Ultrastructural, immunocytochemical and morphometric characterization of liver peroxisomes in gray mullet, Mugil cephalus

Peroxisomes of the hepatocytes of gray mullets, Mugil cephalus, were characterized cytochemically and immunocytochemically using antibodies against the peroxisomal proteins catalase and palmitoyl-coenzyme A (CoA) oxidase. In addition, morphometric parameters of peroxisomes were investigated depending on the hepatic zonation, the age of the animals and the sampling season. Mullet liver peroxisomes were reactive for diaminobenzidine, but presented a marked heterogeneity in staining intensity. Most of the peroxisomes were spherical or oval in shape, although irregular forms were also observed. Their size was heterogeneous, with profile diameters ranging from 0.2 to 3 microm. Peroxisomes tended to occur in clusters, usually near the mitochondria and lipid droplets. They also showed a very close topographical relationship to the smooth endoplasmic reticulum. Mullet liver peroxisomes did not contain cores or nucleoids as rodent liver peroxisomes, but internal substructures were observed in the matrix, consisting of small tubules about 60 nm in diameter and larger semicircles 120 nm in diameter. The volume density of peroxisomes was higher in periportal hepatocytes of mullets sampled in summer than in pericentral hepatocytes, indicating that mullet peroxisomes vary depending on physiological and environmental conditions. By immunoblotting, the mammalian antibodies cross-react with the corresponding proteins in whole homogenates of mullet liver. Paraffin sections immunostained with the antibodies against catalase and palmitoyl-CoA oxidase showed a positive reaction corresponding to peroxisomes localized in the hepatocyte cytoplasm. In agreement, the ultrastructural study revealed that catalase and palmitoyl-CoA oxidase are exclusively localized in the peroxisomal matrix in fish hepatocytes, showing a dense gold labeling. The presence of the peroxisomal beta-oxidation enzyme palmitoyl-CoA oxidase in peroxisomes indicated that these organelles play a key role in the lipid metabolism of fish liver.     

Zonal heterogeneity of peroxisome proliferation and morphology in rat liver after gemfibrozil treatment

The effect of gemfibrozil on the fine structure of peroxisomes across the rat liver lobule was investigated by light and electron microscopy using the alkaline diaminobenzidine (DAB) medium for the visualization of catalase peroxidatic activity. The oral administration of gemfibrozil for 2 weeks induces a striking heterogeneity in the lobular distribution of peroxisomes. The size and shape of peroxisomes, variety of matrix modifications, catalase content, and position within the cell, are functions of the zonal localization of the hepatocytes. The largest and most numerous peroxisomes were found in the centrilobular region indicating that these cells are most sensitive to peroxisome proliferation. On the other hand, the greatest variety of peroxisome shapes and matrix alterations (tubules and plates) was seen more peripherally in the mid-zonal and periportal regions. The larger, round centrilobular peroxisomes stained less intensely than the elongated peroxisomes found more peripherally, indicating a discrepancy between peroxisome size and catalase content. A distinct population of small irregularly shaped peroxisomes, lacking matrix specializations and containing variable catalase content, was found in the mid-zonal region. Peroxisomes in the centrilobular region were located within areas of the cell containing SER and glycogen while those in the more peripheral region were relegated to areas of the cytoplasm separate from RER and SER. In addition to modifications of peroxisomes, gemfibrozil treatment resulted in a proliferation and formation of whorled configurations of SER. This was particularly evident in the mid-zonal region, where single peroxisomal profiles could be seen surrounded by whorls of SER membranes. The results suggest that rat liver hepatocytes of the centrilobular region are the most sensitive to peroxisome proliferation and those of the periportal area are most susceptible to peroxisome matrix alterations after gemfibrozil treatment.     

Catalase in guinea pig hepatocytes is localized in cytoplasm, nuclear matrix and peroxisomes

We have compared the intracellular localization of catalase and another peroxisomal marker enzyme, alpha-hydroxy acid oxidase (HAOX), in the livers of guinea pig and rat using immunoelectron microscopy and subcellular fractionation combined with immunoblotting and enzyme activity determination. Antibodies against both enzymes were raised in rabbits and their specificities established by immunoblotting. By immunoelectron microscopy, gold particles representing antigenic sites for catalase were found in guinea pig hepatocytes not only in peroxisomes but also in the cytoplasm and the nuclear matrix. In rat liver, however, catalase was localized exclusively in peroxisomes with no cytoplasmic labeling. Moreover, in both species HAOX was found only in peroxisomes. Subcellular fractionation revealed that purified peroxisomes from both species contained comparable levels of each, catalase and HAOX activities. The total catalase activity, however, was substantially higher in guinea pig and most of this excess catalase was in the cytosolic fraction with some activity also in nuclei. In rat liver, 30 to 40% of both enzymes and in guinea pig liver 30% of HAOX were recovered in the supernatant fraction implying that the fragility of peroxisomes in both species is quite comparable. These observations establish the occurrence of extraperoxisomal catalase in guinea pig liver. The catalase in the cytoplasm and nucleus of liver parenchymal cells is most probably involved in scavenging of H2O2, protecting the cell against toxic and mutagenic effects of this noxious agent.     

Biogenesis of peroxisomes: immunocytochemical investigation of peroxisomal membrane proteins in proliferating rat liver peroxisomes and in catalase-negative membrane loops

Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554-564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment.     

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.     

Cytochemical localization of catalase and several hydrogen peroxide-producing oxidases in the nucleoids and matrix of rat liver peroxisomes

Synopsis The distribution of catalase, amino acid oxidase, -hydroxy acid oxidase, urate oxidase and alcohol oxidase was studied cytochemically in rat hepatocytes. The presence of catalase was demonstrated with the conventional diaminobenzidine technique. Oxidase activities were visualized with methods based on the enzymatic or chemical trapping of the hydrogen peroxide produced by these enzymes during aerobic incubations.All enzymes investigated were found to be present in peroxisomes. Catalase activity was found in the peroxisomal matrix, but also associated with the nucleoid. After staining for oxidase activities the stain deposits occurred invariably in the peroxisomal matrix as well as in the nucleoids. In all experiments the activity of both catalase and the oxidases was confined to the peroxisomes. The presence of a hydrogen peroxide-producing alcohol oxidase was demonstrated for the first time in peroxisomes in liver cells.The results imply that the enzyme activity of the nucleoids of rat liver peroxisomes is not exclusively due to urate oxidase. The nucleoids obviously contain a variety of other enzymes that may be more or less loosely associated with the insoluble components of these structures.     

Cytochemical and biochemical demonstration of an ATPase in membranes of human peroxisomes.

We demonstrated a neutral Mg-ATPase activity in human peroxisomal membranes. To establish the precise experimental conditions for detection of this ATPase, both cytochemical and biochemical characterizations were first carried out in liver peroxisomes from control and cipofibrate-treated rats. The results demonstrated an Mg-ATPase reaction in both normal and proliferated peroxisomes. The nucleotidase activity, with marked preference for ATP, was sensitive to the inhibitors N-ethylmaleimide and 7-chloro-4-nitro-benzo-2-oxadiazole (NBDCl). An ultrastructural cytochemical analysis was developed to evaluate the peroxisomal localization, which localized the reaction product to the peroxisomal membrane. These characteristics can help to differentiate the peroxisomal ATPase from the activity found in mitochondria and endoplasmic reticulum. The conditions established for detecting the rat peroxisomal ATPase were then applied to human peroxisomes isolated from liver and skin fibroblasts in culture. A similar Mg-ATPase activity was readily shown, both cytochemically and biochemically, in the membranes of human peroxisomes. These results, together with previous evidence, strongly support the presence of a specific ATPase in the human peroxisomal membrane. This ATPase may play a crucial role in peroxisome biogenesis.     

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".     

Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes

Subcellular distribution of pentose-phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+-dependent dehydrogenases of the pentose-phosphate pathway (glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate) caused a 1.5-2.5-fold increase in the amount of glucose-6-phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased the phosphogluconate dehydrogenase, but did not alter glucose-6-phosphate dehydrogenase activity in the cytosolic fraction. The results obtained indicate that the enzymes of the non-oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose-phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose. Similar to catalase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose-6-phosphate and 0.10-0.12 mM for 6-phosphogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose-6-phosphate dehydrogenase was inhibited by palmitoyl-CoA, and to a lesser extent by NADPH. Peroxisomal glucose-6-phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose-phosphate cycle dehydrogenases in rat liver peroxisomes is discussed.     

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)     

Degradation of excess peroxisomes in mammalian liver cells by autophagy and other mechanisms

Here we discuss the mechanisms for the degradation of excess peroxisomes in mammalian hepatocytes which include (a) autophagy, (b) the action of peroxisomal Lon protease and (c) the membrane disrupting effect of 15-lipoxygenase. A recent study using Atg7 conditional-knock-out mice revealed that 70–80% of excess peroxisomes are degraded by the autophagic process. The remaining 20–30% of excess peroxisomes is most probably degraded by the action of peroxisomal Lon protease. Finally, a selective disruption of the peroxisomal membrane has been shown to be mediated by 15-lipoxygenase activity which is followed by diffusion of matrix proteins into the cytoplasm and cytoplasmic proteolysis. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1–4, 2008.     

Investigation of peroxisomal lipid beta-oxidation enzymes in guinea pig liver peroxisomes by immunoblotting and immunocytochemistry.

We investigated the immunoreactivity of the peroxisomal lipid beta-oxidation enzymes acyl-CoA oxidase, trifunctional protein, and thiolase in guinea pig liver and compared it with that of homologous proteins in rat, using immunoblotting of highly purified peroxisomal fractions and monospecific antibodies to rat proteins. In addition, the immunocytochemical localization of beta-oxidation enzymes in guinea pig liver was compared with that of catalase. All antibodies showed crossreactivity between the two species, indicating that these peroxisomal proteins have been well conserved, although all exhibited some differences with respect to molecular size and, in the case of acyl-CoA oxidase, in frequency of the immunoreactive bands. In the latter case, a distinct second band in the 70 KD range was observed in guinea pig, in addition to the regular band due to subunit A present in rat liver. This novel band could be due either to trihydroxycoprostanoyl-CoA oxidase or to the non-inducible branched chain fatty acid oxidase described recently. All three beta-oxidation enzymes were immunolocalized by light and electron microscopy to the matrix of peroxisomes, in contrast to catalase, which is also found in the cytoplasm and the nucleus of hepatocytes in guinea pig liver.     

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase in rat liver. The effects of di-(2-ethylhexyl)phthalate, a peroxisome proliferator

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase (isomerase) was investigated in rat liver. Livers of di-(2-ethylhexyl)phthalate (DEHP)-treated or untreated rats were perfusion-fixed and embedded in Epon or Lowicryl K4M. By light microscopy, reaction deposits for the enzyme were present in the cytoplasmic granules of hepatocytes and interlobular bile duct epithelium. Weak staining was noted in sinus-lining cells. After administration of DEHP, the granular staining of the hepatocytes was markedly enhanced, whereas the staining reaction of the sinus-lining cells decreased. The isomerase staining pattern was quite similar to that of long-chain acyl-CoA dehydrogenase (a mitochondrial marker), but different from that of catalase (a peroxisomal marker). Under electron microscopy, gold particles for isomerase were seen to be confined mainly to mitochondria of the hepatocytes, the bile duct epithelial cells and sinus-lining cells. Peroxisomes were weakly labeled. After DEHP administration, the peroxisomes were markedly induced, but the mitochondria were not. Quantitative analysis showed that the induction of the peroxisomal isomerase was only 2-fold whereas the mitochondrial isomerase was enhanced about 5-fold, 40 times as high as the peroxisomal enzyme. The results show that the mitochondria are the main intracellular site for isomerase and the peroxisomes a minor site. The mitochondrial isomerase of the rat liver is markedly induced by peroxisome proliferators, DEHP and clofibrate.     

Insulin-degrading enzyme exists inside of rat liver peroxisomes and degrades oxidized proteins

Insulin-degrading enzyme (IDE) was detected by immunoblot analysis in highly purified rat liver peroxisomes. IDE in the peroxisomal fraction was resistant to proteolysis by trypsin and chymotrypsin under conditions where the peroxisomal membranes remained intact. After sonication of the peroxisomal fraction, IDE was recovered in the supernatant fraction. Further, the localization of IDE in the peroxisomes was shown by immunoelectron microscopy. In addition, IDE isolated from peroxisomes degraded insulin as well as oxidized lysozyme as a model substrate for oxidized proteins. These results suggest that IDE exists in an active form in the matrix of rat liver peroxisomes and is involved in elimination of oxidized proteins in peroxisomes.     

Phosphoinositide synthesis and degradation in isolated rat liver peroxisomes

Analyzing peroxisomal phosphoinositide (PId(#)) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P(2) and PtdIns(3,5)P(2). PtdIns3P was hardly detected in vitro, however, was observed in vivo after [(32)P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of phosphatase inhibitors enhanced the amount of PtdIns4P. The results obtained provide evidence that isolated rat liver peroxisomes synthesize PIds and suggest the association of PId 4-kinase and PId 5-kinase and PId 4-phosphatase activities with the peroxisomal membrane.     

Peroxisome proliferation and oxidative stress mediated by activated oxygen species in plant peroxisomes

The existence of a relationship between clofibrate-induced peroxisome proliferation and oxidative stress mediated by activated oxygen species was studied in intact peroxisomes purified from Pisum sativum L. plants. Incubation of leaves with 1 mM clofibrate produced a remarkable increase in the peroxisomal activity of acyl-CoA oxidase and, to a lesser extent, of xanthine oxidase, whereas there was a nearly complete loss of catalase activity and a decrease in Mn-superoxide dismutase. Ultrastructural studies of intact leaves showed that clofibrate induced a five- and twofold proliferation of the peroxisomal and mitochondrial populations, respectively, in comparison with those in control leaves. Prolonged incubation with clofibrate produced considerable alterations in the ultrastructure of cells. In peroxisomal membranes, the NADH-induced generation of O2- radicals, as well as the lipid peroxidation of membranes, increased as a result of treatment of plants with clofibrate. In intact peroxisomes treated with this hypolipidemic drug, the H2O2 concentration was higher than in peroxisomes from control plants. These results demonstrate that clofibrate stimulates the production of activated oxygen species (O2- and H2O2) inside peroxisomes, as well as the lipid peroxidation of peroxisomal membranes. This effect is concomitant with a decrease of catalase and Mn-SOD activities, the main peroxisomal enzymatic defenses against H2O2 and O2-, and indicates that in the toxicity of clofibrate, at the level of peroxisomes, an oxidative stress mechanism mediated by activated oxygen species is involved.     

Selective cytochemical localization of peroxidase, cytochrome oxidase and catalase in rat liver with 3,3'-diaminobenzidine

In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specificity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15-30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylte buffer pH 6.5 and 0.02% H2O2. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) buffer at pH 10.5 and 0.15% H2O2. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes.

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.     

Structure, composition, physical properties, and turnover of proliferated peroxisomes. A study of the trophic effects of Su-13437 on rat liver

Peroxisome proliferation has been induced with 2-methyl-2-(p-[1,2,3,4-tetrahydro-1-naphthyl]-phenoxy)-propionic acid (Su-13437). DNA, protein, cytochrome oxidase, glucose-6-phosphatase, and acid phosphatase concentrations remain almost constant. Peroxisomal enzyme activities change to approximately 165%, 50%, 30%, and 0% of the controls for catalase, urate oxidase, L-alpha-hydroxy acid oxidase, and D-amino acid oxidase, respectively. For catalase the change results from a decrease in particle-bound activity and a fivefold increase in soluble activity. The average diameter of peroxisome sections is 0.58 +/- 0.15 mum in controls and 0.73 +/- 0.25 mum after treatment. Therefore, the measured peroxisomal enzymes are highly diluted in proliferated particles. After tissue fractionation, approximately one-half of the normal peroxisomes and all proliferated peroxisomes show matric extraction with ghost formation, but no change in size. In homogenates submitted to mechanical stress, proliferated peroxisomes do not reveal increased fragility; unexpectedly, Su-13437 stabilizes lysosomes. Our results suggest that matrix extraction and increased soluble enzyme activities result from transmembrane passage of peroxisomal proteins. The changes in concentration of peroxisomal oxidases and soluble catalase after Su-13437 allow the calculation of their half-lives. These are the same as those found for total catalase, in normal and treated rats, after allyl isopropyl acetamide: about 1.3 days, a result compatible with peroxisome degradation by autophagy. A sequential increase in liver RNA concentration, [14C]leucine incorporation into DOC-soluble proteins and into immunoprecipitable catalase, and an increase in liver size and peroxisomal volume per gram liver, characterize the trophic effect of the drug used. In males, Su-13437 is more active than CPIB, another peroxisome proliferation-inducing drug; in females, only Su-13437 is active.     

The peroxisome (microbody) membrane: effects of detergents and lipid solvents on its ultrastructure and permeability to catalase

Synopsis The effects of detergents, organic lipid solvents, and several adjuvants used in cell fractionation on the ultrastructure of the peroxisomal (microbody) membrane and its permeability to catalase have been investigated. Chopper sections of glutaraldehyde-fixed liver were incubated in the presence of various agents, followed by cytochemical staining for catalase and processed for electron microscopy. Catalase activity was also determined biochemically in the incubation medium. Marked catalase diffusion was found after treatment with 1% or 0.5% Triton X-100 or deoxycholate, as well as with 50% ethanol or acetone or 20% propanol ort-butanol. In contrast, 1% digitonin and lower concentrations of the above agents, as well as sucrose or glycerine caused selective diffusion of catalase from a limited population of peroxisomes. Tieatment with 10% polyvinylpyrrolidone (PVP), which has been used as a protective agent in the isolation of microbodies, did not produce any alteration in the fine structure and cytochemical appearance of peroxisomes. These findings concur with earlier biochemical studies on freshly isolated peroxisomes and demonstrate the susceptibility of microbodies, even in glutaraldehyde-fixed rat liver to the effects of various agents which affect the microbody membrane. A close correlation between the ultrastructural integrity of the microbody membrane and its permeability to catalase has been found. The significance of these observations for the assessment of the permeability characteristics of the microbody membrane is discussed.