Peroxisomal oxidation of thiazolidine carboxylates in firefly fat body, frog retina, and rat liver and kidney.

D-amino acid oxidase is a widely distributed peroxisomal enzyme whose principal natural substrates are still unknown. Thiazolidine carboxylates, their derivatives and relatives, and the intermediates in their metabolism are among the more plausible substrate candidates. Using a cytochemical procedure, we have explored the distribution of peroxide-generating enzymatic activity against two thiazolidine carboxylates. We find that these compounds are effective substrates for peroxisomal oxidation in a variety of tissues that contain peroxisomal D-amino acid oxidase. Reaction was seen in the "classical" peroxisomes of rat liver and kidney, the peroxisomes of the fat body of firefly and of Drosophila and the peroxisomes of frog retina. Interestingly, both with the thiazolidine compounds and with more traditional D-amino acid oxidase substrates, the fireflies' photocyte granules, which are peroxisomes, lack activity.     

D-aspartate oxidase in rat, bovine and sheep kidney cortex is localized in peroxisomes.

D-Aspartate oxidase (EC 1.4.3.1) was assayed in subcellular fractions and in highly purified peroxisomes from rat, bovine and sheep kidney cortex as well as from rat liver. During all steps of subcellular-fractionation procedures, D-aspartate oxidase co-fractionated with peroxisomal marker enzymes. In highly purified preparations of peroxisomes, the enrichment of D-aspartate oxidase activity over the homogenate is about 32-fold, being comparable with that of the peroxisomal marker enzymes catalase and D-amino acid oxidase. Disruption of the peroxisomes by freezing and thawing released more than 90% of the enzyme activity, which is typical for soluble peroxisomal-matrix proteins. Our findings provide strong evidence that in these tissues D-aspartate oxidase is a peroxisomal-matrix protein and should be added as an additional flavoprotein oxidase to the known set of peroxisomal oxidases.     

Cytochemical and immunocytochemical study on the peroxisomes of rat liver after administration of a hypolipidemic drug, MLM-160

After administration of a hypolipidemic drug, MLM-160, to male rats, liver peroxisomes were studied by biochemical, cytochemical, and immunocytochemical methods. The activities of D-amino acid oxidase, glycolate oxidase, and urate oxidase increased 2 to 3-fold by the treatment. The increase of the oxidases was confirmed by immunoblotting analysis. By light microscopy, immunoreaction for catalase was present in the cytoplasmic granules of hepatocytes. The stained granules formed some clusters and overlapped each other after MLM-160 treatment. However, immunostaining for D-amino acid oxidase and urate oxidase was present in discrete fine granules which did not overlap each other. By electron microscopy, many peroxisomes showed ring-like extensions and cavitation of the matrix, often giving the appearance of a peroxisome-within-a-peroxisome. In many cases, these unusual peroxisomes seemed to be interconnected with each other. Within the peroxisomes, the catalase was localized in the matrix. Urate oxidase was associated with the crystalloid cores. D-amino acid oxidase was localized focally in a small part of the matrix where the catalase was mostly negative. In conclusion, the administration of MLM-160 to male rats induces some peroxisomal oxidases, accompanying the appearance of unusual peroxisomes. The precise localization of peroxisomal enzymes suggested that there are subcompartments within the liver peroxisomes as shown in rat kidney peroxisomes.     

Peroxisomes in human colon carcinomas. A cytochemical and biochemical study.

The presence of peroxisomes and their enzymic content were investigated and compared in healthy and neoplastic human colon epithelial cells using cytochemical studies at the ultrastructural level as well as biochemical analyses. Catalase-positive organelles were found to be more numerous in normal than in colonic neoplastic cells. Biochemical assays revealed that no D-aminoacid oxidase or L-alpha-hydroxyacid oxidase activity was detected in normal or tumor tissues. The specific activities of catalase, fatty-acyl CoA oxidase and enoyl-CoA hydratase/3 hydroxyacyl-CoA dehydrogenase (the so-called peroxisomal bifunctional enzyme of the beta-oxidation system) were found to be diminished in carcinoma cells compared with the control tissue. The fall in catalase activity correlated well with tumor stage according to Dukes, suggesting that this peroxisomal enzyme could be used as a potential prognostic marker.     

The effect of clofibrate on amphibian hepatic peroxisomes.

Liver peroxisomes of two anuran amphibian species, Rana esculenta and Xenopus laevis, were studied in untreated and in clofibrate-treated adults by means of complementary technical approaches, ie, ultrastructural cytochemistry, cell fractionation and marker enzyme activity assays. In untreated adults, hepatic peroxisomes were found to be very scarce in Xenopus when compared to Rana. Activities of catalase, D-amino acid oxidase and of the three first enzymes of the peroxisomal beta-oxidation system were detected in the light mitochondrial fractions enriched in peroxisomes and prepared from livers of both species. Administration of clofibrate at a daily dose level of 60 mg (Rana) and 90 mg (Xenopus) during ten days induced a drastic peroxisome proliferation in Rana hepatocytes but had no visible effect on the hepatic peroxisomal population of Xenopus. The catalase activity and the peroxisomal beta-oxidation system of liver cells were enhanced in Rana as well as in Xenopus. The hepatic D-amino acid oxidase specific activity was increased in Rana whereas it remained rather constant in Xenopus. Taking advantage of the behaviors of Rana and Xenopus hepatic peroxisomes, the molecular mechanisms of clofibrate induction are now investigated in the target liver cells of the two amphibian species.     

Kinetic properties of rat kidney D-amino acid oxidase associated with peroxisomes

In contrast to hog kidney D-amino acid oxidase, the v vs s plots of D-amino acid oxidase in homogenized rat kidney did not have the form of a rectangular hyperbola, and showed an apparent negative cooperativity. After subcellular fractionation of rat kidney, both of the oxidases in the supernatant fraction and the peroxisomal fraction showed Michaelis-Menten type kinetics. The Km values for D-alanine and D-proline of the peroxisomal fraction were significantly lower than those of the supernatant fraction. The partially purified enzyme from the peroxisomal fraction showed the same kinetic properties as the supernatant fraction. These facts suggest that the two types of rat kidney D-amino acid oxidase were originally identical and that some interaction between the enzyme and peroxisomes is physiologically important for the function of the enzyme.     

Cytochemical localization of a D-amino acid oxidizing enzyme in peroxisomes of Drosophila melanogaster

A peroxide generating oxidase is demonstrated cytochemically in the peroxisomes of adult and larval Drosophila melanogaster, Oregon R and Rosy-506 strains. This enzyme activity is demonstrable using D-pipecolate or D-proline, but not L-proline, as substrate and is inhibited by kojic acid. Thus this enzyme shares cytochemical characteristics with vertebrate D-amino acid oxidase.     

Pipecolic acid is oxidized by renal and hepatic peroxisomes. Implications for Zellweger's cerebro-hepato-renal syndrome (CHRS)

Increased levels of pipecolic acid have been reported in patients with cerebro-hepato-renal syndrome (CHRS) of Zellweger and the general deficiency of peroxisomal function has been implicated in its pathogenesis. We have therefore investigated the capacity of normal peroxisomes to metabolize pipecolic acid. Highly purified peroxisomes were obtained from rat liver and rat and beef kidney cortex by a recently developed method using metrizamide gradients and a vertical rotor. These preparations oxidized D,L-pipecolic acid as evidenced by the measurement of H2O2 production. Incubation with either the D- or L-isomer revealed that almost exclusively D-pipecolate is oxidized. The specific activities proved to be 20-50 times higher in renal than in hepatic peroxisomes. A commercially available crystalline suspension of D-amino acid oxidase from porcine kidney also oxidized the pipecolic acid with the following rates 54:36:1 respectively for D-:,D,L-:L-isomers. Incubation of vibratome sections of rat kidney and liver in a medium containing D-pipecolic acid and cerous ions, revealed electron-dense deposits over the matrix of peroxisomes confirming the localization also by fine structural cytochemistry. These observations demonstrate the capability of mammalian peroxisomes to oxidize pipecolic acid and suggest that the absence or deficiency of peroxisomal D-amino acid oxidase may be implicated in the pathogenesis of hyperpipecolatemia in Zellweger's CHRS.     

A new peroxisomal disorder with enlarged peroxisomes and a specific deficiency of Acyl-CoA oxidase (pseudo–Neonatal adrenoleukodystrophy)

In the present paper two siblings are presented with clinical manifestations very similar to those of patients affected by neonatal adrenoleukodystrophy. In contrast to neonatal adrenoleukodystrophy patients, hepatic peroxisomes in these siblings were enlarged in size and not decreased in number. Accumulation of very-long-chain fatty acids (VLCFA) was associated with an isolated deficiency of the fatty acyl-CoA oxidase, the enzyme that catalyzes the first step of the peroxisomal beta-oxidation. Plasma levels of di- and trihydroxy-coprostanoic acid, phytanic acid, and pipecolic acid were normal; furthermore, acyl-CoA:dihydroxyacetone phosphate acyltransferase activity in cultured fibroblasts was also found to be normal. The clinical, biochemical, and cytochemical features found in these two siblings are compared with those seen in two other disorders characterized by the absence of a decreased number of hepatic peroxisomes and the presence of VLCFA: (1) pseudo-Zellweger syndrome (deficiency of peroxisomal thiolase activity) and (2) X-linked childhood adrenoleukodystrophy (deficiency of activation of lignoceric acid). Review of the different biochemical defects possible in very-long-chain fatty-acid oxidation reveals different clinical pictures of varying severity, depending on the level at which the biochemical defect occurs.     

Effect of clofibrate application on morphology and enzyme content of liver peroxisomes.

Male albino rats (Sprague Dawley) were fed for 2-6 weeks on a diet containing 0.75% clofibrate. Liver cell fractions obtained from these animals were assayed for peroxisomal enzymes. In the cell homogenate the catalase activity was doubled, whereas the activity of urate oxidase was found to be only slightly depressed. The activity of carnitine acetyltransferase increased several times. In liver peroxisomes purified by isopycnic gradient centrifugation the specific activity of urate oxidase decreased appreciably showing that peroxisomes formed under the proliferative influence of clofibrate are not only modified with respect to their morphological characteristics but also to their enzymic equipment. This is also obvious from the changes in peroxisomal carnitine acetyltransferase activity which was enhanced by clofibrate to more than the fivefold amount. In purified mitochondria this enzyme was even more active: clofibrate advances both, the peroxisomal and the mitochondrial moiety of carnitine acetyltransferase. Morphological and cytochemical studies showed an increase in the number of microbodies and as compared to the controls microbodies were lying in groups more frequently. Small particles located closely adjacent to "normal" sized peroxisomes were found particularly after short feeding periods. While the number of coreless microbodies increased studies gave no clear evidence for an increase in marked shape irregularities of the peroxisomes.     

Postnatal Development and Isolation of Peroxisomes from Brain

We analyzed the postnatal peroxisome development in rat brain by measuring the enzyme activities of catalase and acyl-CoA oxidase and beta-oxidation of [1-14C]lignoceric acid. These enzyme activities were higher between 10 and 16 days of postnatal life and then decreased. We developed and compared two different methods for isolation of enriched peroxisomes from 10-day-old rat brain by using a combination of differential and density gradient centrifugation techniques. Peroxisomes in Percoll (self-generating gradient) banded at a density of 1.036 +/- 0.012 g/ml and in Nycodenz continuous gradient at 1.125 +/- 0.014 g/ml. Acyl-CoA oxidase, D-amino acid oxidase, L-pipecolic acid oxidase, and dihydroxyacetone phosphate acyltransferase activities and activities for the oxidation of very long chain fatty acid (lignoceric acid) were almost exclusively associated with catalase activity (a marker enzyme for peroxisomes) in the gradient. The postnatal increase in peroxisomal activity with the onset of myelination and the presence of enzyme for the biosynthesis of plasmalogens and oxidation of very long chain fatty acid (both predominant constituents of myelin) suggest that brain peroxisomes may play an important role in the assembly and turnover of myelin.     

The primary structure of the flavoprotein D-aspartate oxidase from beef kidney.

The complete primary structure of the peroxisomal flavoenzyme D-aspartate oxidase from beef kidney has been determined by analyses of the peptides obtained through fragmentation of the carboxymethylated protein with trypsin, CNBr, heptafluorobutyric acid/CNBr and Staphylococcus aureus V8 protease. The protein consists of a single polypeptide of 338 residues, accounting for a M(r) of 37,305 for the apoprotein. A form of the enzyme lacking Lys-338 and therefore ending with Pro-337 has been detected. The N-terminal residue is blocked. Seven cysteines and no disulfide bridges are present. Residue 228 can be either Ile or Val. Thus, D-aspartate oxidase presents two types of heterogeneity in the polypeptide chain in addition to the one already described concerning the possible content of FAD or 6-hydroxyflavin adenine dinucleotide. Comparison of the primary structure of D-aspartate oxidase with other known sequences reveals that D-aspartate oxidase is homologous with D-amino acid oxidase (another flavo-oxidase) and does not present significant sequence similarities with any other protein, including flavoenzymes.     

Studies on peroxisomes III. Further studies on the intraparticulate localization of peroxisomal components in the liver of the rat

The peroxisome-rich fraction prepared from rat liver homogenate was treated by various procedures and the behavior of the peroxisomal core on sucrose density gradient centrifugation was investigated.Peroxisomes were destroyed by various treatments, such as pH 9.0, VirTis blender, sonication and deoxycholate, resulting in the solubilization of catalase from the particles. Urate oxidase was not solubilized at all such treatments. Although D-amino acid oxidase was solubilized by treatments with deoxycholate and VirTis blender, this enzyme was found to be resistant to solubilization by treatment with pH 9.0 or sonication, in contrast to catalase.When the peroxisomal core was investigated, using urate oxidase activity as a marker, its density proved to be changed when submitted to various treatments. These results indicated that the peroxisomes consist of four compartments: a catalase-containing compartment (matrix), a urate oxidase containing compartment (core), a D-amino acid oxidase containing compartment and a low density compartment which is proposed for the first time in the present paper. Furthermore, it was also found that the last two compartments seem to be bound to the core, though the binding might be weak.     

Enzymologic study of the structural organization of the matrix or rat liver peroxisomes]

The effect of ionic strength and pH on the release of some enzymes of the matrix of peroxisomes in rat's liver was studied. Catalase, L ALpha-hydroxy acid oxidase, isocitrate dehydrogenase, glycerophosphate dehydrogenase and lactate dehydrogenase were easily released from the particles during their lysis and treatment with 0.16 M KCl, whereas urate oxidase, NADH cytochrome c reductase and D-amino acid oxidase were not solubilized. After the solubilization of peroxisomal membrane by 0.2% Triton X-100, the remaining core contained about 50% amino acid oxidase activity, and had 1.28--1.30 g/cm3 density. These results suggest that D-amino acid oxidase associates with urate oxidase in the peroxisomal core.     

Unusual responses of rat hepatic and renal peroxisomes to RMI 14, 514, a new hypolipidemic agent.

RMI 14, 514 ([5-tetradecycloxy]-2-furancarboxylic acid) represents a new class of hypolipidemic agents which cause unusual ultrastructural changes in liver of male rats and in selected peroxisomal enzymes in liver and kidney of both sexes. Among the principal ultrastructural changes in peroxisomes of male rat liver were (a) cavitation and compartmentalization of the matrix, often giving the appearance of a peroxisome-within-a-peroxisome, and (b) narrow, dense extensions of canaliculi or cisterns from the periphery of the peroxisome, forming partial circlets or surrounding irregular areas of cytoplasm. The unusual enzyme responses were (a) elevation of catalase activity in liver and kidney in female rats, (b) increased activity of three hydrogen peroxide-producing oxidases (urate oxidase, L-alpha-hydroxy acid oxidase, and D-amino acid oxidase) in the liver of both sexes, and (c) elevation of activity of the last two oxidases in male kidney. The peculiar ultrastructural changes in liver peroxisomes combined with the responses of selected peroxisomal enzymes represent unusual modulations or adaptations of these organelles to a hypolipidemic agent, the effects of which have not been reported extensively.     

Liver and kidney peroxisomes in lactating rats and their pups after treatment with ciprofibrate. Biochemical and morphometric analysis.

We administered the hypolipidemic drug ciprofibrate to lactating rats and examined the enzymatic content and ultrastructural features of liver and kidney peroxisomes, both in treated animals and in their pups. The peroxisomal morphometric parameters, in particular, were measured in specimens submitted to the cytochemical reaction for the marker enzyme catalase. In liver of treated rats, the activities of peroxisomal enzymes involved in the fatty acid catabolism were significantly increased, while D-amino acid oxidase activity was lower than in controls; increments were also found in relative volume and pleiomorphism degree of the peroxisomal compartment, where a catalase dilution was supposed to occur. In the kidney, the treatment induced generalized increases of all examined enzymes; values significantly higher than controls were found in peroxisomal relative volume and numerical density, while the peroxisomal mean diameter practically did not change. The two organs, moreover, were affected by the drug in an age-dependent way, the pups being more responsive than the adults. The organ- and age-specific responses to the drug are interpreted as possibly related to the tissue-specific distribution of the peroxisomal proliferator activated receptor isotypes.     

Immunocytochemical localization of urate oxidase, fatty acyl-CoA oxidase, and catalase in bovine kidney peroxisomes

We investigated the localization of urate oxidase, peroxisomal fatty acyl-CoA oxidase, and catalase in bovine kidney by immunoblot analysis and protein A-gold immunocytochemistry, using the respective polyclonal monospecific antibodies raised against the enzymes purified from rat liver. By immunoblot analysis, these three proteins were detected in bovine kidney and bovine liver homogenates. Subcellular localization of these three enzymes in kidney was ascertained by protein A-gold immunocytochemical staining of Lowicryl K4M-embedded tissue. Peroxisomes in bovine kidney cortical epithelium possessed crystalloid cores or nucleoids, which were found to be the exclusive sites of urate oxidase localization. The limiting membrane, the marginal plate, and the matrix of renal peroxisomes were negative for urate oxidase staining. In contrast, catalase and fatty acyl-CoA oxidase were found in the peroxisome matrix. These results demonstrate that, unlike rat kidney peroxisomes which lack urate oxidase, peroxisomes of bovine kidney contain this enzyme as well as peroxisomal fatty acyl-CoA oxidase.     

Ultrastructural localization of D-amino acid oxidase in microperoxisomes of the rat nervous system.

A recently developed procedure for the localization of D-amino acid oxidase (D-AAO) has been used to investigate the distribution of this enzyme in rat nervous tissue. Initial studies were carried out on kidney to validate the methods. The cytochemically demonstrable enzyme in kidney is inhibited by kojic acid, a known competitive D-AAO inhibitor. Omission of the catalse inhibitor, aminotriazole, from the cytochemical medium produces a marked diminution of D-AAO reaction product in kidney peroxisomes. This would be expected if catalase and D-AAO are present in the same particles. In brain, kojic acid-inhibitable D-AAO is demonstrable in numerous bodies within astrocytes especially in the cerebellum, a brain region known from biochemistry to contain particularly high levels of the oxidase. In preparations incubated for catalase, far fewer positive bodies are seen in the cerebellum. Moreover, omission of aminotriazole has little evident effect on the D-AAO reaction. Thus, the oxidase-containing cerebellar bodies may be relatively poor in catalse. In contrast, several nervous system cell types that contain relatively numerous catalase-positive bodies, contain none with detectable D-AAO. Such heterogeneity of peroxisome enzyme content is in accord with reports from biochemical studies of brain.     

Influence of subtotal hepatectomy on peroxisomes and peroxisomal enzymes of rat liver and isolated liver cell fractions.

The activities of peroxisomal enzymes of rat liver were followed 1 to 10 days after subtotal (60-70%) hepatectomy in homogenates prepared from regenerating livers and in cell fractions isolated from them. Catalase activity was found to be depressed in the total liver homogenate (H) as well as in the mitochondrial (M) and soluble (S) fractions, while it did not change appreciably in the microsomal (Mc) and lysosomal (L) fractions. Alpha-hydroxyacid oxidase behaved in a similar fashion. In contrast to these enzymes, urate oxidase activity remained unchanged in H, whereas it was decreased in M and increased in L and Mc during the first 5 days after operation. These results agree well with the assumption that microbody proliferation is initiated by the fragmentation of large peroxisomes. The different relations of peroxisomal enzyme activities during regeneration time are discussed with respect to the possible existence of various kinds of peroxisomes with different enzyme equipments and with different turnover rates. Biochemical examinations ions were paralleled to morphological and histochemical studies. An early increase in number of peroxisomes was found to occur during the first day after partial hepatectomy, which is accompanied by decrease in particle size. During the first mitotic wave (24-36 hrs post op.) the number of peroxisomes per cell was reduced to about the half. After this time number and size of the particles began to increase. Positive staining of ribosomes was frequently observed in the vicinity of peroxisomes after the application of the cytochemical catalase reaction (alkaline diaminobenzidine medium). This phenomenon is interpreted to represent rather a diffusion artifact than the cytochemical identification of newly synthesized catalase.