FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2059574370> ?p ?o ?g. }

• W2059574370 endingPage "23069" @default.
• W2059574370 startingPage "23055" @default.
• W2059574370 abstract "Fibrates are known to induce peroxisome proliferation and the expression of peroxisomal β-oxidation enzymes. To analyze fibrate-induced changes of complex metabolic networks, we have compared the proteome of rat liver peroxisomes from control and bezafibrate-treated rats. Highly purified peroxisomes were subfractionated, and the proteins of the matrix, peripheral, and integral membrane subfractions thus obtained were analyzed by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry after labeling of tryptic peptides with the iTRAQ reagent. By means of this quantitative technique, we were able to identify 134 individual proteins, covering most of the known peroxisomal proteome. Ten predicted new open reading frames were verified by cDNA cloning, and seven of them could be localized to peroxisomes by immunocytochemistry. Moreover, quantitative mass spectrometry substantiated the induction of most of the known peroxisome proliferator-activated receptor α-regulated peroxisomal proteins upon treatment with bezafibrate, documenting the suitability of the iTRAQ procedure in larger scale experiments. However, not all proteins reacted to a similar extent but exerted a fibrate-specific induction scheme showing the variability of peroxisome proliferator-activated receptorα-transmitted responses to specific ligands. In view of our data, rat hepatic peroxisomes are apparently not specialized to sequester very long chain fatty acids (C22–C26) but rather metabolize preferentially long chain fatty acids (C16–18). Fibrates are known to induce peroxisome proliferation and the expression of peroxisomal β-oxidation enzymes. To analyze fibrate-induced changes of complex metabolic networks, we have compared the proteome of rat liver peroxisomes from control and bezafibrate-treated rats. Highly purified peroxisomes were subfractionated, and the proteins of the matrix, peripheral, and integral membrane subfractions thus obtained were analyzed by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry after labeling of tryptic peptides with the iTRAQ reagent. By means of this quantitative technique, we were able to identify 134 individual proteins, covering most of the known peroxisomal proteome. Ten predicted new open reading frames were verified by cDNA cloning, and seven of them could be localized to peroxisomes by immunocytochemistry. Moreover, quantitative mass spectrometry substantiated the induction of most of the known peroxisome proliferator-activated receptor α-regulated peroxisomal proteins upon treatment with bezafibrate, documenting the suitability of the iTRAQ procedure in larger scale experiments. However, not all proteins reacted to a similar extent but exerted a fibrate-specific induction scheme showing the variability of peroxisome proliferator-activated receptorα-transmitted responses to specific ligands. In view of our data, rat hepatic peroxisomes are apparently not specialized to sequester very long chain fatty acids (C22–C26) but rather metabolize preferentially long chain fatty acids (C16–18). Peroxisomes, often described as multipurpose organelles, are involved in numerous biochemical pathways of lipid and peroxide metabolism. The existence of more than 15 peroxisomal inherited diseases like the Zellweger syndrome or X-linked adrenoleukodystrophy, which are often lethal, emphasizes the vital importance of these organelles (1Raymond G.V. Curr. Opin. Pediatr. 1999; 11: 572-576Crossref PubMed Scopus (14) Google Scholar, 2Moser H.W. Front. Biosci. 2000; 5 (–D306): D298Crossref PubMed Google Scholar, 3Schluter A. Fourcade S. Domenech-Estevez E. Gabaldon T. Huerta-Cepas J. Bertthommler G. Ripp R. Wanders R.J. Poch O. Pujol A. Nucleic Acids Res. 2007; 35 (–D822): D815Crossref PubMed Scopus (65) Google Scholar). Cells are able to remodel the protein composition of their peroxisomes and to adjust the number of these organelles in response to physiological or environmental stimuli to cope with the metabolic needs. Consequently, peroxisomes are regarded as highly modular organelles, with different protein composition depending on the metabolic function or cell type. Fibrates, used as hypolipidemic drugs, induce both the proliferation of peroxisomes as well as the β-oxidation of fatty acids (4Lazarow P.B. De Duve C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2043-2046Crossref PubMed Scopus (1180) Google Scholar). These alterations of the metabolic functions of peroxisomes are accompanied by changes of proteins involved in the biogenesis of the organelles as well as in enzymes for the breakdown of fatty acids (5Lazarow P.B. Fujiki Y. Mortensen R. Hashimoto T. FEBS Lett. 1982; 150: 307-310Crossref PubMed Scopus (17) Google Scholar, 6Fahimi H.D. Reinicke A. Sujatta M. Yokota S. Ozel M. Hartig F. Stegmeier K. Ann. N. Y. Acad. Sci. 1982; 386: 111-135Crossref PubMed Scopus (120) Google Scholar). Several investigators in the past have used fibrate-induced peroxisome proliferation as a model to analyze selected aspects of peroxisome biogenesis or lipid metabolism, using techniques like Western blotting, enzyme assays, immunocytochemistry, Northern blotting, or in situ hybridization (6Fahimi H.D. Reinicke A. Sujatta M. Yokota S. Ozel M. Hartig F. Stegmeier K. Ann. N. Y. Acad. Sci. 1982; 386: 111-135Crossref PubMed Scopus (120) Google Scholar, 7Leighton F. Coloma L. Koenig C. J. Cell Biol. 1975; 67: 281-309Crossref PubMed Scopus (132) Google Scholar, 8Moody D.E. Reddy J.K. J. Cell Biol. 1976; 71: 768-780Crossref PubMed Scopus (108) Google Scholar, 9Beier K. Voölkl A. Hashimoto T. Fahimi H.D. Eur. J. Cell Biol. 1988; 46: 383-393PubMed Google Scholar, 10Chen N. Crane D.I. Biochem. J. 1992; 283: 605-610Crossref PubMed Scopus (14) Google Scholar, 11Kovacs W. Stangl H. Voölkl A. Schad A. Fahimi H.D. Baumgart E. Eur. J. Biochem. 2001; 268: 4850-4859Crossref PubMed Scopus (26) Google Scholar). However, complex rearrangements of the entire peroxisomal proteome could not be analyzed with these techniques. In this respect, mass spectrometry represents a straightforward alternative, particularly because of the development of more sensitive analyzers and the introduction of isotope labeling reagents (12Ong S.E. Mann M. Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1321) Google Scholar). Thus, it is now possible to characterize the different physiological states of an organelle structure by top-down quantitative mass spectrometry because of its ability to identify hydrophobic polypeptides, like integral membrane proteins (13Wu C.C. MacCoss M.J. Howell K.E. Yates J.R. II I Nat. Biotechnol. 2003; 21: 508-510Crossref PubMed Scopus (88) Google Scholar, 14Goshe M.B. Blonder J. Smith R.D. J. Proteome Res. 2003; 2: 153-161Crossref PubMed Scopus (76) Google Scholar, 15Bagshaw R.D. Mahuran D.J. Callahan J.W. Mol. Cell. Proteomics. 2005; 4: 133-143Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 16Schirmer E.C. Gerace L. Trends Biochem. Sci. 2005; 30: 551-558Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), to investigate quantitative changes of individual proteins, even of low abundance, by the use of stable isotope-labeled tags (17Andersen J.S. Mann M. EMBO Rep. 2006; 7: 874-879Crossref PubMed Scopus (166) Google Scholar), and to record modulations of selected pathways in response to exogenous stimuli. Last but not least, it enables the identification of previously undetected proteins and their integration into a cellular network as a basis for further functional studies. Because of their small proteome, peroxisomes represent a model of comparably low complexity suitable to use quantitative MS 3The abbreviations used are: MS, mass spectrometry; ACOX1, acyl-CoA oxidase 1; d-PBE, peroxisomal D-bifunctional enzyme; l-PBE, peroxisomal l-bifunctional enzyme; Tysnd1, trypsin domain containing 1; ACBD, acyl-CoA binding domain; MS/MS, tandem mass spectrometry; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropane-sulfonic acid; GFP, green fluorescent protein; PPAR, peroxisome proliferator-activated receptor; ALDP, adrenoleukodystrophy protein. in a functional approach. By using this technique, we have analyzed the peroxisomal proteome in response to fibrate treatment with the aim to elucidate complex proteome rearrangements, to identify undetected proteins, and to define their subcellular location and organelle-specific functions. In our analysis we isolated peroxisomes from bezafibrate-treated and control rats by means of density gradient centrifugation using a standardized procedure (18Voölkl A. Fahimi H.D. Eur. J. Biochem. 1985; 149: 257-265Crossref PubMed Scopus (101) Google Scholar). To further increase the precision of the analysis, isolated peroxisomes were carefully subfractionated into matrix and peripheral as well as integral membrane protein fractions to enrich low abundant proteins. The proteins were directly digested with trypsin and labeled with iTRAQ reagent for quantitative liquid chromatography-MS/MS analysis. Thus, we were able to identify 134 proteins, including 15 new predicted open reading frames. Ten of them could be verified by cDNA cloning, and seven were shown to localize at least partially to peroxisomes. The quantitative assessment of protein abundance in response to the fibrate treatment revealed no new fibrate-induced protein species. Convincing evidence, however, is provided by the quite distinct induction rates of long chain fatty acid synthetase and PMP70 in contrast to very long chain fatty acid synthetase and ALDP and the finding that long chain fatty acids (C16–C18) rather than very long chain fatty acids (C22–C26) are the primary substrates of rat liver peroxisomes after fibrate treatment. Maintenance and Bezafibrate Treatment of Rats—Female Sprague-Dawley rats (200–300 g) were kept in accordance with the guidelines of the humane care and use of laboratory animals at the Zentrale Versuchstieranlage, University of Heidelberg, Germany. Each of 50 animals was fed for 10 days with 20 g of Altromin 1324 (Altromin International, Lage, Germany) containing 1 mg/g bezafibrate (Roche Diagnostics), equaling a dose of ∼75 mg/kg body weight. The control group of 70 animals was fed with the same amount of untreated Altromin 1324. After the 10-day treatment, the animals were starved overnight and anesthetized with chloral hydrate. Subsequently, livers were excised, weighed, and minced in ice-cold homogenization buffer (250 mm sucrose, 5 mm MOPS, 1 mm EDTA, 0.1% ethanol, 2 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 1 mm ɛ-aminocaproic acid, pH 7.2). Isolation and Subfractionation of Peroxisomes—Homogenization of the tissue and subcellular fractionation was performed according to Voölkl and Fahimi (18Voölkl A. Fahimi H.D. Eur. J. Biochem. 1985; 149: 257-265Crossref PubMed Scopus (101) Google Scholar) with a few modifications described by Weber and co-workers (19Weber G. Islinger M. Weber P. Eckerskorn C. Voelkl A. Electrophoresis. 2004; 25: 1735-1747Crossref PubMed Scopus (49) Google Scholar). Finally, purified peroxisomes were washed once with gradient buffer (5 mm MOPS, 1mm EDTA, 0.1% ethanol, 2 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 1 mm ɛ-aminocaproic acid, pH 7.2) and stored at –80 °C. For subfractionation into matrix, peripheral, and integral membrane compartments, peroxisomes were thawed and centrifuged at 30,000 × g, resulting in supernatant S1 and pellet P1. P1 was resuspended in TVBE buffer (1 mm Na2CO3, 1 mm EDTA, 0.1% ethanol, 0.01% Triton X-100, pH 7.6), supplemented to 0.1% Triton and sonicated with two 15-s pulses at 100 watts for quantitative membrane disruption. Thereafter, a second centrifugation step at 100,000 × g for 30 min, resulted in a first membrane pellet (P2) and supernatant S2. To remove peripherally attached proteins, P2 was suspended in 250 mm KCl and centrifuged again at 100,000 × g (S3 and P3). Subsequently, P3 was suspended in 100 mm Na2CO3 and kept on ice for 30 min. After a final centrifugation at 100,000 × g, the supernatant (S4), containing mainly the core protein urate oxidase (>90%), was discarded, and the integral membrane pellet (P4) was washed and stored in gradient buffer. For the matrix fraction, S1 and S2 were combined, concentrated with VivaSpin columns (MW 5000, Sartorius, Goettingen, Germany), and dialyzed in gradient buffer. Supernatant S3 was concentrated and dialyzed in the same way to obtain a fraction of peripherally attached membrane proteins. iTRAQ Labeling and Liquid Chromatography-MS/MS—To ensure that equal protein amounts were subjected to iTRAQ labeling, first protein concentrations of the subfractions were determined by the Bradford method. For a more precise comparison of protein amounts in the membrane fractions, aliquots of all three subfractions were run on an SDS gel and stained with Coomassie Blue. Thereafter, staining intensity of the protein pattern was compared by densitometric analysis of the gel (Quantity One Software, Bio-Rad). Peroxisomal proteins (100 μg per sample) were trypsin-digested (membrane pellets were first solubilized in 0.8% RapiGest, Waters) and tagged with iTRAQ reagents according to the protocol provided by the manufacturer (Applied Biosystems). The iTRAQ-labeled peptides were resolved in the first-dimensional liquid chromatography with a polysulfoethyl A column. Peptides were eluted with a linear gradient of 0–500 mm KCl in 20% acetonitrile, 10 mm KH2PO4, pH 2.9, over 25 min at a flow rate of 50 μl/min. Fractions were collected at 1-min intervals, dried, and redissolved in 30 μl of 0.1% trifluoroacetic acid. For the second-dimensional liquid chromatography, 10 μl were injected into a capillary C18 column (150 × 100 mm inner diameter. column) at 500 nl/min and separated with a linearly increasing concentration of acetonitrile from 5 to 50% in 30 min and to 100% in 5 min. The eluent was mixed with matrix (7 mg of α-cyanohydroxycinnaminic acid in 1 ml of 50% acetronitrile, 0.1% trifluoroacetic acid, 10 mm dicitrate ammonium) delivered at a flow rate of 1.5 ml/min and deposited off-line to the Applied Biosystems metal target every 15 s for a total of 192 spots, and analyzed on an ABI 4700 proteomics analyzer. Peptide CID was performed at 1 kV; the collision gas was nitrogen. MS/MS spectra were collected from 5000 laser shots. The peptides with signal to noise ratio above 50 at the MS mode were selected for MS/MS experiment; a maximum of 30 MS/MS was allowed per spot. The precursor mass window was 180 relative resolution (at full width half-maximum). MS/MS spectra were searched against the rat and mice data bases (Celera Discovery System, CDS) using GPS Explorer (ABI) and Mascot (MatrixScience) with trypsin specificity and fixed iTRAQ modifications at lysine residue and the N termini of the peptides. Mass tolerance was 100 ppm for precursor ions and 0.5 Da for fragment ions; one missed cleavage was allowed. For each MS/MS spectrum, a single peptide annotation with the highest Mascot score was retrieved. CDS protein sequence redundancy was removed by clustering the precursor protein sequences of the retrieved peptides using the cluster algorithm Cd hit (20Li W. Godzik A. Bioinformatics (Oxf.). 2006; 22: 1658-1659Crossref PubMed Scopus (6447) Google Scholar) at a sequence similarity threshold of 90%. Subsequently, all peptides were matched against the obtained protein clusters; those peptides that matched more than one protein cluster represent common sequences between proteins and were not considered for protein identification and quantification. For quantification, peak areas for each iTRAQ signature peak were obtained and corrected according to the manufacturer’s instruction to account for the differences in isotopic overlap. To compensate for the possible variation in the starting amount between the samples, the individual peak areas of each iTRAQ signature peak were log2-transformed to obtain a normal distribution and then normalized to the total peak area of this signature peak. Low signature peaks generally have larger variation, which may compromise the quantitative analysis of the proteins. Therefore, the iTRAQ signature peaks lower than 2000 were removed from quantitation. Within an MS experiment a Student’s t test was used over bezafibrate versus control groups. In the mass spectrometry, samples of peripheral and integral membrane proteins were run in parallel to obtain quantitative information on proteins retained at the membrane after carbonate stripping (data not shown). Because most of the proteins detected in the peripheral fraction were found in the matrix as well but specifically enriched proteins were not detected, the data for these subgroups were combined in Table 4. The ratio obtained for individual proteins identified in both groups was used to calculate a conjoint value for the induction in both subfractions to reduce data complexity. The mass spectrometry of the peroxisomal matrix was done independently, in a double experiment, and hence the quantitative measurements are shown as autonomous data. As a threshold for proteins presented, we used the identification of at least three peptides with the ion score of the highest peptide match >95% or two peptides with the highest peptide match >99%. For completion, some known peroxisomal proteins with identification rates below the threshold (indicated in Table 4) were included in the lists, because of the high probability of detection in a fraction of isolated peroxisomes. Nevertheless, only proteins detected with at least three peptides (peak area > 2000) were considered for quantification.TABLE 4List of protein identifications Open table in a new tab Immunoblotting of Selected Peroxisomal Proteins and Enzyme Assays—Western blotting was performed according to the semi-dry method using polyvinylidene difluoride membranes (21Kyhse-Andersen J. J. Biochem. Biophys. Methods. 1984; 10: 203-209Crossref PubMed Scopus (2159) Google Scholar). Antibodies used for the immunodetection of selected proteins are of rabbit origin and were raised as published previously (uricase, PMP70, catalase, l-bifunctional enzyme, hydroxyacid oxidase, and acyl-CoA oxidase 1) or kind gifts from T. Hashimoto, Shinshu University School of Medicine, Nagano, Japan (long chain fatty acid CoA ligase 1, PMP22, and Pex11α), J. Adamski, GSF-Neuherberg, Germany (d-bifunctional enzyme), and G. Dodt, University of Tuebingen, Germany (Pex14). The polyclonal antibodies against GRP78/BiP and PECI were purchased from BD Biosciences. The activity of acyl-CoA oxidase 1 (ACOX1) was determined according to Small et al. (22Small G.M. Burdett K. Connock M.J. Biochem. J. 1985; 227: 205-210Crossref PubMed Scopus (286) Google Scholar) and total β-oxidation activities as described by Lazarow and De Duve (4Lazarow P.B. De Duve C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2043-2046Crossref PubMed Scopus (1180) Google Scholar). Enzyme activities of catalase, esterase, cytochrome c oxidase, and acid phosphatase were performed as described previously (18Voölkl A. Fahimi H.D. Eur. J. Biochem. 1985; 149: 257-265Crossref PubMed Scopus (101) Google Scholar). Plasmids and Cloning of Expression Vectors for Newly Identified Peroxisomal Proteins—For the cloning of newly identified potential peroxisomal proteins, total rat liver RNA was isolated using RNeasy mini kit (Qiagen, Hilden, Germany) and reversetranscribed into cDNA by SuperScript III reverse transcriptase (Invitrogen). Primers spanning the whole predicted open reading frame of the genes of interest were synthesized at MWG (Muönchen, Germany). PCR amplification of the target cDNAs was performed using Pfx polymerase (Invitrogen), and the amplicon was directly cloned into TOPO II vectors. After sequencing of the inserts (GATC, Konstanz, Germany), the open reading frames were isolated and ligated into pCMV-Tag vectors (Stratagene, Amsterdam, The Netherlands) for generation of fusions with the Myc epitope. For expression of Myc fusion proteins in mammalian cell lines, the coding regions were under control of the cytomegalovirus promoter. Except for protein sequences with predicted PTS1 signals at the C terminus where only N-terminal cDNA versions were constructed, both C- and N-terminally tagged versions were generated (see Table 1). For the analysis of wild-type protein variants, open reading frames of XP_237252.1 (ACBD5.2) and PMP52 were cloned into pCDNA3.1 vectors. In the case of XP_237252.1 (ACBD5) the 5′-end of the cDNA was determined using the GeneRacer RACE-PCR kit (Invitrogen). For cotransfection experiments, plasmid pDsRed (Clontech) or pGHL252 were used to visualize transgenic cells. The plasmid pGHL252 contains the sequence encoding a fusion protein (RFP-PTS1) of the red fluorescent protein (from pDsRed) with a peroxisomal targeting signal SKL under control of the murine ROSA26 promoter as well as an expression cassette for selection of bleomycin in prokaryotic and eukaryotic cells.TABLE 1Plasmid constructs used for microscopic investigationsProtein nameAbbreviation used in textProtein accession numberGenBank™ accession number of cDNAWild typeN-terminal tagC-terminal tagAcyl-CoA dehydrogenase family member 11Q80XL6XM_236582–Myc–δ3,δ2-Enoyl-CoA isomeraseNP_001009275BC088178.1–Myc–Zinc-binding alcohol dehydrogenase domain containing 2Zn-ADHXP_214526XM_001060611–Myc–Mosc domain containing protein 2MOSC2O88994AF095741.1–MycMycTrypsin domain containing 1Tysnd1XP_345107.1XP_001056113.1–Myc–PMP52PMP52NP_001013918NM_001013896+MycMycShort chain dehydrogenase 7bDhrs7bXP_213317.1BC086453.1–MycMycDhrs7b.2EF445633MycMycAcyl-CoA-binding protein ACBD5ACBD5.1XP_237252.1EF026991+MycMycACBD5.2EF026992MycMycPotential dienelactone hydrolaseNP_001008770.1BC088459.1–MycMycLactamase, β2NP_663356NM_001024247.1–Myc–Ezrin-radixin-moesin-binding phosphoproteinEBP 50Q9JJ19AF154336.1–Myc– Open table in a new tab Cell Culture and Cell Lines—Chinese hamster ovary (CHO) cells and CHO-derived cells were maintained in Ham’s F-12 medium supplemented with 10% fetal calf serum (PAA, Coölbe, Germany) in a fully humidified incubator at a temperature of 37 °C and an atmosphere of 5% CO2 in air. For visualization of peroxisomes in living cells, CHO cells were transfected with a mixture of plasmid pVgRXR (Invitrogen) and of plasmid pGHL97 (23Islinger M. Luöers G.H. Zischka H. Ueffing M. Voölkl A. Proteomics. 2006; 6: 804-816Crossref PubMed Scopus (54) Google Scholar) that contains an expression cassette for a GFP-PTS1 fusion protein under control of the murine phosphoglycerate kinase promoter. Cell clones were analyzed by fluorescence microscopy for expression of the GFP-PTS1 fusion protein, and positive clones were isolated for further analysis. One of the resulting cell clones with homogeneous expression of the GFP-PTS1 was named BGL69 and was used in this study. Subcellular Localization of Epitope-tagged Proteins—For colocalization studies of epitope-tagged proteins with peroxisomes, expression vectors were transiently transfected into BGL69 cells cultured on glass coverslips using the Gene Porter system (Peqlab, Erlangen, Germany) according to the manufacturer’s recommendations. After transfection, coverslips were washed with PBS and then fixed at room temperature in 4% freshly depolymerized paraformaldehyde in 0.15 m HEPES, pH 7.4, for 15 min. Cells were washed and permeabilized with 0.2% Triton X-100 and 0.2% Tween 20 in PBS. To reduce nonspecific binding of antibodies, cells were blocked for 30 min with Roti-Block (Roth, Karlsruhe, Germany) in PBS. Roti-Block was also used for dilution of specific primary and Cy3-labeled secondary antibodies. Cells were incubated with monoclonal primary antibodies against the Myc epitope (9E10, kindly provided by M. Schrader, University of Marburg, Germany) for 1 h. After washing the coverslips three times for 5 min, they were incubated with the Cy3-labeled goat anti-mouse antibodies (Dianova, Hamburg, Germany) for 1 h. Control stainings were performed according to the same protocol except that the primary antibodies were omitted. After a final washing, the coverslips were mounted with 50% glycerol in PBS containing 1.5% (w/v) n-propyl gallate as an antifade. For visualization of mitochondria, living CHO cells were incubated in Ham’s F-12 medium supplemented with 0.5 μm MitoTracker Red (Invitrogen) for 30 min followed by an incubation in regular medium for 15 min. Cells were fixed and propagated for immunocytochemistry as described. For costaining of the Myc epitope in Mitotracker-labeled cells, the monoclonal anti-Myc antibodies were visualized using a Cy2-labeled goat anti-mouse antiserum (Dianova, Hamburg, Germany). Visualization of the endoplasmic reticulum with Concanavalin A was performed as described recently (23Islinger M. Luöers G.H. Zischka H. Ueffing M. Voölkl A. Proteomics. 2006; 6: 804-816Crossref PubMed Scopus (54) Google Scholar). Light microscopic analysis was carried out on a Leica DMRE fluorescence microscope (Leica, Wetzlar, Germany) with standard filters for detection of fluorescein isothiocyanate and Cy3. Digital images were acquired using a DXM1200F digital camera system with the Nikon ACT-1 software. Fibrates are known to induce hepatomegaly, to stimulate peroxisome proliferation, and to induce the enzymes involved in the β-oxidation of fatty acids (6Fahimi H.D. Reinicke A. Sujatta M. Yokota S. Ozel M. Hartig F. Stegmeier K. Ann. N. Y. Acad. Sci. 1982; 386: 111-135Crossref PubMed Scopus (120) Google Scholar). Accordingly, the bezafibrate treatment of animals was found to cause an increase in liver weight of 18.8% compared with untreated control animals (9.24 ± 1.06 versus 7.78 ± 0.64, p < 0.001). A more pronounced effect of the drug treatment was observed at the organellar level. Total peroxisomal protein increased from 26.5 to 102.4 μg/g liver, reflecting a 3.9-fold augmentation. Because this ratio was more or less consistent in all peroxisomal subfractions (matrix, 4.2, integral membrane fraction 3.3; peripheral membrane fraction, 3.7), peroxisomes were increased primarily in their number and to a lesser extent in size. In Table 2, the properties of peroxisomes isolated in up to 10 experiments are summarized. According to the values concerning catalase and β-oxidation activities, the peroxisomal fractions obtained are highly enriched (purity of ∼95%), with an average contamination by mitochondria and by endoplasmic reticulum of 2%, and hence are expected to meet the requirements for proteome analysis. To evaluate also the purity and contamination of peroxisomes isolated from bezafibrate-treated animals, we used the marker enzymes cytochrome c oxidase (mitochondria), esterase (endoplasmic reticulum), and acid phosphatase (lysosomes) to compare the former with “control peroxisomes.” Because of the increase in peroxisome number in response to the treatment, most contaminating organelles decreased. In detail, the activities of cytochrome c oxidase were reduced by the factor of 3.4 and of esterase by 1.92, whereas that of acid phosphatase was augmented by the factor of 2.3 (Table 3), although no lysosomal proteins could be identified in the MS analysis.TABLE 2Properties of the peroxisome fraction (PO) purified from livers of untreated ratsEnzymeNo. of exp.Units/g liverUnits/mg liver proteinRecovery in PORelative specific activitySpecific activityPurity/contaminationmg/g liver% total liverUnits/mg PO protein%Protein10265.36 ± 81.840.28 ± 0.08Catalase1053.86 ± 11.34202.96 × 10-39.96 ± 1.9237.67 ± 4.287.6595.31Lipid β-oxidation40.951 ± 0.2553.58 × 10-311.18 ± 3.2536.32 ± 5.090.13091.89β-Glucuronidase410.12 ± 1.5638.14 × 10-30.014 ± 0.010.069 ± 0.0560.0030.139Esterase6326.15 ± 65.461.2290.014 ± 0.010.093 ± 0.070.1141.99Cytochrome c oxidase833.10 ± 9.45124.74 × 10-30.018 ± 0.020.09 ± 0.050.0111.82Lactate dehydrogenase2448.15 ± 6.981.6890.060.2940.497 Open table in a new tab TABLE 3Marker enzymes activities in isolated peroxisomes of bezafibrate-treated and control animalsEnzymeControlBezafibrateUnits/mgUnits/mgCatalase8.58.0ACOX 1 (C18)24.1324.83ACOX 1 (C24)1.130.30β-Oxidation (C18)16.98 (milliunits/mg)45.45 (milliunits/mg)β-Oxidation (C24)0.30 (milliunits/mg)0.22 (milliunits/mg)Unspecific esterase0.1400.073Acidic phosphatase0.0960.223Cytochrome c oxidase0.0170.005 Open table in a new tab To improve the precision of our MS analysis, isolated peroxisomes were subfractionated into so-called matrix, peripheral, and integral membrane fractions prior to MS/MS. Because peroxisomes exhibit a quite unique ratio of membrane to matrix (usually 1:10) and, moreover, are characterized by a portion of “poorly soluble” matrix proteins, which do not easily separate from the membrane (24Alexson S.E. Fujiki Y. Shio H. Lazarow P.B. J. Cell Biol. 1985; 101: 294-304Crossref PubMed Scopus (90) Google Scholar), we had to adapt our subfractionation protocol to obtain a membrane preparation as pure as possible. For this purpose, we first disrupted the organelles by freezing and thawing in the presence of 0.075% Triton X-100, followed by sonication in 0.1% Triton X-100. Using this procedure, the matrix fraction was indeed enriched with the poorly soluble matrix proteins, e.g. the bifunctional enzymes (supplemental Fig. S1a), which was not possible with lower Triton X-100 concentrations. Yet, the Triton treatment also solubilized the integral membrane proteins to a minor extent (supplemental Fig. S1b), which was accepted to avoid contamination of the membrane fraction by matrix proteins. High salt KCl treatment is typically used for extraction of peripheral membrane proteins. However, because a complete clearance of poorl" @default.
• W2059574370 created "2016-06-24" @default.
• W2059574370 creator A5006363548 @default.
• W2059574370 creator A5018627313 @default.
• W2059574370 creator A5037003034 @default.
• W2059574370 creator A5060424166 @default.
• W2059574370 creator A5082400216 @default.
• W2059574370 date "2007-08-01" @default.
• W2059574370 modified "2023-10-12" @default.
• W2059574370 title "Rat Liver Peroxisomes after Fibrate Treatment" @default.
• W2059574370 cites W1497620344 @default.
• W2059574370 cites W1603279882 @default.
• W2059574370 cites W1736640447 @default.
• W2059574370 cites W1826460582 @default.
• W2059574370 cites W1838916330 @default.
• W2059574370 cites W1963530308 @default.
• W2059574370 cites W1965171820 @default.
• W2059574370 cites W1965580938 @default.
• W2059574370 cites W1966079498 @default.
• W2059574370 cites W1967573492 @default.
• W2059574370 cites W1970805949 @default.
• W2059574370 cites W1975558168 @default.
• W2059574370 cites W1987876984 @default.
• W2059574370 cites W1993303930 @default.
• W2059574370 cites W1993959069 @default.
• W2059574370 cites W1996050234 @default.
• W2059574370 cites W2003481405 @default.
• W2059574370 cites W2005411425 @default.
• W2059574370 cites W2005833247 @default.
• W2059574370 cites W2008202623 @default.
• W2059574370 cites W2010783362 @default.
• W2059574370 cites W2014879410 @default.
• W2059574370 cites W2015124930 @default.
• W2059574370 cites W2019148902 @default.
• W2059574370 cites W2020214584 @default.
• W2059574370 cites W2022525198 @default.
• W2059574370 cites W2024028178 @default.
• W2059574370 cites W2028538636 @default.
• W2059574370 cites W2030850427 @default.
• W2059574370 cites W2033603842 @default.
• W2059574370 cites W2042042511 @default.
• W2059574370 cites W2046855853 @default.
• W2059574370 cites W2060772102 @default.
• W2059574370 cites W2061237536 @default.
• W2059574370 cites W2061361921 @default.
• W2059574370 cites W2063731065 @default.
• W2059574370 cites W2063834877 @default.
• W2059574370 cites W2066656769 @default.
• W2059574370 cites W2074876603 @default.
• W2059574370 cites W2075688260 @default.
• W2059574370 cites W2077141296 @default.
• W2059574370 cites W2081312507 @default.
• W2059574370 cites W2084486974 @default.
• W2059574370 cites W2091649205 @default.
• W2059574370 cites W2092934795 @default.
• W2059574370 cites W2093023633 @default.
• W2059574370 cites W2093048036 @default.
• W2059574370 cites W2094000826 @default.
• W2059574370 cites W2094974920 @default.
• W2059574370 cites W2095619127 @default.
• W2059574370 cites W2101641567 @default.
• W2059574370 cites W2112606108 @default.
• W2059574370 cites W2121546157 @default.
• W2059574370 cites W2127924963 @default.
• W2059574370 cites W2128697397 @default.
• W2059574370 cites W2130425468 @default.
• W2059574370 cites W2130452115 @default.
• W2059574370 cites W2133459569 @default.
• W2059574370 cites W2150158059 @default.
• W2059574370 cites W2155051500 @default.
• W2059574370 cites W2156125289 @default.
• W2059574370 cites W2156330912 @default.
• W2059574370 cites W2234188892 @default.
• W2059574370 cites W4250989610 @default.
• W2059574370 cites W5739282 @default.
• W2059574370 cites W87093425 @default.
• W2059574370 doi "https://doi.org/10.1074/jbc.m610910200" @default.
• W2059574370 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17522052" @default.
• W2059574370 hasPublicationYear "2007" @default.
• W2059574370 type Work @default.
• W2059574370 sameAs 2059574370 @default.
• W2059574370 citedByCount "110" @default.
• W2059574370 countsByYear W20595743702012 @default.
• W2059574370 countsByYear W20595743702013 @default.
• W2059574370 countsByYear W20595743702014 @default.
• W2059574370 countsByYear W20595743702015 @default.
• W2059574370 countsByYear W20595743702016 @default.
• W2059574370 countsByYear W20595743702017 @default.
• W2059574370 countsByYear W20595743702018 @default.
• W2059574370 countsByYear W20595743702019 @default.
• W2059574370 countsByYear W20595743702020 @default.
• W2059574370 countsByYear W20595743702021 @default.
• W2059574370 countsByYear W20595743702022 @default.
• W2059574370 countsByYear W20595743702023 @default.
• W2059574370 crossrefType "journal-article" @default.
• W2059574370 hasAuthorship W2059574370A5006363548 @default.
• W2059574370 hasAuthorship W2059574370A5018627313 @default.
• W2059574370 hasAuthorship W2059574370A5037003034 @default.

• next