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Influence of sound stimulation on plasma membrane H+-ATPase activity
Colloids and Surfaces B: Biointerfaces 25 (2002) 183– 188 www.elsevier.com/locate/colsurfb
Influence of sound stimulation on plasma membrane H+-ATPase activity Bochu Wang a,*, Hucheng Zhao a, Xiujuan Wang a, Chuanren Duan a, Daohong Wang a, Akio Sakanishi b a
Key Lab for Biomechanics and Tissue Engineering under the State Ministry of Education, College of Bioengineering, Chongqing Uni6ersity, Chongqing 400044, People’s Republic of China b Biological and Chemical Engineering, Faculty of Engineering, Gunma Uni6ersity, Gunma, Japan Received 28 September 2001; accepted 7 November 2001
Abstract Influence of sound stimulation on H+-ATPase activity is studied in this paper. The experimental results show that PM H+-ATPase activity of Chrysanthemum callus is increased apparently by sound stimulation. The effects of sound stimulation on Ca2 + in cell wall are partially diminished of by chelating them, which demonstrates that the increase of PM H+-ATPase activity depends on Ca2 + under sound stimulation. The PM H+-ATPase activity of C. callus that were stimulated by sound wave comes back its initial level after dephosphorylation. These results indicate that probably Ca2 + -dependent phosphorylation raises PM H+-ATPase activity under sound stimulation. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sound stimulation; Plasma membrane; H+-ATPase activity
1. Introduction PM H+-ATPase, a kind of glycoprotein across membranes, plays an important role in the processes of growth and development of plants. It pumps H+ out of cells depending on ATP to create an electrochemical H+ gradient across membranes and proton electrochemical H+ gradient, which drives sub-cotransfer system of plasma membranes and becomes main power of ion and * Corresponding author. Tel.: + 86-23-65-10257; fax: + 8623-64-102507. E-mail address: [email protected] (B. Wang).
nutrition substances in and out of cells. Other important physiological and biochemical processes, such as growth, development, turgor pressure and maintenance of plasma pH, are also regulated by PM H+-ATPase [1–3]. Just because of these important physiological functions, studies on the structure and function of PM H+-ATPase have been improved greatly during 20 years after plant cells was found. The structure and transport activity of PM H+-ATPase and hydrolysis mechanism of ATP are revealed by biochemical research. By means of molecular research, the amino acid sequences of active position of PM H+-ATPase have been known and its clone has
0927-7765/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 1 ) 0 0 3 2 0 - 4
184
B. Wang et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 183–188
been obtained in some plants. And it has been recognized that PM H+-ATPase of advanced plants, coded by polygene, can be regulated on the several levels of transcription, translation and posttranslation. The expression of the genes has organ difference. PM H+-ATPase exists as isoenzyme and its function fields are made ultimately certain. As the structure and the function of PM H+-ATPase are clearing in the recent years, most of studies have focused on its function regulation mechanism and its action in the processes of environment signal transfer. One of important characteristics of PM H+-ATPase is that it functions as the target of cell function regulation, and its activity is affected by light, plant hormone, fungal toxin and other environment factors [6–8]. Thus, it can be seen that PM H+-ATPase plays an important role in taking on environment stimulation. Then how does environment stimulation change PM H+-ATPase activity? There are few reports whether the PM H+-ATPase activity changes are due to its structure varieties directly caused by the environment stimulation or a series of physiological and biochemical reactions caused by mechanical stimulation. Its regulation mechanism is probably the theory basis for exploring pesticide and regulating the growth and development of plants. Sound wave is a specific form of alternating stress, which has great effects on plant growth [9], therefore, the changes of PM H+-ATPase and the reasons for its changes are analyzed under sound stimulation in this work.
Fig. 1.
2. Materials and methods
2.1. Materials Chrysanthemum calluses were inoculated in conical flasks with 25 ml MS medium (supplemented with 1.0 mg/l BA, 0.5 mg/l NAA). The calluses were cultured in illumination incubator at 26 °C.
2.2. Sound stimulation Alternating stimulation field was achieved by sound generator (Fig. 1), which was designed in our laboratory. Exuberantly growing C. calluses were cut into even cubes and inoculated in mediums. Inoculated calluses were stimulated by sound wave with a certain intensity (100 db) and frequency (1000 Hz) two times per day for 2 weeks, and each time for 30 min. Control group were placed in the same environment with stressed one.
2.3. Separation and purification of plasma membrane C. calluses were cut into 1 mm3 cubes and homogenized in a blender with 0.25 mol/l sucrose, 10% (v/v) glycerol, 2 mmol/l EGTA, 1 mmol/l DTT and 0.5% BSA. This homogenate was filtered through four layers of cheesecloth and centrifuged at 12 000× g for 10 min. The supernatant was centrifuged at 50 000× g for 30 min and the pellet was suspended in 0.25 M sucrose, 1 mM
B. Wang et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 183–188
185
DTT and 5 mM (pH 6.5) Tris– Mes. The plasma membranes were purified in 6.2% dextran T-500 and PEG-3350.
2.4. Purification of plasma membrane H+-ATPase The purified membranes were washed with 0.2% Triton x-100 and 0.5 M KCl and the ultimate concentration of membrane proteins was 0.1 mg/ml. The mixture was centrifuged at 11 500× g for 45 min. The pellet was suspended (the concentration of membrane proteins was 0.5 g/l) and treated with 0.6% (w/v) Zw at room temperature for 5 min. It was centrifuged at 1500× g for 30 min after supplemented with sodium cholate (0.9% (w/v) as ultimate concentration). The upper phases mixed with saturate ammonium sulfate solution were placed for 0.5 h and centrifuged at 50 000×g for 10 min. The new phase was mixed with saturate ammonium sulfate solution again and placed through night at 4 °C. The mixture was centrifuged at 50 000×g for 10 min. The lower phase was suspended and centrifuged at 11 500×g for 2 h, and the pellet was kept in liquid nitrogen.
2.5. Measurement of ATPase acti6ity Membrane proteins (10 mg reacted with 3 mM ATP, 3 mM MgSO4, 100 mM KCl and 25 mM BTP-Mes (pH 6.5) at 38 °C for 20 min. The reaction was stopped by 0.5 ml 10% SDS. The solution, added 2 ml 1% ammonium molybdate (1.6 M HCl) and 0.1 ml 0.2% naphthionic acid (compounded 12% NaHSO4 with 1.25% Na2SO3 was placed at room temperature for 30 min and make colorimetry at 700 nm. The unit of ATPase activity is mmol Pi/mg pro/h.
2.6. Dephosphorylation reaction PM H+-ATPase (300 mg) was added with 5 ml of 30 mM Tris – HCl buffer (pH 8.5), 1.5 mM EGTA and 0.1 M KCl. The mixture was centrifuged at 200 000×g for 15 min. The pellet was suspended in 40 ml of 30 mM Tris – HCl (pH 8.5), 0.1 M KCl and 1 mM MgSO4. Then a certain
Fig. 2. Effect of sound stimulation on the H+-ATPase activity of plasma membrane from C. callus.
amounts (mg) of alkaline phosphates were added. The reaction was stopped after 20 min with 20 ml buffer of 30 mM Hepes–Tris (pH 7.0), 1.5 mM EGTA and 0.1 M KCl. This suspension was centrifuged as above and the pellets were collected for measurements.
2.7. Enzyme clea6age treatment of trypsin Enzyme cleavage of plasma membranes was treated by the method of Palmgren [10]. Plasma membrane proteins (20 mg), added with 2 mg of trypsin, were mixed with 20 ml of 25 mM Tris – Mes (pH 7.5), 2 mM DTT, 5 mM EDTA and 0.25 M sucrose at 20 °C. The reaction was stopped after 10 min with 20 ml buffer of 30 mM Hepes– Tris (pH 7.0), 1.5 mM EGTA and 0.1 M KCl.
3. Results
3.1. Influence of sound stimulation on PM H+-ATPase acti6ity As shown in Fig. 2, PM H+-ATPase activity of C. callus increased under stimulation. But it was inhibited if sound stimulated plasma membrane vesicles of Chrysanthemum directly (Fig. 3). It indicated that the cause of the increase of PM H+-ATPase under sound stimulation lied in a series of physiological and biochemical reactions rather than the change of constellation of enzyme. Some studies have demonstrated that the regulation of most of enzyme activity was mediated by
186
B. Wang et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 183–188
Fig. 5. Effect of EGTA on the H+-ATPase activity of C. callus under sound stimulation. Fig. 3. Influence of sound stimulation on W-ATPase activity of plasma membrane vesicle isolated from C. callus.
other factors, for example, the second messenger. As important messengers in cells, Both Ca2 + and CaM participate in and regulate most of physiological processes together. Accordingly, the influence of Ca2 + at different concentrations on PM H+-ATPase activity is studied. It can be seen from Fig. 4 that sound stimulation can increase PM H+-ATPase activity whether there are Ca2 + in medium or not. Hence, do the increasing of PM H+-ATPase of the callus need Ca2 + ? To answer this question, EGTA was chelated with Ca2 + of cell wall. EGTA is a kind of chelator specialized in Ca2 + , which cannot go through plasma membranes and diminishes only Ca2 + of cell wall. The calluses were cultured in medium with EGTA and it is PM H+-ATPase activity was measured after sound stimulation for 2 weeks. Fig. 5 shows that the increase of sound stimulation on PM H+-ATPase activity is partly diminished when Ca2 + is chelated. It indicates
Fig. 4. Effect of calcium in the matrix on a PM H+-ATPase activity of C. callus.
that the changes of PM H+-ATPase depend on Ca2 + . To demonstrate the mechanism of the influence of sound on PM H+-ATPase activity, we made PM H+-ATPase of stressed group reacted with alkaline phosphatase. The result suggested that the activity of stressed group by dephosphorylation was close to that of control group. That is, the effect of sound stimulation was removed. It indicated that the effect of sound stimulation on PM H+-ATPase activity was achieved through Ca2 + -dependent phosphorylation (Fig. 6).
4. Discussion Studies on physiological and biochemical function of PM H+-ATPase have shown that its activity can be affected by light, hormone, and fungal toxin. Consequently, the growth and development of plants is affected [6–8]. At the same time, it has
Fig. 6. Effect of alkaline phosphatase on the H+-ATPase activity of PM vesicles from Chrysanthemum under sound stimulation.
B. Wang et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 183–188
been found in our experiments that PM H+-ATPase activity could also be changed by sound stimulation, which indicates that PM H+-ATPase similarly plays an important role in response to mechanical stress of C. callus. In the experiment, we stimulated directly plasma membrane vesicles by sound wave and mensurated changes of PM H+-ATPase activity. However, the metrical results are converse to those of stimulation group in vivo (Fig. 2). These results suggest that the changes of PM H+-ATPase activity under sound stimulation may be due to a series of physiological and biochemical reactions rather than the changes of constellation of PM H+-ATPase caused by mechanical stimulation. Ca2 + , an important second messenger in the cell, regulates the structure and function of effector proteins by connecting with target proteins in ‘stimulation – response’ coupling system, thereby, affects physiological and biochemical processes of plant cells [2 –5]. Extent research findings demonstrate that mechanical stress can increase steadily the concentration of intracellular Ca2 + . Then whether is the increase of PM H+-ATPase activity associated with Ca2 + and CaM or not? It was found that the promotion of sound stimulation on PM H+-ATPase activity of callus was partly diminished by chelating Ca2 + of cell wall with chelator specialized in Ca2 + (Fig. 5), which implies Ca2 + is concerned with the influence of sound stimulation on PM H+-ATPase activity and Ca2 + of cell wall, as a Ca2 + storage outside cell, plays an important role in sound stimulation. Sound stimulation can enhance the concentration of intracellular Ca2 + . Protein kinaseor phosphorylase are activated when the concentration of intracellular Ca2 + increases to its critical value. Thus, PM H+-ATPase activity increased through phosphorylation. It is showed in experiment that PM H+-ATPase activity falls obviously after dephosphorylating PM H+-ATPase of stressed group, which suggests the promotion of PM H+-ATPase activity is also due to phosphorylation. In the recent years research has indicated that reversible phosphorylation processes of proteins has an important effect in the of signal transfer in cells of animals and prokaryotes. The processes are not only the common channel of intracellular signals,
187
but also the center of interaction among intracellular messenger systems [11,12]. Though acquisition of plant cells is less than that of animals and prokaryotes. It is distinctly showed in the experiment that reversible phosphorylation processes probably have similar functions in the signal transfer in plant cells. Protein kinase includes many families and species, each of them has specific zymolyte and mediates specific process of signal transfer. In this study induction mechanism was demonstrated for the first time that Ca2 + -dependent protein kinase, which is one of Ca2 + signal receptors in plant cells, changes PM H+ATPase activity by transferring sound signals to PM H+-ATPase regulated by it. As a result, the responses of cells to sound stimulation are achieved. The regulation of PM H+-ATPase activity is very complex. Its activity change involves in covalence bedeck under sound stimulation. Further research are needed to find whether it involves in shearing bedeck, and the cause of the activity changes is the affinity of PM H+-ATPase to zymolyte or the maximum reaction velocity under sound stimulation.
References [1] R. Serrano, Structure and function of plasma membrane ATPase, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40 (1989) 61 – 94. [2] B. Michelef, M. Boutry, The plasma membrane H+-ATPase. A highly regulated enzyme with multiple physiological functions, Plant Physiol. 108 (1993) 1 – 6. [3] R. Gibrat, J.P. Grouzis, J. Rigaud, et al., Potassium stimulation of com root plasma lemma ATPase by glucose. Interaction between domains and reconstituted vesicles with purified ATPase, Plant Physiol. 93 (1990) 1183 – 1189. [4] J.F. Hrper, L. Manney, N.D. Dewitt, et al., The Arbidopsis thaliana plasma membrane H+-ATPase multigene family, J. Biol. Chem. 265 (1990) 13601 – 13608. [5] N.N. Ewing, B. Bennet, Assesment of the number of P-type H+-ATPase gene in tomato, Plant Physiol. 106 (1994) 547 – 557. [6] M.G. Palmgren, Regulation of plant plasma membrane H+-ATPase activity, Physiol. Plant 83 (1991) 314 – 323. [7] A.B. Marre, The proton pumps of the plasma lemma and the tonoplast of higher plants, J. Bioenerg. Biomembr. 17 (1985) 1 – 21.
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B. Wang et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 183–188
[8] R. Serrano, Plasma membrane ATPase, in: C. Larsson, I.M. Moller (Eds.), The Plant Plasma Membrane, Structure, Function and Molecular Biology, Springer, Berlin, 1990, pp. 127 – 153. [9] K. Shen, B. Xi, The research of thermodynamic phase behavior of tobacco cell under the alternative stress, Acta Biophys. Sin. 15 (1999) 579 –583. [10] M.G. Palmgren, Regulation of plant plasma membrane H+-ATPase activity, Physiol. Plant 83 (1991) 314 – 323.
[11] T. Kinoshita, M. Nishimura, K. Shimazaki, Cytosolic concentration of Ca2 + regulates the plasma membrane H+-ATPase in guard cell of fava bean, Plant Cell 7 (1995) 1333 – 1342. [12] T.M. Yuasa, Ca2 + -dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: partial purification and Ca2 + -dependent association of enzyme to the microsomes, Arch. Biochem. Biophys. 296 (1992) 175 – 182.
Influence of sound stimulation on plasma membrane H+-ATPase activity Bochu Wang a,*, Hucheng Zhao a, Xiujuan Wang a, Chuanren Duan a, Daohong Wang a, Akio Sakanishi b a
Key Lab for Biomechanics and Tissue Engineering under the State Ministry of Education, College of Bioengineering, Chongqing Uni6ersity, Chongqing 400044, People’s Republic of China b Biological and Chemical Engineering, Faculty of Engineering, Gunma Uni6ersity, Gunma, Japan Received 28 September 2001; accepted 7 November 2001
Abstract Influence of sound stimulation on H+-ATPase activity is studied in this paper. The experimental results show that PM H+-ATPase activity of Chrysanthemum callus is increased apparently by sound stimulation. The effects of sound stimulation on Ca2 + in cell wall are partially diminished of by chelating them, which demonstrates that the increase of PM H+-ATPase activity depends on Ca2 + under sound stimulation. The PM H+-ATPase activity of C. callus that were stimulated by sound wave comes back its initial level after dephosphorylation. These results indicate that probably Ca2 + -dependent phosphorylation raises PM H+-ATPase activity under sound stimulation. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sound stimulation; Plasma membrane; H+-ATPase activity
1. Introduction PM H+-ATPase, a kind of glycoprotein across membranes, plays an important role in the processes of growth and development of plants. It pumps H+ out of cells depending on ATP to create an electrochemical H+ gradient across membranes and proton electrochemical H+ gradient, which drives sub-cotransfer system of plasma membranes and becomes main power of ion and * Corresponding author. Tel.: + 86-23-65-10257; fax: + 8623-64-102507. E-mail address: [email protected] (B. Wang).
nutrition substances in and out of cells. Other important physiological and biochemical processes, such as growth, development, turgor pressure and maintenance of plasma pH, are also regulated by PM H+-ATPase [1–3]. Just because of these important physiological functions, studies on the structure and function of PM H+-ATPase have been improved greatly during 20 years after plant cells was found. The structure and transport activity of PM H+-ATPase and hydrolysis mechanism of ATP are revealed by biochemical research. By means of molecular research, the amino acid sequences of active position of PM H+-ATPase have been known and its clone has
0927-7765/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 1 ) 0 0 3 2 0 - 4
184
B. Wang et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 183–188
been obtained in some plants. And it has been recognized that PM H+-ATPase of advanced plants, coded by polygene, can be regulated on the several levels of transcription, translation and posttranslation. The expression of the genes has organ difference. PM H+-ATPase exists as isoenzyme and its function fields are made ultimately certain. As the structure and the function of PM H+-ATPase are clearing in the recent years, most of studies have focused on its function regulation mechanism and its action in the processes of environment signal transfer. One of important characteristics of PM H+-ATPase is that it functions as the target of cell function regulation, and its activity is affected by light, plant hormone, fungal toxin and other environment factors [6–8]. Thus, it can be seen that PM H+-ATPase plays an important role in taking on environment stimulation. Then how does environment stimulation change PM H+-ATPase activity? There are few reports whether the PM H+-ATPase activity changes are due to its structure varieties directly caused by the environment stimulation or a series of physiological and biochemical reactions caused by mechanical stimulation. Its regulation mechanism is probably the theory basis for exploring pesticide and regulating the growth and development of plants. Sound wave is a specific form of alternating stress, which has great effects on plant growth [9], therefore, the changes of PM H+-ATPase and the reasons for its changes are analyzed under sound stimulation in this work.
Fig. 1.
2. Materials and methods
2.1. Materials Chrysanthemum calluses were inoculated in conical flasks with 25 ml MS medium (supplemented with 1.0 mg/l BA, 0.5 mg/l NAA). The calluses were cultured in illumination incubator at 26 °C.
2.2. Sound stimulation Alternating stimulation field was achieved by sound generator (Fig. 1), which was designed in our laboratory. Exuberantly growing C. calluses were cut into even cubes and inoculated in mediums. Inoculated calluses were stimulated by sound wave with a certain intensity (100 db) and frequency (1000 Hz) two times per day for 2 weeks, and each time for 30 min. Control group were placed in the same environment with stressed one.
2.3. Separation and purification of plasma membrane C. calluses were cut into 1 mm3 cubes and homogenized in a blender with 0.25 mol/l sucrose, 10% (v/v) glycerol, 2 mmol/l EGTA, 1 mmol/l DTT and 0.5% BSA. This homogenate was filtered through four layers of cheesecloth and centrifuged at 12 000× g for 10 min. The supernatant was centrifuged at 50 000× g for 30 min and the pellet was suspended in 0.25 M sucrose, 1 mM
B. Wang et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 183–188
185
DTT and 5 mM (pH 6.5) Tris– Mes. The plasma membranes were purified in 6.2% dextran T-500 and PEG-3350.
2.4. Purification of plasma membrane H+-ATPase The purified membranes were washed with 0.2% Triton x-100 and 0.5 M KCl and the ultimate concentration of membrane proteins was 0.1 mg/ml. The mixture was centrifuged at 11 500× g for 45 min. The pellet was suspended (the concentration of membrane proteins was 0.5 g/l) and treated with 0.6% (w/v) Zw at room temperature for 5 min. It was centrifuged at 1500× g for 30 min after supplemented with sodium cholate (0.9% (w/v) as ultimate concentration). The upper phases mixed with saturate ammonium sulfate solution were placed for 0.5 h and centrifuged at 50 000×g for 10 min. The new phase was mixed with saturate ammonium sulfate solution again and placed through night at 4 °C. The mixture was centrifuged at 50 000×g for 10 min. The lower phase was suspended and centrifuged at 11 500×g for 2 h, and the pellet was kept in liquid nitrogen.
2.5. Measurement of ATPase acti6ity Membrane proteins (10 mg reacted with 3 mM ATP, 3 mM MgSO4, 100 mM KCl and 25 mM BTP-Mes (pH 6.5) at 38 °C for 20 min. The reaction was stopped by 0.5 ml 10% SDS. The solution, added 2 ml 1% ammonium molybdate (1.6 M HCl) and 0.1 ml 0.2% naphthionic acid (compounded 12% NaHSO4 with 1.25% Na2SO3 was placed at room temperature for 30 min and make colorimetry at 700 nm. The unit of ATPase activity is mmol Pi/mg pro/h.
2.6. Dephosphorylation reaction PM H+-ATPase (300 mg) was added with 5 ml of 30 mM Tris – HCl buffer (pH 8.5), 1.5 mM EGTA and 0.1 M KCl. The mixture was centrifuged at 200 000×g for 15 min. The pellet was suspended in 40 ml of 30 mM Tris – HCl (pH 8.5), 0.1 M KCl and 1 mM MgSO4. Then a certain
Fig. 2. Effect of sound stimulation on the H+-ATPase activity of plasma membrane from C. callus.
amounts (mg) of alkaline phosphates were added. The reaction was stopped after 20 min with 20 ml buffer of 30 mM Hepes–Tris (pH 7.0), 1.5 mM EGTA and 0.1 M KCl. This suspension was centrifuged as above and the pellets were collected for measurements.
2.7. Enzyme clea6age treatment of trypsin Enzyme cleavage of plasma membranes was treated by the method of Palmgren [10]. Plasma membrane proteins (20 mg), added with 2 mg of trypsin, were mixed with 20 ml of 25 mM Tris – Mes (pH 7.5), 2 mM DTT, 5 mM EDTA and 0.25 M sucrose at 20 °C. The reaction was stopped after 10 min with 20 ml buffer of 30 mM Hepes– Tris (pH 7.0), 1.5 mM EGTA and 0.1 M KCl.
3. Results
3.1. Influence of sound stimulation on PM H+-ATPase acti6ity As shown in Fig. 2, PM H+-ATPase activity of C. callus increased under stimulation. But it was inhibited if sound stimulated plasma membrane vesicles of Chrysanthemum directly (Fig. 3). It indicated that the cause of the increase of PM H+-ATPase under sound stimulation lied in a series of physiological and biochemical reactions rather than the change of constellation of enzyme. Some studies have demonstrated that the regulation of most of enzyme activity was mediated by
186
B. Wang et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 183–188
Fig. 5. Effect of EGTA on the H+-ATPase activity of C. callus under sound stimulation. Fig. 3. Influence of sound stimulation on W-ATPase activity of plasma membrane vesicle isolated from C. callus.
other factors, for example, the second messenger. As important messengers in cells, Both Ca2 + and CaM participate in and regulate most of physiological processes together. Accordingly, the influence of Ca2 + at different concentrations on PM H+-ATPase activity is studied. It can be seen from Fig. 4 that sound stimulation can increase PM H+-ATPase activity whether there are Ca2 + in medium or not. Hence, do the increasing of PM H+-ATPase of the callus need Ca2 + ? To answer this question, EGTA was chelated with Ca2 + of cell wall. EGTA is a kind of chelator specialized in Ca2 + , which cannot go through plasma membranes and diminishes only Ca2 + of cell wall. The calluses were cultured in medium with EGTA and it is PM H+-ATPase activity was measured after sound stimulation for 2 weeks. Fig. 5 shows that the increase of sound stimulation on PM H+-ATPase activity is partly diminished when Ca2 + is chelated. It indicates
Fig. 4. Effect of calcium in the matrix on a PM H+-ATPase activity of C. callus.
that the changes of PM H+-ATPase depend on Ca2 + . To demonstrate the mechanism of the influence of sound on PM H+-ATPase activity, we made PM H+-ATPase of stressed group reacted with alkaline phosphatase. The result suggested that the activity of stressed group by dephosphorylation was close to that of control group. That is, the effect of sound stimulation was removed. It indicated that the effect of sound stimulation on PM H+-ATPase activity was achieved through Ca2 + -dependent phosphorylation (Fig. 6).
4. Discussion Studies on physiological and biochemical function of PM H+-ATPase have shown that its activity can be affected by light, hormone, and fungal toxin. Consequently, the growth and development of plants is affected [6–8]. At the same time, it has
Fig. 6. Effect of alkaline phosphatase on the H+-ATPase activity of PM vesicles from Chrysanthemum under sound stimulation.
B. Wang et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 183–188
been found in our experiments that PM H+-ATPase activity could also be changed by sound stimulation, which indicates that PM H+-ATPase similarly plays an important role in response to mechanical stress of C. callus. In the experiment, we stimulated directly plasma membrane vesicles by sound wave and mensurated changes of PM H+-ATPase activity. However, the metrical results are converse to those of stimulation group in vivo (Fig. 2). These results suggest that the changes of PM H+-ATPase activity under sound stimulation may be due to a series of physiological and biochemical reactions rather than the changes of constellation of PM H+-ATPase caused by mechanical stimulation. Ca2 + , an important second messenger in the cell, regulates the structure and function of effector proteins by connecting with target proteins in ‘stimulation – response’ coupling system, thereby, affects physiological and biochemical processes of plant cells [2 –5]. Extent research findings demonstrate that mechanical stress can increase steadily the concentration of intracellular Ca2 + . Then whether is the increase of PM H+-ATPase activity associated with Ca2 + and CaM or not? It was found that the promotion of sound stimulation on PM H+-ATPase activity of callus was partly diminished by chelating Ca2 + of cell wall with chelator specialized in Ca2 + (Fig. 5), which implies Ca2 + is concerned with the influence of sound stimulation on PM H+-ATPase activity and Ca2 + of cell wall, as a Ca2 + storage outside cell, plays an important role in sound stimulation. Sound stimulation can enhance the concentration of intracellular Ca2 + . Protein kinaseor phosphorylase are activated when the concentration of intracellular Ca2 + increases to its critical value. Thus, PM H+-ATPase activity increased through phosphorylation. It is showed in experiment that PM H+-ATPase activity falls obviously after dephosphorylating PM H+-ATPase of stressed group, which suggests the promotion of PM H+-ATPase activity is also due to phosphorylation. In the recent years research has indicated that reversible phosphorylation processes of proteins has an important effect in the of signal transfer in cells of animals and prokaryotes. The processes are not only the common channel of intracellular signals,
187
but also the center of interaction among intracellular messenger systems [11,12]. Though acquisition of plant cells is less than that of animals and prokaryotes. It is distinctly showed in the experiment that reversible phosphorylation processes probably have similar functions in the signal transfer in plant cells. Protein kinase includes many families and species, each of them has specific zymolyte and mediates specific process of signal transfer. In this study induction mechanism was demonstrated for the first time that Ca2 + -dependent protein kinase, which is one of Ca2 + signal receptors in plant cells, changes PM H+ATPase activity by transferring sound signals to PM H+-ATPase regulated by it. As a result, the responses of cells to sound stimulation are achieved. The regulation of PM H+-ATPase activity is very complex. Its activity change involves in covalence bedeck under sound stimulation. Further research are needed to find whether it involves in shearing bedeck, and the cause of the activity changes is the affinity of PM H+-ATPase to zymolyte or the maximum reaction velocity under sound stimulation.
References [1] R. Serrano, Structure and function of plasma membrane ATPase, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40 (1989) 61 – 94. [2] B. Michelef, M. Boutry, The plasma membrane H+-ATPase. A highly regulated enzyme with multiple physiological functions, Plant Physiol. 108 (1993) 1 – 6. [3] R. Gibrat, J.P. Grouzis, J. Rigaud, et al., Potassium stimulation of com root plasma lemma ATPase by glucose. Interaction between domains and reconstituted vesicles with purified ATPase, Plant Physiol. 93 (1990) 1183 – 1189. [4] J.F. Hrper, L. Manney, N.D. Dewitt, et al., The Arbidopsis thaliana plasma membrane H+-ATPase multigene family, J. Biol. Chem. 265 (1990) 13601 – 13608. [5] N.N. Ewing, B. Bennet, Assesment of the number of P-type H+-ATPase gene in tomato, Plant Physiol. 106 (1994) 547 – 557. [6] M.G. Palmgren, Regulation of plant plasma membrane H+-ATPase activity, Physiol. Plant 83 (1991) 314 – 323. [7] A.B. Marre, The proton pumps of the plasma lemma and the tonoplast of higher plants, J. Bioenerg. Biomembr. 17 (1985) 1 – 21.
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B. Wang et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 183–188
[8] R. Serrano, Plasma membrane ATPase, in: C. Larsson, I.M. Moller (Eds.), The Plant Plasma Membrane, Structure, Function and Molecular Biology, Springer, Berlin, 1990, pp. 127 – 153. [9] K. Shen, B. Xi, The research of thermodynamic phase behavior of tobacco cell under the alternative stress, Acta Biophys. Sin. 15 (1999) 579 –583. [10] M.G. Palmgren, Regulation of plant plasma membrane H+-ATPase activity, Physiol. Plant 83 (1991) 314 – 323.
[11] T. Kinoshita, M. Nishimura, K. Shimazaki, Cytosolic concentration of Ca2 + regulates the plasma membrane H+-ATPase in guard cell of fava bean, Plant Cell 7 (1995) 1333 – 1342. [12] T.M. Yuasa, Ca2 + -dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: partial purification and Ca2 + -dependent association of enzyme to the microsomes, Arch. Biochem. Biophys. 296 (1992) 175 – 182.