Response of mung bean invertase to fluoride 177
RESPONSE OF MUNG BEAN INVERTASE TO FLUORIDE
K Ouchi,a,b M-H Yu,c A Shigematsua
Chiba, Japan, and Bellingham, USA
SUMMARY: Invertase from mung bean (Vigna radiata) seedlings treated with varying concentrations of NaF was partially purified by two-dimensional high-performance liquid chromatography (HPLC). Two protein fractions exhibiting invertase activity (Invertase I and II) were obtained. The in vivo activities of both isozymes from NaF-treated seedlings were decreased in a concentration-dependent manner. However, Invertase II was much more sensitive to NaF than Invertase I.
Keywords: F-exposure, HPLC separation, Invertase, Isozymes, Mung bean seedlings.
INTRODUCTION
The importance of invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) in the germinating seed is well recognized, and its presence has been reported in various plant species.1-7 Three forms of invertase occur in plants: acid, alkaline, and neutral invertases. Acid invertase has been isolated from both the cell wall and the cytoplasm. Miron and Schaffer8 observed that the soluble acid invertase activity in the green-fruited Lycopersicon hirsutum Humb. and Bonpl. declined proportionally with the rise in sucrose content while the cell-wall bound acid invertase activity remained constant throughout development.
Several factors have been shown to influence invertase activity, including aging, hormones, wounding, and light in terminally differentiated tissues.9-11 Singh and Knox2 isolated Lilium pollen invertase and showed that the activity was unaffected by 5 mM each of ZnSO4, KI, NaF, MgCl2, CaCl2, and Na3BO3. However, they demonstrated that 1 mM each of HgCl2 and p-chloromercuribenzoate (PCMB) strongly inhibited the enzyme activity, suggesting that the catalytic activity of the enzyme involved SH groups.
In our previous study, we observed the occurrence of two isoforms of acid invertase in mung bean (Vigna radiata) seedlings, with molecular weights of approximately 30 kD and 45 kD, respectively.12 In contrast to the finding of Singh and Knox with Lilium pollen,2 we found that mung bean seedlings exposed to NaF resulted in marked decreases in invertase activity, and that the decreases were NaF concentration-dependent.12 Furthermore, in vitro studies showed that the activity was enhanced by Ca2+ ions, suggesting that the invertase inhibition by F may be due, in part, to its interaction with Ca. Recently, we studied the properties of the two isozymes from mung bean radicles and found that they exhibited different sensitivities toward NaF.
MATERIALS AND METHODS
Germination of Seeds. Mung bean seeds, purchased locally, were briefly treated with 0.5 M H2SO4 and soaked in distilled water for 24 hr. The seedlings were placed in petri dish lined with filter paper, and treated with 0 (control), 0.2, 0.5, 1.0 or 2.0 mM NaF for 72 hr. At the end of the treatment, 50 radicles from each treatment group were randomly selected for determination of fresh weight. They were then stored at -80°C until use.
Preparation of Crude Enzyme Extract. Frozen tissues were first ground in a mortar and pestle and then homogenized in 0.2 M NaCl and 0.2 mM Pefabloc SC, a serine protease inhibitor, in a cold glass homogenizer. The homogenate was centrifuged at 10,000 g for 1 hr at 4°C, and the resultant supernatant was used as crude enzyme extract.
High Performance Liquid Chromatography (HPLC). An on-line column switching HPLC system (Figure 1) was used for separation and partial purification of invertase. The system consisted of Hitachi model L-5000 gradient controller (Tokyo Japan), a model L-6000 pump, a model 655 A-11 pump, a model L-4300 UV monitor, a Rheodyne Model 7125 liquid chromatographic injector (Cotati, CA, USA), and a Sensyu-Kagaku (Tokyo, Japan) model EIE002 six-way rotary valve.
Asahipak GS-520H gel filtration column (250 x 7.6 mm ID) (Column 1, Figure 1) (Showa-Denko, Tokyo, Japan) was used for initial sample purification. The analytical column (Column 2, Figure 1) was a TSK-gel DEAE-5PW anion-exchange column (75 x 7.5 mm ID) (TOSHO, Tokyo, Japan).
The two columns were equilibrated with 50 mM Tris-HCl buffer, pH 7.5 (Buffer 1). Four hundred µL of crude enzyme extract was applied onto Column 1 and the protein was eluted with Buffer 1 using an isocratic method, at a flow rate of 1.0 mL/min. Proteins in the eluates were determined by measurement of the absorbance in a spectrophotometer at 280 nm, and the fractions were tested for invertase activity. Since invertase activity was detected in fractions eluted from Column 1 between 5.5 min and 7.5 min following sample injection, the six-way valve was switched at 5.5 min to shift the eluate onto Column 2. Column 2 was initially eluted with Buffer 1, and at 10 min it was shifted to Buffer 2 (0.4 M NaCl-50 mM Tris-HCl buffer, pH 7.5), at a flow rate of 1.0 mL/min. The absorption of the fractions resulting from Column 2 elution was determined in a spectrophotometer at 225 nm. A 0.5 mL fraction was collected at an interval of 30 seconds for testing invertase activity.
Assay of Invertase. Invertase activity was determined by the method of Dey13 with a slight modification. One hundred µL of the eluate was added to a test tube containing 100 µL of 0.1 M sodium acetate buffer (pH 4.4), and the mixture was incubated at 30°C for 3 min. This was followed by addition of 50 µL of 0.5 M sucrose and the mixture was incubated at the same temperature for 1 hr. Enzyme reaction was terminated by the addition of 300 µL of 0.2 M potassium phosphate and the resultant mixture was heated in boiling water for 3 min. The amount of glucose produced during the enzyme reaction was determined by the glucose oxidase-o-dianisidine method. Protein in each fraction was determined by the method of Bradford.14 Specific activity of invertase is defined as nmole glucose produced per mg protein per min.
RESULTS
A typical elution profile of the crude extract for the initial purification is shown in Figure 2, whereas the anion-exchange chromatography of the fractions containing invertase is presented in Figure 3. The elution profile showed several fractions exhibiting invertase activity to constitute two peaks, or two isozymes, with elution times of about 20 and 22 min, and are hereafter referred to as Invertases I and II, respectively. A 70-fold purification was obtained for isozymes from the control seedlings, and the specific activities for Invertases I and II were 1700 and 2000 nmol/mg/min, respectively.
The relationship between seedling growth and invertase activity in vivo was studied by plotting the average length of the radicles and invertase activity against NaF concentrations used in the exposure. As shown in Figure 4, the two parameters closely correlated with each other.
The elution pattern of the extracts from the control and NaF-treated seedlings was similar to each other. However, the in vivo invertase activity of the extract from NaF-treated seedlings was decreased compared with the control, and the decreases were NaF concentration-dependent (Table 1). Furthermore, a marked difference in the extent of enzyme inhibition was found between the two isozymes. For seedlings exposed to 2 mM NaF, the decrease for Invertases I and II was 30% and 41%, respectively (Table 1).
To study the effects of NaF on in vitro invertase activity, eluates representing Invertases I and II of the control sample were used in the enzyme assay. Each of the assay mixtures contained 0, 1, 5, or 10 mM NaF. The mixture was incubated for 60 min as previously, and at an interval of 20 min an aliquot was removed from the mixture for testing invertase activity. The results showed that the two isozymes responded differently to added NaF. Whereas the activity of Invertase I remained unaffected, that of Invertase II was depressed (Figures 5a and b).
Figure 2. Gel filtration chromatography of mung bean extract.
A 400 µL crude enzyme extract was placed onto the Asahipak GS-520 H
column and eluted with 50 mM Tris-HCl buffer (pH 7.5) at 1.0 mL/min.
Invertase activity is expressed as nmol/min/fraction (0.25 mL).
Table 1. Effect of NaF on in vivo activities of invertase isozymes.
Specific activity (nmol/mg/min)NaF
mM / Invertase I
/ Percent of
control / Invertase II
/ Percent of
control
0 / 55.6 ± 1.7 / - / 55.2 ± 0.4 / -
0.2 / 50.9 ± 0.8* / 92 / 50.1 ± 5.9 / 91
1.0 / 44.1 ± 1.4* / 79 / 43.0 ± 4.8* / 78
2.0 / 39.1 ± 4.8* / 70 / 32.7 ± 1.7* / 59
Values are means ± S.D., n = 3. *P <0.05.
Figure 3. Anion exchange chromatography of mung bean extract.
Invertase activity is expressed as nmole/min/fraction (0.5 mL).
Figure 4. Comparison between radicle growth and invertase activity in vivo.
DISCUSSION
Separation of invertase by two-dimensional HPLC confirmed the presence of two forms of invertase in mung bean seedlings (Figures 2 and 3). This observation agrees with earlier reports by Arai et al4 and Yu et al12 In the present study, F exposure depressed invertase activity in vivo, in a concentration-dependent manner (Table 1). However, a marked difference was evident when the decreases in enzyme activity exhibited by the two isozymes were compared. For example, at 1 mM NaF the reduction of specific activity for Invertase II was 2% greater than for Invertase I. But, at 2 mM NaF the reduction for Invertase II was 16% greater than for Invertase I (Table 1).
Differences in sensitivity toward F were again manifested by the two isozymes in in vitro studies where assay mixture contained added NaF at varying concentrations. Whereas the activity of Invertase I in reaction mixtures containing added NaF was similar to that of the control during the 60 min incubation period, that of Invertase II was depressed. In this case, for reasons unknown, the decreases were not concentration-dependent (Figures 5a and b).
As mentioned previously, several factors including aging,9,10 hormones, wounding, and light in terminally differentiated tissues were shown to affect invertase activity.11 Ranwala et al6 observed that the activities of acid and neutral invertases in the mesocarp of developing muskmelon (Cucumis melo L. cv Prince) fruit declined with maturation of the fruit. Acid invertase activity was found to be high in immature fruits, and it declined rapidly and concomitantly with the accumulation of sucrose as the fruit matured. By contrast, Singh and Knox2 reported no increase in invertase activity during germination of Lilium pollen, suggesting that the enzyme is not an active participant in germination. Duke et al5 also indicated that invertase was not essential for primary root growth although they admitted that there were probably advantages to its presence. Our results with mung bean radicles clearly pointed out a close relationship between root growth and invertase activity (Figure 4).
Singh and Knox,2 in their study of invertase from Lilium pollen, showed that the enzyme activity was unaffected by 5 mM each of ZnSO4, KI, NaF, MgCl2, CaCl2, and Na3BO3. However, they demonstrated that 1 mM each of HgCl2 and PCMB, a sulfhydryl group inhibitor, strongly inhibited the enzyme activity, suggesting that the catalytic activity of the enzyme involved SH groups. Lin and Sung15 reported that one form of the isozymes from rice leaves was inhibited by metal ions including Pb2+, Zn2+, Cu2+, Hg2+, and Ag+. However, because the enzyme was not inhibited by PCMB and PMSF, a serine protease inhibitor, the authors concluded that cysteine and serine did not participate directly in the active-site catalytic reaction of the enzyme.
Results of our in vivo study have clearly shown F inhibition of invertase in geminating mung bean seeds. Although the mechanism by which F inhibits the enzyme is not known, it may involve removal of the cofactor Ca by F, as is the case with a number of other enzymes including amylase in mung bean seedlings.18 Furthermore, our results strongly point to marked differences in sensitivities that the two isozymes manifested toward F. The reason for the differences is unclear. It is possible, however, that they may be due to the size of the individual isozymes and the different amounts of Ca2+ ions bound to each of the two molecules. Since a preliminary study indicates that the molecular size of Invertase II is smaller than that of Invertase I, it is reasonable to assume that its bound Ca level may be lower, and is thus more susceptible to F exposure. Supporting this interpretation is the observation that both isozymes were inhibited in vivo by NaF, and that the inhibition became greater with increases in NaF concentrations used in the treatment of the seedlings (Table 1).
ACKNOWLEDGEMENT
M-H Yu thanks the Institute of Whole Body Metabolism for the scholarship during the course of this study.
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Published by the International Society for Fluoride Research
Editorial Office: 17 Pioneer Crescent, Dunedin 9001, New Zealand
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