Symposium Papers |
Correspondence to: Alfred Völkl, Dept. of Anatomy and Cell Biology II, U. of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany.
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Summary |
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Peroxisomes (POs) are a heterogenous population of cell organelles which, in mammals, are most abundant in liver and kidney. Although they are usually isolated by differential and density gradient centrifugation, isolation is hampered by their high fragility, sensitivity to mechanical stress, and their sedimentation characteristics, which are close to those of other major organelles, particularly microsomes. Consequently, until now only the so-called "heavy" POs with a buoyant density of 1.221.24 g/cm3 have been highly purified from rat liver, whereas the other subpopulations also present in that tissue have escaped adequate characterization. The purification of these subpopulations has become an essential task in view of the functional significance of POs in humans, and the putative importance of peroxisomal subpopulations in the biogenesis of this organelle. Here we used an alternative novel approach to density gradient centrifugation, called immune free flow electrophoresis (IFFE). IFFE combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle complexed to an antibody against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided that the pH of the electrophoresis buffer is adjusted to pH ~8.0, the pI of IgG molecules. Because of this reduced electrophoretic mobility, IgG-coupled particles can be separated in an electric field from those that are noncoupled and hence more mobile. The IFFE technique has been recently applied for isolation of regular POs ( = 1.221.24 g/cm3) from a light mitochondrial fraction of rat liver. We succeeded in isolating different subpopulations of POs by applying IFFE to heavy, light, and postmitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The PO subfractions obtained differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic POs.
(J Histochem Cytochem 47:11111117, 1999)
Key Words: isolation, peroxisome subpopulations, immune free flow electrophoresis
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Introduction |
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The heterogeneity of peroxisomes (POs) is a well-known phenomenon that is substantiated or at least supported by several lines of evidence: (a) the demonstration of distinct types of the organelle exhibiting differences in size, shape, and protein composition even in the same tissue (
Free flow electrophoresis (FFE) has occasionally been used to purify lysosomes, endosomes, and Golgi vesicles as well as mitochondrial subcompartments (
The first approach in making use of this combination has been termed antigen-specific electrophoretic cell separation (ASECS) and was introduced for the isolation of human T- and B-lymphocyte subpopulations (
In this study we used IFFE also for the separation of POs from a heavy mitochondrial (HM) and a postmitochondrial (PM) preparation (
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Materials and Methods |
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Reagents
3-(N-morpholino)propanesulfonic acid (Mops), phenylmethylsulfonyl fluoride (PMSF), and dithiothreitol (DTT) were purchased from Sigma (Munich, Germany) and 5-((N-2,3-dihydroxypropylacetamido)-2,4,6-tri-iodo-N,N'-bis(2,3-dihydrox-ypropyl))-isophthalamide (Nycodenz) from Life Technologies (Eggestein, Germany). All other reagents were from Merck (Darmstadt, Germany) and of the highest grade available.
Antibodies
The preparation of antibodies against peroxisomal enzymes, i.e., catalase, acyl-CoA oxidase (AOx), and urate oxidase (UOx) and assessment of their monospecificity have been established previously (
A synthetic peptide of the C-terminal 10 amino acids of rat PO membrane protein PMP 70 was coupled to thyroglobulin (
The polyclonal antibodies against the rat hepatic PO membrane proteins PMP 22 and Pex 11 and that against PAF 2 and Pex 5 were kindly provided by Drs. T. Hashimoto (Matsumoto, Japan), T. Osumi (Osaka, Japan), and S. Subramani (San Diego, CA), respectively.
Treatment of Animals
Female SD rats weighing about 250 g were obtained from the Zentrale Versuchstieranlage (University of Heidelberg) where they were kept in accordance with the guidelines of the humane care and use of laboratory animals of the FRG. They were fasted overnight, anesthetized with chloral hydrate, and livers were drained of blood by perfusion with saline (0.9% NaCl, w/v), subsequently excised, dried with filter paper, and weighed. After removal of the connective tissue, they were minced in ice-cold homogenization buffer (HB): 250 mM sucrose, 5 mM Mops, 1 mM EDTA, 0.1% ethanol, 2 mM PMSF, 1 mM DTT, 1 mM -aminocaproic acid.
Subfractionation of Liver Homogenates and Preparation of Heavy, Light, and Postmitochondrial Fractions
Homogenization of the minced tissue and subcellular fractionation by differential centrifugation were performed according to an established method (-aminocaproic acid (
= 1.20 g/cm3). It was spun in a fixed-angle type rotor (Ti 45; Beckman) at 37,000 x g for 20 min and the fluffy layer banding on top of the cushion, comprising microsomes, lysosomes, and peroxisomes was reloaded onto a fresh Nycodenz cushion and recentrifuged at 55,000 x g for 20 min in the same rotor. The pellet collected was resuspended in ice-cold HB (1.2 ml) using a glass rod and was designated the postmitochondrial fraction (PM). Aliquots of fractions HM, LM, and PM were processed by IFFE as detailed below. In addition, highly purified POs were isolated from an LM fraction by metrizamide density gradient centrifugation (
Incubation of Fractions HM, LM, and PM with Anti-PMP 70 Antibody
One-ml aliquots of the HM, LM, and PM fractions corresponding to about 0.251 g of liver were mixed with 1 ml HB containing 1 mg polyclonal anti-PMP 70 antibody, 1 mg of BSA, and leupeptin (1 µM), and the mixtures were incubated at room temperature for >1 hr to immunocomplex the POs of these fractions. Immunoprecipitates and noncomplexed particles were collected by centrifugation for 20 min at 1950 x g (HM), 25,500 x g (LM), and 55,000 x g (PM), and the pellets were suspended in 2 ml medium buffer (MB), pH 8.0, containing 250 mM sucrose, 10 mM triethanolamine, 10 mM acetic acid, and 0.25 mg/ml bovine serum albumin.
Free Flow Electrophoresis
Free flow electrophoresis was performed employing the Octopus instrument (Dr. G. Weber; Kirchheim/Heimstetten, Germany). Just before each run, the separation chamber was washed with MB and electrophoresis was subsequently conducted at 4C in the same buffer with a field of 1000 V and 100 mA and a flow rate of ~5 ml/fraction/hr. Samples were perfused into the separation chamber at ~2 ml/hr and fractions were collected through a 96-channel peristaltic pump. Individual runs took approximately 6090 min. Optical densities (ODs) of fractions were measured at 280 nm and they were concentrated by centrifugation for 20 min at g forces stated above. Pellets were suspended in HB and stored at -80C for enzyme analysis, SDS-PAGE, and immunoblotting.
Protein Assay
Protein was assayed using the Coomassie Blue binding method (
SDS-PAGE and Immunoblotting
SDS-PAGE was performed under reducing conditions using 12.5% polyacrylamide gels (Laemmli 1970). Polypeptides resolved were transferred to nitrocellulose (
Densitometric Analysis of Immunoblots
Specific signals revealed by ECL, representing the immune complexes of matrix and membrane proteins of PO derived from LM, HM, and PM fractions as well as from an LM density gradient centrifugation (LMgrad-PO), were analyzed by densitometry and ratios of intensities were calculated, with the PMP 22 signal serving as reference for the membrane proteins while the soluble PO matrix enzymes catalase and acyl-CoA oxidase were compared to the insoluble urate oxidase.
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Results |
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Isolation of Peroxisomal Subpopulations by IFFE
The electropherogram shown in Figure 1 exemplifies the fractionation of an LM fraction by means of IFFE. The respective peroxisomes isolated, LM-PO1 collected in fractions 3234 and LM-PO2 in fractions 3739, are indicated by circles. The distribution of protein across all fractions, reflected by the optical density at 280 nm (OD280) and the determination of catalase, acyl-CoA oxidase, and urate oxidase activities (not shown) consistently reveal a clear-cut separation of nonperoxisomal organelles (first peak) and peroxisomes (second peak and shoulder). These findings, which are in line with corresponding results recently reported (
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Immunoblotting of the Subpopulations Isolated
PO subpopulations isolated by IFFE from an HM, LM, and PM preparation of a rat liver homogenate were analyzed by SDS-PAGE (Figure 2) and subsequent Western blotting (Figure 3 Figure 4 Figure 5) in comparison to highly purified POs (LMgrad-PO) obtained by concomitant metrizamide density gradient centrifugation of the same LM preparation. Whereas the polypeptide patterns of POs immunoisolated from the LM and PM fractions largely coincide with the LMgrad-PO, that of HM- POs does not (Figure 2). This might be due to polypeptides from contaminating organelles, although HM-PO might also represent a unique PO subpopulation.
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Immunoblotting using antibodies against PO matrix enzymes (catalase, acyl-CoA oxidase, urate oxidase) as well as membrane proteins (PMP 22, PMP 70, Pex 11, Pex 5, and PAF 2) revealed differences between the protein-specific signals in a given subpopulation (Figure 3 Figure 4 Figure 5). More importantly, however, each protein investigated specifically varied in its signal intensity between LM-, HM-, PM-, and LMgrad-PO, suggesting a different composition of these PO populations. This interpretation is substantiated by the densitometric analysis of the blots and the calculation of relative intensities, with the UOx and PMP 22 signals, respectively, serving as references. As is indicated by the data depicted in the graphs related to the corresponding blots (lower halves of Figure 3 Figure 4 Figure 5), individual PO subpopulations indeed differ (a) in the ratios of the soluble matrix enzymes catalase and acyl-CoA oxidase to the insoluble matrix enzyme urate oxidase (Figure 3), (b) in the ratios of the membrane proteins PMP 70 and Pex 11 to the most abundant protein constituent of the PO membrane, i.e., PMP 22 (Figure 4), and (c) particularly in the PMP 22-related ratios of Pex 5 and PAF 2, proteins considered to contribute to the import of PO matrix enzymes. It should be noted in this context, that the highly purified POs (LMgrad-PO) obtained by density gradient centrifugation of an LM preparation contain only a minor amount of Pex 5 and PAF 2, which is almost consistent with the LM PO2 fraction (Figure 5).
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Discussion |
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Distinct Subpopulations of POs Can Be Isolated by IFFE from the Main Subcellular Fractions of Rat Liver
In this study we have used IFFE to isolate POs from an LM (crude peroxisomal) fraction of rat liver, exactly as we described recently (
The POs immunoisolated as described above are expected to differ markedly in their buoyant densities, considering that heavy, light, and postmitochondrial fractions were recovered at quite divergent g forces, i.e., 1950, 25,000, and 55,000 x g, respectively. Indeed, in earlier studies PO fractions have been collected at densities other than ~1.24 g/cm3 (
The Isolated Subpopulations Exhibit Significant Differences in Composition of the Matrix and Membrane Proteins
To figure out whether distinct physicochemical properties account for the distribution of POs to the hepatic subfractions investigated or whether they truly differ in their protein composition, we have used polyclonal antibodies directed against peroxisomal matrix and membrane proteins and immunoblotting to compare HM-, LM-, PM-, and LMgrad-POs (Figure 3 Figure 4 Figure 5). It should be noted that we did not load identical quantities of HM, LM, and PM POs to the SDS gels, taking into account the heterogeneous composition in protein of these fractions; they putatively differ in their ratios of peroxisomal to contaminating organellar (e.g., mitochondrial, microsomal) proteins, and albumin and IgG protein, which are routinely present in the separation medium, probably stick to various extents to the different organelles. Therefore, we analyzed the immunoblots by densitometry and calculated the corresponding ratios to either the peroxisome membrane protein PMP 22 or the insoluble matrix protein UOx. The data presented in the lower halves of Figure 3 Figure 4 Figure 5 provide strong evidence that HM-PO, LM-PO1, LM- PO2, and PM-POs are distinct subpopulations because of the divergent proportions of their matrix and membrane proteins. Therefore, the ratios of the soluble matrix enzymes catalase and acyl-CoA oxidase to the insoluble urate oxidase are quite similar in LM-PO1 and LM-PO2, as well as in LMgrad-PO, yet markedly differ in PM- and HM-POs (Figure 3). This might just reflect different degrees of extraction due to the divergent g forces applied to pellet HM-, LM-, and PM-POs. Indeed, POs are quite fragile organelles and PO matrix proteins are commonly extracted at quite different rates; in particular, catalase leaks out quite easily (
Differences were also observed in the distribution of the membrane proteins PMP 70, Pex 11, and PMP 22 (Figure 4). Whereas the latter was present in all fractions, albeit in different concentrations, only quite low amounts of PMP 70 and Pex 11 could be detected in HM-POs. Distinct ratios of Pex 11 and PMP 70 to PMP 22 were found in LM PO1 and LM PO2, in line with the distribution of Pex 5 and PAF 2 (Figure 5), which are mostly enriched in LM-PO1. In view of the role attributed to these proteins, which are considered as constituents of the PO import mechanism (
The observations presented here gain particular importance in view of the recent debate on the role of the endoplasmic reticulum (ER) in the biogenesis of POs (
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Acknowledgments |
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Supported by the Deutsche Forschungsgemeinschaft (SFB 601/A8).
We wish to thank Drs Hashimoto (Matsumoto, Japan), Osumi (Osaka, Japan), and Subramani (San Diego, CA) for providing us with the antibodies against PMP 22, PAF 2, and Pex 5.
Received for publication March 26, 1999; accepted March 30, 1999.
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