Peroxisomes from the heavy mitochondrial fraction: isolation by zonal free flow electrophoresis and quantitative mass spectrometrical characterization
Peroxisomes are a heterogeneous group of organelles fulfilling reactions in a variety of metabolic pathways. To investigate if functionally different subpopulations can be found within a single tissue, peroxisomes from the heavy mitochondrial fraction (HM-Po) of the rat liver were isolated and compa...
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| Hauptverfasser: | , , , , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
4 January 2010
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| In: |
Journal of proteome research
Year: 2010, Jahrgang: 9, Heft: 1, Pages: 113-124 |
| ISSN: | 1535-3907 |
| DOI: | 10.1021/pr9004663 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1021/pr9004663
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| Verfasserangaben: | Markus Islinger, Ka Wan Li, Maarten Loos, Sven Liebler, Sabine Angermüller, Christoph Eckerskorn, Gerhard Weber, Afsaneh Abdolzade, and Alfred Völkl |
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| 520 | |a Peroxisomes are a heterogeneous group of organelles fulfilling reactions in a variety of metabolic pathways. To investigate if functionally different subpopulations can be found within a single tissue, peroxisomes from the heavy mitochondrial fraction (HM-Po) of the rat liver were isolated and compared to “classic” peroxisomes from the light mitochondrial fraction (LM-Po) using iTRAQ tandem mass spectrometry. Peroxisomes represent only a minor although significant proportion of the heavy mitochondrial fraction (2700gmax) precluding a straightforward isolation by standard protocols. Thus, a new fractionation scheme suitable for a subsequent mass spectrometrical analysis was developed using a combination of centrifugation techniques and zonal free flow electrophoresis. On the basis of the iTRAQ-measurement, a variation of the peroxisomal protein pattern between both fractions could be determined and further confirmed by immunoblotting and enzyme activity assays for selected proteins: whereas peroxisomes from the light mitochondrial fraction contain high amounts of β-oxidation enzymes, peroxisomes from the heavy mitochondrial fraction were dominated by enzymes fulfilling other functions. Among other findings, HM-Po was characterized by a high abundance of D-amino acid oxidase. This observation can be mirrored at the ultrastructural level, where tissue sections of liver peroxisomes show a heterogeneous staining for the enzymes activity, when visualized by the cerium technique. | ||
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