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Title: Biochemical characterization of soluble acid and alkaline invertases from shoot 1

of etiolated pea seedlings 2

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Running title: Purification of soluble invertases from pea seedling 4

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Authors: Donggiun Kim1, So Yun Park2, Youngjae Chung3, Jongbum Park1, 6

Sukchan Lee4*, Taek-Kyun Lee2* 7

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1 Department of Biological Science, Silla University, Busan, 617-736, Korea 9

2 South Sea Environment Research Department, Korea Ocean Research and Development 10

Institute, Geoje, 656-830, Korea 11

3 Department of Life Science and Biotechnology, Shin Gyeong University, Hwaseong 445-741, 12

Korea 13

4 Department of Genetic Engineering, Sungkyunkwan University, Suwon, 440-746, Korea 14

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* Authors for Co-correspondence. 16

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Dr. Taek-Kyun Lee 18

Tel: +82-55-639-8630 19

Fax: +82-55-639-8639 20

Email:tklee@kordi.re.kr 21

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Dr. Sukchan Lee 23

Tel: +82-31-290-7866 24

Fax: +82-31-290-7870 25

Email: sukchan@skku.ac.kr 26

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Abstract 32

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Soluble invertase was purified from pea (Pisum sativum L.) by sequential procedures 34

entailing ammonium sulfate precipitation, DEAE-Sepharose column, Con-A- and Green 35

19- Sepharose affinity columns, hydroxyapatite column, ultra-filtration, and Sephacryl 36

300 gel filtration. The purified soluble acid (SAC) and alkaline (SALK) invertases had a 37

pH optimum of 5.3 and 7.3, respectively. The temperature optimum of two invertases 38

was 370C. The effects of various concentrations of Tris-HCl, HgCl2, and CuSO4 on the 39

activities of the two purified enzymes were examined. Tris-HCl and HgCl2 did not 40

affect SAC activity whereas 10 mM Tris-HCl and 0.05 mM HgCl2 inhibited SALK 41

activity by about 50%. SAC and SALK were inhibited by 4.8 mM and 0.6 mM CuSO4 42

by 50%, respectively. The enzymes display typical hyperbolic saturation kinetics for 43

sucrose hydrolysis. The Kms of SAC and SALK were determined to be 1.8 and 38.6 44

mM, respectively. The molecular masses of SAC shown by SDS-PAGE and 45

immunoblotting were 22 kDa and 45 kDa. The molecular mass of SALK was 30 kDa. 46

Iso-electric points of the SAC and SALK were estimated to be about pH 7.0 and pH 5.7, 47

respectively. 48

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Keywords: Soluble acid invertase, Soluble alkaline invertase, Purification, 51

Characterization, Pea 52

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Introduction 63

Sucrose is synthesized in the photosynthetic tissues, from where it is transported 64

to the non-photosynthetic tissues of the plants. In the heterotrophic parts, sucrose is 65

cleaved either by invertase (β-D-fructosfuranosidase, EC 3.2.1.26) or by sucrose 66

synthase (EC 2.4.1.13). Invertase catalyses the hydrolysis of sucrose into D-glucose and 67

D-fructose, the main forms of carbon and energy supply in plant metabolism. Therefore 68

invertase has been widely studied, especially in plants but in other organism such as 69

yeast and fungus for basic biochemical and physiological studies as well as several 70

industrial applications (Muramatsu and Nakakuki 1995; Sanchez et al. 2001). Although 71

possible physiological roles of this enzyme have intrigued biologists for more than one 72

hundred years, purification and characterization of the iso-enzyme and localization of 73

the activity in cell components have been still undergoing actively in many different 74

plants (Roitsch and Gonzalez, 2004; Barratt et al., 2009). 75

Invertases are widely distributed in the plant world and numerous studies 76

describing their presence have been published (Bruskova et al. 2004; Hashizume et al. 77

2003; Huang et al. 2008; Isla et al. 1995; Lin et al. 1999; Liu et al. 2006; Vorster and 78

Botha 1998). Traditionally, invertases have been classified as ‘soluble’, readily 79

extractable from plant tissues in buffer solutions, and ‘insoluble’, extractable only in 80

buffer solutions containing high concentrations of salt. Within these classes, convention 81

recognizes ‘acid’ invertases, having optimum activity ranging from pH 3.5 to 5.6, and 82

‘neutral’ or ‘alkaline’ invertases with optimum activity close to pH 6.8-8.0 (ap Rees 83

1988). 84

Soluble acid invertases (SACs) have the following common characteristics (Faye 85

et al. 1981; Hashizume et al. 2003; Isla et al. 1995; Konno et al. 1993; Milling et al. 86

1993; Tang et al. 1996; Walker and Pollock 1993). They are generally N-glycosylated 87

proteins with molecular weights ranging between 23 kDa and 68 kDa and pH optima 88

about 3.5 to 5.6. Their pI values range between 3.2 with 9.3 and Km for sucrose has 89

been reported between 0.65 to 16 mM. Some researchers have suggested that SACs 90

appears to be confined to the vacuole (Leigh et al. 1979; Unger et al. 1992), but others 91

have suggested that it is also located in the free space of cytoplasm (Fahrendorf and 92

Beck 1990). Ricardo and ap Rees demonstrated high activity of SACs in elongating 93

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organs and in expanding cells in young seedlings (Ricardo and Ap Rees 1970). In 94

Phaseolus vulgaris, the major sucrose-hydrolyzing enzyme is a SAC that is most active 95

during the early stages of leaf growth (Morris and Arthur 1984). During leaf 96

development in grape fruit, young leaves contain high SACs activity while the 97

predominant invertase activity of mature leaves is SALK (Schaffer 1986). 98

Soluble alkaline invertases (SALKs) which has been isolated and purified from 99

citrus fruit, soybean nodules, pea leaves, chicory roots and carrot suspension culture, are 100

all non-glycosylated proteins with molecular weights ranging between 60 kDa and 70 101

kDa by SDS-PAGE (Chen and Black 1992; Ende and Laere 1995; Lee and Sturm 1996; 102

Lin et al. 1999; Morell and Copeland 1984). The isolated active enzyme is commonly 103

described as a tetramer or octomer with pH optimum about 6.5 to 8.0. The localization 104

of SALK is not yet clear. Although plant invertases have been extensively studied from 105

both physiological and biochemical viewpoints, important information about 106

purification and characterization of SALKs from pea is still limited. 107

This study describes the purification procedures and biochemical characterizations 108

of SAC and SALK from pea seedlings. These two soluble invertases were purified to 109

homogeneity, characterized and their properties were analyzed. 110

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Results 113

114

Purification of invertases 115

The procedures described below represent the methods from extensive 116

preliminary experimentation. As a general practice, the tissue was ground to powder in 117

liquid nitrogen. The ground tissue was processed for the extraction of one group of 118

invertases, acid or alkaline, at one time because of the logistic difficulty for 119

simultaneous performance of two separate protocols. 120

SAC was extracted from the powdered tissue in citrate/phosphate pH 6.2. An 121

enriched SAC preparation was collected by ammonium sulfate precipitation (45-70% 122

saturation). The precipitated protein was dissolved in 10 mM 4-(2-hydroxyethyl)-1-123

piperazineethanesulfonic acid (HEPES) buffer (pH 6.0) and dialyzed free of sulfate at 124

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4oC. The dialysate was passed through a CM-Sepharose column equilibrated with 10 125

mM HEPES buffer (pH 6.0). After washing the column with the same buffer, the bound 126

activity was eluted with a linear increasing gradient of NaCl. The acid enzyme activity 127

eluted as a single peak (Fig. 1A). Fractions containing high enzyme activity were 128

combined and applied to Con-A (Fig. 1B) which selectively binds N-glycosylated 129

protein. SAC was bound by the column and was eluted with 0.2 M methyl mannoside. 130

Fractions containing high enzyme activity were combined, dialyzed, and subjected to 131

Green19-Sepharose affinity chromatography. After washing this column with the same 132

buffer, the bound activity was eluted with a linear increasing gradient of NaCl (Fig. 1C). 133

The overall purification for SAC was 313-fold (Table 1). 134

SALK was initially extracted from the powdered tissue with citrate/phosphate (30 135

mM, pH 7.2) containing proteinase inhibitors. An enriched SALK preparation was 136

collected by ammonium sulfate fractionation as the precipitate from a 25-45% 137

saturation fraction. The precipitate was dissolved in 10 mM HEPES buffer (pH 7.2) for 138

DEAE-Sepharose column and dialyzed free of sulfate at 4oC. The dialysate was passed 139

through a DEAE-Sepharose column equilibrated with 10 mM HEPES buffer (pH 7.2). 140

After washing the column with the same buffer the bound protein was eluted with a 141

linear increasing gradient of NaCl. Two peaks of activity were observed (Fig. 2A). The 142

first peak was shown to be SAC by pH optimum measurement. This fraction was 143

discarded. Fractions from the second peak containing high enzyme activity were 144

combined and passed through Con-A- and Green 19- Sepharose affinity columns to 145

remove any contaminating SAC, which binds to both those ligands, as well as any other 146

proteins which may be bound. The eluate containing SALK was concentrated on an 147

hydroxyapatite column. The protein bound to hydroxyapatite was eluted by an 148

increasing gradient of phosphate buffer (Fig. 2B). The pooled fractions containing 149

SALK were concentrated by ultra-filtration and subjected to Sephacryl 300 gel filtration. 150

The elution of invertase activity reflects a protein molecular mass only slightly lower 151

than the exclusion limit of the Sephacryl 300 (500 kDa) (Fig. 2C). The overall 152

purification for SALK was 50-fold (Table 2). 153

154

Optimum pH and temperature 155

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The optimum pH of each separated invertase was determined (Fig. 3A). SAC 156

showed activity in the range from pH 4 to 7.5 and SALK showed activity in the range 157

from pH 5 to 9. SAC and SALK were most active at pH 5.3 and pH 7.3, respectively. 158

Each activity of SAC and SALK from 20oC to 70oC was examined (Fig. 3B). Both 159

soluble invertases were most active at 37oC. 160

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Effects of inhibitors 162

The effect of increasing concentrations of Tris-HCl, HgCl2, and CuSO4 on the 163

activity of the two purified enzymes was examined. The effect of a number of reagents, 164

Tris-HCl, CuSO4, and HgCl2, which was reported to influence invertase activity (Ende 165

and Laere 1995; Lee and Sturm 1996; Obenland et al. 1993), was tested by assaying 166

invertase activity in the presence of various concentrations of the reagents. Tris-HCl did 167

not affect SAC activity up to 14 mM whereas 10 mM Tris-HCl inhibited SALK activity 168

by about 50%. (Fig. 4A). SAC activity was not affected by HgCl2 but SALK was 169

inhibited by 0.05 mM HgCl2 by 50% and strongly inactivated by 0.1 mM HgCl2 (Fig. 170

4B). The effect of CuSO4 was less clear but this salt appears to have more inhibitory 171

role to SALK than the SAC (Fig. 4C). SAC was inhibited by 4.8 mM CuSO4 about 50%. 172

However, SALK was much more sensitive than SAC and inhibited by 0.6 mM CuSO4 173

up to 50%. 174

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Kinetic properties 176

The numerical value of Km is of interest for several reasons. It provides a means 177

of comparing enzymes from different sources and it also offers a measure of the affinity 178

of an enzyme for its substrate. A low Km reflects a high affinity (Segel 1976). The Km 179

values for the four enzyme preparations using sucrose as substrate were determined by 180

Hanes-Woolf plots. 181

The Hanes-Woolf equation is: [S]/V = 1/Vmax x [S] + Km/Vmax. 182

Plotting [S]/V against [S] the intercept on the [S] axis gives Km (Markus et al. 1976). 183

Typical Michaelis-Menten kinetics was observed when the activities of the two 184

purified invertases were measured in sucrose concentrations up to 100 mM (Fig. 5A and 185

5B). Each Km value of two enzymes for sucrose was determined. The Kms of SAC and 186

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SALK were determined to be 1.8 and 38.6 mM, respectively. 187

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Substrate Specificity 189

To be hydrolyzed by invertase (EC 3.2.1.26: β-fructofuranosidase) a substrate 190

should contain an unsubstituted β-D-fructofuranosyl residue. The ability of each of the 191

two purified invertase isoenzymes to hydrolyze a range of oligosaccharides was 192

examined. The results, shown in Table 3, are given as a percentage of the substrate 193

hydrolyzed relative to sucrose. The oligosaccharides tested were raffinose (Gal-α-1,6-194

Glu-β-1,2-Fru), melezitose (Glu-α-1,3-Fru-α-1,2-Glu) and trehalose (Glu-α-1,1-Glu). 195

Raffinose, a β-fructofuranoside, was hydrolyzed to about 40% of the rate at which 196

sucrose (Glu-β-1,2-Fru) was hydrolyzed. Melezitose, an α-fructofuranoside, was about 197

5% hydrolysed and trehalose, an α-glucopyranoside, not at all (Table 3). Hence, all of 198

the sucrose-hydrolyzing enzymes isolated here appeared to be typical β-199

fructofuranosidases. 200

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Molecular weight determinations 202

During purification of SAC, 22 kDa and 45 kDa polypeptides increased slightly in 203

intensity from lane 1 to 4. Polyclonal antibody preparations against carrot soluble-, 204

carrot insoluble-, and yeast acid- invertase reacted with these 22 kDa and 45 kDa bands 205

(Fig. 6A) (Lauriere et al. 1988; Schwientek et al. 1995; Unger et al. 1992). 206

During the purification of SALK, particularly a 30 kDa single polypeptide 207

increased in intensity after gel filtration. This polypeptide reacted with a polyclonal 208

antibody against sugar beet SALK bound specifically with this band (Fig. 6B) (ap Rees 209

1988). 210

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Iso-electric point (pI) determination 212

The purified invertases were electrophoresed on agarose or polyacrylamide 213

isoelectric focusing gels. The enzymes were resolved by either Coomassie Blue staining 214

for protein or activity staining for invertase. 215

SALK was electrophoresed on IEF-PAGE. Gels were stained either with 216

Coomassie Blue or with the invertase activity assay (Fig. 7). Since invertase activity 217

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was readily visualized the iso-electric point was determined to be about pH 5.7. In 218

contrast, SAC activity was not detectable after IEF-PAGE. As an alternative approach, 219

SACs were electrophoresed on an horizontal agarose gel at pH 7.0 (Fig. 8). An assay of 220

the liquid from the sample strongly showed SAC activity. It was concluded from this 221

that SAC has a pI of close to 7.0 and was therefore immobile during the electrophoresis. 222

223

Discussion 224

225

Purification of pea soluble alkaline and acid invertases 226

The purification of SAC from a great many plant species has been reported. SAC 227

purification has a common precipitation step: 45-70% saturated ammonium sulfate, 228

followed by cation exchange chromatography and Con-A adsorption chromatography. 229

Most researchers have reported SAC to have a low pI (approx. 5) and to be highly N-230

glycosylated in the Arabidopsis leaves, potato tubers, Urtica leaves, Beet storage roots 231

and carrot seedling (Burch et al. 1992; Fahrendorf and Beck 1990; Leigh et al. 1979; 232

Tang et al. 1996; Unger et al. 1992). However, the pea SAC, although probably 233

glycosylated, has an apparent pI of about 7.0 (Fig. 8). 234

The SALK was extracted from pea tissue by participating in the 25-45% saturated 235

ammonium sulfate same as the purification of soluble neutral and alkaline invertases 236

from carrot (Lee and Sturm 1996). In the present study the enzyme activity was bound 237

by DEAE anion exchange chromatography which was to be expected from the pI of 5.7 238

found by IEF. In contrast to SAC, the pea SALK did not bind to Con-A and is therefore 239

unlikely to be glycosylated. This is consistent with results reported for SALK isolated 240

from cultured carrot cells (Stommel and Simon 1990), from soybean hypocotyls (Chen 241

and Black 1992), from chicory root (Ende and Laere 1995), and from the suspension 242

cultured carrot cells (Lee and Sturm 1996). Although no precise pI values have been 243

reported for the chicory and carrot enzymes, anion exchange chromatography was 244

utilized in each case during purification (Ende and Laere 1995; Lee and Sturm 1996). 245

The overall purification for the pea SALK was only 50 fold and the recovery of activity 246

was poor (Table 1). This result is similar to the data reported for soy bean nodule (40 247

fold), soy bean hypocotyl (98-fold) and chicory root which was about 80 fold (Chen and 248

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Black 1992; Ende and Laere 1995; Morell and Copeland 1984). Difficulty in 249

purification and low stability in vitro was also reported to occur during carrot SALK 250

purification (Lee and Sturm 1996). It has been suggested that in some cases the absence 251

of glycosylation may contribute to this lack of stability. A similar problem occurred in 252

efforts to purify yeast non-glycosylated invertase (Lampen 1971). Glycosyl moieties 253

have been shown to help dissociation and give stability to some enzymes in the in vitro 254

condition (Esmon et al. 1987; Schulke and Schmid 1988; Tammi et al. 1987). 255

256

Characterization of pea soluble alkaline and acid invertases 257

The two different enzymes have distinct pH optima. SAC and SALK have pH 258

optima similar to the physiological pH values expected in their presumed locations in 259

the plant. Studies of the inhibition of the pea enzyme activity by Hg, Cu, and Tris-HCl, 260

showed that these isozymes have properties consistent with those reported for other 261

invertases. The strong inhibition of both invertases by Tris is in agreement with other 262

reports (ap Rees 1988; Chen and Black 1992; Lee and Sturm 1996; Morell and 263

Copeland 1984). SALK activities from soybean nodules and carrot suspension cultured 264

cells were strongly inhibited by Cu as were the pea SALK in the present study (Lee and 265

Sturm 1996; Morell and Copeland 1984). SALK was completely inhibited by Hg, 266

suggesting that one or more reduced sulfhydryl groups may be essential for the activity. 267

This result, too, is consistent with other reports (Chen and Black 1992; Ende and Laere 268

1995; Morell and Copeland 1984). 269

The kinetic properties of the enzymes were similar to those of other plants. 270

Apparently, all of enzymes' response to the increasing sucrose concentrations followed 271

Michaelis-Menten kinetics. The Km value of SAC (1.8 mM) showed this enzyme to 272

have more than 20-fold in affinity for sucrose than that of SALK (Km = 38.6 mM). This 273

is consistent with data collected in an previous review in which a comparison of Km 274

values of several plant invertases showed that the SAC commonly had a higher affinity 275

for sucrose than the alkaline enzyme (Avigad 1982). 276

Published data on the native molecular mass of purified SALK describe relatively 277

high molecular sizes. Soybean SALK was reported to be a 240 kD homotetramer 278

composed of four identical subunits (Chen and Black 1992). SALKs from broad bean 279

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and chicory root are similar to that from soybean (ap Rees 1988; Ende and Laere 1995). 280

One of the carrot SALKs was reported to be a homo-octamer composed of eight 281

identical subunits (57 kDa) in native form (Lee and Sturm 1996). The purified pea 282

SALK was shown to contain a single polypeptide band (30 kDa) by SDS-PAGE and 283

immunoblotting. The sugar beet alkaline antibodies reacted with this pea SALK 284

polypeptide. A broad elution pattern from Sephacryl 300 made it difficult to estimate a 285

native molecular mass but the relatively early elution of activity suggested a molecular 286

size larger than the 30 kDa size found by SDS-PAGE. The pea SALK showed a single 287

band of activity with an iso-electric point of pH 5.7 on an IEF gel (Fig.7). The existence 288

of an oligomeric structure in the native protein may contribute to the difficulties 289

experienced in purifying this enzyme. 290

Using immunoblotting to identify SAC polypeptides, three bands of different 291

molecular mass were visualized after SDS-PAGE of the purified pea activity. Two major 292

bands of 45 kDa and 22 kDa were identified with different antibodies (antibodies 293

against carrot soluble and insoluble acid invertase). In addition, a faint band with a 294

molecular weight of about 67 kDa cross-reacted with the carrot insoluble acid antibody 295

(Fig. 6). In spite of the use of protease inhibitors during the purification steps, the 45 296

kDa and 22 kDa bands might be due to proteolytic cleavage of a larger polypeptide 297

from SAC. Such a breakdown has been reported to occur during the purification of the 298

carrot SAC (Unger et al. 1994; Unger et al. 1992). In that case, after analysis by SDS-299

PAGE the carrot protein appeared with molecular masses of 68, 43 and 25 kDa. Amino 300

acid sequence analysis and immunological studies proved that the 43 kDa and 25 kDa 301

polypeptides were fragments of the larger polypeptide. Proteolytic processing or 302

degradation have also been reported for invertase from tomato, from barley, and from 303

potato (Burch et al. 1994; Obenland et al. 1993; Yelle et al. 1991). The pea SAC in this 304

study did not resolve into more than one band on agarose gel electrophoresis (Fig.8). 305

Generally, neutral and alkaline invertase were not glycosylated and they used 306

sucrose for sole substrate. Therefore these two invertases were reported as no more 307

fructofuranosidase (Sturm et al., 1999; Roitsch and Gonzalez, 2004). However, in this 308

study both soluble invertases showed the β-fructofuranosidase activity in the selective 309

manner, displaying clear substrate preference for sucrose as a β-fructofuranosidase. Lee 310

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and Sturm (1996) reported that soluble neutral and alkaline invertase were purified from 311

carrot suspension cells and neutral invertase used sucrose as well as raffinose and 312

stachylose for its substrates. However alkaline invertase reacted with only sucrose for 313

its substrate. In addition, the optimumal pH for neutral invertase was pH 6.8 and SALK 314

showed pH 7.3 for their optimal pH in this study. It means that SALK could be an 315

neutral invertase rather than alkaline invertas by the biochemical characterizations. 316

Moreover This β-fructofuranosidase activity distinguishes between α- and β-linked 317

fructose residues and is unable to hydrolyze glucose linkages (Table 3). This establishes 318

the isolated enzymes as true invertases and not the α-glucosidase, sucrase (Dahlqvist 319

1984). In this work, we purified and investigated the biochemical kinetic properties of 320

SALK and SAC in rapidly growing etiolated pea seedlings. Functional and 321

physiological analysis of these two invertases from pea seedlings are currently doing 322

because these invertases have different biochemical properties compared to others 323

reported previously and it is also possible that these enzymes may have unknown roles 324

in plant growth and development. 325

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Materials and methods 328

Plant material 329

Seeds of the garden pea, Pisum sativum L. cv. Little Marvel (dwarf) or Alaska 330

(tall), were planted and grown in the greenhouse at Sungkyunkwan University, Korea. 331

To obtain etiolated tissue, pea seeds were surface-sterilized by washing in 10% (v/v) 332

Clorox (commercial solution of calcium hypochlorite) solution for 10 min before 333

rinsing in sterile distilled water and planted in autoclaved vermiculite. The seeds were 334

grown at room temperature in the dark for 7 days before treatment with 15 μM 335

gibberellic acid (GA3) solution. The sprayed plants were harvested after 2 days. The 336

required tissues were harvested separately, weighed, and stored at -80oC. 337

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Reagents 339

All common reagents were of analytical grade. Acrylamide (electrophoresis grade), 340

N,N’-methylene-bisacrylamide, N,N,N’,N’-tetramethyl-ethylenediamine (Temed) and 341

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ammonium persulfate were purchased from Sigma-Aldrich Korea. Sodium dodecyl 342

sulfate (SDS) was purchased from United States Biochemical Co.. Molecular weight 343

markers, Low Range (from 6.5 kDa to 66 kDa) and High Range (36 kDa to 205 kDa) 344

were obtained from Sigma. Ampholytes were the Pharmalyte brand obtained from Sigma. 345

Low EEO agarose was purchased from Fisher Biotech. 346

347

Invertase assay 348

Invertase activity in tissue extracts or column separation fractions was determined 349

by measuring the amount of reducing sugars by sucrose hydrolysis. For the standard 350

assay of invertase, final volume of digest solution was 1 mL buffer, 50 mM 351

citrate/phosphate, pH 6.5 contained 100 mM sucrose, and 0.1 U of enzyme. Invertase 352

assay was initiated by the addition of enzyme. The mixture was incubated at 370C for 60 353

min, followed by the addition of 1 mL of the dinitrosalicylate reagent (1% [w/v] 3,5-354

dinitrosalicylic acid, 1.6% [w/v] sodium hydroxide and 30% [w/v] sodium potassium 355

tartrate) which also served to stop the reaction (Arnold 1965). This mixture was heated 356

in boiling water bath for 10 min, cooled to room temperature and the absorbance was 357

measured at 560 nm using a Beckman DU-40 spectrophotometer. The reducing sugar 358

produced by invertase activity reacts with the dinitrosalicylic acid reagent generating a 359

red-orange color. A standard curve was prepared for an equi-molar mixture of glucose 360

and fructose. A linear relationship between absorbance and glucose/fructose content 361

covers the range from 0 μM to 1000 μM glucose or fructose per assay. One unit (U) of 362

invertase activity was defined as the formation of 1 μmol of reducing sugar from 363

sucrose per minute at 37oC. Specific activity was expressed as units of invertase activity 364

per milligram of protein per min. 365

For the assay in non-denaturing electrophoresis gels, gels were placed in a 366

solution (100 mL) containing 0.1 M sucrose, 200 mM citrate/phosphate buffer, pH 7.0, 367

1 mg/mL phenazine methosulfate, 1 mg/mL nitro blue tetrazolium, and 8 units/mL 368

glucose oxidase and incubated at 37oC in the dark for 1-3 hours. Invertase isoforms 369

show up on such gels as red-purple bands identifying glucose produced by sucrose 370

hydrolysis (Gabriel and Wang 1969). 371

372

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Protein assay 373

The protein content of extract solutions and column fractions was determined 374

using the procedure of Bradford (Bradford 1976). Reagents were obtained from Bio-375

Rad (Bio-Rad Laboratories Inc. CA). Bovine serum albumin (BSA) was used as the 376

standard protein in the range 0-100 μg/assay. As a rough indicator of protein content in 377

fractions collected from column chromatography, the absorbance of the solutions at 280 378

nm was measured. 379

380

Crude extract preparation 381

All procedures were carried out at 4oC. Fresh or cold-stored tissue was frozen 382

with liquid nitrogen and powdered in a Sorval blender (Omni-Mixer 117350). 500 g of 383

the powder was stirred with 2,500 ml of extraction buffer. The tissue extraction buffer 384

was 30 mM citrate/phosphate containing 0.2% β-mercapto-ethanol and 1 mM phenyl 385

methyl sulfonyl fluoride (PMSF) and 1 mM benzamidine as protease inhibitors). PMSF 386

was added from a stock solution (100 mM in 100% isopropanol). The extraction buffer 387

was adjusted to pH 6.5 for the extraction of SAC and to pH 7.2 for SALK extraction 388

because preliminary results showed that SALK was precipitated around pH 6.0. The 389

homogenate was vacuum-filtered through 2 layers of cheesecloth. Polyvinyl 390

polypyrrolidone (1% [w/v]) was added to the filtrate, stirred for 30 min, and centrifuged 391

at 10,000 g for 30 min. The supernatant was used for the purification of soluble 392

invertases. 393

394

Ammonium sulfate precipitation 395

Preliminary experiments showed that soluble invertase activity determined at pH 396

7.0 in citrate/phosphate buffer was present in the 10-70% saturation ammonium sulfate 397

precipitates. After redissolving these fractions, repeated ammonium sulfate fractionation 398

showed that the 25-45% saturation contained the highest specific activity and 30-50% 399

contained the highest total activity of invertase measured at pH 8 in citrate/ phosphate 400

assay buffer. This was considered to be SALK. In similar experiments assaying 401

invertase with pH 5 citrate/phosphate buffer, the 50-70% saturation fractions contained 402

the highest specific activity and total activity of what was considered to be SAC. 403

14

Therefore, for routine purification, after rejecting the initial precipitation from 10% 404

saturated ammonium sulfate the supernatant was brought to 70% saturation and the 405

precipitate collected. After dissolving this precipitate in extraction buffer, the 25-45% 406

saturation fraction was collected for further purification of the SALK and the 45-70% 407

fraction collected for the SAC. 408

409

Ion exchange chromatography 410

DEAE-Sepharose anion-exchange chromatography was an effective step for 411

removal of contaminating proteins and SACs because SALK activity was bound tightly 412

to the DEAE matrix after the column was equilibrated with HEPES buffer (10 mM 413

HEPES containing 0.2% β-mercapto-ethanol, 1 mM PMSF and 1 mM benzamidine, 414

adjusted to pH 6.8 with 1 M sodium hydroxide). After washing the unbound proteins 415

from the column with equilibration buffer the SALK activity was eluted with a gradient 416

of 0-0.3 M NaCl. CM-Sepharose cation-exchange chromatography was used for SAC 417

and for removing other contaminating proteins. The sample, dissolved in 10 mM 418

HEPES buffer, pH 6.0 was applied to the column (10 x 2.5 cm) pre-equilibrated with the 419

same buffer. After washing the unbound proteins from the column with equilibration 420

buffer the SAC activity was eluted with a gradient of 0-0.5 M NaCl. 421

422

Absorption chromatography 423

Hydroxyapatite chromatography was used for further purification of invertase. 424

The sample, dissolved in 20 mM potassium phosphate buffer (pH 6.8) was applied to 425

the column (10 x 2.5 cm) pre-equilibrated with the same buffer. After washing the 426

unbound proteins from the column with equilibration buffer, invertase activity was 427

eluted with a gradient of 20-300 mM potassium phosphate buffer, pH 6.8. 428

Concanavalin-A sepharose (Con-A) chromatography was used for both SALK and 429

SAC, taking advantage of the fact that Con-A adsorbs glycosylated proteins (Faye et al. 430

1981). The sample dissolved in Con-A buffer (10 mM HEPES, pH 6.8, containing 0.2% 431

β-mercaptoethanol, 1 mM PMSF, 1 mM benzamidine, 500 mM NaCl, 1 mM MnCl2, 432

and 1 mM CaCl2) was applied to the column (10 x 1.5 cm) pre-equilibrated with the 433

same buffer. After washing the unbound proteins from the column with equilibration 434

15

buffer, invertase activity was eluted with a gradient of 0-200 mM methyl-D-mannose in 435

the same buffer. Binding of SAC to Con-A Sepharose suggests that the enzyme is an N-436

glycosylated protein containing mannose residues. SALK activity was not bound by 437

Con-A. 438

439

Reactive Green-19 affinity chromatography 440

For the Reactive Green-19 Affinity chromatography small column (10 x 1.5 cm) 441

equilibrated with 10 mM HEPES, pH 7.2 was used. Bound proteins were eluted by a 442

gradient of 0-0.5 M NaCl. SAC is bound by Green 19. SALK is not. 443

444

Gel filtration chromatography 445

The two soluble invertases were further purified by gel filtration chromatography 446

(Sephacryl 300). Column (90 cm x 1.2 cm) was equilibrated with HEPES buffer (10 447

mM HEPES, pH 6.8, containing 0.2% β-mercaptoethanol, 1 mM PMSF, 1 mM 448

benzamidine, 500 mM NaCl). The sample used was the major peak of invertase 449

obtained after Green 19 for SALK or SAC. 450

451

Concentration methods 452

Lyophilization was done as described by Everse and Stolzenbach where the 453

sample was shell frozen in a special lyophilization flask and dried using a Labconco 454

Freeze-dryer Model 4451F (Everse and Stolzenbach 1971). Concentration was also 455

done by ultrafiltration using Centriprep 10 concentrators (Amicon, W.R. Grace & Co.). 456

Solutes with molecular weights greater than the membrane cut-off (3 kDa) remain in the 457

sample container (retentate) and become increasingly concentrated. Samples were put 458

into each concentrator and centrifuged in a refrigerated IEC C-4B centrifuge at 3000 459

rpm until the volume of the retentate was 2-3 mL The filtrate was discarded after being 460

assayed for invertase activity to control for faulty filtration membranes. The 461

concentrated solutions were stored or used in further chromatographic separations. 462

463

Enzyme characterization 464

The optimum pH for purified enzyme was determined over a pH range 4 to 10. 465

16

Incubation of sucrose at pH values of 3.5 or lower under the conditions used resulted in 466

some non-enzymic hydrolysis. The assay mixture contained 100 μL of enzyme solution 467

and 900 μL of reaction buffer supplemented by 50 mM citrate/phosphate buffer, pH 6.5 468

and 50 mM sucrose to make 1 mL solution. The reaction was conducted for 30 min at 469

30oC. 470

The optimum temperature for activity of purified enzyme was determined over the 471

range 0oC to 70oC at the optimum pH for each isozyme. Reaction mixtures (minus 472

enzyme) were equilibrated at each temperature prior to initiation of the reaction by 473

addition of enzyme. Reactions were conducted for 30 min at different temperatures. 474

The substrate specificity of purified enzyme was tested by assaying for reducing 475

sugars after incubation with sucrose (Glu-β-1,2-Fru), raffinose (Gal α-1,6-Glu-β-1,2-476

Fru), melezitose (Glu α-1,3-Fru α-1,2-Glu), and trehalose Glu α-1,1-Glu). Enzyme 477

activity was assayed by measuring the production of reducing sugars. Substrate 478

concentration was 50 mM. The amount of substrate hydrolysis was compared to that of 479

sucrose at 100%. 480

For kinetic measurements the reaction mixtures consisted of a constant amount of 481

enzyme, a range of sucrose concentration, in a constant volume of 50 mM 482

citrate/phosphate, pH 6.5 buffer. The purified protein (0.1 U) was incubated with 483

increasing sucrose concentration. The reactions were carried out for 30 min at 37oC. 484

Each incubation pH for SALK and SACs was pH 7.3 and pH 5.3, respectively. The Km 485

value was calculated by using Hanes-Woolf plots (Segel 1976). 486

487

Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) 488

SDS-PAGE was carried out with the Hoefer Mini Slab Gel Unit (Hoefer Scientific 489

Instrument Inc. CA) according to the method of Laemmli with a final gel concentration 490

of 12% acrylamide from monomer stock solution (30% acrylamide, 2.7% N,N’-491

methylene- bisacrylamide) (Laemmli 1970). The separating gel contained 2 mL 492

monomer stock solution, 0.75 ml running gel buffer (1.5 M Tris-HCl, pH 8.8), 1 mL 493

10% SDS, 50 μL initiator (10% ammonium persulfate), 6 μL Temed, and 1.6 mL 494

double-distilled water. The stacking gel contained 250 μL monomer stock solution, 400 495

μL stacking gel buffer, 25 μL, 10 % SDS, 12 μL initiator (10% ammonium persulfate), 496

17

2.5 μL Temed, and 1 mL double distilled water. Samples containing 5-20 μg proteins in 497

less than 10 μL of denaturing buffer were loaded to each well in the polymerized gel. 498

The gel was electrophoresed at room temperature at 80 volts for the first 10 min, and at 499

100 volts thereafter until the marker dye front reached 1 cm from the bottom of the gel. 500

For staining of proteins, the gel was immersed in Coomassie Blue solution (0.125% 501

[w/v] Coomassie Brilliant Blue R-250, 50% [v/v] methanol, 10% [v/v] glacial acetic 502

acid) for more than 3 h at room temperature. The gel was destained for 1 h in solution I 503

(50% [v/v] methanol, and 10% [v/v] glacial acetic acid) and for 1-2 days in solution II 504

(5% [v/v] methanol, 7% [v/v] glacial acetic acid). The stained gel was photographed 505

immediately or vacuum-dried for records. 506

507

Iso-Electric Focusing-Poly-Acrylamide Gel Electrophoresis (IEF-PAGE) 508

IEF was performed using vertical tube gels or mini-slab gels. The gels were made 509

by a modification of the method described in the Hoeffer Scientific Instrument protocol 510

book. The slab gel contained 1.7 mL SDS-PAGE monomer stock solution (which 511

contains no SDS), 0.9 mL glycerol, 0.5 mL ampholytes (wide range, pH 3-10), 35 μL 512

initiator (10% ammonium persulfate), 25 μL Temed, and 0.6 mL double-distilled water. 513

The gel mixture was degassed before the addition of Temed and initiator. The gel was 514

pre-electrophoresed at 80 volts for 10 min before adding the samples to each well. After 515

loading the gel was electrophoresed at 100 volts for 2 h in a cold room (4oC). Following 516

electrophoresis, the gel was stained with Coomassie Blue solution for protein after 517

fixation or, without fixation, with the glucose assay for invertase activity. For 518

immunoblotting after IEF, one lane of gel was cut and protein was transferred to 519

nitrocellulose paper. For staining of proteins the gel was fixed in solution contained 520

20% (w/v) trichloroacetic acid for about 60 min. and post-fixed with a solution 521

contained 40% (v/v) ethanol, 10% (v/v) acetic acid and 0.25% SDS before staining in 522

Coomassie Blue solution (0.125% [w/v] Coomassie Brilliant Blue R-250, 40% [v/v] 523

ethanol, 10% [v/v] glacial acetic acid) for more than 3 h at room temperature. The gel 524

was destained for 1 h in a solution containing 40% [v/v] ethanol, and 10% [v/v] glacial 525

acetic acid. The stained gel was photographed immediately or vacuum-dried for records. 526

527

18

Agarose gel electrophoresis 528

Agarose gels (1 cm thick, 10 x 8 cm gel) contained 1% (w/v) agarose (low EEO 529

agarose) were electrophoresed at 4 oC for 3 h at 100 volts in 50 mM citrate/phosphate 530

buffer (pH 7.0). Following electrophoresis, the gel was stained using the glucose assay 531

for invertase activity. 532

533

Western blotting/ immunoblotting 534

Protein samples resolved in each of the gel systems were electrophoresed on 535

denaturing SDS- or non-denaturing IEF-polyacrylamide gels. The electrophoresed 536

protein was transferred to nitrocellulose membrane using the protein blot apparatus 537

(Hoefer Transfer TE 22 unit). After electrophoresis, the gel and nitrocellulose 538

membrane were soaked in electro-transfer buffer (25 mM Tris, 190 mM glycine, 0.15% 539

[w/v] SDS and 20% [v/v] methanol) (Towbin et al. 1979). The gel was put in 540

the ’sandwich assembly’ that consisted of the nitrocellulose membrane on one side of 541

the gel, followed by 3 layers of 3 MM Whatman filter paper on either side and the 542

outermost layer consisting of a layer of sponge on both sides. The sandwich assembly 543

was placed in the Hoefer Transfer unit in such a way that the nitrocellulose membrane 544

side faced the anode. The transfer was performed at 50 volts for 2 hours. The membrane 545

was cut in strips to separate the duplicate samples and treated with blocking buffer 546

contained 8% BSA in TPBS (10 mM sodium phosphate, pH 9.5, with 0.9% NaCl and 547

0.1% Tween 20). The membranes were incubated with diluted serum (1:1000) or anti-548

invertase antibodies (Table 2-1). 549

These antibodies were used at various dilutions in blocking buffer at room 550

temperature with constant rocking agitation for 2 hours. Following three washes of 5 551

min each with TPBS buffer, the membranes were incubated with diluted (1:2000) 552

secondary antibodies conjugated with alkaline phosphatase in blocking buffer for 1 hour 553

under constant gentle rocking agitation. Sequential washes of the membranes were for 5 554

min each with 1) TPBS buffer, 2) PBS buffer (10 mM sodium phosphate with 0.9% 555

NaCl), 3) TBS buffer (100 mM Tris/HCl with 0.9% NaCl) at pH 9.5. The washed 556

membranes were treated with alkaline phosphatase assay solution. After color 557

development, the membranes were rinsed with distilled water, air dried, and stored in a 558

19

dessicator until photographed. 559

560

Acknowledgements 561

This work was partly supported by grants from the Korea Ocean Research & 562

Development Institute (No. PE98474) and by grants from BioGreen 21 Project funded 563

by Rural Development Administration of Korea (No. 20070401-034-028-009). 564

565

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22

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Physiol. 95, 1026-1035.683

23

Table 1. Purification of pea soluble acid invertase. 684

Purification Step Volume

(mL) Total Protein

(mg) Total Activity

(U) Specific Activity

(U/mg) Fold

Purification

Crude Extract 2500 5294 282 0.05 1 45% - 70% AS 430 1657 103.6 0.06 1.17 CM 16 6.05 8.7 1.45 27.2 Con-A 6 1.5 4. 2.67 50 Green 19 4 0.124 2.063 16.64 313

685

24

Table 2. Purification of pea soluble alkaline invertase. 686

Purification Step Volume

(mL) Total Protein

(mg) Total Activity

(U) Specific Activity

(U/mg) Fold

Purification

Crude Extract 2500 5294.00 85.8 0.0162 1.0 25% - 45% AS DEAE 20 8.59 1.9 0.2200 13.6 Hydroxyapatite 10 3.27 1.3 0.3884 24.0 Gel Filtration 6 0.39 0.3 0.8200 50.6

687

25

Table 3. Substrate specificity of the purified invertases. 688

Substrate Alkaline invertase (%) Acid invertase (%)

Sucrose Melezitose Raffinose Trehalose

100 4.56 38.89 N/D

100 3.12 40.06 N/D

N/D=Not detected

689

26

Figure Legends 690

Fig. 1. Purification of pea soluble alkaline invertase. A, DEAE-Sepharose column 691

chromatogram; B, hydroxyapatite column chromatogram; C, gel filtration 692

chromatogram. Pi=Inorganic phosphate. All experiments were performed 3-5 times and 693

results were represented by averages of individual data. 694

Fig. 2. Purification of pea soluble acid invertase. A, CM-Sepharose column 695

chromatogram; B, Con-A-Sepharose column chromatogram; C, Green 19 696

chromatogram. All experiments were performed 3-5 times and results were represented 697

by averages of individual data. 698

Fig. 3. Effect of pH (A) and temperature (B) on activity of pea soluble alkaline and acid 699

invertases. A, effect of pH on activity of pea invertases; B, effect of reaction 700

temperature on activity of pea invertases. All data were adjusted relative to maximum 701

activity (100%) for each enzyme. All experiments were performed 3-5 times and results 702

were represented by averages of individual data. 703

Fig. 4. Effects of Tris-HCl (A), HgCl2 (B), and CuSO4 (C) on activity of pea alkaline 704

and acid invertases. Each assay was pre-incubated with enzyme for 5 min before 705

substrate (50 mM sucrose) was added to the reaction mixture. Results are expressed 706

as % initial activity. All experiments were performed 3-5 times and results were 707

represented by averages of individual data. 708

Fig. 5. Saturation curves of soluble alkaline (A) and acid (B) invertase from Pisum 709

sativum L. for sucrose. The insets show the Lineweaver–Burk plot. All experiments 710

were performed 3-5 times and results were represented by averages of individual data. 711

Fig. 6. Molecular weight dertermination of pea soluble alkaline (A) and acid (B) 712

invertases from SDS-PAGE and immunoblotting. Approximately 20 μg of protein were 713

added in lanes. SDS-PAGE was carried out in 10 % (w/v) polyacrylamide gels. Alkaline 714

(A) and acid (B) invertases were stained with Coomassie Blue (lane A2 and B2), 715

immunostained with anti-sugar beet alkaline invertase antibody (lane A3) and anti-716

27

carrot insoluble acid invertase antibody (lane B3). High and low molecular markers are 717

given on the lane A1 and B1. 718

Figure 7. IEF-PAGE analysis of protein samples of purified soluble alkaline invertase 719

activity. Lane 1, standard pI marker, pH 5.6; lane 2, standard pI marker, pH 5.9; lane 3, 720

soluble alkaline invertase preparation stained with Coomassie Blue; lane 4, soluble 721

alkaline invertase preparation stained for invertase activity. The arrow indicates the 722

estimated pI for the invertase activity in comparison with the standard markers. 723

Figure 8. Agarose gel electrophoresis of soluble acid invertase. The arrows indicate the 724

location of invertase activity. Electrophoresis was carried out in citrate/phosphate buffer 725

(50 mM, pH 7.0) for 2 hours before staining for invertase activity. 726

Fig. 1 Kim et al.

Fraction number10 20 30

Spe

cific

act

ivity

(U)

0.0

0.5

1.0

1.5

2.0

Am

ount

of p

rote

in (m

g)

0

1

2

3

4

5

NaC

l (M

)

0.0

0.1

0.2

0.3

0.4

0.5Total activityTotal proteinNaCl

Fraction number5 10 15

Spe

cific

act

ivity

(U)

0.0

0.3

0.6

0.9

1.2

Am

ount

of p

rote

in (m

g)

0.0

0.2

0.4

0.6

0.8

1.0

Man

nose

(M)

0.00

0.05

0.10

0.15

0.20Total activityTotal proteinMannose

Fraction number5 10 15 20

Spe

cific

act

ivity

(U)

0.0

0.1

0.2

0.3

0.4

Am

ount

of p

rote

in (m

g)

0.0

0.1

0.2

0.3N

aCl (

M)

0.0

0.1

0.2

0.3

0.4

0.5Total activityTotal proteinNaCl

A

B

C

Spec

ific

activi

ty (U/m

g)

Spec

ific

activi

ty (U/m

g)

Spec

ific

activi

ty (U/m

g)

Fig. 2 Kim et al.

Fraction number20 40 60

Spe

cific

act

ivity

(U)

0.0

0.2

0.4

0.6

0.8

Am

ount

of p

rote

in (m

g)

0

2

4

6

8

10

NaC

l (M

)

0.00

0.05

0.10

0.15

0.20

0.25

0.30Total activityTotal proteinNaCl

Fraction Number10 20 30

Spe

cific

act

ivity

(U)

0.0

0.1

0.2

0.3

Am

ount

of p

rote

in (m

g)

0.0

0.4

0.8

1.2

1.6

2.0

Inor

gani

c ph

osph

ate

(M)

0.0

0.1

0.2

0.3Total activityTotal proteinPi

Fraction Number5 10 15 20 25 30

Spe

cific

act

ivity

(U)

0.0

0.1

0.2

0.3

0.4

Am

ount

of p

rote

in (m

g)

0.0

0.5

1.0

1.5

2.0Col 9 vs Col 10 Col 9 vs Col 11

A

B

C

Spec

ific

activi

ty (U/m

g)

Spec

ific

activi

ty (U/m

g)

Spec

ific

activi

ty (U/m

g)

Fig. 3 Kim et al.

pH4 5 6 7 8 9

Rel

ativ

e en

zym

e ac

tivity

(%)

0

20

40

60

80

100

120 SACSALKA

Temperature (oC)20 30 40 50 60 70

0

20

40

60

80

100

120 SACSALKB

Fig. 4 Kim et al.

Concnetration of Tri-HCl (mM)

0 2 4 6 8 10 12 14

Rel

ativ

e en

zym

e ac

tivity

(%)

0

20

40

60

80

100

120

Concentration of HgCl2 (mM)

0.00 0.05 0.10 0.15 0.200

20

40

60

80

100

120

Concentration of CuSO4 (mM)

0 2 4 60

20

40

60

80

100

120SACSALK

A B CSACSALK

SACSALK

Fig. 5 Kim et al.

[S] mM0 20 40 60 80 100

V (U

/mgp

rote

in/m

in)

0

1

2

3

4

5

[S] mM0 10 20 30 40 50 60

V (U

/mgp

rote

in/m

in)

0.00

0.01

0.02

0.03

A B

1/S-0.04 0.00 0.04 0.08 0.12

1/V

0.3

0.6

0.9

1.2

1/S0 5 10 15 20

1/V

200

400

600

Fig. 6 Kim et al.

kDa

8466

5545

36

2924

A B1 2 3 4kDa

66

45

36

2924

20

14.26.5

1 2 3

1 2 3 4

pI 5.9

5.65.7

Fig. 7 Kim et al.

Fig. 8 Kim et al.

+ -