Comparison of intraperoxisomal localization form and properties of amphibian (Rana catesbeiana) uricase with those of other animal uricases

Liver uricase of bull frog (Rana catesbeiana) was present as the soluble form in the peroxisomal matrix and consisted of four identical subunits with a molecular weight of 30,000. These properties were identical with those of fish liver uricase but differed from mammalian liver uricase. Purified uricase from the frog liver was insoluble in hypertonic, hypotonic and detergent solutions at pH 6-9. This insolubility was the same as mammalian liver uricase but differed from fish liver uricase; fish uricase was soluble in these solutions. The frog liver uricase did not cross-react immunologically with both uricases of fish and mammalian liver. An immunological cross-reactivity of liver uricase was observed among amphibia.     

Biogenesis of peroxisomes in regenerating rat liver. I. Sequential changes of catalase and urate oxidase detected by ultrastructural cytochemistry

The biogenesis of peroxisomes has been investigated in the model of regenerating rat liver after partial hepatectomy using ultrastructural cytochemical staining methods: catalase as a marker of the peroxisomal matrix and uricase for the cores. The peroxisomes in regenerating rat liver showed several distinctive features: a) marked variation in shape and size, e.g., peroxisomes with tail-like extensions and tortuously elongated rod-shaped ones, b) formation of peroxisomal clusters and, c) interconnections between adjacent peroxisomes suggesting cleavage or budding. Whereas the reaction product for catalase was present at all intervals after hepatectomy in the matrix of all peroxisomes, the pattern of localization of uricase case varied with the time. It was confined to the cores in controls and at 10 days after the operation, while at 24 and 48 h it showed, in addition, a diffuse reaction in the matrix of some peroxisomes. In interconnected apparently dividing peroxisomes, the core with positive uricase reaction was present only in one half, while the other half was devoid of the reaction product. Similarly, the diffuse uricase staining was confined to the half which contained the core with the other half remaining unstained. These observations are consistent with the concept that new peroxisomes are formed from preexisting ones by budding and segmentation. While catalase is transferred uniformly to all new segments, uricase is compartmentalized in certain portions, of the apparently growing "peroxisomal reticulum".     

Cloning of rabbit uricase cDNA reveals a conserved carboxy-terminal tripeptide in three species

cDNA clones encoding uricase have been isolated from a rabbit liver cDNA library. The nucleotide sequences of the cDNAs have been determined and those of the rat uricase cDNA have been revised. In all three uricases, the carboxy-terminal tripeptides are Ser-Arg/Lys-Leu sequences, which have recently been suggested as an essential element of peroxisomal targetting signals for many but not all peroxisomal proteins.     

Degradation of uric acid to urea and glyoxylate in peroxisomes.

The distribution of enzymes involved in purine degradation in fish and crustaceous liver was examined by centrifugation in a sucrose density gradient. In mackerel, yellow mackerel, and prawn liver and mantis club hepatopancreas, uricase and allantoinase were located only in the peroxisomes and in the soluble fraction from broken peroxisomes, and allantoicase was located only in the peroxisomes. Uricase and allantoinase seem to be located in the peroxisomal matrix and allantoicase in the peroxisomal membrane. Adenase, guanase, and xanthine oxidase were present only in the soluble fraction of mackerel liver.     

Uricase protein sequences: conserved during vertebrate evolution but absent in humans

Uricase is a peroxisomal liver enzyme that catalyzes the oxidation of uric acid to allantoin during purine catabolism. It is present in vertebrates in most species of fish, amphibians, and mammals but its enzymatic activity is absent in hominoids. We have used Western blot analysis in a comparative study to establish a homology among uricases from different species of vertebrates. Using antibodies against denatured rat liver uricase, we have been able to detect for the first time cross-reactivity with the uricase of species ranging in the evolutionary scale from fish to primates (macaque). Our results suggest that these uricases have a common evolutionary origin. Our conclusion is also supported by the fact that uricase from different species exhibits identical tissue, subcellular localization, and similarity of molecular weights. This study was extended to include human liver samples. Using the same approach but with a more sensitive detection system (alkaline phosphatase instead of peroxidase), we did not detect polypeptide species related to rat uricase in human fetal or adult liver samples, which indicates that during hominoid evolution, the mutational event responsible for the loss of uricase activity in humans precluded formation of a translatable uricase mRNA.     

Determination of the cross-points of rat liver peroxisomes,peroxisomal core and the core components by cross-partition

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same crosspoint, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, d-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2:1. The theoretical value of cross-point of the core calculated from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

Determination of the cross-points of rat liver peroxisomes, peroxisomal core and the core components by cross-partition.

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same cross-point, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, D-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2 : 1. The theoretical value of cross-point of the core calculated from from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

Characterization of liver-specific expression of rat uricase using monoclonal antibodies and cloned cDNAs

Tissue distribution of uricase (urate oxidase, EC 1.7.3.3) was studied by immunoblotting and RNA slot blot analysis. For immunoblotting, highly specific monoclonal antibodies against rat liver uricase were obtained, and for mRNA detection, a cloned uricase cDNA was used. Among seven tissues studied, uricase was immunologically detected only in the liver. The contents of uricase in other tissues, i.e., brain, thymus, heart, spleen, kidney and lactating mammary gland, were estimated to be less than 2% of that in the liver. Uricase mRNA was also detected only in the liver. The steady-state level of the mRNA in the isolated hepatocytes was relatively constant during the 8-day culture period when compared with those of other mRNAs expressed in the liver, suggesting a unique control mechanism of its expression.     

Effects of antilipolytic agents on rat liver peroxisomes and peroxisomal oxidative activities

The mechanisms involved in the inhibitory effects of antilipolytic agents on rat liver peroxisomal fatty acid oxidative activity have been explored. Treatment of fasting rats with antilipolytic drugs (either 3,5-dimethylpyrazole (12 mg/kg body weight) or Acipimox (25 mg/kg body weight)) resulted in a decrease in free fatty acid and glucose plasma levels within 5–10 and in a significant increase in the plasma glucagon to insulin ratio within 15. Changes in the fatty acid oxidative activity appeared with a 2.5–3 h delay and were then very rapid (a 30–40% decrease in the activity occured in additional 2 h). Many peroxisomal enzyme activities (including non-β-oxidative activities such as uricase and D-amino acid oxidase) exhibited similar changes with the same delay. Simultaneously with the enzyme changes, at the electron microscope level many autophagic vacuoles were detected in the liver cells, often containing peroxisomal structures. Glutamine, an inhibitor of proteolysis in vivo, prevented the decrease in enzyme activities. It was concluded that the decrease in peroxisomal enzyme activities may be the consequence of enhanced peroxisome degradation due to the stimulation of autophagic processes in liver cells.     

Localization of urate oxidase in the crystalline cores of rat liver peroxisomes by immunocytochemistry and immunoblotting

We investigated the immunocytochemical localization of urate oxidase by light and electron microscopy. Rabbits were immunized with urate oxidase prepared from rat liver and the resulting antibody was further purified by affinity chromatography. Immunoblotting of the antigen revealed a single band of Mr 32,500 daltons, consistent with a subunit of uricase. The same band was observed in immunoblots prepared from a total peroxisome fraction and in its subfraction containing the cores, but not in the matrix portion. Immunostaining of 1-micron sections with the antibody against uricase followed by protein A-gold-silver showed fine granules in hepatocytes, which exhibited distinct fluorescence when examined in a microscope equipped with epifluorescence illumination. Incubation of ultra-thin sections of rat liver, embedded in Lowicryl K4M, LR White, or Epon, with the anti-uricase antibody followed by protein A-gold showed prominent labeling of the crystalline cores, with no reaction in the surrounding peroxisomal matrix. In contrast, the core region was spared whereas the matrix was heavily labeled in sections incubated with an antibody against catalase. Direct incubation of cores, isolated by centrifugation, with the anti-uricase antibody followed by protein A-gold revealed gold particles on the surface of isolated cores, with rare particles within the lumen of the polytubular structures that make up the cores. Specificity of the immunolabeling was established in sections incubated with an IgG fraction from pre-immunized rabbits. These observations demonstrate that in normal rat liver urate oxidase is exclusively associated with the crystalline cores in peroxisomes.     

Evolution of urate-degrading enzymes in animal peroxisomes

The end product of purine metabolism varies from species to species. The degradation of purines to urate is common to all animal species, but the degradation of urate is much less complete in higher animals. The comparison of subcellular distribution, intraperoxisomal localization forms, molecular structures, and some other properties of urate-degrading enzymes (urate oxidase, allantoinase, and allantoicase) among animals is described. Liver urate oxidase (uricase) is located in the peroxisomes in all animals with urate oxidase. On the basis of the comparison of intraperoxisomal localization forms, mol wt, and solubility of liver urate oxidase among animals, it is suggested that amphibian urate oxidase is a transition form in the evolution of aquatic animals to land animals. Allantoinase and allantoicase are different proteins in fish liver, but the two enzymes form a complex in amphibian liver. The subcellular localization of allantoinase and allantoicase varies among fishes. Hepatic allantoinase is located both in the peroxisomes and in the cytosol in saltwater fishes, and only in the cytosol in freshwater fishes. Hepatic allantoicase is located on the outer surface of the, peroxisomal membrane in the mackerel group and in the peroxisomal matrix in the sardine group. Amphibian hepatic allantoinase-allantoicase complex is probably located in the mitochondria. On the basis of previous data, changes of allantoinase and allantoicase in molecular structure and intracellular localization during animal evolution may be as follows: Fish liver allantoinase is a single peptide with a mol wt of 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 mol wt of 48,000, and is located in the peroxisomal 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.     

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).     

Immunogold localization of nodule-specific uricase in developing soybean root nodules

Immunogold labeling was used to study the time of appearance and distribution of a nodule-specific form of uricase (EC 1.7.3.3) in developing nodules of soybean (Glycine max (L.) Merr.) inoculated with Bradyrhizobium japonicum. The enzyme was detected in thin sections of tissue embedded in either L R White acrylic resin or Spurr's epoxy resin, by employing a polyclonal antibody preparation active against a subunit of soybean nodule uricase. Antigenicity was better preserved in L R White resin, but ultrastructure was better maintained in Spurr's. Uricase was first detectable with protein A-gold in young, developing peroxisomes in uninfected cells, coincident with the release of Bradyrhizobium bacteroids from infection threads in adjacent infected cells. As the peroxisomes enlarged, labeling of the dense peroxisomal matrix increased. Gold particles were never observed over the paracrystalline inclusions of peroxisomes, however. Despite a close association between enlarging peroxisomes and tubular endoplasmic reticulum, uricase was not detectable in the latter. In mature nodules, labeling of uricase was limited to the large peroxisomes in uninfected cells. Small peroxisome-like bodies present in infected cells did not become labeled.Abbreviations BSA bovine serum albumin - Da dalton - ER endoplasmic reticulum - IgG immunoglobulin G     

Studies on peroxisomes. VI. Relationship between the peroxisomal core and urate oxidase.

The peroxisomal core from the liver of rats was purified 450-fold as a marker of urate oxidase [EC 1.7.3.3.] activity. This preparation has a high specific activity of urate oxidase but not of other peroxisomal enzymes: D-amino acid oxidase [EC 1.4.3.3.], L-alpha-hydroxy acid oxidase [EC 1.1.3.15], or catalase [EC 1.11.1.6]. No activity of marker enzymes for other subcellular particles; cytochrome c oxidase [EC1.9.3.1] (mitochondria), acid phosphatase [EC 3.1.3.2] (lysosomes), or glucose-6-phosphatase [EC 3.1.3.9] (microsomes), was detected in this preparation. The core obtained showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the position of the band was found to correspond to a molecular weight 35,000. When the peroxisomal core was subjected to treatment at various pH's with 0.1 M carbonate buffer, urate oxidase was almost completely solubulized at pH 11.0, although approximately 35% of the core protein still remained in the pellet After solubilization of the core at pH 11.0, the specific activity of urate oxidase in the supernatant increased about 1.6 times; the density of the insoluble protein remaining in the pellet was identical with the that of the original core on sucrose density gradient centrifugation.     

Selective induction of peroxisomal enzymes by the hypolipidemic drug bezafibrate. Detection of modulations by automatic image analysis in conjunction with immunoelectron microscopy and immunoblotting

Quantitative immunoelectron microscopy in conjunction with quantitative analysis of immunoblots have been used to study the effects of bezafibrate (BF), a peroxisome-proliferating hypolipidemic drug, upon six different enzyme proteins in rat liver peroxisomes (Po). Antibodies against following peroxisomal enzymes: catalase, urate oxidase, alpha-hydroxy acid oxidase, acyl-CoA oxidase, bifunctional enzyme (hydratase-dehydrogenase) and thiolase, were raised in rabbits, and their monospecificities were confirmed by immunoblotting. Female Sprague-Dawley rats were treated for 7 days with 250 mg/kg/day bezafibrate and liver sections were incubated with the appropriate antibodies followed by the protein A-gold complex. The labeling density for each enzyme was estimated by automatic image analysis. In parallel experiments immunoblots prepared from highly purified peroxisome fractions of normal and BF-treated rats were incubated with the same antibodies. The antigens were visualized by an improved protein A-gold method including an anti-protein A step and silver amplification. The immunoblots were also quantitated by an image analyzer. The results revealed a selective induction of beta-oxidation enzymes by bezafibrate with thiolase showing the most increase followed by bifunctional protein and acyl-CoA oxidase. The labeling density for catalase and alpha-hydroxy acid oxidase was reduced, confirming fully the quantitative analysis of immunoblots which in addition revealed reduction of uricase. These observations demonstrate that hypolipidemic drugs induce selectively the beta-oxidation enzymes while other peroxisomal enzymes are reduced. The quantitative immunoelectron microscopy with automatic image analysis provides a versatile, highly sensitive and efficient method for rapid detection of modulations of individual proteins in peroxisomes.     

Purification and characterization of peroxisomal apo and holo alanine:glyoxylate aminotransferase from bird liver.

Alanine:glyoxylate aminotransferase has been reported to be present as the apo enzyme in the peroxisomes and as the holo enzyme in the mitochondria in chick (white leghorn) embryonic liver. However, surprisingly, birds were found to be classified into two groups on the basis of intraperoxisomal forms of liver alanine:glyoxylate aminotransferase. In the peroxisomes, the enzyme was present as the holo form in group 1 (pigeon, sparrow, Java sparrow, Australian budgerigar, canary, goose, and duck), and as the apo form in group 2 (white leghorn, bantam, pheasant, and Japanese mannikin). In the mitochondria, the enzyme was present as the holo form in both groups. The peroxisomal holo enzyme was purified from pigeon liver, and the peroxisomal apo enzyme from chicken (white leghorn) liver. The pigeon holo enzyme was composed of two identical subunits with a molecular weight of about 45,000, whereas the chicken apo enzyme was a single peptide with the same molecular weight as the subunit of the pigeon enzyme. The peroxisomal holo enzyme of pigeon liver was not immunologically cross-reactive with the peroxisomal apo enzyme of chicken liver, the mitochondrial holo enzymes from pigeon and chicken liver, and mammalian alanine:glyoxylate aminotransferases 1 and 2. The mitochondrial holo enzymes from both pigeon and chicken liver had molecular weights of about 200,000 with four identical subunits and were cross-reactive with mammalian alanine:glyoxylate aminotransferase 2 but not with mammalian alanine:glyoxylate aminotransferase 1.     

Developmental changes of adenosine deaminase, xanthine oxidase, and uricase in mouse tissues

The activities of adenosine deaminase, xanthine oxidase, and uricase were followed in the liver, kidney, stomach, and intestine during pre- and postnatal development of the mouse. Results indicated that some type of coordinate control exists between the uricase and the xanthine oxidase levels in liver, stomach, intestine, and kidney. No coordinate control was seen between adenosine deaminase and xanthine oxidase in liver and kidney. The developmental changes between the intestine and stomach xanthine oxidase and adenosine deaminase were found to be related. The results obtained were consistent with the idea that intermediate metabolites in a pathway play some role in controlling the level of enzymes further down the pathway.A superificial resemblance in the timing of changes in feeding habits and changes in enzyme levels during development was found. Results of artifical change of feeding habit indicate that the control of enzyme levels was inherent rather than the result of dietary change.     

Postnatal development of peroxisomal and mitochondrial enzymes in rat liver.

Subcellular organellles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks. The peroxisomal and mitochondrial fatty acid beta-oxidation and the peroxisomal and supernatant activities of catalase and glycerol-3-phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively. Fatty acid beta-oxidation in both peroxisomes and mitochondria and peroxisomal glycerol-3-phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal beta-oxidation accounted for at least 10% of the total beta-oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial beta-oxidation after two weeks of age. The greatest change in beta-oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much beta-oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol-3-phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week. Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5- to 3-fold, and all of the increased activity was in the supernatant fraction.     

EPR spin trapping of a radical intermediate in the urate oxidase reaction

Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin. The chemical mechanism of the urate oxidase reaction has not been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPR spectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (Porcine Liver, Bacillus Fastidiosus, Candida Utilitis) was performed in the presence of 5-diethoxyphosphoryl-5-methyl-pyrroline-N-oxide (DEPMPO) as spin trap. DEPMPO was added to reaction mixture and a radical adduct was observed in all cases. Therefore, for the first time, the presence of a radical intermediate in the uricase reaction was experimentally proved.     

Selective inhibition of hepatic peroxisomal fatty acid beta-oxidation by enoximone.

Although beta-oxidation of fatty acids occurs in both peroxisomes and mitochondria, beta-oxidizing enzymes in these organelles have distinct differences in their specifity and sensitivity to inhibitors. In this study, the effects of the phosphodiesterase inhibitor enoximone on hepatic peroxisomal and mitochondrial beta-oxidation were investigated. In liver homogenates from control rats, cyanide-insensitive peroxisomal beta-oxidation of palmitoyl-CoA was inhibited progressively by increasing concentrations of enoximone. Similar results were obtained in liver homogenates from rats pretreated with the known peroxisomal proliferator diethylhexylphthalate. In contrast, mitochondrial beta-oxidation of palmitoyl-CoA was not inhibited by enoximone. These data show that enoximone selectively inhibits basal as well as induced peroxisomal, but not mitochondrial, beta-oxidation of the CoA thioester of long-chain fatty acids. The availability of specific inhibitors of peroxisomal beta-oxidation should prove useful in elucidating regulatory mechanisms operative in this pathway in normal as well as in proliferated peroxisomes.