Title: Biochemical characterization of soluble acid and … · · 2013-04-191 Title: Biochemical...
Transcript of Title: Biochemical characterization of soluble acid and … · · 2013-04-191 Title: Biochemical...
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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:[email protected] 21
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Dr. Sukchan Lee 23
Tel: +82-31-290-7866 24
Fax: +82-31-290-7870 25
Email: [email protected] 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
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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
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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
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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
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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
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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
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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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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.
+ -