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 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 (2700g(max)) 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 beta-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.
Islinger
Markus
M
Department of Anatomy and Cell Biology, Ruprecht-Karl University, 69120 Heidelberg, Germany. Markus.Islinger@urz.uni-heidelberg.de
Li
Ka Wan
KW
Loos
Maarten
M
Liebler
Sven
S
Angermüller
Sabine
S
Eckerskorn
Christoph
C
Weber
Gerhard
G
Abdolzade
Afsaneh
A
Völkl
Alfred
A
D000704:Analysis of Variance; D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D003605:D-Amino-Acid Oxidase / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods*; D005260:Female; D007553:Isotope Labeling; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000737:chemistry* / Q000378:metabolism; D020675:Peroxisomes / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
D003605:D-Amino-Acid Oxidase