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1 Research Institute for Sustainable Humanosphere, Kyoto University, Uji, Kyoto 611-0011, Japan
2 University of North Sumatra, Jl. Bioteknologi No. 1 Kampus USU, Medan 20513, Indonesia
3 Department of Applied Biological Chemistry, University of Osaka Prefecture, Sakai, Osaka 599-8231, Japan
Correspondence
Takefumi Hattori
thattori{at}rish.kyoto-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Present address: Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Present address: Nisshin Foods Inc., Tokyo, Japan.
Present address: Environmental and Biotechnological Frontier Engineering, Fukui University of Technology, Fukui, Japan.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The glyoxylate intermediate is further condensed with acetyl-CoA, yielding malate by catalysis of MS (Wong & Aji, 1956). Thus, for maintaining the TCA cycle, the GLOX cycle enables a net synthesis of C4 carboxylic acids from a carbon source of C2 compounds (Kornberg & Madsen, 1957; Kornberg & Krebs, 1957; Kornberg, 1966), such as acetate, ethanol, and fatty-acid-derived acetyl-CoA.
In general, micro-organisms induce ICL as an adaptive enzyme for gluconeogenesis only when they are grown on C2 compounds (Vanni et al., 1990). However, when glucose is present together with these non-fermentable compounds, expression of an ICL-encoding gene is repressed, or, in some cases, the ICL protein is further inactivated, which is called catabolite repression or inactivation (Herrero et al., 1985; López-Boado et al., 1988; Fernández et al., 1992; De Lucas et al., 1994; Ordiz et al., 1996; Amor et al., 2000; Schüller, 2003; López et al., 2004).
In contrast to the above biochemical features of ICLs, both ICL and MS occur as constitutive enzymes in a wide variety of glucose-grown wood-destroying basidiomycetes, including Fomitopsis palustris (Munir et al., 2001a). Furthermore, F. palustris accumulates oxalate in the culture fluid, producing a yield of 80 % based on the amount of glucose consumed during vegetative growth (Munir et al., 2001b). The acid formed plays a crucial role in the wood-decaying process (Dutton & Evans, 1996; Shimada et al., 1997). Biochemical analysis of the unique oxalate fermentation of F. palustris has led to the conclusion that the fungus acquires biochemical energy for growth by oxidizing glucose to oxalate. In such an acid fermentation process, ICL, shared by the GLOX and TCA cycles, plays a major role, whereas neither isocitrate dehydrogenase (IDH) nor 2-oxoglutarate dehydrogenase (ODH) plays a significant role (Munir et al., 2001b). Thus, the constitutive key enzyme ICL replenishes the intermediates glyoxylate and succinate to the GLOX and TCA cycles, respectively (Munir et al., 2001b). The role of F. palustris ICL is contrasted with the hither-to-known function of ICL required for gluconeogenesis in other micro-organisms.
In view of this unique carbon metabolism in F. palustris, one question arises: in which organelle in the cell of F. palustris are the GLOX enzymes cytochemically compartmentalized? Microbial ICL is generally found in microbodies called peroxisomes (Osumi et al., 1978; Valenciano et al., 1996, 1998; Titorenko et al., 1998; Maeting et al., 1999) or glyoxysomes (Kionka & Kunau, 1985), with a few exceptions (Taylor et al., 1996; Chaves et al., 1997; Ono et al., 2003). In the case of basidiomycetes, the ICL of the litter-decomposing Coprinus lagopus grown on acetate has been found to occur in peroxisomes by subcellular fractionation analysis (O'sullivan & Casselton, 1973). However, those authors detected ICL activity from a lighter buoyant density fraction than the mitochondrial fraction, which contradicts results reported for several other micro-organisms. The ICL of Coprinus cinereus acu-7 has been proposed to be peroxisomal based on the presence of the peroxisome target signal (PTS1) (Subramani, 1993) at the C terminus (Chaure et al., 1997), without direct experimental evidence. Furthermore, localization of the induced ICLs has been investigated for micro-organisms grown on C2 compounds, but, to the best of our knowledge, the ICL localization for glucose-grown micro-organisms has not been reported.
Therefore, we were motivated to investigate the intracellular localization of the GLOX cycle enzymes, and other enzymes involved in oxalate fermentation, in glucose-grown F. palustris. Previously, we purified and characterized ICL from F. palustris (Munir et al., 2002), which is termed FPICL1 in this study. We report here the characterization of FPICL1 cDNA encoding FPICL1, and peroxisomal localization of FPICL1 based on subcellular fractionation and immunocytochemical experiments using anti-FPICL1 antibody. The localization of MS, acetyl-CoA synthase (ACS; EC 6.2.1.1) and oxaloacetate acetylhydrolase (OXA; EC 3.7.1.1) was also determined. The results are discussed in relation to possible transportation of metabolites between subcellular sites, and also in relation to the constitutive metabolic coordination of the TCA and GLOX cycles with the oxalate biosynthesis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Fresh mycelia were processed for the experiments described below.
Amino acid sequencing.
Purified ICL protein (approx. 80 µg; Munir et al., 2002) was digested with lysylendopeptidase, and the resulting peptides were separated by HPLC on a reverse-phase C18 column. N-terminal amino acid sequences were determined by use of an ABI Precise 491 automated protein sequencer (Applied Biosystems). Protein concentrations were determined by the method of Bradford (1976), using BSA as a standard.
Isolation of cDNA encoding FPICL1.
Total RNA was extracted from the mycelia of glucose-grown F. palustris by use of a QuickPrep Total RNA Extraction kit (Amersham Biosciences), according to the manufacturer's instructions. PolyA+ RNA was isolated from the total RNA using Oligotex-dT30<Super>(Takara Bio), and cDNA was synthesized with a Timesaver cDNA synthesis kit (Amersham Biosciences), followed by size fractionation into pools containing more than 0.5 kb. The cDNA pool was cloned into a lambda ZAP II vector containing the whole phagemid pBluescript SK sequence (Stratagene), and packaged into 
phage extract (Gigapack III packaging extract kit; Stratagene). The cDNA library was constructed with 3.5x105 p.f.u. µg–1.
In order to prepare probe for the screening of FPICL1, PCR was performed with the cDNA, prepared as described above, as a template, and a pair of primers, (P1, 5'-GGTCACATGGCTGGTAAGGT-3'; and P2, 5'-GACCGGCGAGAGTGATGAAC-3'), which were designed from the sequences within the Aspergillus nidulans ICL gene (acuD; GenBank accession no. X62696). We screened cDNA encoding the putative protein, including the internal amino acid sequence of FPICL1. The amplified fragment was subsequently used as an [
-32P]dATP-labelled probe for cDNA library screening by standard plaque hybridization procedures (Sambrook et al., 1989). Because the obtained cDNA fragment lacked the 5' region, we performed PCR using a cDNA library that had been newly constructed by Takara Bio as a template, and a pair of primers (P3, 5'-GAAAGAAAAAAAATATACCCCAGC-3'; and P4, 5'-ACCTTGCCCGCCATATGCCCGCACTTCTT-3'), in order to determined the sequence of the 5' end of the cDNA. DNA sequencing was performed by Big Dye terminator v2.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems) and an ABI PRISM 377 DNA sequencer (Applied Biosystems).
Subcellular fractionation.
Cell organelles were obtained from the protoplast homogenate by differential centrifugation. Approximately 100 g (wet wt) of mycelia were harvested by filtration through cheesecloth, and washed well with distilled water. Mycelial mats were suspended in a lysing buffer [50 mM maleate, pH 6.0, containing 0.6 M mannitol, 1 mM EDTA and 5 % (w/v) Lysing Enzyme (Sigma-Aldrich)], and gently shaken at 28 °C. After 2–3 h, protoplast formation was complete, and the lysate was chilled to 4 °C. The protoplasts were collected at 4 °C by a low-speed centrifugation, resuspended in a protoplast buffer (50 mM Tris/HCl, pH 7.5) containing 1 M sorbitol, 1 mM EDTA and 1 mM PMSF, and homogenized in a Potter-Elvehjem homogenizer with a loosely fitting Teflon pestle. The homogenate was centrifuged at 1500 g for 10 min to remove cell debris and nuclei. The post-nuclear supernatant (PNS) was filtered through Miracloth (Calbiochem), and then centrifuged at 20 000 g for 20 min, to yield pellet (20KgP) and supernatant (20KgS) fractions. The 20KgP fraction, consisting mainly of mitochondria and peroxisomes, was resuspended in a suspension buffer (50 mM Tris/HCl, pH 7.5) containing 0.5 M sucrose, 1 mM EDTA and 1 mM PMSF, and layered onto a discontinuous sucrose density-gradient [1 ml 60 % (w/v) sucrose, 2 ml each of 50, 46, 43, and 41 % sucrose, and 1 ml 35 % sucrose, in 10 mM Tris/HCl (pH 7.5)]. The gradient solution was centrifuged at 10 0000 g for 5 h at 4 °C in a Beckman SW40 Ti swinging-bucket rotor. Fractions of 0.75 ml were collected from the bottom of the tube, and analysed for various enzyme activities. The sucrose concentration of each fraction was determined refractometrically at 25 °C. Each operation was done in triplicate.
Enzyme assays.
All enzyme assays were performed spectrophotometrically at 30 °C by use of a Shimadzu UV-1650P spectrophotometer. ICL and MS activities of a glyoxylate cycle marker were determined by the method of Dixon & Kornberg (1959). ICL activity was assayed by measurement of the increase in absorbance at 324 nm due to the formation of the phenylhydrazone derivative of glyoxylate produced from isocitrate. MS activity was determined on the basis of the consumption of acetyl-CoA, with a decrease in absorbance at 340 nm. Catalase (EC 1.11.1.6) activity, as a peroxisomal marker, was determined by measuring the decomposition of H2O2 by the method of Aebi (1983). Succinate dehydrogenase (SDH; EC 1.3.5.1) activity as a mitochondrial marker was measured on the basis of a modified method of Moore & Ewaze (1976). Malate dehydrogenase (MDH; EC 1.1.1.37) activity was assayed as described by Labrou et al. (1997). ACS activity was assayed according to the method of Heather & Fairhurst (1998). OXA activity was determined by measurement of the decrease in absorbance at 255 nm due to the hydrolysis of oxaloacetate (Lenz et al., 1976). One unit of enzyme activity is defined as the amount of enzyme that catalyses the formation or decomposition of 1 µmol product or substrate, respectively, per minute, under the conditions described.
Generation of anti-FPICL antibody.
To obtain the pure protein [terminated with an SKL tripeptide representing a peroxisome targeting signal 1 (PTS1)] for generating an anti-FPICL1 antibody, the pET-32 Ek/LIC expression system (Novagen) was used. A DNA fragment encoding C-terminal 336 aa (out of a total of 539 aa) was amplified by PCR with the primer pairs designed from the FPICL1 sequences, and subcloned into the expression vector. The truncated protein is expressed as the fusion protein with thioredoxin, H-tag and S-tag at the N terminus. Escherichia coli strain Origami B (DE3) was cultured in Luria–Bertani medium supplemented with 100 µg ampicillin ml–1 at 30 °C, and protein expression was induced by IPTG added at a final concentration of 0.1 mM. Cells were harvested by centrifugation, and suspended in 20 mM Tris/HCl (pH 7.0) containing 0.3 M NaCl and 20 mM imidazole. After sonication, cell lysates were subjected to affinity chromatography using a Ni+-nitrilotriacetate (NTA) column, and the purified fusion protein was incubated with enterokinase to remove the N-terminal tags. The enterokinase coexisting in the sample was captured with EKapture Agarose (Novagen). The recombinant protein without a tag was rescued from the fraction passed through the Ni+-NTA column. SDS-PAGE analysis (Laemmli, 1970) revealed a protein band with an apparent molecular mass of 39 kDa, which corresponded to the deduced size of the FPICL1 truncation (not shown).
The homogeneous protein (1.2 mg), emulsified with the same volume of Freund's complete adjuvant (2 ml total volume), was used to immunize rabbits. This was followed by two additional booster injections. One week after the second injection, a blood sample was collected to measure the titre of the antibody by ELISA. After the last injection, the rabbit was killed under anaesthesia. Blood was centrifuged at 3000 g for 10 min, and the antiserum fraction was pooled, and stored at –80 °C until use.
Western blotting.
Proteins were separated on an SDS-PAGE gel containing 15–25 % polyacrylamide, and electrotransferred to PVDF membranes (Immobilon-P; Millipore). The membrane was blocked in 0.5 % (w/v) skim milk in PBS-T (0.05 % Tween-20 in PBS), and then incubated with rabbit polyclonal antibody raised against FPICL1 (1 : 3000 dilution in PBS-T) or control rabbit serum (1 : 2000 dilution in PBS-T). After washing in PBS-T, the membrane was incubated with horseradish-peroxidase-conjugated donkey anti-rabbit IgG secondary antibody (1 : 5000 dilution in PBS-T). The immunoreactive proteins were detected by chemiluminescence with the ECL detection system (Amersham Biosciences), according to the manufacturer's instructions. Preparation of cell-free extracts from F. palustris, and purification of the native enzyme, were performed by the methods of Munir et al. (2002).
Electron microscopy and immunocytochemistry.
F. palustris mycelia grown on glucose medium were pre-fixed in 2.5 % (v/v) glutaraldehyde and 1.5 % (w/v) paraformaldehyde in 20 mM PIPES (pH 7.2) for 3 h at 4 °C. Pre-fixed samples prepared were washed three times with the buffer, and fixed overnight at 4 °C in 1 % (w/v) OsO4 in 20 mM PIPES (pH 7.2). After dehydration in a graded ethanol series at room temperature, the samples were embedded in Spurr's resin (Spurr, 1969), and serially sectioned on a Reichert ultramicrotome (Ultracut E; Reichert-Jung). Ultrathin sections mounted on nickel grids were stained with uranyl acetate at room temperature, followed by lead citrate staining. For cytochemical staining of catalase, a glutaraldehyde/paraformaldehyde-fixed sample was incubated for 3 h at 4 °C with diaminobenzidine (DAB; 1 mg ml–1) in 100 mM Tris/HCl (pH 10.5), containing 0.1 % H2O2, before OsO4-fixation. In control experiments with a catalase inhibitor, 3-amino-1,2,4-triazole, a pre-fixed sample was incubated for 30 min with this chemical, and then transferred to DAB solution.
For immunoelectron microscopy, mycelia were fixed overnight at 4 °C in 0.2 % glutaraldehyde and 4 % paraformaldehyde in 20 mM PIPES (pH 7.2), containing 0.5 mM CaCl2. Samples were washed three times with the buffer, dehydrated in a graded ethanol series, and embedded in LR-White resin (London Resin). Ultrathin sections were cut with an ultramicrotome. The sections were blocked in 0.1 % BSA in PBS for 30 min at room temperature, and incubated for 1 h with anti-FPICL1 antiserum (1 : 30 dilution in PBS) or non-immune rabbit serum (1 : 40 dilution in PBS). After washing in PBS, the sections were incubated for 1 h with goat anti-rabbit IgG antibody conjugated with 15 nm gold particles (1 : 20 dilution in PBS-T). The sections were washed in distilled water, and then stained with uranyl acetate. After staining, all sections were examined with a JOEL 1200 EX transmission electron microscope at 100 kV.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). All of the 10 polypeptides obtained from FPICL1 by digestion with lysylendopeptidase were found in this deduced amino acid sequence. The hexapeptide KKCGHM, highly conserved as an ICL signature in the sequences of most ICLs reported to date, was found in the sequence at aa 201–206. The deduced polypeptide had a predicted molecular mass of 59 735 Da, which is similar to the estimated molecular mass for the purified FPICL1 (Munir et al., 2002). By analogy with acu-D, the ICLs of A. nidulans (Gainey et al., 1992), D111, D165, D167 and E194 are suggested to be a cluster for the Mg2+-binding site. The predicted amino acid sequence was compared with those of the ICLs of Phanerochaete chrysosporium, C. cinereus (Chaure et al., 1997), A. nidulans (Gainey et al., 1992) and Saccharomyces cerevisiae (Schöler & Schüller, 1993), which revealed 83, 79, 63 and 54 % identity, respectively (Fig. 1). Interestingly, FPICL1 was found to contain the motif KRGT at positions 39–42. This is a putative cAMP-dependent protein kinase phosphorylation site for the T residue. A similar site has been found in C. cinereus acu7 (Chaure et al., 1997), and ICL1 sequences of S. cerevisiae (Fernández et al., 1992) and Candida tropicalis (Atomi et al., 1990). In particular, S. cerevisiae ICL1 has been proposed to be phosphorylated at T53, followed by inactivation in the presence of glucose (López-Boado et al., 1988; Ordiz et al., 1996). However, the role of the motif KRGT in FPICL1 remains to be assessed. The C-terminal amino acids SKL, representing PTS1 (Subramani, 1993), suggest a peroxisomal localization of FPICL1 in F. palustris.
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). Furthermore, greater than 80 % of catalase activity was recovered from the pellet fraction (20KgP) containing mitochondria and peroxisomes, whereas 20 % was from the supernatant fraction (20KgS) (Fig. 2d). The result strongly supports the localization of catalase in microbodies, but not in the cytosol. Moreover, the type of microbody that was stained with DAB was distinct from mitochondria, as shown in Fig. 3(a, b), but it was not stained with DAB in the presence of a catalase inhibitor (data not shown) (Dijken et al., 1975; Fahimi & Baumgart, 1999). Furthermore, the fact that catalase has been commonly used as a peroxisomal marker enzyme supports the peroxisomal localization of catalase in F. palustris. The finding is in accordance with oleate-grown A. nidulans (Valenciano et al., 1996), but is different from glucose-grown S. cerevisiae, in which catalase is widely distributed in mitochondria, peroxisomes and cytosol (Petrova et al., 2002).
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). Furthermore, FPICL1 and MS were not cytosolic. Greater than 60 % of FPICL1, and 70 % of MS, activities were recovered from the fraction containing microbody (20KgP), whereas only 10 % each of the key enzymes was from the supernatant fraction containing cytosol (20KgS) (Fig. 2d).
The peroxisomal localization of FPICL1 was further confirmed by immunocytochemical analysis with rabbit antiserum containing anti-FPICL1 antibodies. We prepared the antiserum which specifically reacted with purified FPICL1. Only one protein band corresponding to the enzyme appeared on the gel loaded with cell-free extracts of F. palustris (Fig. 4a). A significant amount of immunogold labelling was located in the peroxisomes, but not in the cytosol or mitochondria (Fig. 4b). Alternatively, peroxisomal ICLs have been reported for Ashbya gossypii grown on C2 compounds (Maeting et al., 1999), C. tropicalis (Tanaka & Ueda, 1993), Yarrowia lipolytica (Titorenko et al., 1998), A. nidulans (Valenciano et al., 1996), and a glyoxysome-like ICL for acetate or oleate-grown Neurospora crassa (Kionka & Kunau, 1985). However, to the best of our knowledge, this investigation provides the first experimental evidence for peroxisomal localization of ICL in a glucose-grown basidiomycete.
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). We found that there was no difference in MDH activity between the cytosolic and the pellet fractions (data not shown), which suggested that MDH isozymes were distributed in various organelles and cytosol in a cell of F. palustris. A similar finding has been reported for S. cerevisiae, in which MDH isozymes are distributed throughout the mitochondria, peroxisomes and cytosol (Steffan & McAlister-Henn, 1992).
Features of carbon metabolism of F. palustris
Although we previously proposed that ICL was shared by the TCA and GLOX cycles because of isocitrate metabolism (Munir et al., 2001b), we propose a revised model of carbon metabolism in relation to oxalate biosynthesis, as shown by the enzyme localizations in Fig. 5. The TCA and GLOX cycles, and acetate recycling, have been proposed to be involved constitutively in carbon metabolism for oxalate fermentation of glucose-grown F. palustris, on the basis of enzyme activities detected from the mycelia catalysing each metabolic step shown in Fig. 5 (Munir et al., 2001b). The findings of peroxisomal FPICL1 and MS suggest a peroxisomal GLOX cycle, although the peroxisomal localization of MDH, citrate synthase and aconitase needs to be demonstrated for conclusive confirmation of a peroxisomal GLOX cycle. On the other hand, the presence of mitochondrial SDH suggests a mitochondrial TCA cycle, although the mitochondrial localization of fumarase, MDH, citrate synthase and aconitase remains to be investigated in order to confirm this. In this context, it is noteworthy that conversion of the C6 carboxylic acid to succinate through the TCA cycle is not thought to be significant, because a lower level of IDH activity than FPICL1 activity, and a lack of ODH activity, have been detected from the mycelia of F. palustris (Munir et al., 2001b). The presence of cytosolic ACS and OXA suggests that acetate recovery (Munir et al., 2001b) occurs in the cytosol, although the presence of cytosolic MDH needs to be demonstrated in order to confirm a cytosolic acetate recovery process. On the other hand, we have not investigated enzymes involved in conversion of glucose to acetyl-CoA via pyruvate.
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). This speculated transportation of succinate is different from the inducible succinate transportation from the cytosol to mitochondria in S. cerevisiae grown on a non-fermentable carbon source (Flores et al., 2000). (2) Isocitrate or citrate transportation from mitochondria to peroxisomes may occur to solve the problem caused by the lesser catabolic activity for C6 carboxylic acid metabolism in the TCA cycle than in the GLOX cycle (Munir et al., 2001b). (3) Acetyl-CoA formed in the cytosol must be transported to mitochondria to be catabolized because of the large amount of acetate produced concomitantly with oxalate (Munir et al., 2001b). A similar process of acetyl-CoA formation and utilization, concomitant with oxalate production, has been proposed for A. niger (Ruijter et al., 1999). Alternatively, acetyl-CoA must be transported to peroxisomes as a substrate for MS and citrate synthase. However, the dual localization of citrate synthase and aconitase in mitochondria and peroxisomes remains to be elucidated, although dual localization of citrate synthase occurs in S. cerevisiae (Lee et al., 2000). These transportations are postulated to occur constitutively, because oxalate fermentation is indispensable for the growth of F. palustris (Munir et al., 2001b). However, further research is needed to elucidate the mechanisms of transportation of the organic acid metabolites within a cell of F. palustris. In conclusion, FPICL1 and MS are key enzymes of the GLOX cycle, and occur constitutively in peroxisomes in F. palustris grown on glucose. An acetate-recovery system operates in the cytosol, and thus organic acid metabolites involved in oxalate biosynthesis are finally oxidized to oxalate.
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ACKNOWLEDGEMENTS |
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Received 21 November 2005;
revised 27 January 2006;
accepted 14 February 2006.
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), sucrose (
); (b) FPICL1 (
), MS (
); (c) SDH (
), catalase (Cat; 
); (d) relative enzyme activities in supernatant fraction (PNS) obtained from centrifugation at 3000 g (black bars). The activities in pellet (20 KgP; white bars) and supernatant (20 KgS; grey bars) fractions obtained from centrifugation at 20 000 g centrifugation. Data points represent means; SEM is given in (d), n=3.
