Peroxisomes of the rat cardiac and soleus muscles increase after starvation. A biochemical and immunocytochemical study

We have investigated the change of catalase activity in the homogenates of rat cardiac and skeletal muscles. After 7 days' starvation, the catalase activity of heart increased about 3-fold and that of soleus muscle enhanced 2-fold higher than that of control rats. Immunoblot analysis of catalase showed a single band in the homogenates of cardiac and soleus muscles and increase of catalase antigen after starvation. Light microscopic immunoenzyme staining showed that after starvation catalase positive granules markedly increased in both the cardiac and soleus muscle. Quantitative analysis of the staining showed that number of the granules per 100 microns 2 of tissue section was about 1.4-fold in the soleus muscle and 1.7-fold in the cardiac muscle after starvation. By electron microscopy of alkaline DAB staining, we confirmed that the granules were peroxisomes, which increased in both number and size. Furthermore, we stained the peroxisomes for catalase by a protein A-gold technique. Labeling density (gold particles/micron 2) of the cardiac and soleus muscles from the starved rat increased approximately 1.4 times as much as that of normal animal. When the numerical density is multiplied by the labeling density, the values are largely consistent with the enhancement of catalase activity. These results show that increase in the catalase activity of the muscle tissue after starvation is caused by increase in number and size of peroxisomes.     

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.     

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.     

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.     

Peroxisomes of the rat cardiac and soleus muscles increase after starvation

Summary We have investigated the change of catalase activity in the homogenates of rat cardiac and skeletal muscles. After 7 days' starvation, the catalase activity of heart increased about 3-fold and that of soleus muscle enhanced 2-fold higher than that of control rats. Immunoblot analysis of catalase showed a single band in the homogenates of cardiac and soleus muscles and increase of catalase antigen after starvation. Light microscopic immunoenzyme staining showed that after starvation catalase positive granules markedly increased in both the cardiac and soleus muscle. Quantitative analysis of the staining showed that number of the granules per 100 m² of tissue section was about 1.4-fold in the soleus muscle and 1.7-fold in the cardiac muscle after starvation. By electron microscopy of alkaline DAB staining, we confirmed that the granules were peroxisomes, which increased in both number and size. Furthermore, we stained the peroxisomes for catalase by a protein A-gold technique. Labeling density (gold particles/m²) of the cardiac and soleus muscles from the starved rat increased approximately 1.4 times as much as that of normal animal. When the numerical density is multiplied by the labeling density, the values are largely consistent with the enhancement of catalase activity. These results show that increase in the catalase activity of the muscle tissue after starvation is caused by increase in number and size of peroxisomes.     

The different effects of the peroxisome proliferators clofibric acid and bis(2-ethylhexyl)phthalate on the activities of peroxisomal oxidases in rat liver

Treatment with peroxisome proliferators induces increased numbers and alterations in the shape of peroxisomes in liver. It ultimately leads to hepatocellular carcinomas induced by the persistent production of high amounts of H2O2 as a result of a dramatical increase in acyl-CoA oxidase activity. The effects of peroxisome proliferators on other peroxisomal oxidase activities are less well documented. In the present study, the distribution patterns of the activity of SdD-amino acid oxidase, SlD-alpha-hydroxy acid oxidase, polyamine oxidase, urate oxidase and catalase activities were investigated in unfixed cryostat sections of liver, kidney and duodenum of rats treated with either clofibrate or bis(2-ethylhexyl)phthalate. The activities of xanthine oxidoreductase, which produces urate, a potent anti-oxidant, and xanthine oxidase, which produces oxygen radicals, were studied as well. The liver was the only organ that was affected by treatment. The number of peroxisomes increased considerably. SdD-Amino acid oxidase and polyamine oxidase activities were completely abolished by the treatment, whereas SlD-alpha-hydroxy acid oxidase activity decreased and urate oxidase activity increased periportally and decreased pericentrally. Total catalase activity increased because of the larger numbers of peroxisomes, but it decreased per individual peroxisome. Xanthine oxidoreductase activity decreased, whereas the percentage of xanthine oxidase remained constant. We conclude that oxidases in rat liver are affected differentially, indicating that the expression of activity of each oxidase is regulated individually. © 1998 Chapman & Hall     

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.     

Effects of dietary carbohydrate intake on antioxidant enzyme activity and copper status in the copper-deficient streptozotocin (STZ) diabetic rat

The aim of this study was to investigate how dietary lactose, compared with sucrose, in association with copper deficiency influences the antioxidant and copper status in the diabetic rat. Two groups of male rats (n = 12) were fed copper-deficient diets containing either 300 g/kg of sucrose or 300 g/kg of lactose in a pair-feeding regime for 35 days. Six rats from each group were injected with streptozotocin to induce diabetes. After a further 16 days the animals were killed and the liver, heart, and kidney removed for the measurement of copper levels and the activities of antioxidant and related enzymes. Diabetes resulted in higher hepatic and renal copper levels compared with controls. The copper content of the heart and kidney in diabetic rats consuming sucrose was also significantly higher than in those consuming lactose. Catalase activity in the liver, heart, and kidney was significantly increased in diabetic rats compared with controls. Hepatic glutathione S-transferase and glucose-6-phosphate dehydrogenase and cardiac copper zinc superoxide dismutase activities were also higher in diabetes. Sucrose, compared with lactose feeding, resulted in higher cytochrome c oxidase and glutathione peroxidase activities in the kidney while glucose-6-phosphate dehydrogenase activity was lower. The combination of lactose feeding and diabetes resulted in significantly higher activities of cardiac managanese superoxide dismutase and catalase and renal manganese superoxide dismutase and glucose-6-phosphate dehydrogenase. These results suggest that sucrose consumption compared with lactose appears to be associated with increased organ copper content and in general decreased antioxidant enzyme activities in copper-deficient diabetic rats.     

Participation of peroxisomes in lipid biosynthesis in the harderian gland of guinea pig.

Peroxisomal enzyme activities in the guinea-pig harderian gland, which has a unique lipid composition, were studied. Activities of catalase, acyl-CoA oxidase and the cyanide-insensitive acyl-CoA beta-oxidation system in this tissue were comparable with those in rat liver. The activities of dihydroxyacetone phosphate acyltransferase (DHAPAT, EC 2.3.1.42) and alkyl-DHAP synthase (EC 2.5.1.26) were appreciable, and the distributions of both activities were consistent with that of sedimentable catalase activity. Glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15), which is localized in both microsomes (microsomal fractions) and mitochondria in the rat liver, was a peroxisomal enzyme in the harderian gland, though the activity was only about one-tenth of the DHAPAT activity. These enzymes had different pH profiles and substrate specificity. The existence of high activities of enzymes of the acyl-DHAP pathway in peroxisomes suggests the physiological significance of peroxisomes in the biosynthesis of glycerol ether phospholipid and 1-alkyl-2,3-diacylglycerol in the guinea-pig harderian gland.     

Studies on peroxisomes. V. Effect of ethyl p-chlorophenoxyisobutyrate on the centrifugal behavior of rat liver peroxisomes.

After Wistar male rats had been fed on a diet containing 0.25% of ethyl p-chlorophenoxyisobutyrate (CPIB) for 28 days, changes in the enzyme activities and centrifugal behavior of rat liver peroxisomes were investigated. (1) Compared with control rats fed on the basal diet, the catalase [EC 1.11.1.6] activity of rat livers after the administration of CPIB increased about 2.5-fold, while urate oxidase [EC 1.7.3.3] activity did not change significantly. Though D-amino acid oxidase [EC 1.4.3.3] activity markedly decreased to approximately one-sixth of the control, the activity of L-alpha-hydroxy acid oxidase [EC 1.1.3.15], a flavin enzyme like D-amino acid oxidase, was not affected significnatly after the administration of CPIB. (2) When the hepatic cells of CPIB-treated rats were fractionated by differential centrifugation, most of the increase of catalase activity appeared in the supernatant fraction. A decrease in the hepatic D-amino acid oxidase activity of CPIB-treated rats was observed in all the fractions. As for the subcellular distribution of the particle-bound enzymes, the specific activities of both catalase and urate oxidase of CPIB-treated rat livers were higher in the light mitochondrial fraction than in other fractions. (3) Sedimentation patterns in a sucrose density gradient did not show any difference between normal peroxisomers, and CPIB-treated ones. (4) In the case of CPIB-treated rats, studies of their sedimentation patterns by Ficoll density gradient centrifugation showed two main particulate peaks containing both catalase and urate oxidase, although only a single peak was observed in the case of control rats.     

Intraparticulate localization of some peroxisomal enzymes related to fatty acid beta-oxidation

Male Wistar rats were given a diet containing 0.05% (w/w) LK-903 (alpha-methyl-p-myristyroxycinnamic acid 1-monoglyceride) for 2 weeks. The activities of four hepatic peroxisomal enzymes involved in the fatty acyl-CoA beta-oxidizing system were determined. The activities of fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase were all increased about three times by administration of LK-903. The intraparticulate localizations of the four enzymes were then investigated by treatment of the purified peroxisomes with Triton X-100, by sonication, and by sucrose-density-gradient centrifugation after Triton X-100 treatment. The results suggest that thiolase is localized in the matrix of peroxisomes, that crotonase and beta-hydroxybutyryl-CoA dehydrogenase are located in the core, and that all or at least part of fatty acyl-CoA oxidase is associated with the core, though its association is weak.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation

Catalase activity was inhibited by aminotriazole administration to rats in order to evaluate the influence of catalase on the peroxisomal fatty acyl-CoA beta-oxidation system. 2 h after the administration of aminotriazole, peroxisomes were prepared from rat liver, and the activities of catalase, the beta-oxidation system and individual enzymes of beta-oxidation (fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase) were determined. Catalase activity was decreased to about 2% of the control. Among the individual enzymes of the beta-oxidation system, thiolase activity was decreased to 67%, but the activities of fatty acyl-CoA oxidase, crotonase and beta-hydroxybutyryl-CoA dehydrogenase were almost unchanged. The activity of the peroxisomal beta-oxidation system was assayed by measuring palmitoyl-CoA-dependent NADH formation, and the activity of the purified peroxisome preparation was found to be almost unaffected by the administration of aminotriazole. The activity of the system in the aminotriazole-treated preparation was, however, significantly decreased to 55% by addition of 0.1 mM H2O2 to the incubation mixture. Hydrogen peroxide (0.1 mM) reduced the thiolase activity of the aminotriazole-treated peroxisomes to approx. 40%, but did not affect the other activities of the system. Thiolase activity of the control preparation was decreased to 70% by addition of hydrogen peroxide (0.1 mM). The half-life of 0.1 mM H2O2 added to the thiolase assay mixture was 2.8 min in the case of aminotriazole-treated peroxisomes, and 4 s in control peroxisomes. The ultraviolet spectrum of acetoacetyl-CoA (substrate of thiolase) was clearly changed by addition of 0.1 mM H2O2 to the thiolase assay mixture without the enzyme preparation; the absorption bands at around 233 nm (possibly due to the thioester bond of acetoacetyl-CoA) and at around 303 nm (due to formation of the enolate ion) were both significantly decreased. These results suggest that H2O2 accumulated in peroxisomes after aminotriazole treatment may modify both thiolase and its substrate, and consequently suppress the fatty acyl-CoA beta-oxidation. Therefore, catalase may protect thiolase and its substrate, 3-ketoacyl-CoA, by removing H2O2, which is abundantly produced during peroxisomal enzyme reactions.     

Evidence for increased peroxidative activity in muscles from streptozotocin-diabetic rats

The ability of cardiac and skeletal muscles from diabetic rats to metabolize superoxide and hydrogen peroxide was determined by the activities of superoxide dismutase (SOD) and catalase, respectively. Male and female Sprague-Dawley rats, 43 days old, were made diabetic with a single intravenous injection of streptozotocin (70 mg/kg body weight). On the 80th day after injection the blood glucose concentration of these rats was increased fourfold, and the plasma insulin concentration was decreased four- to fivefold compared to controls. Body weights of male diabetic rats were 61% and those of female diabetic rats were 66% of their ad libitum-fed controls. The seven different skeletal muscles examined weighed less in the diabetic rats than in controls of the same age and body weight. The hearts of the diabetic rats weighed more than those of controls of the same age and body weight. Comparison to the body weight controls allowed the distinction of specific effects due to lack of insulin from effects due to retardation in muscle growth. Increased catalase activity in all muscles examined from diabetic rats (plantaris, gastrocnemius, and heart) suggested a response in catalase activity similar to that of starved rats. SOD activity was not altered in the diabetic rat skeletal muscles and erythrocytes, but was somewhat decreased in the heart.     

Characterization of peroxisomes in Tetrahymena pyriformis

Peroxisomes from Tetrahymena pyriformis contained catalase, d-amino acid oxidase, cyanide-insensitive fatty acyl-CoA oxidizing system, carnitine acetyltransferase, isocitrate lyase, leucine:glyoxylate aminotransferase and phenylalanine:glyoxylate aminotransferase. These activities, except carnitine acetyltransferase, were found at the highest levels in the light mitochondrial fraction, whereas the highest activity of carnitine acetyltransferase was found in the micotchondrial fraction. Sucrose density gradient centrifugation showed that the density of peroxisomes was approx. 1.228 g/ml and that of mitochondria was approx. 1.213 g/ml. When the light mitochondrial fraction was treated with deoxycholate or by freeze-thawing, most of the activities of catalase and isocitrate lyase were solubilized, whereas about half of the original activity of aminotransferase remained in the pellet fraction. Addition of fatty acid and clofibrate increased the activities of the cyanide-insensitive fatty acyl-CoA oxidizing system and isocitrate lyase in the peroxisomes. The activity of catalase was slightly increased by glucose and clofibrate; leucine:glyoxylate aminotransferase activity was significantly increased by clofibrate treatment.     

Peroxisomal beta-oxidation enzyme proteins in the Zellweger syndrome

The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very long-chain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal beta-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low Mr were seen in the patients. With anti-(oxoacyl-CoA thiolase), high Mr cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal beta-oxidation enzymes.     

Peroxisomes in digestive gland cells of the mussel Mytilus galloprovincialis Lmk. Biochemical, ultrastructural and immunocytochemical characterization.

The present study was undertaken because of the paucity of information on peroxisomes in molluscs and the increasing importance of these organisms as sensitive indicators of environmental pollution. Peroxisomes were identified by electron microscopy in all three main cell types of the digestive gland of the bivalve mollusc Mytilus galloprovincialis Lmk. They stained weakly with the alkaline diaminobenzidine reaction but showed distinct immunolabeling with an antibody against mammalian catalase by the postembedding protein A-gold procedure. In addition, mussel digestive gland peroxisomes were isolated by differential and metrizamide-density gradient centrifugation, and a 30-fold enrichment of catalase and a 20-fold enrichment of palmitoyl-CoA oxidase was obtained over the initial homogenate. By Western blotting, isolated peroxisomes crossreacted with antibodies to catalase and, furthermore, specific and prominent labeling of isolated peroxisomes was also demonstrated in thin sections incubated with anti-catalase antibodies. These observations establish that peroxisomes in molluscan digestive gland contain the peroxisomal marker enzymes catalase and acyl-CoA oxidase and that they can be labeled by cytochemical and immunocytochemical techniques. Further studies of alterations of molluscan peroxisomes by environmentally relevant xenobiotics are warranted.     

Separate peroxisomal oxidases for fatty acyl-CoAs and trihydroxycoprostanoyl-CoA in human liver

Fatty acyl-CoAs as well as the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids are beta-oxidized in peroxisomes. The first reaction of peroxisomal beta-oxidation is catalyzed by acyl-CoA oxidase. We recently described the presence of two fatty acyl-CoA oxidases plus a trihydroxycoprostanoyl-CoA oxidase in rat liver peroxisomes (Schepers, L., P. P. Van Veldhoven, M. Casteels, H. J. Eyssen, and G. P. Mannaerts. 1990. J. Biol. Chem. 265: 5242-5246). We have now developed methods for the measurement of palmitoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase in human liver. The activities were measured in livers from controls and from three patients with peroxisomopathies. In addition, the oxidase activities were partially purified from control livers by ammonium sulfate fractionation and heat treatment, and the partially purified enzyme preparation was subjected to chromatofocusing, hydroxylapatite chromatography, and gel filtration. In earlier experiments this allowed for the separation of the three rat liver oxidases. The results show that human liver, as rat liver, contains a separate trihydroxycoprostanoyl-CoA oxidase. In contrast to the situation in rat liver, no conclusive evidence was obtained for the presence of two fatty acyl-CoA oxidases in human liver. Our results explain why bile acid metabolism is normal in acyl-CoA oxidase deficiency, despite a severely disturbed peroxisomal fatty acid oxidation and perhaps also why, in a number of other cases of peroxisomopathy, di- and trihydroxycoprostanic acids are excreted despite a normal peroxisomal fatty acid metabolism.     

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.     

Immunocytochemical analysis of soluble epoxide hydrolase and catalase in mouse and rat hepatocytes demonstrates a peroxisomal localization before and after clofibrate treatment

The intracellular localization of soluble epoxide hydrolase and catalase was investigated in hepatocytes from untreated and clofibrate-treated male C57B1/6 mice and from untreated male Sprague-Dawley rats. Polyclonal rabbit antibodies directed against purified mouse liver cytosolic epoxide hydrolase and rat liver catalase were used and their specificity ascertained by Ouchterlony immunodiffusion and immunoblotting. The IgG fraction was purified and incubated with cryosections of isolated hepatocytes or liver tissue, priorly fixed in 4% paraformaldehyde, and protein-A gold conjugates were used to visualize the antigen-antibody reaction. The soluble form(s) of epoxide hydrolase was found to be localized in the matrix of peroxisomes in hepatocytes from normal and clofibrate-treated mice and normal rats. No significant reactivity was found against plasma membrane, nuclei, mitochondria, the Golgi apparatus, endoplasmic reticulum, lysosomes, or cytosol. Catalase was also localized to peroxisomes in all samples investigated. Accordingly, both the catalase and the epoxide hydrolase activities routinely recovered in the high-speed supernatant after subfractionation of rat and mouse liver tissue mostly seemed to be due to extensive matrix leakage from peroxisomes, and this phenomenon may also be found in other species. Rat hepatocytes contained less epoxide hydrolase than mouse hepatocytes, as judged by both immunocytochemical labeling and biochemical data. Clofibrate treatment of mice decreased the labeling density of epoxide hydrolase and catalase in hepatocytes peroxisomes, as expected, and more unlabeled peroxisomes were observed.     

Immunogold localization of peroxisomal enzymes in Epon-embedded liver tissue. Enhancement of sensitivity by etching with ethanolic sodium hydroxide

Epon-embedded biological materials exhibit well preserved ultrastructure but generally weak antigenicity. Brief etching of ultrathin Epon sections with resin solvent, ethanolic sodium hydroxide, brought about a nearly 2-fold increase in the immunogold labeling density of rat and human hepatic peroxisomes after incubation with antisera against catalase and 3 enzymes of lipid beta-oxidation: acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase. The etching was superior to pretreatment with an oxidant, sodium metaperiodate. Despite some deterioration of the cellular ultrastructure, the obtained enhancement of the sensitivity of the protein A-gold method may be helpful in cases when the antigenicity to be detected is strongly inhibited by epoxy resin embedding.