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Satoshi Sano, Masami Ueda, Sakihito Kitajima, Toru Takeda, Shigeru Shigeoka, Norihide Kurano, Shigetoh Miyachi, Chikahiro Miyake, Akiho Yokota, Characterization of Ascorbate Peroxidases from Unicellular Red Alga Galdieria partita, Plant and Cell Physiology, Volume 42, Issue 4, 15 April 2001, Pages 433–440, https://doi.org/10.1093/pcp/pce054
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Abstract
Galdieria partita, a unicellular red alga isolated from acidic hot springs and tolerant to sulfur dioxide, has at least two ascorbate peroxidase (APX) isozymes. This was the first report to demonstrate that two isozymes of APX are found in algal cells. Two isozymes were separated from each other at the hydrophobic chromatography step of purification and named APX-A and APX-B after the elution order in the chromatography. APX-B accounted for 85% of the total activity. Both isozymes were purified. APXs from Galdieria were monomers whose molecular weights were about 28,000, similar to stromal APX of higher plants. APX-A cross-reacted with monoclonal antibody raised against APX of Euglena gracilis in immunoblotting, but APX-B did not, although the antibody can recognize all other APXs tested. The amino-terminal sequences of APX-A and -B from Galdieria had some homology with each other but little homology with those from other sources. Their Km values for ascorbate and hydrogen peroxide were comparable with those of APX from higher plants. Unlike the green algal enzymes, the donor specificities of Galdieria APXswere as high as those of plant chloroplastic APX. On the contrary, these APXs reduced tertiary-butyl hydroperoxide as an electron acceptor as APXs from Euglena and freshwater Chlamydomonasdo. The inhibition of APX-A and -B by cyanide and azide, and characteristics of their light absorbance spectra indicated that they were heme peroxidases.
(Received December 25, 2000; Accepted February 5, 2001).
Introduction
Active oxygen species, such as superoxide anion radical, hydrogen peroxide, hydroxyl radical and singlet oxygen, are toxic to cells. Since the production of such molecular species is inevitable to living cells, effective, rapid scavenging of active oxygens is essential in protecting cells from oxidative damage. In photosynthetic organisms, superoxide is formed by the univalent photoreduction of dioxygen in PSI on thylakoid membranes as photosynthesis is saturated by light (Miyake and Yokota 2000). Hydrogen peroxide, which is formed from superoxide by the disproportionation catalyzed by superoxide dismutase (SOD), is reduced to water by ascorbate peroxidase (APX) using ascorbate (AsA) as the electron donor. Oxidized ascorbate is reduced to ascorbate by ferredoxin, monodehydroascorbate reductase and dehydroascorbate reductase (Asada 1999, Shimaoka et al. 2000).
APX is a member of the class I superfamily of heme peroxidases (Welinder 1992). In higher plants, four isozymes of APX with different cellular locations have been characterized; namely, stromal (Chen and Asada 1989, Nakano and Asada 1987), thylakoid-bound (Miyake et al. 1993), cytosolic (Chen and Asada 1989, Dalton et al. 1987, Elia et al. 1992, Mittler and Zilinskas 1991) and microbody APXs (Yamaguchi et al. 1995). In green algae, APX has also been found to function as an enzyme for scavenging of hydrogen peroxide. However, the subcellular localization and the properties of APX in some green algae differ from those of higher plants. In Euglena gracilis, APX is localized only in the cytosol (Shigeoka et al. 1980a, Shigeoka et al. 1980b). Freshwater Chlamydomonasreihardtii C9 grown in the selenium-free medium and halotolerant Chlamydomonas sp. W80 contain APX only in the stroma of chloroplasts (Takeda et al. 1997, Takeda et al. 2000). Chlorella vulgaris has been found to contain only one isozyme of APX (Takeda et al. 1998). APX has also been found in the zooxanthella (Lesser and Shick 1989), but the APX has not been characterized well. The algae that have two or more isozymes of APX have never been found. With respect to non-photosynthetic organisms, APX is found in the protozoan Trypanosoma cruzi (Boveris et al. 1980) and in bovine eyes (Wada et al. 1998).
Galdieria partita is a unicellular red alga inhabiting acidic, sulfur-containing hot springs. Galdieria has an ability to grow with aeration of 100% CO2 at 50°C and pH 1 in the presence of 5 Pa sulfur dioxide (SO2) or 5 Pa nitrogen dioxide (Hasegawa 1994). The resistance to such a high SO2 concentration has never been observed not only in higher plants but also in algae. The toxicity of SO2 is derived from SO32– generated from SO2 with cellular constituents. Active oxygen species are thought to be involved in the toxicity as the secondary toxic substances (Tanaka 1994). In higher plants, SO2 comes into leaves through the stomata and dissolves in intercellular space. SO32– in aqueous solution penetrates the plasma membranes of mesophyll cells and finally reaches organelles including chloroplasts. Furihata et al. (1997) found that uptake of sulfite into Chlorella cells through plasma membranes occurs by simple diffusion of the unionized form (H2SO3) through the lipid bilayer. Sulfite is present as its unionized form at low pHs, where Galdieria grows, and the red algal cells are expected to be easily filled with sulfite. Thus, it is highly probable that Galdieria has a higher ability to scavenge active oxygens. Firstly, we measured activities of enzymes participating in scavenging of active oxygens in the cell extract of G. partita. Activities of the enzymes involved in scavenging active oxygens were detected. We purified and characterized two isozymes of APX from G. partita. This is the first report demonstrating the occurrence of two isozymes of APX in algae and their purification from non-green photosynthetic organisms.
Materials and Methods
Culture of Galdieria
Galdieriapartita was isolated from the acidic hot spring along the shore on the Kodakara island of the Tokara Islands in Japan (Hasegawa 1994). Galdieria was grown in the Allen’s autotrophic medium (Allen 1959) containing 0.003% EDTA-2Na at pH 2.0 under continuous illumination (90 µmol photonsm–2 s–1) at 45°C with aeration of air. Cells at the log phase of growth, about 3 weeks after inoculation, were harvested by centrifugation at 10,000×g for 20 min. The pellet was resuspended in water and centrifuged again. The washed cells were frozen by liquid nitrogen and stored at –80°C.
Preparations of the extracts of Galdieria and spinach leaves and of the recombinant enzyme for stromal APX of spinach
Frozen cells (0.2 g) of Galdieria and spinach were homogenized in cooled pestles and mortars with 2 ml of 10 mM phosphate buffer (pH 7.0) containing 1.0 mM EDTA and 1.0 mM AsA. After the homogenate was centrifuged at 18,000×g at 4°C for 10 min, the supernatant was passed through HiTrap Desalting (Amersham Pharmacia Biotech, Uppsala, Sweden) for removing low-molecular-weight oxidoreductants. The fraction containing proteins was used as the crude extract. For fractionation of Galdieria cells, the homogenate was centrifuged at 1,000×g at 4°C for 10 min and the supernatant was centrifuged at 10,000×g at 4°C for 10 min again. After the centrifugation, the second supernatant was withdrawn as the soluble fraction. The pellet was suspended in the same medium and washed three times. The pellet finally obtained was the crude thylakoid fraction.
To prepare the recombinant stromal APX protein of spinach, Escherichia coli strain BL21(DE3) harboring its transit sequence-deleted cDNA in the pET3a expression vector (Yoshimura et al. 1998) was grown at 37°C in 300 ml of LB medium supplemented with 50 mg liter–1 of ampicillin. At the growth stage giving light absorbance at 600 nm at 0.5, isopropyl-β-d-thiogalactoside was added to a final concentration of 1 mM. When the absorbance reached 1.5, the cells were harvested by centrifugation and stored at –80°C. The E. coli cells were resuspended in 4 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 1 mM AsA and 1 mM phenylmethylsulfonylfluoride and disrupted by sonication. This lysate was centrifuged at 20,000×g for 20 min. The supernatant was applied onto a PD-10 column (Amersham Pharmacia) equilibrated with the above buffer to remove low molecular weight components from the cells. The eluate was concentrated with a Microcon (Millipore, Bedford, MA, U.S.A.) and used as the recombinant stromal APX of spinach.
Assays of enzymes and protein
The activity of APX was determined in 50 mM MES-KOH (pH 6.0 or 6.5) or 50 mM potassium phosphate buffer (pH 7.0) containing 0.5 mM AsA and 0.1 mM hydrogen peroxide and enzyme in a total volume of 1 ml (Nakano and Asada 1981). The hydrogen peroxide-dependent oxidation of AsA was followed by monitoring the decrease in absorbance at 290 nm assuming an absorption coefficient of 2.8 mM–1 cm–1. The amount of APX that can oxidize 1 µmol of AsA at 25°C for 1 min is defined as 1 unit of enzyme.
The assay mixture with other electron donors was the same as that for AsA-oxidation activity except that one of the following electron donor was added to the reaction mixture in place of AsA (Chen and Asada 1989); d-isoascorbate (2.8 mM–1 cm–1 at 290 nm), NAD(P)H (6.2 mM–1 cm–1 at 340 nm), pyrogallol (2.47 mM–1 cm–1 at 430 nm), 3,3′-diaminobenzidine (3.73 mM–1 cm–1 at 460 nm) and guaiacoll (26.6 mM–1 cm–1 at 470 nm) and glutathione. The rate of the hydrogen peroxide-dependent oxidation of the donors was determined with the absorption coefficients cited in parentheses. In the use of glutathione as the electron donor, the rate of its oxidation was measured by the coupling method with NADPH-glutathione reductase and NADPH (Little et al. 1970). The alkylhydroperoxide-dependent oxidation of AsA was also determined with 1.0 mM tert-butylhydroperoxide.
The activities of catalase (Bergmeyer 1983), SOD (Flohè and Ötting 1984), monodehydroascorbate reductase (Hossain and Asada 1985), dehydroascorbate reductase (Hossain and Asada 1984) and glutathione reductase (Shigeoka et al. 1987) were measured by the methods of the references cited.
The concentration of protein was determined by the method of Bradford (1976) with bovine serum albumin as the standard.
Purification of APX from Galdieria
The purification procedures were carried out at 4°C and the column chromatographies were done with a Fast-Protein Liquid Chromatography system (Amersham Pharmacia Biotech). Cells of Galdieria (5 g) were suspended in 100 ml of buffer A [50 mM potassium phosphate buffer (pH 6.5) containing 1.0 mM EDTA, 1.0 mM AsA and 1.0 mM phenylmethylsulfonylfluoride]. The suspended cells were disrupted by Bead-Beater (Biospec Products, Bartlesville, OK, U.S.A.) for 15 s four times with cooling intervals of 45 s. After centrifugation at 10,000×g for 20 min, the pellet containing unbroken cells and cell debris was suspended in buffer A and disrupted again. The supernatants from three extractions were pooled together and fractionated by ammonium sulfate between 40 and 80% saturation. The precipitated protein was dissolved in 50 ml of buffer B [10 mM potassium phosphate buffer (pH 6.5) containing 1.0 mM EDTA and 1.0 mM AsA] supplemented by ammonium sulfate at 40% saturation. This enzyme solution was loaded onto a column (26 mm i.d. × 300 mm) of TSKgel Butyl-Toyopearl 650 (Tosoh, Tokyo, Japan) equilibrated with the buffer B containing ammonium sulfate at 40% saturation. The column was developed with a linear gradient of 40–0% saturation of ammonium sulfate at the flow rate of 0.5 ml min–1. The fractions containing the activity of APX were pooled and ammonium sulfate was added to 90% saturation. The precipitate of proteins obtained by centrifugation at 10,000×g for 20 min was dissolved in buffer B containing 0.15 M KCl. This solution was gel-filtrated with a HiLoad 26/60 Superdex 75 prep grade column (Amersham Pharmacia Biotech) equilibrated with the buffer B containing 0.15 M KCl. The active fractions were pooled as purified APX.
Immunoblotting
SDS-PAGE was done by the method of Laemmli (1970) and proteins were transferred onto polyvinyliden difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, U.S.A.) by the semi-dry method. The membrane was reacted with the mouse monoclonal antibody raised against APX of Euglena as the first antibody (Ishikawa et al. 1996b) and then alkaline phosphatase-conjugated anti-mouse immunogloblin G goat immunogloblin G (Promega, Madison, WI, U.S.A.) as the second antibody. The alkaline phosphatase reaction was proceeded according to the manufacturer’s manual.
Estimation of molecular weight of APXs purified from Galdieria
The molecular weight of native APX purified form Galdieria was measured using a Superdex 200 column 10/30 (Amersham Pharmacia Biotech) previously calibrated with the following reference proteins: ribulose 1,5-bisphosphate carboxylase (mol wt 550,000), catalase (240,000), glucose 6-phosphate dehydrogenase (140,000), bovine serum albumin (66,000) and cytochrome c (12,000). The column was developed with 10 mM potassium phosphate buffer (pH 7.0) containing 0.15 M KCl, 1.0 mM AsA and 1.0 mM EDTA.
Determination of amino-terminal sequence of Galdieria APX
The purified enzyme was separated with SDS-PAGE and transferred onto PVDF membrane. The membrane was stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 solution in 50% (v/v) methanol and destained with 50% (v/v) methanol. The protein spot corresponding to the molecular weight was analyzed by an automated Edman degradation protein sequencer, model 492 (PE Biosystems, Foster City, CA, U.S.A.).
Results and Discussion
The crude extract of the Galdieria cells contained the activities of enzymes involved in scavenging of active oxygens, such as SOD, catalase, APX, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase (data not shown). The occurrence of these activities suggests that active oxygen species generated in the cells of Galdieria are scavenged by the same scavenging enzyme system as that found in higher plants and green algae. The specific activity of APX in the crude extract of Galdieria was 6 to 32 units (mg protein)–1. The APX activity was detected only in the soluble fraction but not in the membrane fraction.
Purification of two isozymes of APX from Galdieria
Purification of APX from Galdieria obtained from 5 g of cells is summarized in Table 1. At the purification step with Butyl-Toyopearl (Fig. 1), two isozymes of APX were separated from each other. Galdieria is the first alga to be shown to contain two or more isozymes of APX, since the green algae reported so far contain only one APX protein (Shigeoka et al. 1980a, Shigeoka et al. 1980b, Takeda et al. 1997, Takeda et al. 1998, Takeda et al. 2000). We called these isozymes APX-A and APX-B in the order of the elution. The elution profile (Fig. 1) show that APX-B accounts for about 85% of the total APX activity in the Galdieria cells. APX-A was purified with a yield of 1.4% and APX-B with a yield of 19% (Table 1). APX-B had a specific activity of 506 units (mg protein)–1, but was still contaminated by a few minor proteins as seen in SDS-PAGE (Fig. 2A). The APX-A preparation showed only one protein band in SDS-PAGE and had twofold higher specific activity than APX-B (Fig. 2A). These specific activities were comparable with those of APXs that have been purified from other photosynthetic organisms (Chen and Asada 1989, Dalton et al. 1987, Elia et al. 1992, Mittler and Zilinskas 1991, Takeda et al. 2000). This study is the first case succeeded in the purification of APX from non-green algae.
The molecular weights of both APXs deduced from SDS-PAGE were 28,000. The molecular weights of the two isozymes determined by native gel-filtration were the same as those deduced by SDS-PAGE (data not shown), indicating that these APXs are monomers. Stromal APX of higher plants is also monomer with the molecular weight of about 30,000 (Chen and Asada 1989), but cytosolic APX is a generally dimer (Chen and Asada 1989, Dalton et al. 1987, Mittler and Zilinskas 1991). APX from Euglena (Ishikawa et al. 1996b), freshwater Chlamydomonas (Takeda et al. 1997), Chlorella (Takeda et al. 1998) and halotorelant Chlamydomonas (Takeda et al. 2000) function as monomers with the molecular weights of 58,000, 32,000, 34,000 and 31,000, respectively. On this point, algal APX seems to be more similar to the stromal than cytosolic isozyme of higher plants.
Fig. 2B shows the immunoblotting of APXs of Galdieria with the monoclonal antibody raised against Euglena APX (Ishikawa et al. 1996a). APX-A showed a cross-reaction with the antibody, but APX-B did not. The monoclonal antibody has been reported to cross-react with APXs from chloroplasts and the cytosol of higher plants and from green algae (Ishikawa et al. 1995, Ishikawa et al. 1996a, Ishikawa et al. 1996b, Takeda et al. 1997, Takeda et al. 1998, Takeda et al. 2000) and to be specific for the common epitope of APXs. APX-B may lack the specific region conserved in APXs from many other sources.
Amino acid sequences of amino-terminal region
Purified APX isozymes were subjected to an Edman degradation to determine the amino-terminal amino acid sequence. The amino acid sequences of APX-A and -B were successfully determined from the amino termini to 20th and 22nd residues, respectively (Fig. 3). Seven residues were identical at the same position between the isozymes. APXs from higher plants share high degrees of homology. Especially ones that reside in the same subcellular compartment have more than 80% of homology. However, it has been reported that the amino-terminal sequences of APXs of some green algae, two species of Chlamydomonas, Euglena and Chlorella, have no significant homology with those of APX isozymes from higher plants and that there is only partial homology even among green algal enzymes (Ishikawa et al. 1996b, Takeda et al. 1997, Takeda et al. 1998). Both of amino-terminal regions of two isozymes from the red alga also exhibited very low degrees of homology to those of APXs known so far. However, in the case of halotolerant Chlamydomonas W80, the whole amino acid sequence of APX deduced from cDNA for it shows about 43–54% homology to APX isozymes of spinach although the sequence of amino-terminus region has a little homology with those of higher plants (Takeda et al. 2000). It may be likely the internal sequences of APX-A and -B may share the common features found in APXs from the other sources.
Enzymatic properties
The Km values of APX-A and APX-B for AsA were 355 µM and 174 µM and those for hydrogen peroxide were 17 µM and 28 µM, respectively (Table 2). APX-A showed a higher affinity for hydrogen peroxide than APX-B. Inversely, APX-B showed a higher affinity for AsA than APX-A. These Km values are similar to thoseof APX isozymes that have ever been purified (Chen and Asada 1989, Mittler and Zilinskas 1991, Miyake et al. 1993, Takeda et al. 1998).
When AsA was the electron donor, the two isozymes showed different pH optima; APX-A had a broad optimum at pH 6.0 to 6.5 and APX-B had a sharper optimum peak at pH 6.5 to 7.0 than APX-A (Table 2).
APX-A and -B showed a high specificity for AsA and no or very low activity with electron donors such as pyrogallol and NADH (Table 2). While the donor specificity of chloroplastic APX isozymes of higher plants are very high, cytosolic APX and green algal APX are able to catalyze the oxidation of pyrogaloll with hydrogen peroxide at appreciable rates (Asada 1994, Ishikawa et al. 1996b, Takeda et al. 1997, Takeda et al. 1998, Takeda et al. 2000). Thus, the donor specificities of APX isozymes from Galdieria were more similar to those of chloroplastic APX rather than those of cytosolic APX. The APXs from Euglena and freshwater Chlamydomonas were known to reduce alkyl hydroperoxides as well as hydrogen peroxide with AsA (Ishikawa et al. 1996a, Takeda et al. 1997) though enzymes of higher plants, Chlorella and halotolerant Chlamydomonas did not (Asada 1994, Takeda et al. 1998, Takeda et al. 2000). APX isozymes from Galdieria were also able to reduce t-butyl hydroperoxide as an electron acceptor in the presence of AsA (Table 2).
Inhibitors
APX is classified as a member of the class I family of heme peroxidases (Welinder 1992). Cyanide and azide inhibited the activity of both APX-A and -B (Table 3), indicating those APX isozymes from Galdieriaare also heme peroxidases (Asada 1994). The inhibition by thiol reagents is one of the characteristic properties of APX to be distinguished from guaiacol peroxidases. It has been proposed that a thiol group participates in the active center (Amako et al. 1994). According to crystallographic analyses of APX from pea, the oxidation of AsA occurs in the vicinity of Cys-32 (Mandelman et al. 1998), which is the only one cysteine residue conserved in APXs from various sources. p-Chloromercuribenzoate strongly inactivated both isozymes of Galdieria. However, iodoacetate, iodoacetamide and 5,5′-dithiobis(2-nitrobenzoic acid) inhibited APX-A about 30% but did not inhibit APX-B (Table 4). These have been observed with APXs from higher plants (Chen and Asada 1989, Nakano and Asada 1987, Miyake and Asada 1992).
Hydroxylamine, p-aminophenol and hydroxyurea are suicide inhibitors for APX (Chen and Asada 1990). The former two reagents also inhibited APXs of Galdieria. Another suicide inhibitor, hydroxyurea, showed different inhibitory actions between both isozymes; APX-B was relatively resistant to this inhibitor (Table 3). APX oxidizes the suicide inhibitors to the respective radicals by hydrogen peroxide and those radicals seem to inactivate APX (Chen and Asada 1990). APX-B may not be able to oxidize hydroxyurea effectively. In immunoblotting, APX-B could not react with the monoclonal antibody raised against Euglena APX, although the antibody has a wide cross-reactivity with APXs from higher plants and green algae. It is considered that the antibody recognizes the common structure among APXs from various sources (Ishikawa et al. 1995, Ishikawa et al. 1996a, Ishikawa et al. 1996b, Takeda et al. 2000). The low sensitivity of APX-B to hydroxyurea might reflect its unique structure, as considered in the reactivity with the monoclonal antibody.
Absorption spectra
The oxidized APX-A and -B gave absorption spectra characteristic to the ferric high-spin state of heme (Paul et al. 1953). The oxidized APX-B showed a Soret peak at 404 nm with the absorption coefficient of 7.0×104 M–1 cm–1, and the peak was shifted to 433 nm by the reduction with dithionite (Fig. 4). In addition to the Soret band, the oxidized enzyme had broad absorption peaks at 502 and 624 nm. These two peaks disappeared and an α-peak newly appeared at 557 nm upon the reduction of the enzyme by dithionite. APX-B showed a little difference in the absorption coefficient at the Soret peaks between their oxidized and reduced forms, as was observed in the case of stromal APXs from tea and spinach (Chen and Asada 1989, Nakano and Asada 1987). The Soret peak of APX-B shifted by reducing and forming cyanide complex toward longer wavelength as compared to that of stromal APX. This is also observed with thylakoid-bound APX from spinach leaves (Miyake et al. 1993). The cyanide complex of the enzyme showed a Soret band at 420 nm and additional absorption peak at 544 nm.
Stability of heme
One of the properties characteristic to APX is its inactivation in an AsA-depleted medium. Under the conditions where the concentration of AsA is lower than 20 µM, the chloroplastic isozymes of APX are inactivated with a half-life of about 30 s, but cytosolic APX has a half-life of an hour or more (Asada 1997). Because this inactivation can be avoided under anaerobic conditions, dioxygen has been assumed to participate in the inactivation. A trace of hydrogen peroxide produced by the autooxidation of AsA brought over with APX from the stock enzyme solution oxidizes APX to its oxidized intermediate (Compound I). If Compound I of APX is not reduced by AsA, it is oxidized and decomposed to the inactivated form (Miyake et al. 1993, Miyake and Asada 1996).
Both of two isozymes from Galdieria in the AsA-depleted medium containing 100 µM hydrogen peroxide under the anaerobic conditions were inactivated at the slow rate, as was the cytosolic APX of higher plants (Amako et al. 1994), while proteins containing recombinant stromal APX showed rapid loss of the APX activity (Fig. 5). These results show that both of the Galdieria APXs are members of the stable isozymes of APX in the absence of AsA, as the cytosolic APX of higher plants. It has been reported that APXs of green algae are also as stable in the absence of AsA as cytosolic APXs of higher plants. It seems that algal APXs are relatively stable to depletion of the electron donor (Miyake et al. 1991, Ishikawa et al. 1996b, Takeda et al. 1997, Takeda et al. 1998, Takeda et al. 2000). It has been reported that APXs of green algae are also as stable in the absence of AsA as cytosolic APXs of higher plants.
Thermal stability
Because G. partita is a thermophilic alga, it is expected that its APX isozymes would be thermostable. The activities of APXs from the cytosol of spinach leaves and Galdieria declined by treating at 60°C for 2 min to about 70% of the control without any heat treatment (Table 4). The treatment at 80°C reduced the activity of cytosolic APX of spinach leaves to 17%, but both APX isozymes from Galdieria still kept about 60% of the original activities. The melting temperature of recombinant APX from higher plant was found to be 49°C (Jones et al. 1998). Cytochrome c peroxidase from yeast, which is also a member of class I peroxidase, has the melting temperature of 62°C (Bujons et al. 1997). It is possible that the thermal stability of APX from Galdieria is higher than that of cytochrome c peroxidase.
Conclusions
Sulfuric oxidants generate active oxygen species and are injurious to photosynthetic organisms (Asada and Kiso 1973). Since G. partita lives in the habitat containing these oxidants, it was expected that the organism might have a more active system for scavenging the active oxygen species than the higher plants easily damaged by the oxidants. In fact, the activity of APX of the cells of Gladieria was higher than in higher plants. Two isozymes of APX purified from the red alga had very similar enzymatic properties to those from higher plants, except that the Galdieria enzymes were much more thermostable than the relatively stable cytosolic form of APX of higher plants. In this context, it is reasoned that the occurrence of a high activity of APX in the Galdieria cells may be related to the observed tolerance to sulfuric oxidants. Comparisons between the subcellular localizations and the primary structures of the two isozymes with those of higher plants and green algae will be the next step to explain the mechanism of the tolerance of G. partita to active oxygen species.
Acknowledgements
This study was partly supported by the Petroleum Energy Center and the Research Association for Biotechnology subsidized by the Ministry of Economy, Trade and Industry of Japan.
Present address: Department of Biological Resource Science, Faculty of Agriculture, Kyoto Prefectural University, Kyoto, 606-8522 Japan.
Corresponding author: E-mail, yokota@bs.aist-nara.ac.jp; Fax, +81-743-72-5569.
Purification steps | Protein (mg) | Activity (units) | Specific activity (units (mg protein)–1) | Yield (%) | Purification (-fold) |
Crude extract | 486 | 6,635 | 13.65 | 100 | 1 |
40% satd. (NH4)2SO4 | 291 | 9,550 | 32.85 | 144 | 2.4 |
Butyl-Toyopearl | |||||
(APX-A) | 3.6 | 424.3 | 117.9 | 6.4 | 8.6 |
(APX-B) | 18.1 | 2,443 | 134.6 | 37 | 9.9 |
Superdex 75 | |||||
(APX-A) | 0.08 | 95.3 | 1,191 | 1.4 | 87 |
(APX-B) | 3.62 | 1,246 | 506 | 19 | 37 |
Purification steps | Protein (mg) | Activity (units) | Specific activity (units (mg protein)–1) | Yield (%) | Purification (-fold) |
Crude extract | 486 | 6,635 | 13.65 | 100 | 1 |
40% satd. (NH4)2SO4 | 291 | 9,550 | 32.85 | 144 | 2.4 |
Butyl-Toyopearl | |||||
(APX-A) | 3.6 | 424.3 | 117.9 | 6.4 | 8.6 |
(APX-B) | 18.1 | 2,443 | 134.6 | 37 | 9.9 |
Superdex 75 | |||||
(APX-A) | 0.08 | 95.3 | 1,191 | 1.4 | 87 |
(APX-B) | 3.62 | 1,246 | 506 | 19 | 37 |
Purification steps | Protein (mg) | Activity (units) | Specific activity (units (mg protein)–1) | Yield (%) | Purification (-fold) |
Crude extract | 486 | 6,635 | 13.65 | 100 | 1 |
40% satd. (NH4)2SO4 | 291 | 9,550 | 32.85 | 144 | 2.4 |
Butyl-Toyopearl | |||||
(APX-A) | 3.6 | 424.3 | 117.9 | 6.4 | 8.6 |
(APX-B) | 18.1 | 2,443 | 134.6 | 37 | 9.9 |
Superdex 75 | |||||
(APX-A) | 0.08 | 95.3 | 1,191 | 1.4 | 87 |
(APX-B) | 3.62 | 1,246 | 506 | 19 | 37 |
Purification steps | Protein (mg) | Activity (units) | Specific activity (units (mg protein)–1) | Yield (%) | Purification (-fold) |
Crude extract | 486 | 6,635 | 13.65 | 100 | 1 |
40% satd. (NH4)2SO4 | 291 | 9,550 | 32.85 | 144 | 2.4 |
Butyl-Toyopearl | |||||
(APX-A) | 3.6 | 424.3 | 117.9 | 6.4 | 8.6 |
(APX-B) | 18.1 | 2,443 | 134.6 | 37 | 9.9 |
Superdex 75 | |||||
(APX-A) | 0.08 | 95.3 | 1,191 | 1.4 | 87 |
(APX-B) | 3.62 | 1,246 | 506 | 19 | 37 |
APX-A | APX-B | |
Km (mM) | ||
AsA | 355 | 174 |
Hydrogen peroxide | 17 | 28 |
Optimum pH | 6.0–6.5 | 6.5–7.0 |
Electron donors a | ||
0.5 M AsA | 100 | 100 |
0.6 mM d-Isoascorbate | 105 | 15 |
0.15 mM NADPH | 10 | 0 |
0.15 mM NADH | 0 | 0 |
20 mM Pyrogallol | 0 | 0 |
20 mM 3,3′-Diaminobenzidine | 0 | 0 |
20 mM Guaiacol | 0 | 5 |
1 mM Glutathione | 23 | 0 |
Electron acceptors b | ||
0.1 mM Hydrogen peroxide | 100 | 100 |
1 mM tert-Butylhydroperoxide | 61 | 12 |
APX-A | APX-B | |
Km (mM) | ||
AsA | 355 | 174 |
Hydrogen peroxide | 17 | 28 |
Optimum pH | 6.0–6.5 | 6.5–7.0 |
Electron donors a | ||
0.5 M AsA | 100 | 100 |
0.6 mM d-Isoascorbate | 105 | 15 |
0.15 mM NADPH | 10 | 0 |
0.15 mM NADH | 0 | 0 |
20 mM Pyrogallol | 0 | 0 |
20 mM 3,3′-Diaminobenzidine | 0 | 0 |
20 mM Guaiacol | 0 | 5 |
1 mM Glutathione | 23 | 0 |
Electron acceptors b | ||
0.1 mM Hydrogen peroxide | 100 | 100 |
1 mM tert-Butylhydroperoxide | 61 | 12 |
a The activity with 0.5 mM AsA was fixed to 100.
b The activity with 0.1 mM H2O2 was fixed to 100.
APX-A | APX-B | |
Km (mM) | ||
AsA | 355 | 174 |
Hydrogen peroxide | 17 | 28 |
Optimum pH | 6.0–6.5 | 6.5–7.0 |
Electron donors a | ||
0.5 M AsA | 100 | 100 |
0.6 mM d-Isoascorbate | 105 | 15 |
0.15 mM NADPH | 10 | 0 |
0.15 mM NADH | 0 | 0 |
20 mM Pyrogallol | 0 | 0 |
20 mM 3,3′-Diaminobenzidine | 0 | 0 |
20 mM Guaiacol | 0 | 5 |
1 mM Glutathione | 23 | 0 |
Electron acceptors b | ||
0.1 mM Hydrogen peroxide | 100 | 100 |
1 mM tert-Butylhydroperoxide | 61 | 12 |
APX-A | APX-B | |
Km (mM) | ||
AsA | 355 | 174 |
Hydrogen peroxide | 17 | 28 |
Optimum pH | 6.0–6.5 | 6.5–7.0 |
Electron donors a | ||
0.5 M AsA | 100 | 100 |
0.6 mM d-Isoascorbate | 105 | 15 |
0.15 mM NADPH | 10 | 0 |
0.15 mM NADH | 0 | 0 |
20 mM Pyrogallol | 0 | 0 |
20 mM 3,3′-Diaminobenzidine | 0 | 0 |
20 mM Guaiacol | 0 | 5 |
1 mM Glutathione | 23 | 0 |
Electron acceptors b | ||
0.1 mM Hydrogen peroxide | 100 | 100 |
1 mM tert-Butylhydroperoxide | 61 | 12 |
a The activity with 0.5 mM AsA was fixed to 100.
b The activity with 0.1 mM H2O2 was fixed to 100.
Inhibitors | APX-A (%) a | APX-B (%) a | |
None | 100 | 100 | |
1 mM | KCN | 0 | 0 |
1 mM | Sodium azide | 21 | 46 |
0.2 mM | p-Chloromercuribenzoic acid | 15 | 27 |
0.1 mM | 5,5′-Dithiobis(2-nitrobenzoic acid) | 74 | 144 |
5 mM | Iodoacetamide | 74 | 99 |
5 mM | Iodoacetate | 70 | 138 |
0.7 mM | p-Aminophenol | 51 | 44 |
0.2 mM | Hydroxylamine | 22 | 13 |
10 mM | Hydroxyurea | 25 | 85 |
Inhibitors | APX-A (%) a | APX-B (%) a | |
None | 100 | 100 | |
1 mM | KCN | 0 | 0 |
1 mM | Sodium azide | 21 | 46 |
0.2 mM | p-Chloromercuribenzoic acid | 15 | 27 |
0.1 mM | 5,5′-Dithiobis(2-nitrobenzoic acid) | 74 | 144 |
5 mM | Iodoacetamide | 74 | 99 |
5 mM | Iodoacetate | 70 | 138 |
0.7 mM | p-Aminophenol | 51 | 44 |
0.2 mM | Hydroxylamine | 22 | 13 |
10 mM | Hydroxyurea | 25 | 85 |
a The activity measured without any inhibitor was fixed at 100.
Inhibitors | APX-A (%) a | APX-B (%) a | |
None | 100 | 100 | |
1 mM | KCN | 0 | 0 |
1 mM | Sodium azide | 21 | 46 |
0.2 mM | p-Chloromercuribenzoic acid | 15 | 27 |
0.1 mM | 5,5′-Dithiobis(2-nitrobenzoic acid) | 74 | 144 |
5 mM | Iodoacetamide | 74 | 99 |
5 mM | Iodoacetate | 70 | 138 |
0.7 mM | p-Aminophenol | 51 | 44 |
0.2 mM | Hydroxylamine | 22 | 13 |
10 mM | Hydroxyurea | 25 | 85 |
Inhibitors | APX-A (%) a | APX-B (%) a | |
None | 100 | 100 | |
1 mM | KCN | 0 | 0 |
1 mM | Sodium azide | 21 | 46 |
0.2 mM | p-Chloromercuribenzoic acid | 15 | 27 |
0.1 mM | 5,5′-Dithiobis(2-nitrobenzoic acid) | 74 | 144 |
5 mM | Iodoacetamide | 74 | 99 |
5 mM | Iodoacetate | 70 | 138 |
0.7 mM | p-Aminophenol | 51 | 44 |
0.2 mM | Hydroxylamine | 22 | 13 |
10 mM | Hydroxyurea | 25 | 85 |
a The activity measured without any inhibitor was fixed at 100.
Spinach cytosolic APX (%) | Galdieria | ||
APX-A (%) | APX-B (%) | ||
4°C | 100 | 100 | 100 |
50°C | 65 | 73 | 77 |
80°C | 17 | 58 | 63 |
Spinach cytosolic APX (%) | Galdieria | ||
APX-A (%) | APX-B (%) | ||
4°C | 100 | 100 | 100 |
50°C | 65 | 73 | 77 |
80°C | 17 | 58 | 63 |
The APX isozymes were incubated at the temperatures indicated for 2 min in the presence of 0.5 mM AsA just before measuring the remaining activities.
Spinach cytosolic APX (%) | Galdieria | ||
APX-A (%) | APX-B (%) | ||
4°C | 100 | 100 | 100 |
50°C | 65 | 73 | 77 |
80°C | 17 | 58 | 63 |
Spinach cytosolic APX (%) | Galdieria | ||
APX-A (%) | APX-B (%) | ||
4°C | 100 | 100 | 100 |
50°C | 65 | 73 | 77 |
80°C | 17 | 58 | 63 |
The APX isozymes were incubated at the temperatures indicated for 2 min in the presence of 0.5 mM AsA just before measuring the remaining activities.
Abbreviations
References
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