Rhizomelic chondrodysplasia punctata: biochemical studies of peroxisomes isolated from cultured skin fibroblasts.

Peroxisomes isolated from cultured skin fibroblasts of two patients with rhizomelic chondrodysplasia punctata (RCDP) and two controls were compared for biochemical studies. These experiments provided the following results: (1) peroxisomes isolated from RCDP-cultured skin fibroblasts had the same density (1.175 g/ml) as control peroxisomes; (2) dihydroxyacetone phosphate acyltransferase activity, the first enzyme in the synthesis of plasmalogens, was deficient (0.5% of control) in RCDP peroxisomes and this activity was not observed in any other region of the gradient; (3) the rate of activation (lignoceroyl-CoA ligase) and oxidation of lignoceric acid was normal in RCDP peroxisomes; and (4) peroxisomes from RCDP contained 3-ketoacyl-CoA thiolase in the unprocessed form (44-kDa protein), whereas control peroxisomes had both processed (41-kDa protein) and unprocessed forms of 3-ketoacyl-CoA thiolase. The presence of both processed and unprocessed 3-ketoacyl-CoA thiolase in control peroxisomes and the unprocessed form in RCDP peroxisomes suggests that processing of 3-ketoacyl-CoA thiolase takes place in peroxisomes. Although the specific activity and percentage of activity of 3-ketoacyl-CoA thiolase in RCDP peroxisomes was only 22-26% of control, the normal oxidation of lignoceric acid in RCDP peroxisomes indicates that unprocessed 3-ketoacyl-CoA thiolase is active. The remaining peroxisomal 3-ketoacyl-CoA thiolase activity in RCDP was observed in a protein fraction (peroxisome ghosts) lighter than peroxisomes. The normal oxidation of fatty acids in peroxisomes and the absence of such activity in peroxisome ghosts (d = 1.12 g/ml) containing peroxisomal proteins in RCDP suggest that RCDP has only one population of functional peroxisomes (d = 1.175 g/ml).     

Identification of superoxide dismutase in rat liver peroxisomes.

In this paper we have investigated whether or not superoxide dismutase is localized in peroxisomes from rat liver. Using an improved method to prepare peroxisomes from clofibrate induced rat livers, we identified superoxide dismutase activity in peroxisomes. This activity was found to be predominantly of the copper-zinc type. The finding of superoxide dismutase activity in peroxisomes makes sense since peroxisomes also contain superoxide generating enzyme activities such as xanthine oxidase.     

Elevated cholesterol and decreased sterol carrier protein-2 in peroxisomes from AS-30D hepatoma compared to normal rat liver

Peroxisomes were isolated from AS-30D hepatoma and compared to normal rat liver cells for the purpose of investigating the cholesterol accumulation in the hepatoma cells. Cholesterol was found to be approximately 10-fold higher relative to protein in AS-30D peroxisomes as compared to peroxisomes from normal liver. The peroxisomes from the hepatoma cells were found to be more stable; catalase was not released from these peroxisomes during isolation or osmotic shock of the peroxisomal fraction. The elevated cholesterol level may stabilize the peroxisomal membrane. Sterol carrier protein-2 (SCP-2) levels were measured using a radioimmunoassay (RIA), which indicated the highest concentration of SCP-2 to be in peroxisomes. Hepatoma peroxisomes had a lower concentration of SCP-2 (2.5 micrograms/mg) than normal liver peroxisomes (8 micrograms/mg). Approximately half of all SCP-2 detected was found to be soluble in both hepatoma and normal rat liver cells. Immunoblots from both rat liver and AS-30D fractions demonstrated the presence of the 14-kDa form of SCP-2. The liver fractions also had a 57-kDa immunoreactive protein, which was barely detectable in the AS-30D fractions. The low abundance of the high molecular weight form of SCP-2 from hepatoma peroxisomes and the lower amounts of SCP-2 detected in the AS-30D peroxisomes may be related to the accumulation of cholesterol in the cells.     

Protein phosphorylation in peroxisomes

The possible presence of phosphorylated proteins in peroxisomes was studied in hepatocytes from nafenopin-treated and normal rats. A 63 kDa phosphorylated protein was consistently and exclusively found in the membrane of peroxisomes from hepatocytes incubated in the presence of 32P-phosphate. The peroxisomes were isolated in metrizamide isopycnic gradients of postnuclear supernatants and were subfractionated by alkaline extraction to separate the membrane and the matrix proteins. Polyacrylamide gel electrophoresis, autoradiography and densitometry were employed to characterize the proteins. The 63 kDa membrane protein copurifies with peroxisomes in metrizamide gradients and apparently can be phosphorylated, in purified peroxisomes, with ATP and catalytic subunit of cAMP-dependent protein kinase.     

Isolation and characterization of peroxisomes from the renal cortex of beef, sheep, and cat

The isolation and characterization of highly purified and structurally well-preserved peroxisomes from the renal cortex of different mammalian species (beef, sheep, and cat) is reported. Renal cortex tissue was homogenized and a peroxisome-enriched light mitochondrial fraction was prepared by differential centrifugation. This was subfractionated by density-dependent banding on a linear gradient of metrizamide (1.12-1.26 g/cm3) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at a mean density of 1.225 cm3. Ultrastructural morphometric examination revealed that peroxisomes made up 97 to 98% of the isolated fractions. By biochemical analysis the contamination with marker enzymes of mitochondria and lysosomes was extremely low. The specific activity of catalase was enriched, depending on the species, between 28- and 38-fold over the homogenate. Peroxisome preparations from all three species exhibited a high but varying level of activity for cyanide-insensitive lipid beta-oxidation. In beef and sheep preparations a small amount of esterase activity cosediments with peroxisomes. These peroxisomes show distinct structural membrane associations with smooth elements of ER. Urate oxidase, a marker enzyme for rat liver peroxisomes, is found only in peroxisomes prepared from beef kidney cortex, with sheep and cat preparations being negative. This correlated with the occurrence of polytubular inclusions in the beef kidney peroxisomes. The large size and the angular shape of isolated peroxisomes as well as the presence of paracrystalline matrical inclusions imply that the majority of peroxisomes are derived from the epithelial cells of the proximal tubule of the kidney cortex. The significant differences found in the characteristics of the renal peroxisomes in three different species investigated, demonstrate the remarkable adaptability and plasticity of this organelle.     

Effect of Hypoxia-Reoxygenation on Peroxisomal Functions in Cultured Human Skin Fibroblasts from Control and Zellweger Syndrome Patients

To delineate the role of peroxisomes in the pathophysiology of hypoxia-reoxygenation we examined the functions of peroxisomes and mitochondria in cultured skin fibroblasts from controls and from patients with cells lacking peroxisomes (Zellweger cells). The loss of peroxisomal functions (lignoceric acid oxidation and dihydroxyacetonephosphate acyltransferase [DHAP-AT] activities) in control cells following hypoxia and hypoxia followed by reoxygenation, suggests that peroxisomes are sensitive to oxidative injury. The sensitivity of peroxisomes to oxidative stress was compared to that of mitochondria by examining the oxidation of palmitic acid (a function of both mitochondria and peroxisomes) in control and Zellweger cell lines, following hypoxia-reoxygenation. The greater loss of activity of palmitic acid oxidation observed in control cells as compared to that seen in Zellweger cells suggests that the peroxisomal β-oxidation system is relatively more labile to hypoxia- reoxygenation induced oxidative stress. This data clearly demonstrates the difference in the response of mitochondria and peroxisomes to oxidative stress.     

Morphometric and cytochemical evaluation of clofibrate-induced peroxisomal proliferation in adult rat hepatocytes cultured on floating collagen gels

The present ultrastructural morphometric and cytochemical studies demonstrate clofibrate induced changes in peroxisomes in adult rat hepatocytes maintained for 14 days in primary culture on floating collagen gels. Catalase activity and the number and diameter of peroxisomes were reduced in hepatocytes cultured for between 2/3 and 7 days. However, hepatocytes cultured for 7-14 days had well-developed peroxisomes containing crystalloid nucleoids. The number of anucleoid peroxisomes in hepatocytes treated with 2 mM Na clofibrate increased with culture age, and by day 14 the number was 2.9 times greater than in freshly isolated hepatocytes. Catalase activity, as well as the number of nucleoid-containing peroxisomes were much greater in treated hepatocytes than in untreated controls, but decreased slightly with culture age. The diameter of peroxisomes was not reduced in the treated cells. These results suggest that the treatment with Na clofibrate is effective both for proliferation and maintenance of peroxisomes and for enhancing catalase activity. In treated hepatocytes, matrical plates were formed in peroxisomes from days 5 to 14 and the number of plate-containing peroxisomes increased with culture age.     

PEROXISOMES IN ABSORPTIVE CELLS OF MAMMALIAN SMALL INTESTINE

Huge numbers of peroxisomes are present in guinea pig duodenum, jejunum, and ileum, and in rat duodenum. The peroxisomes have been studied by light and electron microscopy, including visualization by incubation in a newly-developed alkaline 3,3' diaminobenzidine (DAB) medium. Electron micrographs of more than 3700 guinea pig peroxisomes have been studied. The diameter of most peroxisomes ranges from 0.15 µ. to 0.25 µ. They often appear in clusters, surrounded by and continuous, in numerous places, with smooth endoplasmic reticulum (ER). The ER is extremely tortuous in these regions. Serial sectioning is valuable for studying the ER-peroxisome relationships but viewing sections at different angles, tilted with a goniometer stage, is more informative. The intimate relations of the two organelles appear the same in tissue fixed in four different fixatives. The peroxisomes may be interpreted as localized dilatations of smooth ER retaining multiple membranous continuities. This interpretation is discussed in light of the turnover data on peroxisomal proteins of rat hepatocytes reported by Poole and colleagues. The very large numbers of peroxisomes in intestinal epithelium lead to speculations concerning their functional significance. They resemble the small peroxisomes described in many other cell types. Although the distinctive relationship of these peroxisomes to the ER is probably more significant than their small size, for practical purposes we propose the term "microperoxisomes" to distinguish these peroxisomes from the better-known larger peroxisomes of liver and kidney.     

Characterization of peroxisomes in glucose-grown Hansenula polymorpha and their development after the transfer of cells into methanol-containing media

Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxisomes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.     

Copper–zinc superoxide dismutase is a constituent enzyme of the matrix of peroxisomes in the cotyledons of oilseed plants

The number and type of isoforms of superoxide dismutase (SOD) and their activities were compared in mitochondria and peroxisomes isolated from cotyledons of three different oilseed seedlings. Mitochondrial and peroxisomal isoforms of SOD could be distinguished in nondenaturing polyacrylamide gels by their differential sensitivities to KCN and/or H₂O₂. The type of SOD was not the same for each organelle in each of the three oilseed species. For example, a single Mn–SOD was found in cotton and cucumber mitochondria, whereas four CuZn–SODs were present in mitochondria from sunflower. At least one CuZn–SOD isoform was found in the peroxisomes of all three species. Cucumber peroxisomes contained both a CuZn–SOD and a Mn–SOD, cotton peroxisomes contained a single CuZn–SOD, whilst four separate CuZn–SODs, but no Mn–SOD were found in sunflower peroxisomes. Using antibodies against CuZn–SOD from watermelon peroxisomes or from chloroplasts of Equisetum , a single polypeptide of c . 16·5 kDa was detected on immunoblots of peroxisomal fractions from the three species. Post-embedment, electron-microscopic double immunogold-labelling showed that CuZn–SOD, with malate synthase used as marker enzyme of peroxisomes, was localized in the matrix of these organelles of all three species. These results suggest that CuZn–SOD is a characteristic matrix enzyme of peroxisomes in oilseed cotyledons.     

The origin and maintenance of mammalian peroxisomes involves a de novo PEX16-dependent pathway from the ER

Peroxisomes are ubiquitous organelles that proliferate under different physiological conditions and can form de novo in cells that lack them. The endoplasmic reticulum (ER) has been shown to be the source of peroxisomes in yeast and plant cells. It remains unclear, however, whether the ER has a similar role in mammalian cells and whether peroxisome division or outgrowth from the ER maintains peroxisomes in growing cells. We use a new in cellula pulse-chase imaging protocol with photoactivatable GFP to investigate the mechanism underlying the biogenesis of mammalian peroxisomes. We provide direct evidence that peroxisomes can arise de novo from the ER in both normal and peroxisome-less mutant cells. We further show that PEX16 regulates this process by being cotranslationally inserted into the ER and serving to recruit other peroxisomal membrane proteins to membranes. Finally, we demonstrate that the increase in peroxisome number in growing wild-type cells results primarily from new peroxisomes derived from the ER rather than by division of preexisting peroxisomes.     

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.     

Involvement of the endoplasmic reticulum in peroxisome formation

The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived.     

Urate oxidase in peroxisomes from maize root tips

Summary Peroxisomes isolated from maize root tips contained urate oxidase, although the supplementary enzymes allantoinase, allantoicase and NADH-glyoxylate reductase were not detected. Some glutamate-oxalacetate transaminase was present in peroxisomes. Enzymes of two other pathways occuring in plant peroxisomes, namely glycolate metabolism and the glyoxylate cycle, were not present. The root peroxisome thus resembles peroxisomes of the Arum spadix and supports the concept that peroxisomes constitute a dynamic and differentiating system.     

Peroxisomes of the nematode Caenorhabditis elegans: distribution and morphological characteristics

We analyzed the distribution and morphological characteristics of peroxisomes in the nematode Caenorhabditis elegans by routine electron microscopy, immunoelectron microscopy, and morphometry. Peroxisomes were mainly contained in the epithelial cells of the digestive tract and pharyngeal gland, but some were observed in other cells. Their shape varied from round to twisted. The matrix of most peroxisomes was coarse and uneven, and contained electron-dense nucleoids and frequently tubular substructures. The diameter of peroxisomes in the gut (0.185 micro m) was smaller than that in pharyngeal gland (0.262 micro m). The volume density of peroxisomes per 100 micro m(2) of cytoplasm was 1.86 in the gut and 1.75 in the pharyngeal gland. After treatment with clofibrate, the diameter of peroxisomes increased approximately 1.11-fold in the gut and 1.2-fold in the pharyngeal gland. The volume density of peroxisomes also increased by 2.2-fold in the gut and 2.6-fold in the pharyngeal gland. The labeling density for catalase-2 was almost identical between gut and pharyngeal gland peroxisomes. The results show that in C. elegans peroxisomes mainly distribute in the epithelial cells of the gut and pharyngeal gland. Peroxisomes of the pharyngeal gland are larger than those of the gut, but peroxisomes of both tissues contain catalase-2 at similar concentrations.     

Metabolism of oxygen radicals in peroxisomes and cellular implications.

Peroxisomes are subcellular respiratory organelles which contain catalase and H2O2-producing flavin oxidases as basic enzymatic constituents. These organelles have an essentially oxidative type of metabolism and have the potential to carry out different important metabolic pathways. In recent years the presence of different types of superoxide dismutase (SOD) have been demonstrated in peroxisomes from several plant species, and more recently the occurrence of SOD has been extended to peroxisomes from human and transformed yeast cells. A copper,zinc-containing SOD from plant peroxisomes has been purified and partially characterized. The production of hydroxyl and superoxide radicals has been studied in peroxisomes. There are two sites of O2- production in peroxisomes: (1) in the matrix, the generating system being xanthine oxidase; and (2) in peroxisomal membranes, dependent on reduced nicotinamide adenine dinucleotide (NADH), and the electron transport components of the peroxisomal membrane are possibly responsible. The generation of oxygen radicals in peroxisomes could have important effects on cellular metabolism. Diverse cellular implications of oxyradical metabolism in peroxisomes are discussed in relation to phenomena such as cell injury, peroxisomal genetic diseases, peroxisome proliferation and oxidative stress, metal and salt stress, catabolism of nucleic acids, senescence, and plant pathogenic processes.     

Isozymes of Superoxide Dismutase in Mitochondria and Peroxisomes Isolated from Petals of Carnation (Dianthus caryophyllus) during Senescence

The balance between reactions involving free radicals and processes which ameliorate their effect plays an important role in the regulation of plant senescence. In this study a method was developed to isolate peroxisomes and mitochondria from carnation (Dianthus caryophyllus L. cv Ember) petals. Based on electron microscopy and marker enzyme levels, the proportion of peroxisomes to mitochondria increases during senescence. The superoxide dismutase (SOD) content of these fractions was examined. Mitochondria and peroxisomes were shown to contain two electrophoretically distinct SODs, a manganese-, and an ironcontaining SOD. The Mn- and Fe-SOD were found to have relative molecular weights of 75,000 and 48,000 and isoelectric points of 4.85 and 5.00, respectively. The presence of a Fe-SOD in mitochondria and peroxisomes is unique because this enzyme is usually located in chloroplasts. The activity of these two isoenzymes decreased during senescence in mitochondria but remained high in peroxisomes from senescent tissue. It is suggested that peroxisomes play a particular role in the process of senescence.     

Yeast peroxisomes multiply by growth and division

Peroxisomes can arise de novo from the endoplasmic reticulum (ER) via a maturation process. Peroxisomes can also multiply by fission. We have investigated how these modes of multiplication contribute to peroxisome numbers in Saccharomyces cerevisiae and the role of the dynamin-related proteins (Drps) in these processes. We have developed pulse-chase and mating assays to follow the fate of existing peroxisomes, de novo-formed peroxisomes, and ER-derived preperoxisomal structures. We find that in wild-type (WT) cells, peroxisomes multiply by fission and do not form de novo. A marker for the maturation pathway, Pex3-GFP, is delivered from the ER to existing peroxisomes. Strikingly, cells lacking peroxisomes as a result of a segregation defect do form peroxisomes de novo. This process is slower than peroxisome multiplication in WT cells and is Drp independent. In contrast, peroxisome fission is Drp dependent. Our results show that peroxisomes multiply by growth and division under our assay conditions. We conclude that the ER to peroxisome pathway functions to supply existing peroxisomes with essential membrane constituents.     

Peroxisomes of alkane-utilizing yeasts metabolic functions and practical aspects

One of the most striking features of alkane-grown yeast cells is conspicuous appearance of peroxisomes in harmony with a high level of catalase. This unique phenomenon was first demonstrated in the authors′ laboratory, and the metabolic functions of peroxisomes in yeasts utilizing alkanes has been estabilished with intact peroxisomes isolated by density gradient centrifugation. The organelles participate in the degradation of fatty acids derived from alkanes to C²-units and the synthesis of gluconeogenic intermediates from C²-units. The abundant appearance of peroxisomes in alkane-utilizing cells has allowed successful production of several useful enzymes including catalase, D-amino acid oxidase, uricase, acyl-CoA oxidase etc. Yeast cells will be an excellent system for investigation the functions and development of peroxisomes because biogenesis of the organelles is induced only by transferring the cells into alkane medium from glucose or ethanol medium.     

Development of crystalline peroxisomes in methanol-grown cells of the yeast Hansenula polymorpha and its relation to environmental conditions

The development of peroxisomes has been studied in cells of the yeast Hansenula polymorpha during growth on methanol in batch and chemostat cultures. During bud formation, new peroxisomes were generated by the separation of small peroxisomes from mature organelles in the mother cells. The number of peroxisomes migrating to the buds was dependent upon environmental conditions. Aging of cells was accompanied by an increase in size of the peroxisomes and a subsequent increase in their numbers per cell. Their ultimate shape and substructure as well as their number per cell was dependent upon the physiological state of the culture. The change in number and volume density of peroxisomes was related to the level of alcohol oxidase in the cells. Development of peroxisomes in cells of batch cultures was accompanied by an increase in size of the crystalline inclusions in the organelles; they had become completely crystalline when the cells were in the stationary phase. Peroxisomes in cells from methanol-limited chemostat cultures were completely crystalline, irrespective of growth rate. Results of biochemical and cytochemical experiments suggested that alcohol oxidase is a major component of the crystalline inclusions in the peroxisomes of methanol-grown Hansenula polymorpha. Possible mechanisms involved in the ultrastructural changes in peroxisomes during their development have been discussed.Abbreviations DAB 3,3-diaminobenzidine - OD optical density (663 nm)