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.     

Peroxisome proliferation in Norway spruce induced by ozone

Summary Catalase (EC 1.11.1.6) activity (both total and specific activity) of particulate fractions of needles of Norway spruce [Picea abies (L.) Karst.] was elevated approximately 2-fold following exposure of trees to 60–70 g/m³ of ozone during the growing season compared to trees receiving charcoal filtered air (about 15 g/m³ ozone). Measurements were from homogenates fractionated into particulate and soluble (supernatent) activities. In contrast, the catalase activity of the supernatant was unchanged in response to ozone treatment. Catalase activity declined as the needles aged comparing current, 1-, and 2-year needles but the ozone-induced increment remained constant. Electron microscope cytochemistry using peroxidatic coupling with 3,3-diaminobenzidine carried out in parallel, revealed catalase-containing peroxisomes both in situ and in the particulate fractions analyzed for catalase activity. The tissue volume occupied by peroxisomes in response to needle age and ozone appeared to vary approximately in proportion to the measured catalase activity. Overall cytochemical reactivity for catalase declined with needle age, but, for all years, was greater in needles of trees receiving air supplemented with ozone compared to those of trees receiving charcoal filtered air.Abbreviations DAB 3,3-diaminobenzidine tetrahydrochloride     

A eukaryote without catalase-containing microbodies: Neurospora crassa exhibits a unique cellular distribution of its four catalases

Microbodies usually house catalase to decompose hydrogen peroxide generated within the organelle by the action of various oxidases. Here we have analyzed whether peroxisomes (i.e., catalase-containing microbodies) exist in Neurospora crassa. Three distinct catalase isoforms were identified by native catalase activity gels under various peroxisome-inducing conditions. Subcellular fractionation by density gradient centrifugation revealed that most of the spectrophotometrically measured activity was present in the light upper fractions, with an additional small peak coinciding with the peak fractions of HEX-1, the marker protein for Woronin bodies, a compartment related to the microbody family. However, neither in-gel assays nor monospecific antibodies generated against the three purified catalases detected the enzymes in any dense organellar fraction. Furthermore, staining of an N. crassa wild-type strain with 3,3'-diaminobenzidine and H(2)O(2) did not lead to catalase-dependent reaction products within microbodies. Nonetheless, N. crassa does possess a gene (cat-4) whose product is most similar to the peroxisomal type of monofunctional catalases. This novel protein indeed exhibited catalase activity, but was not localized to microbodies either. We conclude that N. crassa lacks catalase-containing peroxisomes, a characteristic that is probably restricted to a few filamentous fungi that produce little hydrogen peroxide within microbodies.     

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.     

Identification and subcellular localization of catalase activity in bovine adrenal medulla and cortex

Catalase activity was detected in homogenates of bovine adrenal cortex and medulla. Analysis by equilibrium density centrifugation in isoosmotic metrizamide-sucrose gradients revealed that 70% of the medullary catalase activity was soluble while most of the remainder was found in a particulate form with a density of 1.175 g/ml. This was distinct from the densities of lysosomes, mitochondria, and chromaffin granules. Catalase activity in adrenal cortex was primarily (90%) soluble with only 6% being particulate, with a density of 1.185 g/ml. d-Amino acid, uric acid, and α-hydroxyacid oxidase activities, often associated with peroxisomes in other tissues, were absent from homogenates and catalase-containing gradient fractions from either cortex or medulla. There was an indication that some catalase activity was associated with chromaffin granules on the basis of density gradient analysis of both medullary homogenates and crude granule preparations. When granule fractions were subjected to osmotic shock, catalase activity distributed between soluble and sedimentable fractions differently from epinephrine and dopamine β-hydroxylase activity. The sedimentable catalase activity remained associated with chromaffin granule membranes upon isopycnic centrifugation. We concluded that catalase activity in both adrenal cortex and medulla was largely cytoplasmic, but that both tissues contained at least some catalase in dense organelles. Catalase activity which may be associated with chromaffin granules represents a small fraction of the total activity in the medulla.     

Isolation and Biochemical Characterization of Peroxisomes from Cultured Rat Glial Cells

Peroxisomes are now recognized to play important cellular functions and its dysfunction leads to a group of neurological disorders. This study reports peroxisomal enzyme activities in cultured glial cells and peroxisomes isolated from cultured oligodendrocytes and C₆ glial cells. Peroxisomal enzyme activities were found to be higher in oligodendroglial cells than in astrocytes or mixed glial cells. We also developed a method for the isolation of peroxisomes from glial cells by a combination of differential and density gradient centrifugation techniques. Peroxisomes from oligodendrocytes in nycodenz gradient were isolated at a density of 1.165 g/ml ± 0.011. Activities of dihydroxyacetone phosphate acyl transferase, -oxidation of lignoceric acid and -oxidation of phytanic acid were almost exclusively associated with the distribution of catalase activity (a marker enzyme for peroxisomes) in the gradient. This protocol should be a resource for studies designed to investigate the structure and function of peroxisomes in brain cells.     

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase

Summary The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semi-thin (0.25 and 1 m) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for lightmicroscopic analysis may account for the differences.The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.     

Peroxisomes as a cellular source of reactive nitrogen species signal molecules

Peroxisomes are single membrane-bounded subcellular organelles with an essentially oxidative type of metabolism and are probably the major sites of intracellular H₂O₂ production. These organelles also generate superoxide radicals () and besides catalase they have a complex battery of antioxidative enzymes. In recent years the existence of l-arginine-dependent nitric oxide synthase (NOS) activity and the generation of the reactive nitrogen species (RNS) nitric oxide (NO) have been demonstrated in plant peroxisomes. The inter-cellular and intracellular NO carrier S-nitrosoglutathione (GSNO) can be generated inside peroxisomes and the presence of this RNS has been demonstrated in peroxisomes from several plant species. This review analyzes the available evidence concerning the properties of the NOS activity and the generation of the RNS messengers NO and GSNO in peroxisomes in the context of the cellular function of these organelles as a source of RNS signaling molecules. The important physiological functions displayed by NO and other RNS in intra- and inter-cellular communication in different organisms indicate that more attention should be payed to the RNS signaling function of peroxisomes in human, animal and fungal cells, where it is very likely that similar mechanisms to those found in plant peroxisomes are also operative.     

Immunocytochemical localization of L-α-hydroxyacid oxidase in dense bar of dumb-bell-shaped peroxisomes of monkey kidney

Localization of the B of L-hydroxyacid oxidase (HOX-B) in monkey kidney peroxisomes was investigated by immunoelectron microscopic techniques. Kidneys of Japanese monkeys,Macaca fuscata, were fixed with 4% paraformaldehyde+0.25% glutaraldehyde and embedded in LR White resin. Thin sections were stained for HOX-B and catalase by the immunogold technique. HOX-B was localized in the marginal plates of normal peroxisomes and the dense bar of dumb-bellshaped peroxisomes. Catalase was detected in the matrix of normal peroxisomes and in the terminal dilatations of dumb-bell-shaped peroxisomes. There were no gold particles indicating presence of catalase associated with the marginal plates or with the dense bars. Immunoblot analysis of monkey kidney homogenate showed that HOX-B has a molecular mass of 42 kDa that was slightly larger than that of rat kidney HOX-B (39 kDa). The results show that the dense bar of dumb-bell-shaped peroxisomes in monkey kidney is composed of at least HOX-B and is a variation of the marginal plates.     

SUBCELLULAR DISTRIBUTION OF d-AMINO ACID OXIDASE AND CATALASE IN RAT BRAIN

—Rat brain d -amino acid oxidase was found to be confined to the hindbrain, distributed more or less equally between cerebellum and medulla. Histochemical staining showed the enzyme to be localized largely in the molecular layer of the cerebellum. After fractionation by means of several distinct density gradient centrifugation procedures exploiting differences in sedimentation coefficient or in density or in both, the enzyme was found to be entirely or almost entirely associated with cytoplasmic particles with a median diameter of the order of 0·2 μm, and a median equilibrium density in aqueous sucrose of 1·18. Comparison with the behavior of cytochrome oxidase and of N-acetyl-β-glucosaminidase indicates that these particles are not mitochondria and are unlikely to be lysosomes. They also differ significantly from the bulk of the catalase-containing particles, which on an average appear to be somewhat smaller. The possibility that they might contain some catalase activity, and thereby qualify as ‘peroxisomes’, can however not be excluded. In any case, they differ profoundly from the peroxisomes of liver or kidney.     

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.     

Actin localization and function in higher plants

Summary Two different cytochemical methods were used to study the localization of uricase (EC 1.7.3.3) and catalase (EC 1.11.1.6) in developing root nodules of soybean (Glycine max) inoculated as seeds withBradyrhizobium japonicum. One of the methods employs DAB (3,3-diaminobenzidine) and detects uricase activity indirectly by coupling it to endogenous catalase activity. The other method utilizes cerium chloride to detect uricase activity directly. These methods were modified to obtain not only a strong staining reaction but also improved ultrastructural preservation. With the indirect DAB method, intense staining indicative of both uricase and catalase activity was obtained in the enlarged peroxisomes of older uninfected cells. Similar staining was observed in enlarging peroxisomes of younger uninfected cells, and in the material of associated sacs whose bounding membranes appear to arise as distensions of the ER. The observations are discussed in relation to the controversial role of the ER in peroxisome biogenesis. Although the small peroxisome-like organelles of infected cells did not give a clearly positive reaction in the indirect DAB method, they reacted positively in the cerium chloride method, and are considered to be peroxisomes.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum     

Catalase-negative peroxisomes in human embryonic liver

Hepatic peroxisomes in human embryos with a menstrual age of 6 and 7 weeks have been examined via catalase cytochemistry. In the younger sample, the organelles show no catalase activity, their matrix being pale and coarsely reticular. In the 7-week specimen, the peroxisome population consists of catalase-positive and catalase-negative organelles. The latter have a morphology identical to that of the 6-week sample and represent 66% of the population. The positive organelles show a pronounced staining hetereogeneity. Together with the simultaneous presence of negative organelles, this might reflect the onset of catalase import into the peroxisomes during this period. Catalase heterogeneity excludes a continuous exchange of matrix contents; moreover, interconnections between peroxisomes have not been observed, and no cluster formation occurs. The data therefore also suggest that catalase is imported into individual, preexisting organelles in embryonic liver. The three peroxisomal -oxidation enzymes become detectable by immunocytochemistry only later during development. Morphological indications for a rapidly dividing population, such as elongated and/or tailed organelles, have not been observed. Morphometry has revealed that, in these early stages, the organelles are significantly smaller than the peroxisomes of fetal and adult human liver.     

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.     

Quantitative immunocytochemical studies on differential induction of serine

Summary Differential induction of serine: pyruvate aminotransferase (SPT) in rat liver parenchymal cells by administration of glucagon or di-(2-ethylhexyl)phthalate (DEHP) was studied using post-embedding immunocytochemical techniques and morphometric methods. Two groups of rats were fasted for 5 days and daily received peritoneal injection of glucagon (300 g/100 g) or physiological saline. Another two groups of rats were fed on laboratory chow with or without 2% DEHP for 2 weeks. Livers were perfusionfixed, cut into tissue sections (50–100 ), and processed to cytochemistry for catalase, immunocytochemistry for SPT, and conventional procedures for electron microscopy. The morphometric analysis showed that glucagon injection has negligible effect on the volume and numerical density and mean diameter of peroxisomes, whereas volume density of mitochondria was decreased by 25%. By DEHP administration peroxisomes were about 3-fold increased in the volume and numerical density. Mitochondria was increased about 40% in the numerical density, but unchanged in the volume density. Light and electron microscopic immunocytochemistry demonstrated that glucagon injection exclusively enhanced mitochondrial SPT, whereas DEHP administration exclusively induced in peroxisomal SPT. Quantitative analysis showed that by the glucagon injection, the labeling density of mitochondria was increased about 4-fold, but that of peroxisomes was 1.6 times as much as control, while by DEHP administration, the labeling density of peroxisomes was enhanced about 3-fold but that of mitochondria was decreased by 13%. The results clearly indicate that glucagon induces mitochondrial SPT, whereas peroxisome proliferator, DEHP induces peroxisomal SPT.     

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase

Summary The feasibility of the application of an electronic image analyzer, the Texture Analysis System (TAS) (Leitz Wetzlar, FRG), for fast automatic ultrastructural morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained selectively with the alkaline DAB procedure for localization of catalase in order to obtain sufficient contrast for automatic detection by TAS. Electron micrographs of ultrathin sections from this material were analyzed both automatically by TAS and manually by using a digitizer tablet connected with an Apple IIe microcomputer. The results showed negligible differences. As far as the speed of the operation is concerned, the image analysis was 4–5 times faster than the manual technique. In further studies, the importance of using DAB-stained sections for accurate morphometric studies of peroxisomes was demonstrated by comparing the results of such DAB-stained preparations with unstained material. This revealed that the numerical density was lower and the average profile diameter higher in unstained sections. The value for volume density was also affected, being about 30% lower in such preparations. It is likely that in unstained preparations small peroxisomes without crystalline nucleoids were frequently not identified as such and were not taken into account in morphometric calculations.These observations establish that computer-controlled electronic image analysis in conjunction with selective cytochemical staining of peroxisomes for catalase provides a fast, accurate and reliable method for ultrastructural morphometric studies of this organelle in rat liver.     

Impairment of peroxisomal β-oxidation system by endotoxin treatment

It is now clear that peroxisomes play a crucial role in many cellular processes, including the -oxidation of very long chain fatty acids. Recently, mammalian peroxisomes have been shown to contain the antioxidant enzymes, superoxide dismutase and glutathione peroxidase, in addition to catalase. The presence of these enzymes in peroxisomes suggests that peroxisomes undergo oxidative stress in normal and disease states. As an indicator of the potential impact of an oxidative stress on peroxisomal functions, we evaluated the effect of endotoxin exposure on the -oxidation enzyme system in rat liver. Peroxisomes were isolated from liver homogenates by differential and density gradient centrifugations. Endotoxin treatment decreased the -oxidation of lignoceric acid to 56% of control values (p<0.01). The specific activity of the rate limiting enzyme in the system, acyl-CoA oxidase, was decreased to 73% of control values (p<0.05). Immunoblot analysis revealed a 25% decrease in the 21KD subunit of the acyl-CoA oxidase protein. In contrast, the protein levels of the other enzymes in the pathway, trifunctional protein and 3-ketoacyl-CoA thiolase, were increased by 10 and 15%, respectively. These findings suggest that impairment of -oxidation of lignoceric acid by endotoxin treatment is due primarily to a reduction in the activity and protein level of the key enzyme, acyl-CoA oxidase. Oxidative stresses such as endotoxin exposure may have deleterious effects on important peroxisomal functions, such as -oxidation of very long chain fatty acids.     

Peroxisomes in guinea pig liver: their peculiar morphological features may reflect certain aspects of lipoprotein metabolism in this species

Summary We have studied the ultrastructural characteristics and the distribution of peroxisomes in guinea pig liver using electron-microscopic cytochemistry for catalase and morphometry. By light microscopy, peroxisomes appear as dark 0.2–0.5 m granules in the cytoplasm of liver parenchymal cells, often forming large clusters that measure up to 5 m across. Rows of single peroxisomes or their aggregates line the sinusoidal surface of hepatocytes. Electron microscopy reveals that clusters of up to 25 individual peroxisomes are usually located in the subsinusoidal region of parenchymal cells. The mean diameter and the volume density of peroxisomes are larger in pericentral than in periportal regions of the liver lobule. Whereas large amounts of lipoprotein particles with a mean diameter of 160 nm (chylomicrons) are present in the Disse space, the cytoplasm of parenchymal cells contains multivesicular bodies and abundant lipid droplets. In addition, the Golgi complexes show distended lipoprotein-filled vesicles suggesting active biosynthesis of lipoproteins. We propose that the unique features of peroxisomes in guinea pig liver, such as cluster formation and alignment along the sinusoidal surface, may be related to the high levels of lipoproteins in the portal circulation and their hepatic catabolism in this species.     

The cytochemical demonstration of catalase and D-amino acid oxidase in the microbodies of teleost kidney cells

Synopsis The distribution of catalase and D-amino acid oxidase, marker enzymes for peroxisomes, was determined cytochemically in the kidney tubules of an euryhaline teleost, the three-spined stickleback.Catalase activity was localized with the diaminobenzidine technique. The presence of D-amino acid oxidase was determined using H₂O₂ generated by the enzyme, D-alanine as a substrate, and cerous ions for the formation of an electron-dense precipitate. Both enzymes appeared to be located in microbodies. The combined presence of these enzymes characterizes the microbodies as peroxisomes. Biochemically and cytochemically, no urate oxidase or glycolate-oxidizing L--hydroxy acid oxidase could be demonstrated.Stereological analysis of the epithelia lining the renal tubules showed that the fractional volume of the microbodies is 5 to 10 times higher in the cells of the second proximal tubules than in the other nephronic segments or the ureter. The fractional volume of the microbodies was similar in kidneys of freshwater and seawater fishes.