Journal of Experimental Botany, Vol. 51, No. 345, pp. 713-719,
April 2000
© 2000 Oxford University Press
A complex containing both trypsin inhibitor and dehydroascorbate reductase activities isolated from mitochondria of etiolated mung bean (Vigna radiata L. (Wilczek) cv. Tainan No. 5) seedlings
Institute of Botany, Academia Sinica, Nankang, Taipei 115, Taiwan, Republic of China
Received 26 August 1999; Accepted 15 December 1999
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Abstract |
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A complex containing trypsin inhibitor (TI) activity was extracted with 0.1 M TRIS buffer (pH 7.9) from trypsin-treated mitochondria of etiolated mung bean seedlings, and further purified with a Superdex 200 FPLC column. This partially purified complex with an Mr about 820 kDa exhibited additional dehydroascorbate (DHA) reductase activity with specific activities of 0.21, 1.53 and 1.54 µmol ascorbate formed min-1 mg-1 protein at pH 6.0, 6.5 and 7.0, respectively, when glutathione was added. Much lower DHA reductase activity (0.013 and 0.026 µmol ascorbate formed min-1 mg-1 protein at pH 6.5 and 7.0, respectively) was found when glutathione was omitted. The isolated complex gave positive results when it was tested by TI activity staining after SDS-PAGE, and could be recognized by a polyclonal antibody which was raised against 38 kDa sweet potato Kunitz-type TI, one of the root storage proteins of sweet potato. The possible physiological functions of this complex with both TI and DHA reductase activities were discussed.
Key words: Dehydroascorbate reductase, glutathione, mitochondria, mung bean, sweet potato, trypsin inhibitor.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Ascorbate plays an important role in protecting plant cells (Dalton et al., 1986
; Kobayashi et al., 1995; Foyer et al., 1997; Mazhoudi et al., 1997) and insect cells (Summers and Felton, 1993) against the action of reactive oxygen species. In plants, peroxide radicles are scavenged via the ascorbate–glutathione pathway, a coupled series of redox reactions involving four enzymes: ascorbate peroxidase (EC 1.11.1.11), monodehydroascorbate (MDA) reductase (EC 1.6.5.4), dehydroascorbate (DHA) reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) (Asada, 1992; Miyake and Asada, 1992; Dalton et al., 1993; De Leonardis et al., 1995). This pathway has been studied mainly in chloroplasts, in which the possible reactive oxygen species produced by PSI during photosynthesis might cause serious damage. However, the ascorbate–glutathione pathway has also been found in the cytosol (Borraccino et al., 1986; Dalton et al., 1986, 1993; Asada, 1992; Elia et al., 1992), mitochondria and peroxisomes (Jimenez et al., 1997, 1998). When ascorbate functions as an antioxidant in cells, it is oxidized to DHA via successive reversible single electron transfers with MDA as a free radical intermediate. MDA radicals have a relatively short lifetime and disproportionate at neutral pH values and above to DHA and ascorbate (Foyer and Mullineaux, 1998). MDA radicals can be reduced directly back to ascorbate by reduced ferredoxin or by NAD(P)H-dependent MDA reductases (Hossain et al., 1984). DHA can be reduced directly by reduced glutathione at alkaline pH values and also by enzymes which catalyse this conversion (DHA reductase). It has been observed that MDA was a sensitive endogenous index of oxidative stress in leaf tissues (Heber et al., 1996). Transgenic tobacco plants expressing antisense RNA of cytosolic ascorbate peroxidase showed increased susceptibility to ozone injury (Orvar and Ellis, 1997).
The first 17 N-terminal amino acid residues of DHA reductase purified from spinach chloroplasts have been found to be identical to those of soybean trypsin inhibitor (TI) (Trumper et al., 1994). Both the reduced (thiol) form of soybean TI, which was produced by treating the oxidized form of soybean TI with dithiothreitol, and the native (thiol) form of spinach chloroplast DHA reductase could reduce DHA to regenerate ascorbate in the presence of glutathione; meanwhile, the oxidized (disulphide) form of spinach chloroplast DHA reductase, which was produced with oxidized glutathione, also exhibited TI activity. It has been suggested that the DHA reductase activities measured in plant extracts might be due to the side reactions of proteins containing redox-active dicysteine sites (Morell et al., 1997) and plant cells do not possess any specific DHA reductase in the chloroplasts. However, DHA reductases that catalyse the reduction of DHA by reduced glutathione have been purified from spinach, potato and rice (Foyer and Halliwell, 1977; Hossain and Asada, 1984; Dipierro and Borraccino, 1991; Kato et al., 1997). Foyer and Mullineaux favour the view that DHA and DHA reductase exist in leaves and other plant tissues (Foyer and Mullineaux, 1998).
Both sweet potato TIs, the major storage proteins of roots (Hou and Lin, 1997b) and dioscorins, the main tuber storage protein of yam (Hou et al., 1999) were shown to have both MDA reductase and DHA reductase activities and may respond to environmental oxidative stresses. In this study, evidence is presented to show that a complex with an Mr about 820 kDa from trypsin-treated mitochondria of etiolated mung bean seedlings, exhibited not only TI activity but also DHA reductase activity.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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A complex isolated from mitochondria of etiolated mung bean seedlings contained trypsin inhibitor activity
Mitochondria were isolated from 3-d-old etiolated mung bean seedlings (Vigna radiata L. (Wilczek) cv. Tainan No. 5). Two hundred µg of 14H mitochondria from the fraction with a sedimentation coefficient value of 1500S was further purified with continuous sucrose gradients (0.6–1.8 M) (Dai et al., 1998
), then treated with 2 mg trypsin at a trypsin/mitochondria (T/M) ratio of 10 at 37 °C for 30 min and then PMSF was added to a final concentration of 2 mM to stop the trypsin reaction (Zwizinski et al., 1984; Ohba and Schatz, 1987; Heins and Schmitz, 1996). After washing five times with 10 mM Tricine-KOH buffer (pH 7.2) containing 0.4 M mannitol and 1 mM EGTA (Dai et al., 1991), the trypsin-treated mitochondria were extracted with 0.1 M `TRIS buffer (pH 7.9) at 4 °C overnight. The extracts were then concentrated with Centriprep 10 (Amicon, USA) to a small volume for further purification using a Superdex 200 FPLC column (Pharmacia, Uppsala, Sweden) and 0.1 M TRIS elution buffer (pH 7.9). The flow rate was 30 ml h-1 and 0.5 ml eluant was saved for each fraction. The active portions with TI activity were collected for further investigations.
Purification of total TIs from etiolated mung bean seedlings
The 3-d-old etiolated mung bean seedlings (Vigna radiata L. (Wilczek) cv. Tainan No. 5) were homogenized four times with 3 vols (v/w) of 100 mM TRIS buffer (pH 7.9) containing 100 mM NaCl for 30 s. The homogenates were filtered through four layers of cheesecloth and centrifuged at 15 000 g for 30 min twice. The supernatants were directly loaded onto a trypsin-Sepharose 4B affinity column according to a method described previously in order to purify mung bean total TIs which were then lyophilized for further use (Hou and Lin, 1997a).
TI activity and protein determinations
TI activity was assayed according to the method of (Lee and Lin, 1995) by the inhibition of trypsin-catalysed hydrolysis of N-benzoyl-L-arginine-4-nitroanilide. Three determinations were averaged for each data point. Protein was determined according to the method of Bradford (Bradford, 1976) using Bio-Rad protein assay kit (Hercules, CA), and bovine serum albumin was used as a standard.
Determination of DHA reductase activity
DHA reductase activity was determined by measuring the absorbance at 265 nm of regenerated ascorbate at pH 6.0, 6.5 and 7.0 according to the method of Trumper et al. (Trumper et al., 1994) with or without 4 mM glutathione and expressed as µmol ascorbate formed min-1 mg-1 protein. Non-enzymatic reduction of DHA by reduced glutathione in phosphate buffers of different pHs was also measured in a separate cuvette at the same time. A standard curve was plotted using 0.1–6 nM of ascorbate.
TI activity, protein and immunostainings
four parts of samples were mixed with one part of sample buffer, namely 60 mM TRIS buffer (pH 6.8) containing 2% SDS, 25% glycerol and 0.1% bromophenol blue without 2-mercaptoethanol at 4 °C overnight. Silver stain kit (Bio-Rad, Hercules, CA) or Coomassie brilliant blue G-250 (Neuhoff et al., 1985) for protein staining was used on SDS-PAGE gels. TI activity staining was carried out on either a discontinuous gel system with 1.5 cm 4% stacking gel and 6.5 cm 7.5% separating gel or a discontinuous gel system with 1.5 cm 4% stacking gel and two layers of separating gels, 7.5% (1.5 cm) and 15% (5 cm), of SDS-PAGE (Hou and Lin, 1998). The polyclonal antibody against 38 kDa TI from sweet potato storage roots (Ipomoea batatas [L.] Lam var. Tainong 57) was raised against rabbit. After electrophoresis, a complex containing trypsin inhibitor activity was electroblotted onto an Immobilon PVDF membrane, and the immunostaining was achieved with an alkaline phosphatase-naphthyl phosphate system (Lin and Lu, 1994).
Chemicals
All chemicals and reagents were of the highest purity available. Trypsin (TPCK-treated, 40 U mg-1) and N-benzoyl-L-arginine-4-nitroanilide were purchased from E Merck Inc. (Darmstadt, Germany); Seeblue prestained markers for SDS-PAGE was from Novex (San Diego, CA); Immobilon PVDF membrane was from Millipore (Bedford, MA). Molecular weight markers for gel filtration including chymotrypsinogen A (25 kDa), bovine serum albumin (67 kDa), catalase (232 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa) were from Pharmacia Biotech AB (Uppsala, Sweden).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The isolated mitochondria of etiolated mung bean seedlings contain TI activity
In order to remove cytoplasmic protein contamination during mitochondria isolations, trypsin was used for the treatment of isolated mitochondria (Zwizinski et al., 1984
; Ohba and Schatz, 1987; Heins and Schmitz, 1996). The isolated 14H mitochondrial fraction in this experiment was similar to that widely used for mitochondrial protein synthesis in the organelle (Leaver et al., 1983; Dai et al., 1991). After trypsin hydrolysis, mitochondria were washed with buffers five times to remove trypsin and cytoplasmic hydrolysates. The remaining protein contents of the last washing were almost undetectable. Figure 1 shows the TI activity and specific TI activity in crude extracts of mung bean seedlings and trypsin-treated mitochondria of mung bean seedlings with different trypsin/mitochondria ratios (T/M ratio, 0 as a control, 10, 25, 50, and 100 as tests). The TI activity and specific TI activity of trypsin-treated mitochondria were all higher than the control except the specific activity with T/M ratio 100. Similar TI activities, which were about 2-fold of the control, were found for all these four treatments with different T/M ratios. Treatment of proteinase K also gave similar results (data not shown). Porin, the outer membrane marker protein of mitochondria, was undetectable by immunostaining when the T/M ratio was higher than 10 (H Dai, unpublished data). It is clear that trypsin hydrolysis could eliminate cytoplasmic protein contamination as well as the marker protein porin. However, when the higher amounts of trypsin were used, the more extractable proteins were obtained. Hence the specific activity of TI became lower. It is possible that the increasing amount of extractable proteins is due to the partial disruption of mitochondrial membrane during trypsin hydrolysis. The control (T/M 0) mitochondria sample exhibited higher TI specific activity than those of crude extracts. All the results of Fig. 1 suggest that the contamination of TI activity from cytoplasm, if any, is negligible.
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Figure 2
shows both protein staining (A) and TI activity staining (B) on 7.5% SDS-PAGE gels of crude extracts of mung bean seedlings (lane 1) and samples of mitochondria prepared with different T/M ratios (lanes 2 to 6 for T/M ratios of 0, 10, 25, 50, and 100, respectively). There were TI activity bands detected in lanes 2 to 6 (the upper part of Fig. 2B, indicated by the arrow). They all have molecular mass above 250 kDa. In contrast, the crude extract of mung bean seedlings (lane 1) exhibited a TI activity band with a molecular mass of about 56 kDa (indicated by the lower arrow). The results of Fig. 2 suggest that the molecule with TI activity from mitochondria was different from the cytoplasmic one, and further support the existence of TI activity in mitochondria. This is the first report that mitochondria exhibited TI activity with a molecular mass higher than 250 kDa.
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A complex from isolated mitochondria of etiolated mung bean seedlings exhibited TI activity
Large amounts of isolated 14H mitochondria with T/M 10 treatment were used for TI extraction and purification as described in Materials and methods.
Figure 3 shows the chromatograms on a Superdex 200 column of TI purification from T/M 10 treated mitochondria. Only one sharp peak containing TI activity was detected, and the molecular mass was estimated to be about 820 kDa using a standard curve with five molecular weight markers (Fig. 3, inset). The specific activity of TI increased 2-fold from 1.43 (mg trypsin inhibited mg-1 protein added) of mitochondrial extracts to 2.82 of the FPLC column-purified fraction. However, a further purification step with a trypsin affinity column after the FPLC column for the TI-containing complex was not successful. It is possible that the active site of trypsin inhibitor is masked by other components of the complex. On the other hand, the total TIs from etiolated mung bean seedlings could be purified using the trypsin affinity column (Hou and Lin, 1997a). Figure 4 shows protein staining (A), activity staining (B) and immunostaining (C) of mung bean total TIs after affinity column purification (lane 1); T/M 10 mitochondria extracts (lane 2); and TI activity peak after the FPLC column purification (lane 3) on discontinuous SDS-PAGE. The gel system included a 1.5 cm 4% stacking gel and two layers of separating gels, 7.5% (1.5 cm) and 15% (5 cm). Immunostaining was done after protein bands were transferred to a PVDF membrane. The polyclonal antibody, which was raised against sweet potato 38 kDa TI, was used as a probe to detect TIs in the mitochondria of mung bean. When activity staining of mung bean total TIs (Fig. 4B, lane 1) was compared with protein staining (Fig. 4A, lane 1), the major TI activity staining came from three protein bands, two were about 8 kDa and one was about 20 kDa. Some minor TI activity bands, such as 38 kDa and 56 kDa, were also found in mung bean total TI preparations. However, the polyclonal antibody of sweet potato TI recognized 20 kDa and 38 kDa mung bean TI, but not 8 kDa low molecular mass TI (Fig. 4C, lane 1).
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Trypsin inhibitors of Bowman-Birk type (about 8 kDa) and Kunitz type (about 20 kDa) were frequently found in legume seeds, but they were different in molecular weight, amino acid sequences, reactive sites, and disulphide linkage numbers (Birk, 1976
; Laskowski and Kato, 1980). It has been observed that the amino acid sequence of sweet potato TI shared high homology to other Kunitz-type TIs (Yeh et al., 1997). The mung bean TIs (20 and 38 kDa), recognized by the polyclonal antibody of sweet potato TI, may be of the Kunitz type. Mitochondrial extracts with T/M 10 treatment contained many protein bands (Fig. 4A
, lane 2), but only one with molecular mass larger than 250 kDa showed both positive TI activity staining (Fig. 4B, lane 2) and positive immunostaining (Fig. 4C, lane 2). In addition, TI activity peak (820 kDa) after FPLC column purification also exhibited both positive TI activity staining (Fig. 4B, lane 3) and immunostaining (Fig. 4C, lane 3) which were identical to the results of T/M 10 treatment (Fig. 4B, C, lane 2). Magnifications of positive TI activity and immunostaining regions of the gels of mung bean mitochondria (Fig. 4D) showed clearer results. These results showed that TI activity in mitochondria with a molecular mass of 820 kDa, which was recognized by a polyclonal antibody of sweet potato, might contain Kunitz type TI(s). Some other protein bands were found on SDS-PAGE gel after sample buffer treatment while only a single peak of TI activity with high molecular weight was found during FPLC column chromatography. It was postulated that a complex containing Kunitz type TI(s) interacting with other proteins was partially disrupted when treated with SDS detergent. There is ongoing work to identify each component of this new TI complex.
A complex isolated from mitochondria of mung bean seedlings exhibited both TI and DHA reductase activities
The physiological functions of this high molecular weight complex with TI activity in mitochondria were not clear. Although proteases were found in mitochondria (Langer and Neupert, 1996; Skulachev, 1996; Gores et al., 1998; Arlt et al., 1998; Ishisaka et al., 1998), the occurrence of proteinaceous proteinase inhibitors in mitochondria has not been reported yet. It is postulated that a complex containing TI activity in mitochondria might regulate some protease activities in mitochondria during the growth of mung bean seedlings.
It has been found that soybean TI exhibited DHA reductase activity(Trumper et al., 1994). Both sweet potato TIs, the major storage proteins of roots (Hou and Lin, 1997b) and dioscorins, the main tuber storage protein of yam (Hou et al., 1999) were shown to have both MDA reductase and DHA reductase activities and may respond to environmental oxidative stresses. Hence, according to the method described previously (Trumper et al., 1994), the partially purified complex from etiolated mung bean mitochondria after the FPLC column, which had a TI specific activity of 2.82 mg trypsin inhibited mg-1 protein, was used to determine the DHA reductase activities at pH 6.0, 6.5 and 7.0, respectively, with or without the addition of 4 mM (final concentration) glutathione. Non-enzymatic reduction of DHA in phosphate buffers of different pHs was measured in a separate cuvette at the same time. The activity was expressed as µmol ascorbate formed min-1 mg-1 protein. Figure 5 shows the results of DHA reductase activity of the purified complex containing TI activity from mung bean mitochondria. With glutathione added, linear time-courses of DHA reductase activities were found at pH 6.0, 6.5 and 7.0 with specific activities of 0.21, 1.53 and 1.54 µmol ascorbate formed min-1 mg-1 protein, respectively (Fig. 5A). They were comparable with DHA reductase (1.3 µmol ascorbate formed min-1 mg-1 protein at pH 7.0) from spinach chloroplast (Trumper et al., 1994) and DHA reductase (0.2 µmol ascorbate formed min-1 mg-1 protein at pH 7.2) from mitochondria of pea leaves (Jimenez et al., 1997, 1998).
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Without the addition of glutathione (Fig. 5B
), much lower DHA reductase activities were found at pH 6.5 and 7.0 with specific activities of 0.013 and 0.026 µmol ascorbate formed min-1 mg-1 protein, respectively. The results were similar to thioredoxin m and f from spinach chloroplast (0.012 and 0.032 µmol ascorbate formed min-1 mg-1 protein, respectively) and the thioredoxin from E. coli (0.024 µmol ascorbate formed min-1 mg-1 protein) without glutathione added (Trumper et al., 1994). It has been demonstrated that the ascorbate–glutathione pathway (including DHA reductase activity) indeed existed in mitochondria and could protect the organelle from hydrogen peroxide damage (Jimenez et al., 1997, 1998). It is possible that the 820 kDa complex containing both TI and DHA reductase activiies performs a parallel pathway to scavenge reactive oxygen species produced by electron transport chains in mitochondria.
It has been argued (Morell et al., 1997) that the DHA reductase activities measured in plant extracts might be due to side reactions of proteins containing redox-active dicysteine sites and plant cells do not possess any specific DHA reductase in chloroplasts. These authors suggested that TI and thioredoxin in spinach chloroplast might play a role of DHA reductase instead of a real one existing. However, Foyer and Mullineaux (Foyer and Mullineaux, 1998) commented that plants do contain ‘true’ DHA reductases because DHA reductases that catalyses the reduction of DHA by reduced glutathione have been purified from rice, spinach and potato (Foyer and Halliwell, 1977; Hossain and Asada, 1984; Dipierro and Borraccino, 1991; Kato et al., 1997). Several other proteins such as glutaredoxins (thiol transferases), protein disulphide isomerases, and even Kunitz-type trypsin inhibitors have been shown to have DHA reductase activity. Nevertheless, the amino acid sequence of the rice DHA reductase is quite distinct from these other eznymes (Kato et al., 1997). Convincing proof was provided that a specific DHA reductase does exist in both photosynthetic and non-photosynthetic tissues, as well as being detectable in barley and rice (Kato et al., 1997). Furthermore, the presence of near perfect matches of the N-terminal data from Kato et al. (Kato et al., 1997) have been found (Foyer and Mullineaux, 1998) with sequences from the Arabidopsis EST cDNA database.
It is proposed that both TI and DHA reductase activities detected in mitochondria of etiolated mung bean seedlings might reside on the same protein molecule, and this deserves further studies. However, the possibility cannot be excluded that plants do contain ‘true’ DHA reductases.
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Acknowledgments |
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Dr YH Lin and Dr H Dai, respectively, want to thank the financial support (NSC88-2311-001-043) from the National Science Council and from the Institute of Botany, Academia Sinica, Republic of China.
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1 To whom correspondence should be addressed. Fax: +886 2 2782 7954. E-mail:boyhlin{at}ccvax.sinica.edu.tw or E-mail:bodaihwa{at}ccvax.sinica.edu.tw
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