ARTICLE |
Correspondence to: Manuel J. Santos, Depto. Biología Celular y Molecular, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile. E-mail: msantos@genes.bio.puc.cl
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Summary |
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We demonstrated a neutral Mg-ATPase activity in human peroxisomal membranes. To establish the precise experimental conditions for detection of this ATPase, both cytochemical and biochemical characterizations were first carried out in liver peroxisomes from control and cipofibrate-treated rats. The results demonstrated an Mg-ATPase reaction in both normal and proliferated peroxisomes. The nucleotidase activity, with marked preference for ATP, was sensitive to the inhibitors N-ethylmaleimide and 7-chloro-4-nitro-benzo-2-oxadiazole (NBDCl). An ultrastructural cytochemical analysis was developed to evaluate the peroxisomal localization, which localized the reaction product to the peroxisomal membrane. These characteristics can help to differentiate the peroxisomal ATPase from the activity found in mitochondria and endoplasmic reticulum. The conditions established for detecting the rat peroxisomal ATPase were then applied to human peroxisomes isolated from liver and skin fibroblasts in culture. A similar Mg-ATPase activity was readily shown, both cytochemically and biochemically, in the membranes of human peroxisomes. These results, together with previous evidence, strongly support the presence of a specific ATPase in the human peroxisomal membrane. This ATPase may play a crucial role in peroxisome biogenesis. (J Histochem Cytochem 50:405414, 2002)
Key Words: organelles, human, peroxisomes, ATPase, fractionation, cytochemistry, biogenesis
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Introduction |
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PEROXISOMES are essential and ubiquitous subcellular organelles, surrounded by a single membrane and containing a granular matrix (
Biochemical evidence for the presence of a neutral Mg-ATPase activity co-sedimenting with rat liver peroxisomes was presented by
In this report we show unequivocal evidence for the presence of an Mg-ATPase located in membranes from human peroxisomes. By using biochemical and cytochemical conditions standarized to detect a rat liver peroxisomal ATPase, we were able to detect this ATPase activity in peroxisomes isolated from human liver and skin fibroblasts in culture.
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Materials and Methods |
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Human Samples
Human liver and skin biopsy specimens were obtained from patients undergoing surgery for uncomplicated gallstone disease. Informed consent from the patients was obtained by following procedures approved by the Ethics Committee of the Medical School of the Catholic University of Chile. Liver function tests were normal in all the cases. Liver samples were kept frozen at -70C and, when needed, a portion of the frozen sample was sectioned on dry ice and analyzed exactly as described by
Rat Liver
Male SpragueDawley rats (200250 g) were employed, fed with standard diet (control rats) or fed for 2 weeks with standard diet containing 50 mg/kg of ciprofibrate to induce peroxisome proliferation (treated rats).
Biochemical Assays
L-fractions, which contain mainly light mitochondria, peroxisomes, lysosomes, and some microsomes, were prepared from human and rat liver homogenates by standard subcellular methodology (
A postnuclear supernatant, containing most membranous organelles, was prepared from fibroblast homogenates and subfractionated in continuous Nycodenz density gradients exactly as previously described (
The ATPase activity was assayed biochemically as previously described (
To control biochemically the effect of fixation on rat liver ATPase activity, the enzyme was measured in both freshly prepared and fixed L-fractions and in purified peroxisomes. The following fixatives were tried: 4% paraformaldehyde, 2% paraformaldehyde, 2% paraformaldehyde plus 0.5% glutaraldehyde, and 4% paraformaldehyde plus 0.5% glutaraldehyde. The latter mixture gave the best preservation of the peroxisomal ATPase activity and was used for the cytochemical studies. The samples were washed for 30 min in 0.01 M PIPES buffer, pH 7.0, with 0.15 M sucrose after fixation and then were resuspended for the assays. For the biochemical controls, the activity of the enzyme was measured after incubation of the suspensions in the presence or absence of lead nitrate under the conditions employed for cytochemistry.
Electron Microscopy
Pellets of subcellular fractions were processed as reported previously to obtain homogeneous sampling (
The cytochemical medium for ATPase was prepared as previously reported (
To control the cytochemical procedure, various conditions were employed: absence of substrate; substitution of adenosine monophosphate or glucose-6-phosphate for ATP; addition of NEM or NBDCl as inhibitors; and preincubation of the specimens for 10 min at 90C instead of 2C before addition of substrate and lead nitrate. Incubations were carried out for 120180 min at 37C. The specimens were rinsed in distilled water and postfixed with 1% osmium tetroxide for 30 min at 2C, dehydrated in ethanol and acetone, and embedded in epon. Thin sections were cut with an MT2 Porter Blum ultramicrotome and were observed (with and without lead nitrate staining) under a Siemens Elmiskop IA electron microscope.
Cytochemical demonstration of ATPase activity was also performed using the cerium chloride method for capturing inorganic phosphate produced by the ATPase activity (
Cytochemistry for catalase was performed exactly as described by
Nycodenz was purchased from Nyegaard, Oslo; ciprofibrate was a gift from SterlingWinthrop Research Institute (Rensselaer, NY). Other biochemical reagents were from Sigma Chemical (St Louis, MO).
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Results |
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To establish the experimental conditions for detection of a human peroxisomal ATPase, several biochemical and cytochemical studies were first carried out using peroxisomes isolated from rat liver.
Rat Liver Peroxisomes
Biochemical Assays.
Peroxisomes were purified from control and cipofibrate-treated rats after subfractionation of an L-fraction in a continuos Nycodenz gradient (Fig 1). Peroxisomal fractions in this type of fractionation experiment are mainly contaminated by mitochondria. An Mg-ATPase activity was found in peroxisomal fractions. Several substrate analogues were tested in peroxisomes and in mitochondrial fractions. Table 1 shows the phosphate-releasing activity of the purified organelles with different substrates at 2 mM concentration. The peroxisome-associated ATPase activity is specific for ATP, and less active with other substrates: ATP>CTP>GTP>UTP>ADP> UDP = AMP. The pattern of activity for the mitochondrial enzyme is different. It shows similar activity with ATP, GTP, and UTP. The ATPase activity in the presence of inhibitors was measured in mitochondria and peroxisomes. These results are shown in Table 2. Strong inhibition of the peroxisomal enzyme with 50 and 100 µM NBDCl was observed, whereas the mitochondria-associated enzyme showed no inhibition. The concentration of NBDCl employed was established from a concentration curve that gave an IC50 of 20 µM for the peroxisomal enzyme and only slight inhibition for the mitochondrial activity with up to 20 µM NBDC1, employing 100200 µg peroxisomal or 60 mitochondrial protein per test (data not shown). NEM 1 mM, as described previously (
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As shown in Fig 1, most of the ATPase activity follows the mitochondrial and endoplasmic reticulum markers, with only small activity apparently associated with peroxisomes. The ATPase-specific activities for the experiment illustrated were 58, 101 and 4.1 mU/mg protein for the L-fraction, the mitochondrial peak, and the peroxisomal peak, respectively. The ATP-ase + NBDCl graph illustrates the relative inhibition by NBDCl of the activity present in each fraction from the gradient. The peroxisomal peak shows 77% inhibition and the mitochondrial peak none, in agreement with the results shown in Table 2. The ATPase activity in the low-density fractions also shows some sensitivity to NBDCl. As discussed below, this might correspond to fragments from damaged mitochondria.
Cytochemical Localization.
Pellets of proliferated peroxisomal fractions, similar to those characterized in Fig 1, were fixed and incubated in cytochemical media. Peroxisomes were identified using the standard cytochemical reaction for catalase. The DAB peroxidation product was confined to the peroxisomal matrix, which characteristically (
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In the presence of glucose-6-phosphate, an intense positive reaction was observed only in the membranous profiles (Fig 2C), apparently corresponding to the activity of glucose-6-phosphatase in endoplasmic reticulum fragments. Addition of NBDCl to the ATPase standard medium specifically inhibited the ATPase reaction associated with peroxisomes and had no effect on the ATPase present in the endoplasmic reticulum membranes (Fig 2D). However, in the presence of NEM the ATPase cytochemical reaction in both the membranous profiles and the peroxisomes was greatly reduced (data not shown).
The results obtained with purified peroxisomes were confirmed in L-fractions obtained from control rat livers. The Mg-ATPase reaction product was also found on the cytoplasmic side of the peroxisomal membranes (Fig 3). A strong ATPase reaction was present in the membranes of the endoplasmic reticulum, whereas few aggregates of product were associated with mitochondria (data not shown). When glucose-6-phosphate was used as substrate, a positive reaction was found only in the endoplasmic reticulum vesicles (data not shown). Addition of NBDCl resulted in a strong specific inhibition of the Mg-ATPase associated with the peroxisomal membrane (data not shown).
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Human Peroxisomes
Biochemical Assays.
The presence of an Mg-ATPase in human peroxisomes was evaluated biochemically and cytochemically using the standarized conditions for rat liver peroxisomes. Peroxisomal fractions isolated from frozen human liver samples showed easily detected Mg-ATPase activity. As shown in Table 3, the specific activity of the human liver Mg-ATPase was about half that of the rat liver. This activity showed a similar pattern of substrate analogues ATP>CTP> GTP>UTP>ADP>UDP = AMP to rat liver (data not presented) and showed a similar pattern of inhibition with NBDCl (Table 3).
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Fig 4 shows the distribution pattern of the enzyme markers in a Nycodenz density gradient after subfractionation of an L-fraction prepared from frozen human liver. Similar to rat liver, most of the ATPase activity follows the mitochondrial distribution, with only very small activity associated with peroxisomal fractions. The NBDCl pattern of inhibition of the ATPase activity in each fraction of the gradient shows that the peroxisomal peak exhibited about 90% inhibition. The ATPase activity in the low-density fractions also shows some sensitivity to NBDCl.
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Peroxisomes were also isolated from normal human fibroblasts in culture. These fibroblasts were homogenized and the postnuclear supernatant was subfractionated in a Nycodenz density gradient designed to separate peroxisomes from the rest of the membranous organelles (
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Cytochemical Localization.
Pellets of peroxisomal fractions obtained from human liver, similar to those characterized in Fig 4, were fixed and incubated in cytochemical media. Peroxisomes were identified using the standard DAB cytochemical reaction for catalase (Fig 5A). A nucleoid type of structure is seen in these human peroxisomes, as previously shown for peroxisomes isolated from frozen human livers (
The presence of Mg-ATPase was also detected in peroxisomes isolated from human fibroblasts in Nycodenz density gradients, as seen in Fig 6. Peroxisomes were identified using the cytochemical DAB method for catalase (Fig 7A). An Mg-ATPase was also detected in the peroxisomal membrane (Fig 7B). This activity was drastically reduced in the presence of the inhibitor NBDCl (Fig 7D). As previously shown for human liver, a strong cytochemical reaction was seen in the endoplasmic reticulum when the cytochemical medium for ATPase was modified by replacing ATP with glucose-6-phosphate (Fig 7C). To confirm our cytochemical results on peroxisomes isolated from human fibroblasts, we performed additional experiments to demonstrate the presence of an ATPase activity in situ. For this, we fixed human skin fibroblasts and carried out the cytochemical cerium chloride method for detecting ATPase activity. As shown in Fig 8B, finer and clearer reaction products are located on peroxisomal membranes. For comparison, the cytochemical demonstration of catalase is also shown (Fig 8A).
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Discussion |
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The experiments presented here demonstrate, for the first time, the presence of an ATPase activity in the membrane of human peroxisomes. The cytochemical reaction product is localized to the peroxisomal membrane. Specifically, the topology of the cytochemical reaction product in subcellular fractions excludes the possibility that the ATPase activity is also localized in the peroxisomal matrix. The cytochemical results, in addition to the detection of peroxisomal and mitochondrial ATPase activities, also show activity associated with the endoplasmic reticulum. Because microsomal profiles can contaminate peroxisomal fractions, this fact should be taken into consideration when results from biochemical experiments in which the analysis is centered mainly on the peroxisomal and mitochondrial activities are interpreted (
The evidence available from subcellular fractionation data does not support the presence of a functional proton-translocating ATPase in peroxisomes, nor does it indicate a putative function. However, evidence for an ATP-dependent structure-linked latency of peroxisomes in situ has been found in fibroblasts (
Initially, the properties of the rat peroxisomal ATPase suggested that it belongs to the vacuolar or V-type ATPases: neutral, NEM sensitive, low activity when Mg2+ is replaced by Ca2+, and insensitive to oligomycin and vanadate (-subunit in rat liver peroxisomes. NBDCl, an alkylating agent, is known to inhibit various ATPases in variable proportions, apparently depending on the enzyme and the conditions employed (
Systematic characterization of the peroxisomal ATPase, its activity, and the extent of its functional similarities with other ATPases requires its purification. The fact that the 70-kD peroxisomal membrane protein is an ATP-binding protein, presumably involved in protein transport (
It has been suggested that an ATPase activity is involved in peroxisome biogenesis, particularly at the level of import of peroxisome proteins to pre-existing mammalian peroxisomes. In vitro and in vivo evidence suggests that ATP hydrolysis is required to efficiently import peroxisomal matrix proteins (
Finally, a precise characterization of the role of this novel human peroxisomal ATPase is required, especially because the short-term regulatory mechanisms of this organelle have not yet been characterized.
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Acknowledgments |
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Supported by project FONDECYT 1980978.
Received for publication February 28, 2001; accepted September 5, 2001.
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