Sweet potato storage root thioredoxin h2 with both dehydroascorbate reductase and monodehydroascorbate reductase activities.

Running title: Thioredoxin h2 with both dehydroascorbate

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reductase and monodehydroascorbate reductase activities

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Title of article:

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Sweet potato storage root thioredoxin h with both

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dehydroascorbate reductase and monodehydroascorbate

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reductase activities

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Authors' name and address:

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Guan-Jhong HUANG

, Hsien-Jung CHEN

, Yuan-Shiun CHANG

, Ming-Jyh SHEU

and 10

Yaw-Huei LIN

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1

Institute of Chinese Pharmaceutical Sciences, China Medical University, Taichung 12

404, Taiwan;

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2

Department of Horticulture, Chinese Culture University, Taipei 111, Taiwan;

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3

Department of Physiology, School of Medicine, China Medical University, Taichung 15

404, Taiwan;

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4

Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, 17

Taiwan;

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19

Corresponding author:

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Dr. Yaw-Huei Lin

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Institute of Plant and Microbial Biology, Academia Sinica, Nankang,

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Taipei 11529, Taiwan, ROC

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FAX: 886 (2) 2782-7954; TEL: 886 (2) 2789-9590 ext. 320

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E-mail: [email protected] 25

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Abstract

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Recombinant thioredoxin h (Trx h2) overproduced in E. coli (M15) was purified 29

by Ni

-chelated affinity chromatography as previously reported (Huang et al., 2004a).

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The molecular mass of Trx h2 is ca. 1.4 kDa determined by SDS (sodium dodecyl 31

sulfate)-PAGE (polyacrylamide gel electrophoresis). Trx h2 had antioxidant activity 32

(Huang et al., 2004b). Trx h2 reduced dehydroascorbate (DHA) in the presence of 33

glutathione to regenerate ascorbate (AsA). However, without glutathione, Trx h2 has 34

very low DHA reductase activity. AsA was oxidized by AsA oxidase to generate 35

monodehydroascorbate (MDA) free radicals. MDA was also reduced by Trx h2 to 36

AsA in the presence of NADH mimicking the MDA reductase catalyzed reaction.

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These data suggest that Trx h2 have both DHA reductase and MDA reductase 38

activities.

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Keywords: Sweet potato storage roots; Thioredoxin h; Dehydroascorbate reductase;

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Monodehydroascorbate reductase;

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INTRODUCTION

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Ascorbic acid (AsA) plays an important role in protecting plant cells against the 45

action of reactive oxygen species (Dalton et al., 1986; Kobayashi et al., 1995). In 46

plants, peroxide-scavenging was accomplished through the AsA–glutathione pathway, 47

a coupled series of redox reactions involving four enzymes: AsA-specific peroxidase 48

(EC 1.11.1.11), monodehydroascorbate (MDA) reductase (EC 1.6.5.4), 49

dehydroascorbate (DHA) reductase (EC 1.8.5.1), and glutathione reductase (EC 50

1.6.4.2) (Dalton et al., 1993; Leonardis et al., 1995). This pathway has been studied 51

mainly in chloroplasts, in which the possible reactive oxygen species produced by PS 52

I during photosynthesis might cause serious damage. However, the AsA–glutathione 53

pathway has also been found in cytosol (Borraccino et al., 1986; Elia et al., 1992), 54

mitochondria (Lunde et al., 2006), and peroxisomes (Jimenez et al., 1997). When AsA 55

functions as an antioxidant in cells, it is oxidized to MDA free radical, and MDA 56

reductase catalyzes the reduction of MDA back to AsA with NAD(P)H (Hossain et al., 57

1984). MDA was a sensitive endogenous index of oxidative stress in leaf tissues 58

(Heber et al., 1996).

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Thioredoxins, the ubiquitous small proteins with a redox active disulfide bridge, 60

are important regulatory elements in a number of cellular processes (Buchanan, 1991;

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Vianey-Liaud et al., 1994). They all contain a distinct active site, WCGPC, which is 62

able to reduce disulfide bridges of target proteins. Initially described as hydrogen 63

carriers in ribonucleotide reduction in E. coli, they were found to serve as electron 64

donors in a variety of cellular redox reaction (Holmgren, 1985). From genome 65

sequencing data, a significant diversity of thioredoxin genes containing five different 66

multigenic families (f, m, x, o and h) was observed (Mestres-Ortega and Meyer, 1999;

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Meyer et al., 2002; Balmer and Buchanan, 2002). The ferredoxin-thioredoxin system 68

(thioredoxins f and m) has been proved to regulate several enzymatic activities 69

associated with photosynthetic CO

assimilation in chloroplasts. Thioredoxin x 70

contains a transit peptide similar to those required for chloroplast and mitochondria 71

targeting; however, its function is not clearly defined (Mestres-Ortega and Meyer, 72

1999). A new type of plant mitochondrial thioredoxin o was also shown to regulate the 73

activities of several mitochondrial proteins by disulfide bond reduction (Laloi et al., 74

2001).

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Thioredoxin h is generally assumed to be cytosolic, which was supported by the 76

absence of a transit peptide in the genes cloned for the isoforms from tobacco (Marty 77

and Meyer, 2001; Brugidou et al., 1993), Arabidopsis (Rivera-Madrid et al., 1993;

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1995), Triticum aestivum (Gautier et al., 1998), germinating wheat seeds (Serrato etal., 79

2001) and barley seed proteome (Kenji et al., 2003). Moreover, the existence of 80

several forms of thioredoxin h detected in spinach leaves (Florencio et al., 1988), and 81

wheat flour (Johnson et al., 1987) supports the view that higher plants possess 82

multiple and divergent thioredoxin genes (Rivera-Madrid et al., 1995). In this study, 83

we present evidence to show that the recombination protein, thioredoxin h2 exhibit 84

both DHA reductase and MDA reductase activities.

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MATERIALS AND METHODS

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Chemicals

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Ascorbic acid, dehydroascorbic acid, electrophoresis grade acrylamide and Bis 89

(N,N′-methylenediacrylamide), TEMED (N,N,N′,N′-tetramethylenediamine) and APS 90

(ammonium persulfate) were from E. Merck Inc. (Germany). Other chemicals and 91

solvents were purchased from Sigma Chemical Company (St. Louis, MO). The low 92

molecular weight kits for electrophoresis were obtained from Pharmacia (Uppsala, 93

Sweden).

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Expression of thioredoxin h2 in E. coli

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Thioredoxin h2 (Gene Bank accession number: AY344228; Trx h2) was expressed 97

in E. coli. The coding sequence was amplified from Trx h2 cDNA using an 98

oligonucleotide (5´-GAG AGG ATC CAA TGG GAG GGG CT-3´), with a BamH I 99

site (underlined) at the putative initial Met redisue, and an oligonucleotide (5´- ATT 100

TGA AGC TTG ATT GAT GCT -3´), with a Hind III site at the 3´ end. The PCR 101

fragment was subcloned in pGEM T-easy vector. And the plasmid was then digested 102

with BamH I and Hind III and subcloned in pQE32 expression vector (QIAexpress 103

expression system, Qiagen). The resulting plasmid, termed pQE-Trx h2, was 104

introduced into E. coli (M15). Cultures of the transformed E. coli (M15) 105

overexpressed a protein of the expected molecular mass, which was purified by 106

affinity chromatography in Ni-NTA columns (Qiagen), according to the manufacturer´

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s instructions.

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DHA reductase activity assay 110

The DHA reductase activity of Trx h2 was assayed according to the method of 111

Trümper et al. (Tru¨mper et al., 1994) with some modifications. Ten milligrams DHA 112

were dissolved in 5.0 ml of 100 mM phosphate buffer with two pH values (pH 6.0 and 113

7.0). The reaction was carried out at 30°C by adding 100 L Trx h2 solution (100 g 114

protein) to 0.9 ml DHA solution with or without 4 mM glutathione. The increase of

115

absorbance at 265 nm was recorded for 5 min. Non-enzymatic reduction of DHA in 116

phosphate buffer was measured in a separate cuvette at the same time. A standard 117

curve was plotted using 0.1– 50 nmol AsA.

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MDA reductase activity assay 120

The MDA reductase activity of Trx h2 was assayed according to Hossain et al.

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(Hossain et al., 1984) by following the decrease in absorbance at 340 nm due to 122

NADH oxidation. MDA free radicals were generated by AsA oxidase (EC 1.10.3.3) in 123

the assay system (Yamazaki and Pette, 1961). The reaction mixtures contained 50 124

mM phosphate buffer (pH 6.0 and 7.0, respectively), 0.33 mM NADH, 3 mM AsA, 125

AsA oxidase (0.9 U), and 200 L Trx h2 solution (200 g protein) in a final volume 126

of 1 ml. Trx h2 solution was replaced with glutathione for controls.

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Protein stainings of thioredoxin h2 in 15% SDS–PAGE gels 129

Trx h2 were examined by protein staining in 15% SDS–PAGE (sodium 130

dodecylsulfate–polyacrylamide gel electrophoresis) gels (Huang et al., 2004c).

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Twenty microliter samples were mixed with 25 L sample buffer containing 60 mM

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Tris buffer (pH 6.8), 2% SDS, 25% glycerol and 0.1% bromothymol blue, with 133

2-mercaptoethanol (2-ME) in a final concentration of 14.4 mM, and heated at 100°C 134

for 5 min for protein staining. Coomassie brilliant blue G-250 was used for protein 135

staining 136

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MDA reductase activity staining in 15% SDS–PAGE gels 138

Trx h2 were examined for MDA reductase by activity stainings in 15%

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SDS–PAGE gels. Diaphorase activity staining for MDA reductase activity of Trx h2 140

was according to the methods of Kaplan and Beutler (Kaplan and Beutler, 1967) in a 141

15% SDS–PAGE gel. After electrophoresis, the gel was washed with 25%

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isopropanol in 10 mM Tris buffer (pH 7.9) twice to remove SDS before activity 143

staining.

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Statistical Analysis. Means of triplicate were calculated. Student’s t test was used for 146

comparison between two treatments. A difference was considered to be statistically 147

significant when p < 0.05.

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RESULTS

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Effect of pH (6.0 and 7.0) on dehydroascorbate reductase activity of thioredoxin 151

h2.

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To express sweet potato Trx h2 in E. coli, the coding sequence of Trx h2 was 153

subcloned in a pQE-32 expression vector so that sweet potato thioredoxin h was 154

produced with a 6x His-tag at the N-terminus. SDS-PAGE analysis of crude extracts 155

from transformed E. coli (M15) showed a high level of a polypeptide with the 156

expected molecular mass (ca. 14 kDa). The expressed protein was purified from crude 157

extracts by Ni

-chelate affinity chromatography, which yielded highly purified 158

His-tagged thiredoxin h (Huang et al., 2004b).

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The purified Trx h2 samples were used to examine DHA reductase activity. Fig. 1 160

shows AsA regeneration ( 265nm) from DHA at both pH 6.0 and 7.0 with (A) or 161

without (B) glutathione. Fig. 1A shows that Trx h2 exhibited DHA reductase activity 162

and could reduce DHA back to AsA. The specific activities of DHA reductase for Trx 163

h2 in the presence of glutathione were 7.17 and 35.91 nmol AsA produced/min/mg 164

protein at pH 6.0 and 7.0, respectively. However, in the absence of glutathione, very 165

low DHA reductase activities of Trx h2 were found (Fig. 1B): only 0.01 and 0.68 166

nmol AsA produced/min/mg protein at pH 6 and 7.0, respectively. Trx h2 acts as a

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GSH-dependent DHA reductase (Fig. 2), and the rate of reduction was closely 168

proportional to the concentration of GSH. There was either a significant increase in 169

the DHA activity treated with 1, 2, 4 and 4  GSH (p < 0.05). It was reported that 170

thioredoxin m and thioredoxin f from spinach chloroplast and thioredoxin from 171

Escherichia coli exhibit very low DHA reductase activities without glutathione [29].

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Effect of pH (6.0 and 7.0) on monodehydroascorbate reductase activity of 174

thioredoxin h.

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MDA was reduced to AsA in coupling with NADH oxidation (Δ A340nm) at pH 6.0 176

and 7.0 when Trx h2 were used as MDA reductase. Trx h2 exhibited MDA reductase 177

activity at pH 6.0 and 7.0 (Fig. 3), with higher activity at pH 6.0 than pH 7.0 in our 178

assay system. Trx h2 acts as a GSH-dependent MDA reductase (Fig. 3), and the rate 179

of reduction was closely proportional to the concentration of GSH.

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Protein and diaphorase activity stainings in 15% SDS–PAGE gels for detection 182

h.

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MDA reductase activity staining of Trx h2 was done for diaphorase activity 184

(Kaplan and Beutler, 1967) on SDS-PAGE gels (Fig. 4). Comparing Fig 5 (A)

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(protein staining) with 4(B) of Trx h2 one can see that the diaphorase activity staining 186

for MDA reductase activity came from 14 kD Trx h2. MDA reductase and DHA 187

reductase were shown to contain free thiol groups in their catalytic sites (Borraccino rt 188

al., 1989). When AsA is the sole hydrogen donor, the AsA peroxidase, guaiacol 189

peroxidase, and AsA oxidase can produce MDA (Kaplan and Beutler, 1967).

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Nonenzymatic oxidations of AsA also produce MDA when cells were under oxidative 191

stress (Hossain et al., 1984). Dimerization of heat shock protein 25 via S–S bond 192

formation can occur in cells in response to various oxidative stresses (Zavialov et al., 193

1998).

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DISCUSSION

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This is the first report showing that TRX h2 displays both DHA reductase and 197

MDA reductase activities with some unique characteristics.

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In many physiological studies DHA reductase is regarded as one of the chloroplast 199

enzymes involved in the protection against oxidative stress. A specific DHA reductase 200

is frequently demanded as part of the enzymatic equipment to avoid oxidative stress.

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In plant extracts a glutathione-dependent DHA reductase activity has been observed 202

(Hossain etal., 1984) which will recycle DHA to ascorbate. An increase of DHA

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reductase activity and an accumulation of DHA have been frequently implied as 204

biochemical indicators of oxidative stress in plant metabolism (Wise, 1995) but a 205

characterization of DHA reductase has remained elusive because of rapid loss of 206

enzyme activity.

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The thioredoxin system is vital for chloroplast metabolism because redox control 208

of at least 12 different enzymes is achieved by the reductive cleavage of regulatory 209

disulfide bridges in these target enzymes (Buchanan, 1991). Trx h2 thiol-disulfide 210

interchanges were found during DHA reduction to regenerate AsA. Thionin was 211

reported to have intermolecular disulfide linkages with other proteins (Pinerio et al., 212

1995). Thiol groups are central to most redox-sensitive processes in the cell, and their 213

redox state controls cellular processes such as growth, differentiation, and apoptosis.

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The intracellular thiol homeostasis is maintained by the thioredoxin systems, which 215

utilize reducing equivalents from NADPH to reduce both protein and low molecular 216

weight disulfides.

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MDA reductase purified from potato was shown to contain thiol groups in their 218

catalytic sites (Leonardis et al., 1995). Fernando et al., (1992) found that thioredoxin 219

can act as a radical scavenger and facilitate the regeneration of oxidatively damaged 220

proteins and TRX h2 might contribute to its antioxidant activities against hydroxyl 221

and peroxyl radicals (Huang et al., 2004b). When AsA is the sole hydrogen donor, the

222

AsA oxidase can produce MDA (Yamazaki and Pette, 1961). Nonenzymatic 223

oxidations of AsA also produce MDA when cells suffer from oxidative stress (Heber 224

et al., 1996). Taking the above results into consideration, we construct a reduction 225

scheme of both dehydroascorbate (DHA) and monodehydroascorbate (MDA) to 226

ascorbate (AsA) catalyzed by Trx h2 of sweet potato roots. DHA and MDA can be 227

reduced to regenerate AsA by Trx h2 in order to prevent oxidative damage to cytosols 228

of sweet potato roots.

229 230

Acknowledgment 231

The authors want to thank the China Medical University for the financial support 232

(CMU95-211).

233 234

References:

235

Balmer, Y. B. B. Buchanan. 2002.Yet another plant thioredoxin. Trends Plant Sci. 7 : 236

191-193.

237

Borraccino, G. S. Dipierro, O. Arrigoni. 1986. Purification and properties of ascorbate 238

free-radical reductase from potato tubers. Planta 167: 521–526.

239

Borraccino, G. S. Dipierro, O. Arrigoni. 1989. Interaction of ascorbate free radical

240

reductase with sulphhydryl reagents, Phytochemistry 28: 715–717.

241

Brugidou, C. I. Marty, Y. Chartier, Y. Meyer. 1993. The Nicotiana tabacum genome 242

encodes two cytoplasmic thioredoxin genes which are differently expressed.

243

Mol. Gen. Genet. 238: 285-293.

244

Buchanan, B.B. 1991. Regulation of CO2 assimilation in oxygenic photosynthesis:

245

The ferredoxin/thioredoxin system. Perspective on its discovery, present status and 246

future development. Arch. Biochem. Biophys. 288: 1-9.

247

Dalton, D.A. S.A. Russell, F.J. Hanus, G.A. Pascoe, H.J. Evans. 1986. Enzymatic 248

reactions of ascorbate and glutathione that prevent peroxide damage in soybean 249

root nodules. Proc. Natl. Acad. Sci. USA 83: 3811–3815.

250

Dalton, D.A. L.M. Baird, L. Langeberg, C.Y. Taugher, W.R. Anyan, C.P. Vance G.

251

Sarath. 1993. Subcellular location of oxygen defense enzymes in soybean 252

(Glycine max [L.] Merr.) root nodules. Plant Physiol. 102: 481–489.

253

Elia, M.R. G. Borraccino, S. Dipierro. 1992. Soluble ascorbate peroxidase from potato 254

tubers. Plant Sci. 85: 17–21.

255

Fernando, M.R. H. Nanri, S. Yoshitake, K. Nagatakuno, S. Minakami. 1992.

256

Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial 257

cell. Eur. J. Biochem. 209: 917-922.

258

Florencio, F.J. B.C. Yee, T.C. Johnson, B.B. Buchanan. 1988. An NADP/thioredoxin 259

system in leaves: purification and characterization of NADP-thioredoxin 260

reductase and thioredoxin h from spinach. Arch. Biochem. Biophys. 26:

261

496-507.

262

Gautier, M.F. V. Lullien-Pellerin, F. Lamotte-Guery, A. de Guirao, P. Joudrier. 1998.

263

Characterization of wheat thioredoxin h cDNA and production of an active 264

Triticum aestivum protein in Escherichia coli. Eur. J. Biochem. 252: 314-324.

265

Heber, U. C. Miyake, J. Mano, C. Ohno, K. Asada. 1996. Monodehydroascorbate 266

radical detected by electron paramagnetic resonance spectrometry is a sensitive 267

probe of oxidative stress in intact leaves. Plant Cell Physiol. 37: 1066–1072.

268

Holmgren, A. 1985. Thioredoxin. Annu. Rev. Biochem. 54: 237-271.

269

Hossain, M.A. Y. Nakano, K. Asada. 1984. Monodehydroascorbate reductase in 270

spinach chloroplasts and its participation in regeneration of ascorbate for 271

scavenging hydrogen peroxide. Plant Cell Physiol. 25: 385–395.

272

Huang, D.J. H.J. Chen, W.C. Hou, Y.H. Lin. 2004a. Isolation and characterization of 273

thioredoxin h cDNA from sweet potato (Ipomoea batatas [L.] Lam. ‘Tainong 57’) 274

storage roots. Plant Sci. 166: 515-523.

275

Huang, D.J. H.J. Chen, W.C. Hou, C.D. Lin, Y.H. Lin. 2004b. Active recombinant 276

thioredoxin h protein with antioxidant activities from sweet potato (Ipomoea 277

batatas [L.] Lam. ‘Tainong 57’) storage roots. J. Agri. Food Chem. 52:

278

4720-4724.

279

Huang, D.J. H.J.Chen, W.C. Hou, T.E. Chen, Y.H. Lin. 2004c. In vitro reduction of 280

trypsin inhibitor by purified NADPH/ Thioredoxin system from sprouts of sweet 281

potato (Ipomoea batatas (L) Lam.) storage roots. Plant Sci. 166: 435-441.

282

Jimenez, A. J.A. Hernandez, L.A. del Rio, F. Sevilla. 1997. Evidence for the presence 283

of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves.

284

Plant Physiol. 114: 275–284.

285

Johnson, T.C. K. Wada, B.B. Buchanan. 1987 A. Holmgren, Reduction of purothionin 286

by the wheat seed thioredoxin system. Plant Physiol. 85: 446-451.

287

Kaplan, J.C. E. Beutler. 1967. Electrophoresis of red cell NADH- and 288

NADPH-diaphorases in normal subjects and patients with congenital 289

methemoglobinemia, Biochem. Biophys. Res. Commun. 29: 605–610.

290

Kenji, M. C. Finnie, O. Ø stergarrrd, B. Svensson. 2003. Identification, cloning and 291

characterization of two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, from the 292

barley seed proteome. Eur. J. Biochem. 270: 2633-2643.

293

Kobayashi, K. S. Tagawa, S. Sano K. Asada. 1995. A direct demonstration of the 294

catalytic action of monodehydroascorbate reductase by pulse radiolysis. J. Biol.

295

Chem. 270: 27551–27554.

296

Leonardis, S.D. G.D. Lorenzo, G. Borraccino, S. Dipierro. 1995. A specific ascorbate 297

free radical reductase isozyme participates in the regeneration of ascorbate for 298

scavenging toxic oxygen species in potato tuber mitochondria. Plant Physiol. 109:

299

847–851.

300

Lunde, C. U. Baumann , N.J. Shirley , D.P. Drew, G.B. Fincher. 2006. Gene structure 301

and expression pattern analysis of three monodehydroascorbate reductase (Mdhar) 302

genes in Physcomitrella patens: implications for the evolution of the MDHAR 303

family in plants. Plant Mol. Biol. 60(2): 259-75.

304

Marty, I. Y. Meyer. 1991. Nucleotide sequence of a cDNA encoding a tobacco 305

thioredoxin. Plant Mol. Biol. 17: 143-147.

306

Mestres-Ortega, D. Y. Meyer. 1999. The Arabidopsis thaliana genome encodes at least

307

four thioredoxins m and a new prokaryotic-like thioredoxin. Gene 240: 307-316.

308

Meyer, Y. F. Vignols, J.P. Reichheld. 2002. Classification of plant thioredoxins by 309

sequence similarity and intron position. Methods Enzymol. 347: 394-402.

310

Laloi, C. N. Rayapuram, Y. Chartier, J.M. Grienenberger, G. Bonnard, Y. Meyer. 2001.

311

Identification and characterization of mitochondrial thioredoxin system in plants.

312

Proc. Natl. Acad. Sci. U S A. 98: 14144-14149.

313

Pinerio, M. I. Diaz, P. Rodriguez-Palenzuela, E. Titarenko, F. Garcia-Olmedo. 1995.

314

Selective disulphide linkage of plant thionins with other proteins. FEBS Lett.

315

369: 239-242.

316

Rivera-Madrid, R. P. Marinho, C. Brugidou, Y. Chartier, Y. Meyer. 1993. Nucleotide 317

sequence of a cDNA clone encoding an arabidopsis thaliana thioredoxin h.

318

Plant Physiol. 102: 327-328.

319

Rivera-Madrid, R. D. Mestres, P. Marinho, J.P. Jacquot, P. Decottignies, M.

320

Miginiac-Maslow, Y. Meyer. 1995. Evidence for five divergent thioredoxin h 321

sequences in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 92:

322

5620-5624.

323

Serrato, A.J. J.L. Crespo, F.J. Florencio, J.C. Francisco. 2001. Characterization of 324

two thioredoxins h with predominant localization in the nucleus of aleurone and 325

scutellum cells of germinating wheat seeds. Plant Mol. Biol. 46: 361-371.

326

Tru¨mper, S. H. Follmann, I. Ha¨berlein. 1994. A novel dehydroascorbate reductase 327

from spinach chloroplasts homologous to plant trypsin inhibitor, FEBS Lett. 352:

328

159–162.

329

Vianey-Liaud, N. K. Kobrehel, Y. Sauvaire, J.H. Wong, B. B. Buchanan. 1994.

330

Lipoic acid in wheat grains. J. Agric. Food Chem. 42: 1110-1114.

331

Wise, R.R. 1995. Chilling-enhanced photooxidation: The production, action and study 332

of reactive oxygen species produced during chilling in the light. Photosynthesis 333

Res. 45: 79-97 334

Yamazaki, I. L.H. Pette. 1961. Mechanism of free radical formation and 335

disappearance during the ascorbic acid oxidase and peroxidase reactions, 336

Biochem. Biophys. Acta 50: 62-69.

337

Zavialov, A.V. M. Gaestel, T. Korpela, V.P. Zav’yalov. 1998. Thiol:disulfide 338

exchange between small heat shock protein 25 and glutathione, Biochem.

339

Biophys. Acta 1388: 123–132.

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甘藷塊根中硫氧化還原蛋白

h

344

壞血酸還原酶的活性

345

黃冠中

陳顯榮

張永勳

許明志

林耀輝

346

1

中國醫藥大學中國藥學研究所 347

2

中山大學生命科學系 348

3

中國醫藥大學醫學系生理科 349

4

中央研究院植物暨微生物研究所 350

在大腸桿菌(M15)中大量表現重組蛋白質硫氧化還原蛋白 h2 (Trx h2)，利用鎳 351

離子螯合之親和性管柱純化。 Trx h2 經 SDS-PAGE 分析其分子量約為 1.4 kDa.

352

由於 Trx h2 具有抗氧化活性。Trx h2 在含有穀胱甘肽時，去氫抗壞血酸 353

(dehydroascorbate, DHA)含量會降低而生成抗壞血酸(ascorbate, AsA)。但是，

354

在不含有穀胱甘肽時，Trx h2 只有非常低 DHA reductase 活性。AsA 經由 AsA 氧 355

化酶氧化生成單去氫抗壞血酸(monodehydroascorbate, MDA) 自由基。MDA 也可 356

經由 Trx h2 而降低了 AsA 生成， 在 NADH 存在時模仿 MDA reductase 催化反 357

應。這結果建議, Trx h2 同時具有去氫抗壞血酸還原酶和單去氫抗壞血酸還原 358

酶的活性。

359

關鍵詞:

甘藷塊根; 硫氧化還原蛋白 h; 去氫抗壞血酸還原酶; 單去氫抗壞血 360

酸還原酶;

361

Figure Legends

362

Figure. 1. Effect of pH (6.0 and 7.0) on dehydroascorbate reductase activity. Purified 363

recombinant protein of thioredoxin h2 was with (A) or without (B) 4 mM 364

glutathione in the reaction mixtures. The reaction was carried out at 30°C by 365

L thioredoxin h2 solution (100 g protein, 100 mM phosphate 366

buffer, pH 7.0 and 6.0) to 0.9 ml DHA solution with or without 4 mM 367

glutathione. Glutathione was used to be a control. The increase of absorbance 368

at 265 nm was recorded for 5 min.

369 370

Figure. 2. Dependence of dehydroascorbate reductase activity of thioredoxin h2 on 371

GSH concentration. L

372

thioredoxin h2 solution (100 g protein, 100 mM phosphate buffer, pH 7.0) to 373

0.9 ml DHA solution with different concentrations of glutathione. The increase 374

of absorbance at 265 nm was recorded for 5 min.

375 376

Figure. 3. Effect of pH (6.0 and 7.0) on monodehydroascorbate reductase activity of 377

thioredoxin h2. The reaction mixtures contained 50 mM phosphate buffer (pH 378

6.0 and 7.0), 0.33 mM NADH, 3 mM AsA, AsA oxidase (0.9 U), and 200 L 379

thioredoxin h2 solution (200 g protein) in a final volume of 1 ml.

380

Thioredoxin h2 solution was replaced with gluthioione for controls.

381 382

Figure. 4. Protein (A) and diaphorase activity (B) stainings in 15% SDS–PAGE gels 383

for detection of monodehydroascorbate reductase activity of thioredoxin h2.

384

The experiments were done twice and a representative one is shown. ‘M’

385

represents the molecular weight marker and 10 g protein was loaded in each 386

well.

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

Figure. 1.

402

Second

0 50 100 150 200 250 300

     nm

0.00 0.02 0.04 0.06 0.08 0.10

0.12 Thioredoxin h2, pH 7.0

Thioredoxin h2, pH 6.0

A.

B.

0.0 0.3 0.6 0.9 1.2 1.5

1.8 Thioredoxin h2, pH 7.0

Thioredoxin h2, pH 6.0 Glutathione, pH 7.0 Glutathione, pH 6.0

A.

403

404

405

406

407

Figure. 2.

408 409

GSH (mM)

0 1 2 3 4 5

    nm

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

r2 =0.985, p<0.05

410

411

412

413

414

415

416

417

418

419

Figure. 3.

420 421 422 423

Time (Second)

0 100 200 300

 A 340 nm

0.000 0.005 0.010 0.015 0.020 0.025 0.030

0.035 Thioredoxin h, pH 6.0

Thioredoxin h, pH 7.0 Glutathione, pH 7.0 Glutathione, pH 6.0

424

425

426

427

428

429

430

431

Figure. 4.

432 433

434

435

436

437