菠菜叶片过氧体甘氨酸转氨酶初探

菠菜叶片过氧体可转变甘油酸或羟基丙酮酸成为丝氨酸。以甘氨酸为氨基供体时,完整过氧体的转变活性比破碎的约高2.5倍,这不是由于完整的膜包被使有效的羟基丙酮酸浓度增加,或由于膜破碎使辅助因子损失,也不是由于过氧体膜的甘氨酸主动运输系统的作用。结果显示,在过氧体中存在甘氨酸转氨酶,完整过氧体中高的转氨速度可能是由于甘氨酸转氨酶在完整的和破碎的过氧体中的构象或构型不同。     

Peroxisomes in sebaceous glands. IV. Aggregates of tubular peroxisomes in the mouse Meibomian gland

The occurrence of peroxisomes, their morphogenesis during the process of sebaceous transformation and their spatial relationship to the endoplasmic reticulum and lipid droplets were investigated by light and electron microscopy after visualization of the peroxidatic activity of catalase using an alkaline diaminobenzidine medium. The morphological alterations of peroxisomes display a characteristic sequence: During cellular differentiation, a remarkable proliferation of exclusively tubular, diaminobenzidine-reactive peroxisomes occurs. As maturation proceeds, an extensive elongation of tubular peroxisomes is seen. Concomitantly, they are densely packed in a regular, hexagonal arrangement and both the diameter and the catalase content gradually decreases. The most conspicuous feature of mature glandular cells are numerous highly organized aggregates of tubular, almost unstained peroxisomes with a diameter of 50 nm, arranged in a hexagonal pattern. They resemble adjacent tubular profiles of smooth endoplasmic reticulum. However, membrane continuities between these two compartments were never observed. During lethal disintegration peroxisomes subsequently decrease in number, probably by rapid sequestration within autophagolysosomes. The role of tubular peroxisomes in the biosynthesis of wax esters in the mouse Meibomian gland is discussed.     

Morphometric cytochemistry of diminution of catalase-containing peroxisomes in copper-loaded liver

Summary The density of hepatocellular catalase-containing peroxisomes was quantified, utilizing a computer-aided image analysing technique, on 1-m thick diaminobenzidine-stained sections. Hepatic copper accumulation following intraperitoneal injection of cupric chloride resulted in a dose-dependent reduction in the density of catalase-containing peroxisomes. A significant correlation between the density of peroxisomes and the activity of hepatic catalase indicated that computer-aided image analysis of peroxisomes stained by the diaminobenzidine technique provided a useful estimate of catalase activity in liver injured by copper. Slight treatment-related differences in the mean diameter of peroxisomes were detected in high-dose but not low-dose rats.     

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.     

Metabolism of glycolate and glyoxylate in intact spinach leaf peroxisomes

Intact and broken (osmotically disrupted) spinach (Spinacia oleracea) leaf peroxisomes were compared for their enzymic activities on various metabolites in 0.25 molar sucrose solution. Both intact and broken peroxisomes had similar glycolate-dependent o₂ uptake activity. In the conversion of glycolate to glycine in the presence of serine, intact peroxisomes had twice the activity of broken peroxisomes at low glycolate concentrations, and this difference was largely eliminated at saturating glycolate concentrations. However, when glutamate was used instead of serine as the amino group donor, broken peroxisomes had slightly higher activity than intact peroxisomes. In the conversion of glyoxylate to glycine in the presence of serine, intact peroxisomes had only about 50% of the activity of broken peroxisomes at low glyoxylate concentrations, and this difference was largely overcome at saturating glyoxylate concentrations. In the transamination between alanine and hydroxypyruvate, intact peroxisomes had an activity only slightly lower than that of broken peroxisomes. In the oxidation of NADH in the presence of hydroxypyruvate, intact peroxisomes were largely devoid of activity. These results suggest that the peroxisomal membrane does not impose an entry barrier to glycolate, serine, and O₂ for matrix enzyme activity; such a barrier does exist to glutamate, alanine, hydroxypyruvate, glyoxylate, and NADH. Furthermore, in intact peroxisomes, glyoxylate generated by glycolate oxidase is channeled directly to glyoxylate aminotransferase for a more efficient glycolate-glycine conversion. In related studies, application of in vitro osmotic stress to intact or broken peroxisomes had little effect on their ability to metabolize glycolate to glycine.     

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.     

Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins

Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.     

Polyunsaturated fatty acid biosynthesis: a microsomal-peroxisomal process.

The synthesis of 22-carbon fatty acids, with their first double bond at position 4, requires the participation of enzymes in both peroxisomes and the endoplasmic reticulum as well as the controlled movement of fatty acids between these two cellular compartments. It has been observed that there is generally an inverse relationship between rates of peroxisomal beta-oxidation vs those for the microsomal esterification of fatty acids into 1-acyl-sn-glycero-3-phosphocholine. With a variety of different substrates it was found that when a fatty acid is produced in peroxisomes, with its first double bond at position 4, its preferred metabolic fate is to move to microsomes for esterification rather than to serve as a substrate for continued degradation. The required movement, and the associated reactions, in peroxisomes and microsomes is not restricted to the synthesis of 4,7,10,13,16-docosapentaenoic acid and 4,7,10,13,16,19-docosahexaenoic acid. When microsomes and peroxisomes were incubated with NAD, NADPH and malonyl-CoA it was found that 6,9,12-octadecatrienoic acid was metabolized to linoleate. Collectively our findings suggest that there may be considerably more recycling of fatty acids between peroxisomes and the endoplasmic reticulum than was previously recognized.     

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.     

Development of peroxisomes in amphibians. III. Study on liver, kidney, and intestine during thyroxine-induced metamorphosis

This investigation was undertaken to study the ontogeny of hepatic, renal, and intestinal peroxisomes and/or microperoxisomes during thyroxine-induced anuran metamorphosis. Catalase activity was localized cytochemically after incubation in DAB medium, and studied biochemically by a spectrophotometric method. Our morphological and biochemical investigations suggest the formation of a new population of peroxisomes during the hormonal treatment. This is obvious especially for microperoxisomes of the intestinal epithelium since the larval tissue is completely replaced by a new layer during thyroxine-induced metamorphosis. For the peroxisomes of hepatocytes and kidney proximal tubule cells, our assumption is based on the following observations: 1) The number of peroxisomes increases in liver and kidney during thyroxine treatment; 2) this proliferation is accompanied by an enlargement of renal peroxisomes; and 3) 16 days after the beginning of the hormonal treatment, 5.4- and 2.4-fold increases are found for the specific activities of hepatic and renal catalase, respectively. A temporal coordination exists between the structure and the metabolism of peroxisomes and mitochondria during thyroxine-induced metamorphosis.     

A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae.

In vivo time-lapse microscopy reveals that the number of peroxisomes in Saccharomyces cerevisiae cells is fairly constant and that a subset of the organelles are targeted and segregated to the bud in a highly ordered, vectorial process. The dynamin-like protein Vps1p controls the number of peroxisomes, since in a vps1Delta mutant only one or two giant peroxisomes remain. Analogous to the function of other dynamin-related proteins, Vps1p may be involved in a membrane fission event that is required for the regulation of peroxisome abundance. We found that efficient segregation of peroxisomes from mother to bud is dependent on the actin cytoskeleton, and active movement of peroxisomes along actin filaments is driven by the class V myosin motor protein, Myo2p: (a) peroxisomal dynamics always paralleled the polarity of the actin cytoskeleton, (b) double labeling of peroxisomes and actin cables revealed a close association between both, (c) depolymerization of the actin cytoskeleton abolished all peroxisomal movements, and (d) in cells containing thermosensitive alleles of MYO2, all peroxisome movement immediately stopped at the nonpermissive temperature. In addition, time-lapse videos showing peroxisome movement in wild-type and vps1Delta cells suggest the existence of various levels of control involved in the partitioning of peroxisomes.     

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.     

Beta-oxidation of butyrate, the short-chain-length fatty acid, occurs in peroxisomes in the yeast Candida tropicalis.

When an n-alkane-utilizable yeast, Candida tropicalis pK233, was cultivated on butyrate, the fatty acid of shortest chain-length for beta-oxidation, as the sole source of carbon and energy, catalase and the enzymes of the fatty acid beta-oxidation system were inducibly synthesized at high levels. As in the alkane-grown cells, the proliferation of peroxisomes was harmonized with the induction of peroxisomal enzymes. The results of subcellular fractionation and immunoelectronmicroscopy indicated the localization of these enzymes in peroxisomes, not in mitochondria. It was suggested that only peroxisomes have a role in fatty acid beta-oxidation in the yeast cells, unlike in mammalian cells, in which cooperation between peroxisomes and mitochondria is essential.     

Observation of the Yarrowia lipolytica peroxisome-vacuole dynamics by fluorescence microscopy with a single filter set

We constructed the fusion of peroxisomal acyl-CoA oxidase 3 and the enhanced yellow fluorescent protein (EYFP) for fluorescent labeling of Yarrowia lipolytica peroxisomes. Using the spectral overlap between EYFP and FM4-64, we developed a procedure for simultaneous observation of Y. lipolytica peroxisomes and vacuoles with the single fluorescein isothiocyanate filter set. Using this procedure we were able to follow the Y. lipolytica peroxisome-vacuole dynamics under pexophagy conditions and show that Y. lipolytica peroxisomes are degraded in the vacuoles by a macropexophagic mechanism.     

Relationship between peroxisomes and endoplasmic reticulum investigated by combined catalase and glucose-6-phosphatase cytochemistry

The theoretical advantages of electron microscopic cytochemistry were utilized to look for evidence of possible connections between peroxisomes and the endoplasmic reticulum in rat liver. Established cytochemical procedures for catalase (peroxisomes) and glucose-6-phosphatase (endoplasmic reticulum) were carried out, and evidence was sought of diffusion of reaction products between the organelles. No such diffusion was observed: lead phosphate was found in the endoplasmic reticulum and in the nuclear envelope but not in peroxisomes; oxidized diaminobenzidine (DAB) was seen only in peroxisomes. In addition, both types of cytochemistry were carried out on the same tissue. The two kinds of reaction product could be distinguished by virtue of their different electron opacities. No mixing of the two reaction products was observed. These results do not support the hypothesis that peroxisomes and endoplasmic reticulum may be connected; rather, they support the idea that the two organelles exist as separate cellular compartments.     

Peroxisomal peripheral membrane protein YlInp1p is required for peroxisome inheritance and influences the dimorphic transition in the yeast Yarrowia lipolytica

Eukaryotic cells have evolved molecular mechanisms to ensure the faithful inheritance of organelles by daughter cells in order to maintain the benefits afforded by the compartmentalization of biochemical functions. Little is known about the inheritance of peroxisomes, organelles of lipid metabolism. We have analyzed peroxisome dynamics and inheritance in the dimorphic yeast Yarrowia lipolytica. Most peroxisomes are anchored at the periphery of cells of Y. lipolytica. In vivo video microscopy showed that at cell division, approximately half of the anchored peroxisomes in the mother cell are dislodged individually from their static positions and transported to the bud. Peroxisome motility is dependent on the actin cytoskeleton. YlInp1p is a peripheral peroxisomal membrane protein that affects the partitioning of peroxisomes between mother cell and bud in Y. lipolytica. In cells lacking YlInp1p, most peroxisomes were transferred to the bud, with only a few remaining in the mother cell, while in cells overexpressing YlInp1p, peroxisomes were preferentially retained in the mother cell, resulting in buds nearly devoid of peroxisomes. Our results are consistent with a role for YlInp1p in anchoring peroxisomes in cells. YlInp1p has a role in the dimorphic transition in Y. lipolytica, as cells lacking the YlINP1 gene more readily convert from the yeast to the mycelial form in oleic acid-containing medium, the metabolism of which requires peroxisomal activity, than does the wild-type strain. This study reports the first analysis of organelle inheritance in a true dimorphic yeast and identifies the first protein required for peroxisome inheritance in Y. lipolytica.     

Peroxisome development in the metanephric kidney of mouse.

The relationship of enzymatic activity to organelle development and organelle number during differentiation of the metanephric kidney in the mouse was approached from several experimental directions. Biochemical analyses of marker enzymes for peroxisomes (catalase and D-amino acid oxidase), mitochondria (cytochrome oxidase) and lysosomes (acid phosphatase) were performed on kidneys at ages from 17 days prenatal to adult. These data were correlated with a morphometric analysis of populations of peroxisomes and mitochondria in differentiating cells of the proximal tubule. Postnatal development of the metanephric kidney was found to be accompanied by a rapid increase in both the specific activity of catalase and the number of peroxisomes per 100 mu2 in the proximal tubule during the first 4 weeks of postnatal growth. Elaboration of the endoplasmic reticulum (ER) was seen to parallel the increase in number of peroxisomes to which segments of ER were often in close apposition. Extensive interactions between segments of ER and peroxisomes were readily visible in 0.5-mu sections viewed in the high voltage electron microscope. In contrast to peroxisomes, neither mitochondria nor lysosomes followed a similar pattern of net organelle increase, suggesting that a defined population density of mitochondria and lysosomes may exist in the proximal tubule at birth, prior to complete development of the kidney.     

Peroxisomes in mouse and human lung: their involvement in pulmonary lipid metabolism

Only sparse information is available from the literature on the peroxisomal compartment and its enzyme composition in mouse and human lungs. Therefore, in the present investigation we have characterized peroxisomes in different cell types of adult mouse (C57BL/6J) and human lungs in a comprehensive study using a variety of light-, fluorescence- and electron microscopic as well as biochemical techniques and by the use of various peroxisomal marker proteins (Pex13p, Pex14p, ABCD3, beta-oxidation enzymes and catalase). In contrast to previous reports, we have found that peroxisomes are present in all cell types in human and mouse lungs. However, they differ significantly and in a cell-type-specific manner in their structure, numerical abundance and enzyme composition. Whereas catalase showed significant differences between distinct cell types, Pex14p proved to be the marker of choice for labeling all lung peroxisomes. In alveolar type II cells and alveolar macrophages peroxisomes contained significant amounts of the lipid transporter ABCD3 and beta-oxidation enzymes, suggesting their involvement in the modification and recycling of surfactant lipids and in the control of lipid mediators and ligands for nuclear receptors of the PPAR family. Possible connections between ROS and lipid metabolism of lung peroxisomes are discussed.     

Structural and cytochemical characterization of three specialized peroxisome types in soybean

Structural and cytochemical comparisons were made between three peroxisome types in soybean [ Glycine max (L.) Merr. cv. Centennial]. Leaf peroxisomes were densely granular organelles with an amorphous nucleoid and were generally located in close proximity to the chloroplasts. Catalase (EC 1.11.1.6) and glycolate oxidase (EC 1.1.3.1) were localized in these peroxisomes although glycolate oxidase was absent from the nucleoid region. Glyoxysomes, present in the etiolated cotyledons, were coarsely granular organelles that were generally in close proximity to lipid bodies. Malate synthase (EC 4.1.3.2), catalase, and glycolate oxidase were present throughout the matrix. Although peroxisomes were found in both infected and uninfected nodule tissue, uninfected interstitial cell peroxisomes were the most developed. These organelles contained a core surrounded by a less electron-opaque periphery that frequently was in close association with (but distinct from) a network of smooth endoplasmic reticulum. Of the enzymes studied, only catalase and urate oxidase (EC 1.7.3.3) were detected in the nodule peroxisomes. Neither enzyme was detected in the peripheral area of the peroxisome. These data indicate that peroxisomes in the three tissue types have organelle associations, internal structures, enzyme constitutions and packaging that reflect their metabolic differences.     

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.