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Effects of sound-wave stimulation on the secondary structure of plasma membrane protein of tobacco cells
Colloids and Surfaces B: Biointerfaces 25 (2002) 29 – 32 www.elsevier.com/locate/colsurfb
Effects of sound-wave stimulation on the secondary structure of plasma membrane protein of tobacco cells H.C. Zhao a,*, J. Wu a, B.S. Xi a, B.C. Wang b b
a Biomechanics Lab., Department of Engineering Mechanics, Tsinghua Uni6ersity, Beijing 100084, PR China Key Lab for Biomechanics and Tissue Engineering under the State Ministry of Education, Chongqing Uni6ersity, Chongqing 400044, PR China
Received 20 August 2001; accepted 1 October 2001
Abstract In this paper, the effects of sound wave on the structure of the protein of tobacco cells were studied by Circular Dichroism spectra (CD). The results show that the change of plasma membrane protein structure is closely related to the strength and frequency of the sound wave. In a certain range of frequency and strength, the sound wave makes significant changes on the membrane protein structure, producing an increase in a-helix and a decrease in b-turn. This proves that the secondary structure of membrane protein is highly sensitive to the stimulation of sound wave. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Secondary structure of membrane protein; Stimulation of sound wave; Signal transduction
1. Introduction The growth of a plant is uncontrollably affected by environmental factors including mechanical stress. So physicists and biologists are concerned about the relation between stress and growth. People have known for a long time that mechanical stimulation has obvious effects on the growth and development of plants. For example, the creeping plant detects the environment on a route via its contact-sensitive stem, stalk, tentacle and root creeps. The stems of some plants becomes * Corresponding author. Post Doctor of Tsinghua University. E-mail address: [email protected] (H.C. Zhao).
short and thick after being cut [1]. As a special form of alternative stress, sound-wave stimulation has obvious effects on the growth of plants. Many researchers have found that lower strength and frequency sound waves not only disrupt the complete structure of the cell, but also enhance the procedure of metabolism and the permeability and selection of the plasma membrane [2–4]. Thus it has been proved that mechanical stress has visible influence on the growth of plants. However, it is not clear how the plant senses the mechanical stimulation and how the mechanical signal is transferred. The plasma membrane is the outer layer of the cell, so it senses the change of environment conditions first [5]. The membrane protein is the executive of membrane functions, so
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 2 9 4 - 6
30
H.C. Zhao et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 29–32
the study of the effect of mechanical stimulation on the structure and function of membrane protein enables us to learn the response procedure of a plant cell to mechanical stimulation. For this reason, this article focuses on studying the effects of sound wave stimulation on the structure of membrane protein with Circular Dichroism (CD). We expect to have a deeper knowledge on the mechanism of mechanical stimulation on the growth of plants. 2. Materials and methods
2.1. The separation of protoplast of tobacco cell Pour 10 ml newly disposed enzyme solution (1.2% cellulose R-10, 0.2% pectase Y-23, 0.1% BSA and 300 mol/l sorbierite was added, pH 5.5) in a plate 9 cm in diameter. Tear the epidermis on the leaf of tobacco and cut to 0.5× 1 cm2 pieces. Let the back of the leaf face down and spread the enzyme solution on it, store at 25 °C. Keep off the sunlight, pump to vacuum for 3 min, and swirl the culture plate gently, let off the protoplast, filter it via a nylon net. Pour it in a centrifugal tube, add 5 ml 0.8 mol/l solution of mannitol, centrifuge for 3 min under 1000 rpm and collect the suspension protoplast. Pour it to a centrifugal tube with a round bottom, add 2– 3 ml CPW13 drop by drop to wash it, mix evenly and add washing solution of CPW13 until the volume is 10 –12 ml, mix evenly, centrifuge for 1 min at 500 rpm, draw the supernatant at best, and suspend it with the washing solution of CPW13 of about 10 –12 ml. Store at 4 °C in a refrigerator, let it deposit naturally, draw most of the supernatant, quantify it and mix evenly and the density of protoplast solution is about 2× 106/ml.
2.3. Measuring the change of membrane protein structure of the tobacco cell with the CD Utilize the CD (type number CD J-500C) of the biological faculty of Tsinghua University, the key Laboratory of membrane and membrane biological engineering under the state. Get the protoplast solution of experimental groups and control group, dilute it with CPW13 solution to 105/ml, take 1 ml, pour into the species cup and put the species cup in the apparatus. The scanning speed is 200 nm/min, repeat for four times. The scope of wavelength is 200–500 nm, at room temperature. These are the detection conditions for recording the spectrum.
3. Results
3.1. The spectrum of tobacco cell Collect the protoplast stimulated for an hour under the sound wave of 400 Hz, 100 dB and the control, dilute them to 105/ml with washing solution of CPW13, measure the spectrum of CD. The results are shown in Fig. 1. The protoplast affected by the sound wave of 400 Hz, 100 dB has a left-move of its negative peak at 213 and 215 nm on the map. On the other hand, there is an obvious change in the morphology of the peak. We know the change of CD may reflect the secondary structure alteration of the membrane
2.2. The effect of alternati6e stress on the protoplast of tobacco cell The alternative stress field was generated by the sound apparatus in the laboratory. Get the protoplast solution, divide it into 10 groups, stimulate them for an hour, use different strength and frequency of sound wave, respectively. The control need not be disposed.
Fig. 1. The effect of sound stimulation on the secondary structure of membrane protein.
H.C. Zhao et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 29–32 Table 1 The effects of constant strength sound wave with different frequency on the membrane protein structure of the tobacco cell
a-helix b-turn
Control
400 Hz
800 Hz
1200 Hz
41.3 58.6
61.6 38.9
54.6 45.4
40.7 59.3
protein. This means that the sound wave stimulation under some strength and frequency leads to change of the secondary structure of the membrane.
3.2. The effects of constant strength sound wa6e with different frequencies on the membrane protein structure of the tobacco cell Under certain strength of 100 dB, three frequencies of sound wave are selected, 400, 800, and 4000 Hz, respectively. They act on the samples for an hour each. The results are shown in Table 1. We could see that the effect of the sound stimulation of different frequencies on the secondary structure of the membrane protein may vary in degree. The change of secondary structure is most obvious at 400 Hz. There is an increase of a-helix and decrease of b-turn.
3.3. The effects of constant frequency sound wa6e with different strengths on the membrane protein structure of the tobacco cell Under a frequency of 400 Hz, three strengths of sound wave are selected, 90, 100, and 110 dB, respectively. They act on the samples for an hour each. The results are shown in Table 2. The results show that the strength of sound wave affects the membrane protein structure to a Table 2 The effects of constant frequency sound wave with different strength on the membrane protein structure of the tobacco cell
a-helix b-turn
Control
90 dB
100 dB
110 dB
41.3 58.7
56.0 44.0
61.1 38.9
42.6 57.4
31
great extent. In a given range, the effect increases with the increase of the sound wave strength. However, the effect may drop back when the strength is too high. In order to confirm that the CD of the protoplast is the reflection of the secondary structure of the membrane protein, we got the plasma membrane at the same time, and scrutinized its CD. The results show that the CD of protoplast and plasma membrane are basically same at the wavelength range (200– 250 nm) we have secured, and the analysis of the secondary structure is almost accordant. So we are sure that the CD of the protoplast of tobacco cell is the real reflection of the secondary structure of membrane protein.
4. Discussion The research concerning the effect of mechanical stress on the growth of a plant has developed to the molecular level in recent years, and some genes related to the mechanical stress have been found [6]. But how the plant senses the mechanical stimulation, and how the mechanical signal transduced to the inside of a cell are not known clearly. The plasma membrane is the outer layer of the cell. It senses the environment condition changes first. The researches showed that the lipid physical state of plasma membrane is highly sensitive to environment stimulation. The stimulation from the environment can lead to the change of functions of membrane protein [7]. Our experiments showed that the structure of membrane protein undergoes obvious change under the sound stimulation under the frequency of 400 Hz and the strength of 90 dB. We could draw this conclusion from the increase of a-helix, and the decrease of b-turn, which demonstrated that the secondary structure of membrane protein is highly sensitive to the stimulation of sound. The a-helix is stretched into the bilayer of the phospholipid. The hydrophobic bonds are formed between the hydrophobic group of the outside of the a-helix and the alkyl of the hydrophobic region of phospholipid bilayer [8]. The b-turn of the secondary structure is extended on the surface of the membrane and forms hydrophobic and static electron
32
H.C. Zhao et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 29–32
with phospholipids [9]. The interaction of phospholipids and protein molecules and the change of molecular structure affects and regulates the fluidity of the whole membrane. The research we carried out years ago showed that stimulation by sound waves at certain frequencies and strengths enable the increase of the fluidity of the cell membrane [2]. From this we may deduce that the change of the secondary structure of membrane protein may lead to the increase of the fluidity of the plasma membrane.
References [1] P. Larson, Stem formation of young larix as influenced by wind and pruning, For. Sci. 11 (1965) 412 – 424. [2] K.L. Sun, The effects of alternative stress on the thermodymical properties of cultured tobacco cells, Acta Biophysica Sinica 15 (1999) 579 –584 (in Chinese).
[3] Z.W. Shen, The secondary structure changes of plant cell wall proteins aroused by strong sound waves using FT-IR, Acta Photonica Sinica 18 (1999) 600 – 602 (in Chinese). [4] G.Y. Cai, The effects of alternative stress on the membrane protein conformations of tobacco cells by circular dichroism, Acta Photonica Sinica 29 (2000) 289 – 297 (in Chinese). [5] Y.L. Yiu, On the comparative stress physiology, in: Beijing Association of Plant Physiology. In: Advances in plant Physiology and Biochemistry, vol. 15, Science Press, Beijing, 1987, pp. 30 – 35 (in Chinese). [6] J. Broom, Rain-, wind-, and touch-induced expression of calmodulin and calmodilin-related genes in Aabbidopsis, Cell 60 (1990) 357 – 364. [7] Qiu Quan-Sheng, Influence of osmotic stress on Lipid physical states of Plasma Membrane from Wheat Roots, Acta Botanica Sinica 41 (1999) 161 – 165 (in Chinese). [8] J. Elzenge, V. Van, Characterization of ion channels in the plasma membrane of epidermel cells of expanding pea leaves, J. Membr. Biol. 137 (1994) 227 – 235. [9] L.D. Tamara, in: David D. Rodert (Ed.), Glycosyl Phosphatidylinositol-Linked Membrane Protein Structure, Biosynthesis and Function, vol. 37, Academic press, 1994, pp. 236– 240.
Effects of sound-wave stimulation on the secondary structure of plasma membrane protein of tobacco cells H.C. Zhao a,*, J. Wu a, B.S. Xi a, B.C. Wang b b
a Biomechanics Lab., Department of Engineering Mechanics, Tsinghua Uni6ersity, Beijing 100084, PR China Key Lab for Biomechanics and Tissue Engineering under the State Ministry of Education, Chongqing Uni6ersity, Chongqing 400044, PR China
Received 20 August 2001; accepted 1 October 2001
Abstract In this paper, the effects of sound wave on the structure of the protein of tobacco cells were studied by Circular Dichroism spectra (CD). The results show that the change of plasma membrane protein structure is closely related to the strength and frequency of the sound wave. In a certain range of frequency and strength, the sound wave makes significant changes on the membrane protein structure, producing an increase in a-helix and a decrease in b-turn. This proves that the secondary structure of membrane protein is highly sensitive to the stimulation of sound wave. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Secondary structure of membrane protein; Stimulation of sound wave; Signal transduction
1. Introduction The growth of a plant is uncontrollably affected by environmental factors including mechanical stress. So physicists and biologists are concerned about the relation between stress and growth. People have known for a long time that mechanical stimulation has obvious effects on the growth and development of plants. For example, the creeping plant detects the environment on a route via its contact-sensitive stem, stalk, tentacle and root creeps. The stems of some plants becomes * Corresponding author. Post Doctor of Tsinghua University. E-mail address: [email protected] (H.C. Zhao).
short and thick after being cut [1]. As a special form of alternative stress, sound-wave stimulation has obvious effects on the growth of plants. Many researchers have found that lower strength and frequency sound waves not only disrupt the complete structure of the cell, but also enhance the procedure of metabolism and the permeability and selection of the plasma membrane [2–4]. Thus it has been proved that mechanical stress has visible influence on the growth of plants. However, it is not clear how the plant senses the mechanical stimulation and how the mechanical signal is transferred. The plasma membrane is the outer layer of the cell, so it senses the change of environment conditions first [5]. The membrane protein is the executive of membrane functions, so
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 2 9 4 - 6
30
H.C. Zhao et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 29–32
the study of the effect of mechanical stimulation on the structure and function of membrane protein enables us to learn the response procedure of a plant cell to mechanical stimulation. For this reason, this article focuses on studying the effects of sound wave stimulation on the structure of membrane protein with Circular Dichroism (CD). We expect to have a deeper knowledge on the mechanism of mechanical stimulation on the growth of plants. 2. Materials and methods
2.1. The separation of protoplast of tobacco cell Pour 10 ml newly disposed enzyme solution (1.2% cellulose R-10, 0.2% pectase Y-23, 0.1% BSA and 300 mol/l sorbierite was added, pH 5.5) in a plate 9 cm in diameter. Tear the epidermis on the leaf of tobacco and cut to 0.5× 1 cm2 pieces. Let the back of the leaf face down and spread the enzyme solution on it, store at 25 °C. Keep off the sunlight, pump to vacuum for 3 min, and swirl the culture plate gently, let off the protoplast, filter it via a nylon net. Pour it in a centrifugal tube, add 5 ml 0.8 mol/l solution of mannitol, centrifuge for 3 min under 1000 rpm and collect the suspension protoplast. Pour it to a centrifugal tube with a round bottom, add 2– 3 ml CPW13 drop by drop to wash it, mix evenly and add washing solution of CPW13 until the volume is 10 –12 ml, mix evenly, centrifuge for 1 min at 500 rpm, draw the supernatant at best, and suspend it with the washing solution of CPW13 of about 10 –12 ml. Store at 4 °C in a refrigerator, let it deposit naturally, draw most of the supernatant, quantify it and mix evenly and the density of protoplast solution is about 2× 106/ml.
2.3. Measuring the change of membrane protein structure of the tobacco cell with the CD Utilize the CD (type number CD J-500C) of the biological faculty of Tsinghua University, the key Laboratory of membrane and membrane biological engineering under the state. Get the protoplast solution of experimental groups and control group, dilute it with CPW13 solution to 105/ml, take 1 ml, pour into the species cup and put the species cup in the apparatus. The scanning speed is 200 nm/min, repeat for four times. The scope of wavelength is 200–500 nm, at room temperature. These are the detection conditions for recording the spectrum.
3. Results
3.1. The spectrum of tobacco cell Collect the protoplast stimulated for an hour under the sound wave of 400 Hz, 100 dB and the control, dilute them to 105/ml with washing solution of CPW13, measure the spectrum of CD. The results are shown in Fig. 1. The protoplast affected by the sound wave of 400 Hz, 100 dB has a left-move of its negative peak at 213 and 215 nm on the map. On the other hand, there is an obvious change in the morphology of the peak. We know the change of CD may reflect the secondary structure alteration of the membrane
2.2. The effect of alternati6e stress on the protoplast of tobacco cell The alternative stress field was generated by the sound apparatus in the laboratory. Get the protoplast solution, divide it into 10 groups, stimulate them for an hour, use different strength and frequency of sound wave, respectively. The control need not be disposed.
Fig. 1. The effect of sound stimulation on the secondary structure of membrane protein.
H.C. Zhao et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 29–32 Table 1 The effects of constant strength sound wave with different frequency on the membrane protein structure of the tobacco cell
a-helix b-turn
Control
400 Hz
800 Hz
1200 Hz
41.3 58.6
61.6 38.9
54.6 45.4
40.7 59.3
protein. This means that the sound wave stimulation under some strength and frequency leads to change of the secondary structure of the membrane.
3.2. The effects of constant strength sound wa6e with different frequencies on the membrane protein structure of the tobacco cell Under certain strength of 100 dB, three frequencies of sound wave are selected, 400, 800, and 4000 Hz, respectively. They act on the samples for an hour each. The results are shown in Table 1. We could see that the effect of the sound stimulation of different frequencies on the secondary structure of the membrane protein may vary in degree. The change of secondary structure is most obvious at 400 Hz. There is an increase of a-helix and decrease of b-turn.
3.3. The effects of constant frequency sound wa6e with different strengths on the membrane protein structure of the tobacco cell Under a frequency of 400 Hz, three strengths of sound wave are selected, 90, 100, and 110 dB, respectively. They act on the samples for an hour each. The results are shown in Table 2. The results show that the strength of sound wave affects the membrane protein structure to a Table 2 The effects of constant frequency sound wave with different strength on the membrane protein structure of the tobacco cell
a-helix b-turn
Control
90 dB
100 dB
110 dB
41.3 58.7
56.0 44.0
61.1 38.9
42.6 57.4
31
great extent. In a given range, the effect increases with the increase of the sound wave strength. However, the effect may drop back when the strength is too high. In order to confirm that the CD of the protoplast is the reflection of the secondary structure of the membrane protein, we got the plasma membrane at the same time, and scrutinized its CD. The results show that the CD of protoplast and plasma membrane are basically same at the wavelength range (200– 250 nm) we have secured, and the analysis of the secondary structure is almost accordant. So we are sure that the CD of the protoplast of tobacco cell is the real reflection of the secondary structure of membrane protein.
4. Discussion The research concerning the effect of mechanical stress on the growth of a plant has developed to the molecular level in recent years, and some genes related to the mechanical stress have been found [6]. But how the plant senses the mechanical stimulation, and how the mechanical signal transduced to the inside of a cell are not known clearly. The plasma membrane is the outer layer of the cell. It senses the environment condition changes first. The researches showed that the lipid physical state of plasma membrane is highly sensitive to environment stimulation. The stimulation from the environment can lead to the change of functions of membrane protein [7]. Our experiments showed that the structure of membrane protein undergoes obvious change under the sound stimulation under the frequency of 400 Hz and the strength of 90 dB. We could draw this conclusion from the increase of a-helix, and the decrease of b-turn, which demonstrated that the secondary structure of membrane protein is highly sensitive to the stimulation of sound. The a-helix is stretched into the bilayer of the phospholipid. The hydrophobic bonds are formed between the hydrophobic group of the outside of the a-helix and the alkyl of the hydrophobic region of phospholipid bilayer [8]. The b-turn of the secondary structure is extended on the surface of the membrane and forms hydrophobic and static electron
32
H.C. Zhao et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 29–32
with phospholipids [9]. The interaction of phospholipids and protein molecules and the change of molecular structure affects and regulates the fluidity of the whole membrane. The research we carried out years ago showed that stimulation by sound waves at certain frequencies and strengths enable the increase of the fluidity of the cell membrane [2]. From this we may deduce that the change of the secondary structure of membrane protein may lead to the increase of the fluidity of the plasma membrane.
References [1] P. Larson, Stem formation of young larix as influenced by wind and pruning, For. Sci. 11 (1965) 412 – 424. [2] K.L. Sun, The effects of alternative stress on the thermodymical properties of cultured tobacco cells, Acta Biophysica Sinica 15 (1999) 579 –584 (in Chinese).
[3] Z.W. Shen, The secondary structure changes of plant cell wall proteins aroused by strong sound waves using FT-IR, Acta Photonica Sinica 18 (1999) 600 – 602 (in Chinese). [4] G.Y. Cai, The effects of alternative stress on the membrane protein conformations of tobacco cells by circular dichroism, Acta Photonica Sinica 29 (2000) 289 – 297 (in Chinese). [5] Y.L. Yiu, On the comparative stress physiology, in: Beijing Association of Plant Physiology. In: Advances in plant Physiology and Biochemistry, vol. 15, Science Press, Beijing, 1987, pp. 30 – 35 (in Chinese). [6] J. Broom, Rain-, wind-, and touch-induced expression of calmodulin and calmodilin-related genes in Aabbidopsis, Cell 60 (1990) 357 – 364. [7] Qiu Quan-Sheng, Influence of osmotic stress on Lipid physical states of Plasma Membrane from Wheat Roots, Acta Botanica Sinica 41 (1999) 161 – 165 (in Chinese). [8] J. Elzenge, V. Van, Characterization of ion channels in the plasma membrane of epidermel cells of expanding pea leaves, J. Membr. Biol. 137 (1994) 227 – 235. [9] L.D. Tamara, in: David D. Rodert (Ed.), Glycosyl Phosphatidylinositol-Linked Membrane Protein Structure, Biosynthesis and Function, vol. 37, Academic press, 1994, pp. 236– 240.