Isolation of Peroxisomes

I. INTRODUCTION
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
A. Instrumentation

1. Perfusion device (self-made)
2. A 30-ml Potter-Elvehjem tissue grinder (Cat. No. 1931 05145) with a loose-fitting Teflon pestle (Cat. No. 1931 05155; clearance 0.10-0.15mm) and a motor-driven homogenizer (Cat. No. 5308 50001) are from Migge, 69123 Heidelberg, FRG.
3. Refrigerated low- and high-speed centrifuge (e.g., Beckman TJ-6 and J 2-21); ultracentrifuge (e.g., Beckman L90) with corresponding rotors (e.g., Beckman JA-20; VTi 50).
4. Refractometer

B. Chemicals
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).

C. Animals
Female rats of 220-250g body weight, starved overnight

FIGURE 1 Flowchart for the isolation of highly purified (>98%) peroxisomes from rat liver.

III. PROCEDURES (FIG. 1)
A. Perfusion and Homogenization
Solutions

1. Homogenization buffer (HB): To make 1 liter, dissolve 85.56g of sucrose (250mM), 1.046g of MOPS (5mM), 0.372 g of EDTA-Na₂ (1mM), and 1 ml of ethanol (0.1%) in distilled water, adjust pH to 7.2 with NaOH, and add water up to 1 liter. Store at 4°C. Prior to use add per 100ml: 0.2µl of 0.1M PMSF, 0.1ml of 1M ε-aminocaproic acid, and 20µl of 1M DTT.
2. Saline (0.9%): To make 1 liter, dissolve 9g of NaCl in distilled water and adjust to a total volume of 1 liter.

Steps

1. Anesthesize the animal (e.g., by ip injection of chloralhydrate).
2. Weigh the animal, open abdominal cavity, and perfuse liver with 0.9% saline via the portal vein until all blood is drained away.
3. Remove liver, dissect connective tissue, and weigh and cut liver into small pieces collected in a Potter tissue grinder held in an ice bath containing 3 ml/g (wet liver weight) of ice-cold HB.
4. Homogenize tissue with a loose-fitting pestle, applying a single down-and-up stroke at 1000rpm.
5. Pour homogenate into a 50-ml centrifuge tube.

B. Subcellular Fractionation
Solution
HB as described in Section III,A.

Steps

1. To remove debris, unbroken hepatocytes, and blood cells, as well as most of nuclei, centrifuge the total homogenate at 70g for 10min in a refrigerated low-speed centrifuge.
2. Carefully pour off the supernatant (loose pellet !), resuspend pellet in 2ml/g of ice-cold HB, rehomogenize, and spin again under the same conditions.
3. Pour off the second supernatant and combine it with the first one (postnuclear supernatant); discard the pellet.
4. Centrifuge postnuclear supernatant at 1950g for 10min in a refrigerated high-speed centrifuge.
5. Decant supernatant (firm pellet), resuspend pellet manually in 1 ml/g of ice-cold HB using a glass rod, and spin again at 1950g. The final pellet (heavy mitochondrial fraction) contains the majority of mitochondria, large microsomal sheets, and some remaining nuclei. The combined supernatants represent the postmitochondrial supernatant.
6. Subject the latter to 25,300g for 20min; remove supernatant, including the reddish fluffy layer by suction; resuspend pellet in about 10 ml of ice-cold HB using a glass rod; and recentrifuge at 25,300g for 15min. Resuspend the final pellet in 5ml of ice-cold HB again by means of a glass rod, which comprises the enriched heavy peroxisomal = light mitochondrial (λ) fraction. The corresponding supernatant may be either used directly to prepare a microsomal fraction and a final supernatant (soluble proteins mostly of cytosolic origin) or processed further for the isolation of "light PO."
7. To this extent it is centrifuged at an integrated relative centrifugal force (RCF) of 4.47 × 10⁵gmin (g_max = 39,000). Resuspend the pellet thus obtained in 5 ml of ice-cold HB using a glass rod; this pellet represents the enriched light peroxisomal fraction (Luers et al., 1993).

C. Metrizamide Density Gradient Centrifugation
Solutions

1. Gradient buffer (GB): To make 1 liter, dissolve 1.046g of MOPS (5 mM), 0.372g of EDTA-Na₂ (1 mM), and 1 ml of ethanol (0.1%) in distilled H₂O, adjust with NaOH to pH 7.2, and add H₂O up to 1 liter. Store at 4°C. Prior to use add per 100ml: 0.2ml of 0.1M PMSF, 0.1 ml of 1M ε-aminocaproic acid, and 20 µl of 1M DTT.
2. Metrizamide solutions (MS)
   1. 60% (w/v) stock solution: Dissolve 60g of metrizamide in GB by stirring. Add GB up to 100ml. Store at 4~
   2. Gradient solutions: To prepare one gradient, take 3.78, 3.38, 3.53, 2.06, and 3.2 ml of the 60% stock solution. Add up GB to 10, 7, 6, 3, and 4ml using a refractometer to definitely adjust densities to 1.12, 1.155, 1.19, 1.225, and 1.26 g/ml, respectively.

Steps
Preparation of a Metrizamide Gradient

1. Layer sequentially 4, 3, 6, 7, and 10ml, respectively, of MS (1.26-1.12g/ml) in a 40-ml centrifuge tube (e.g., Quick-seal polyallomer, Beckman) to form a discontiuous gradient.
2. Immediately freeze the gradient in liquid nitrogen and store it at -20°C.
3. Thaw the gradient quickly at room temperature using a metallic stand, thus transforming the step gradient into one with an exponential profile.

Gradient Centrifugation

1. Layer 5ml of either the light or the heavy enriched peroxisomal fraction (corresponding to one liver of approximately 5-6 g) on top of the thawed gradient and seal the tube.
2. Centrifuge gradients in a vertical-type rotor (e.g., Beckman VTi50) at an integrated force of 1.256 × 10⁶g min (g_max= 39,000) using slow acceleration/deceleration modes. Under the conditions employed, highly purified heavy peroxisomes band at 1.23-1.24g/ml and light peroxisomes band at 1.20-1.21 g/ml.
3. Recover the peroxisomal fraction by means of a fraction collector; alternatively, the gradient tube can be punctured and the fraction aspirated by a syringe. Store fraction at -80°C.
4. To remove metrizamide, which interferes with the determination of some peroxisomal enzymes (e.g., urate oxidase) or of protein (Lowry method), dilute the peroxisome fraction about 10-fold with HB followed by centrifugation at 25,000 and 39,000 g, respectively, to pellet the organelles.

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.

FIGURE 2 Electron microscopic appearance of isolated heavy peroxisomes after fixation in glutaraldehyde and osmium. The fraction consists almost exclusively of peroxisomes (PO) with only a rare mitochondrion (M). Many peroxisomes contain urate oxidase cores (arrowheads). Bar: 1µm.

FIGURE 3 A preparation comparable to that in Fig. 2 but incubated with alkaline 3,3'-diaminobenzidine for localization of catalase (Fahimi, 1969). Note the electron-dense reaction product of catalase over the matrix of most peroxisomes. This illustrates the absence of catalase leakage, confirming their integrity. Bar: 1µm

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.

FIGURE 4 SDS-PAGE of highly purified heavy peroxisomes from rat (R) and guinea pig (G) liver. A 10-12.5% resolving gel was used, and the amounts of protein loaded per lane were (R) 2.4µg and (G) 5.4µg. Silver staining of polypeptide bands. Mr standards: BSA (66kDa), ovalbumin (45kDa), and trypsinogen (24kDa). Note the distinct differences in the polypeptide patterns between rat and guinea pig peroxisomes.

FIGURE 5 SDS-PAGE of highly purified peroxisomes from control (Co) and bezafibrate-treated (Bz) rat liver. A 10-15% resolving gel was used, and 5.0µg of protein was loaded per each lane. Silver staining of bands. Peroxisomal polypeptides indicated by an arrowhead are PH, trifunctional protein; Cat, catalase; and UOX, urate oxidase. Note the induction of PH and the concomitant reduction of Cat and UOX in peroxisomes of Bz-treated rats. Also note that the control preparations, R in Fig. 4 and Co in Fig. 5, are not identical and that different resolving gels have been used.

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.

Acknowledgments
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.

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