Isolation of Peroxisomes
The investigation of unique functional and structural aspects of peroxisomes (PO) requires the preparation of highly purified fractions of this organelle. This is, however, hampered by two serious problems: (1) the relative paucity of PO (2% of total liver protein) and (2) their considerable fragility. Thus, mild homogenization conditions minimizing mechanical, hydrostatic, and osmotic stress have to be sustained.
In general, the isolation of PO is accomplished in three steps: (a) homogenization of the tissue or disruption of the cells; (b) subfractionation of the homogenate by differential centrifugation usually according to the classical scheme of de Duve et al. (1955); and (c) isolation of purified peroxisomes by density gradient centrifugation of the so-called light mitochondrial (λ) fraction.
Homogenization is commonly carried out in an isotonic medium (e.g., see Section III,A,1) at low salt concentrations to avoid aggregation. Addition of a chelator (e.g., EDTA), however, is feasible, as it prevents the aggregation of microsomes that may contaminate PO. Moreover, the homogenization buffer (HB) should be supplemented with antioxidants (dithiothreitol, DTT), as well as protease inhibitors (phenylmethylsulfonyl fluoride, PMSF; ε-aminocaproic acid) to block oxidative and proteolytic activities in the course of the isolation procedure.
For the purification of PO by density gradient centrifugation, three approaches have been developed. In the classic procedure (Leighton et al., 1968), sucrose gradients and the specialized type Beaufay rotor were employed. A self-generating Percoll gradient in conjunction with a vertical rotor is used for the isolation of PO under isotonic conditions (Neat et al., 1980). The most straightforward approach to obtain highly purified PO makes use of iodinated gradient media such as metrizamide, nycodenz or Optiprep in combination with a vertical rotor (Völkl and Fahimi, 1985; Hartl et al., 1985; van Veldhoven et al., 1996). In the latter media, PO band at the quite high density of 1.24 g/cm3 because of their permeability to low molecular weight compounds (van Veldhoven et al., 1983), well separated from lysosomes as well as from mitochondria and microsomes.
The method described in this article is a modification of the protocol established for the isolation of highly purified (>98%) PO from normal rat liver (Völkl and Fahimi, 1985). Meanwhile, it has been applied to livers and kidneys of several other mammalian species (Fahimi et al., 1993; Zaar et al., 1992), as well as for the isolation of PO from cell cultures (Schrader et al., 1994). With an additional differential centrifugation step, even peroxisomal subpopulations may be isolated according to this protocol, as has been demonstrated by Lüers et al. (1993).
II. INSTRUMENTATION AND MATERIALS
Morpholinopropane sulfonic acid (MOPS, Cat. No. 29 836), PMSF (Cat. No. 32 395), and DTT (Cat. No. 20 710) are from Serva (Heidelberg, FRG). Ethanol (Cat. No. 8006) and NaCl (Cat. No. 0277) are from J. T. Baker (Deventer, Netherlands). EDTA (Cat. No. E 2628-2) is from Max Keller (Mannheim, FRG), ε-aminocaproic acid (Cat. No. 62 075) is from Riedel-de Haen (Seelze, FRG), sucrose (Cat. No. 4621) is from Roth (Karlsruhe, FRG), and metrizamide (Cat. No. 10o 1983N) is from Life Technologies (76344 Eggenstein- Leopoldshafen).
Female rats of 220-250g body weight, starved overnight
III. PROCEDURES (FIG. 1)
A. Perfusion and Homogenization
B. Subcellular Fractionation
HB as described in Section III,A.
C. Metrizamide Density Gradient Centrifugation
Preparation of a Metrizamide Gradient
IV. OBSERVATIONS AND COMMENTS
The properties of the heavy peroxisomal fraction are listed in Table I. Estimated by the specific peroxisomal reference enzymes, it shows a purification rate of about 38-fold over the original homogenate. More than 95% of the total protein content of this fraction is contributed by peroxisomes (Völkl and Fahimi, 1985), with mitochondria and microsomes accounting for about 2% each and lysosomes for less than 1%. This is confirmed by electron microscopy, which shows that peroxisomes make up 98-99% of the fraction (Figs. 2 and 3). Many peroxisomes contain the typical inclusions of urate oxidase in the matrix (Fig. 2), but some extruded free cores are also found between the organelles. The electron-dense cytochemical reaction product of catalase after the incubation of filter preparations in the alkaline 3,3'-diaminobenzidine medium (Fahimi, 1969) is seen over the matrix of the majority of peroxisomes (Fig. 3), demonstrating their integrity and the absence of leakage of catalase.
The polypeptide pattern (SDS-PAGE) of rat liver heavy peroxisomes is shown in Fig. 4 (lane R) confirming their high degree of purity because of the absence of bands typical for mitochondria and microsomes. It also shows distinct differences in protein composition of hepatic peroxisomes between rat (R) and guinea pig (Fig. 4, lane G). The selective induction of specific peroxisomal proteins, such as the trifunctional protein (PH) in rats treated with hypolipidemic fibrates (lane Bz) with the concomitant reduction of catalase (Cat) and urate oxidase (UOx), is apparent in Fig. 5.
The extended procedure outlined here has been successfully employed to isolate heavy and light peroxisome subpopulations from normal and regenerating rat liver differing in density, size, shape, and enzymatic composition (Lüers et al., 1993). Moreover, peroxisome subsets as divergent as the former have been also obtained from the human heptoma cell line HepG2 (Schrader et al., 1994). However, further subpopulations, which are also present in those tissues, escape purification by conventional gradient centrifugation, most probably because their sedimentation properties are close to those of other subcellular organelles, particularly microsomes. Nevertheless, their purification has become an essential task in view of the functional significance of PO in humans in general (Wanders et al., 1995) and the putative importance of peroxisomal subpopulations in the biogenesis of this organelle in particular (Erdmann et al., 1997; Titorenko et al., 1997).
As an alternative to density gradient centrifugation, immune free flow electrophoresis (IFFE) has been introduced and applied successfully for the purification not only of regular PO (ρ = 1.22-1.24 g/cm3) from a light mitochondrial fraction of rat liver, but also of PO from heavy and postmitochondrial fractions (Völkl et al., 1997, 1999). IFFE combines the advantages of eletrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle, complexed with an antibody directed against the cytoplasmic domain of one of its integral membrane proteins, is diminshed greatly, provided the pH of the electrophoresis buffer is adjusted to pH-8.0, which is the pI of IgG molecules. The PO isolated by IFFE from the diverse hepatic subcellular fractions differed in their composition of matrix and membrane proteins as has been revealed by immunoblotting, thus supporting the view that IFFE is a valid method for the purification of distinct subsets of the peroxisomal compartment.
The original work in the laboratory of the authors has been supported by grants of the Deutsche Forschungsgemeinschaft, Bonn, FRG (Fa 146/1-3; Vo 317/3-1; SFB 352 and Vo 317/4-1) and Landesforschungsschwerpunkt- Programm of the State of Baden-Württemberg, FRG.
De Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R., and Appelmans, F. (1955). Intracellular distribution patterns of enzymes in rat liver tissue. Biochem. J. 60, 604-617.
Erdmann, R., Veenhuis, M., and Kunau, W. H. (1997). Peroxisomes: Organelles at the crossroads. Trends Cell Biol. 7, 400-407.
Fahimi, H. D. (1969). Cytochemical localization of peroxidatic activity of catalase in rat hepatic microbodies (peroxisomes). J. Cell Biol. 43, 275-288.
Fahimi, H. D., Baumgart, E., Beier, K., Pill, J., Hartig, F., and Völkl, A. (1993). Ultrastructural and biochemical aspects of peroxisome proliferation and biogenesis in different mammalian species. In "Peroxisomes: Biology and Importance in Toxicology and Medicine" (G. G. Gibson and B. Lake, eds.), pp. 395-424. Taylor and Francis, London.
Hartl, F. U., Just, W. W., Köster, A., and Schimassek, H. (1985). Improved isolation and purification of rat liver peroxisomes by combined rate zonal and equilibrium density centrifugation. Arch. Biochem. Biophys. 237, 124-134.
Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Coffey, J. W., Fowler, S., and De Duve, C (1968). The large-scale preparation of peroxisomes, mitochondria and lysosomes from the livers of rats injected with Triton WR-1339. J. Cell Biol. 37, 482-513.
Lüers, G., Hashimoto, T., Fahimi, H. D., and Völkl, A. (1993). Biogenesis of peroxisomes : Isolation and characterization of two distinct peroxisomal populations from normal and regenerating rat liver. J. Cell Biol. 121, 1271-1280.
Neat, C. E., Thomassen, M. S., and Osmundsen, H. (1980). Induction of peroxisomal β-oxidation in rat liver by high-fat diets. Biochem. J. 186, 369-371.
Schrader, M., Baumgart, E., Völkl, A., and Fahimi, H. D. (1994). Heterogeneity of peroxisomes in human hepatoblastoma cell line HepG2: Evidence of distinct subpopulations. Eur. Cell Biol. 64, 281-294.
Titorenko, V., Ogrydziak, D. M., and Rachubinski, R. A. (1997). Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica. Mol. Cell Biol. 17, 5210-5226.
Van Veldhoven, P., Debeer, L. J., and Mannaerts, G. P. (1983). Waterand solute- accessible spaces of purified peroxisomes. Biochem. J. 210, 685-693.
Van Veldhoven, P., Baumgart, E., and Mannaerts, G. P. (1996). Iodixanol (Optiprep), an improved density gradient medium for the isoosmotic isolation of rat liver peroxisomes. Anal. Biochem. 237, 17-23.
Völkl, A., and Fahimi, H. D. (1985). Isolation and characterization of peroxisomes from the liver of normal untreated rats. Eur. J. Biochem. 149, 257-265.
Völkl, A., Mohr, H., and Fahimi, H. D. (1999). Peroxisomal subpopulations of the rat liver: Isolation by immune free flow electrophoresis. J. Histochem. Cytochem. 47, 1111-1117.
Völkl, A., Mohr, H., Weber, G., and Fahimi, H. D. (1997). Isolation of rat hepatic peroxisomes by means of free flow electrophoresis. Electrophoresis 18, 774-780.
Wanders, R. J. A., Schutgens, R. B. H., and Barth, P. G. (1995). Peroxisomal disorders: A review. J. Neuropathol. Exp. Neurol. 54, 726-739.
Zaar, K. (1992). Structure and function of peroxisomes in the mammalian kidney. Eur. J. Cell Biol. 59, 233-254.
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