified from mackerel liver were compared with the peroxisomal soluble matrix allantoicase purified from sardine liver ( Table 11). The two enzymes were not distinguishable with respect to the number and molecular weight of the subunit and pH optimum. In contrast, they differed in K,,, for allantoate, isoelectric point, and electrophoretic mobility.
On Ouchterlony double diffusion analysis (17), the antibody against sardine allantoicase produced a single band of precipitin against both sardine allantoicase and mackerel allantoicase (Fig. 7, Miniprint). However, the antibody produced a spur between the two precipitin lines.
Molecular weights of allantoicase subunits in the peroxisomal extracts of other fish livers were examined by immunoblotting (18) using the antibody against sardine liver allantoicase. Yellow mackerel was examined as a representative of the sardine group with the peroxisomal soluble matrix allantoicase, and grunt as a representative of the mackerel group with the peroxisomal membrane-bound allantoicase. In each case, the molecular weight of the allantoicase subunit was identical with those of sardine or mackerel allantoicase subunit.

DISCUSSION
We have reported that allantoinase and allantoicase are different proteins in marine fish and invertebrate liver, whereas the two enzymes form a complex in amphibian liver (4,121. This allantoinase-allantoicase complex consists of four subunits (48,000 x 2 + 54,000 x 2). Allantoinase activity of allantoinase-allantoicase complex is from the large subunit (allantoinase subunit) with a M, = 54,000, and allantoicase activity of the allantoinase-allantoicase complex is from the small subunit (allantoicase subunit) with a MI = 48,000. Fish allantoinase is a single peptide with MI = 48,000, and fish allantoicase consists of two identical subunits each with a MI = 54,000. The molecular weight of the large subunit of amphibian allantoinase-allantoicase complex is identical with mackerel liver allantoinase, and the small subunit is identical with the subunit of the peroxisomal soluble allantoicase from sardine liver (12). On the basis of intraperoxisomal localization of hepatic allantoicase, 13 fishes were classified into two groups: the sardine group with allantoicase within the peroxisomal soluble matrix and the mackerel group with allantoicase bound to the peroxisomal membrane. The peroxisomal matrix allantoicase of the sardine group and the peroxisomal membrane-bound allantoicase of the mackerel group were not distinguishable in number and molecular weight of the subunit.
Recently, we found that the subcellular localization of allantoinase varies among fishes, and amphibian allantoinaseallantoicase complex is located only in the cytosol: hepatic allantoinase is located both in the peroxisomes and in the cytosol in marine fishes (sardine, mackerel, grunt, bonito, young yellowtail, flying fish, and yellow mackerel), and only in the cytosol in fresh water fishes (crucian carp, sunfish, large mouth bass, gray mullet, carp, and pale chub)? On the basis of the present and previous data (2)(3)(4)12), changes of allantoinase and allantoicase in the molecular structure and intracellular localization during animal evolution are shown in Fig. 8. Fish liver allantoinase is a single peptide with a M, = 54,000 and is located both in the peroxisomes and in the cytosol, or only in the cytosol. Fish liver allantoicase consists of two identical subunits with a M4 = 48,000 and is located in the peroxisomal soluble matrix or on the outer surface of the peroxisomal membrane. The evolution of fishes to amphibia resulted in the dissociation of allantoicase into subunits and in the association of allantoinase with the subunit of allantoicase. This amphibian enzyme was lost by further evolution.