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Changes in the surface charge of cells induced by electrical pulses
127
Bioelectrochemistry and Bioenergetics, 22 (1989) 127 -133 A section of J. Electroanal. Chem., and constituting Vol. 276 (1989) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
Changes in the surface charge of cells induced by electrical pulses * T.Ch. Tomov and I.Ch. Tsoneva Central Laboratory of Biophysics, Burg. Acad. Sci., G. Bonchev str. bl. 21, Sofia-1113 (Bulgaria) (Received 4 March 1989; in revised form 12 June 1989)
ABSTRACT The effect of electrical pulses with exponential shape on the adsorbing capacity of the cell surface of yeasts was studied experimentally. Pulses with amplitudes of l-l.6 kV/cm are shown to potentiate the adsorption of 9-aminoacridine (cationic dye) and of methylated serum albumin (polycation), reducing the adsorption of unmethylated serum albumin (with predominance of negative charges). The effects last for no less than 10 min. The change in the adsorption capacity is assumed to be due to a change in the surface charge of the cell membranes. The applicability of indicators having different molar mass for studying the surface charge when there is electroporation, is discussed.
INTRODUCTION
When an electric field is applied to a cell suspension, a transmembrane potential A@, is induced across the cell membrane. When the membrane resistance is much higher than the resistance of the medium and of the cytoplasm, according to Laplace’s formula A+,,,= 1 SEr cos cp where E is the field intensity, r is the cell radius and cp is the angle between the field vector and the membrane surface element. In accordance with the theory, the induction of A@,, has been demonstrated experimentally by Gross et al. [l]. Laplace’s transmembrane potential exists only during the inducing pulse (i.e. when E # 0), though under certain conditions (pulse amplitude and duration, composition of the medium, etc.), it can induce longer-lasting effects-ion transport l Presented at the IVth International A. Frumkin Symposium “Bioelectrochemistry row”, Suzdal, U.S.S.R., 24-28 October 1988.
(1302-4598/89/$03.50
0 1989 Elsevier Sequoia S.A.
Today and Tomor-
128
[2], reversible or irreversible poration [3], appearance of an electrically induced long-lived membrane potential [4,5], electrofusion [6,7] and electrotransfection [8]. The effectiveness of the fusion and transfection depends on the surface charge (on the electrostatic repulsion) of the membranes. On the other hand, there exists a correlation between the surface charge and the transmembrane potential [9,10]. It may be expected that electrical pulses inducing fusion of the cells and a long-lived membrane potential also induce changes in the surface charge of the cell membrane. The aim of the present work is to seek experimentally the influence of exogenous electrical pulses on the surface charge of free cells. The experimental object (yeasts) was chosen because: (1) the electrically induced membrane potential has been studied in yeasts [4,5]; (2) the yeasts (intact or in the form of protoplasts) are frequently used as objects for electrofusion [ll-141; (3) yeast suspensions are convenient for work from a methodological point of view.
MATERIAL AND METHODS
The yeast Saccharomyces cerevisae AH215, in an exponential growth phase, was investigated. The yeast was cultivated in YEP medium (1% yeast extract, 2% peptone, 2% glucose). Prior to the experiment the cells were washed thrice in a medium with low ionic strength: 250 mM sucrose, 1 mM Tris+ HCL, pH = 7.4. Bovine serum albumin (BSA) was labelled with 1311 by the method described in ref. 15. To 20 mg BSA dissolved in 1 ml phosphate buffer (20 mM, pH = 7.5) we added consecutively with stirring 10 MB9 K13*I without carrier, 0.05 ml 10% chloramine-T solution (oxidation of iodine and iodination of tyrosine groupes), 0.1 ml 10% sodium methabisulphite (reduction of the unreacted iodine) and 0.05 ml 1 M KI (desorption of the adsorbed unreacted 13rI). The radioisotope-labelled albumin (IBSA) was isolated from the reaction mixture by means of column gel chromatography on Sephadex G 25 in a medium of 20 mM Tris+ HCL, pH = 7.5. Methylated IBSA (IMBSA) was obtained by applying the method described in refs. 16 and 17 with modifications: 2 ml ether was added to 5 mg IBSA dissolved in 0.5 ml 20 mM Tris, pH = 7.5. The protein precipitate was sedimented by centrifugation for 5 min at 5000 g. The supematant was discarded and the pellet was washed thrice with methanol, after which it was resuspended in 3 ml methanol; 0.01 ml 10 M HCl was added and the suspension was stored at room temperature. Three days later the precipitate (i.e. IMBSA) was washed thrice with methanol and was dissolved in 0.5 ml 10 mM Tris+ HCl, pH = 7.6. A yeast suspension with a concentration of approximately lo8 cells/ml in a basic medium with composition 250 mM sucrose and 1 mM Tris, pH = 7.4, was subjected to electrical pulses with exponential shape (capacitor discharge). For this purpose the suspension was poured into a 1 X 1 cm glass fluorimetric cuvette, in which platinum foil electrodes were placed on two opposite walls. The electrodes were connected to the device shown in Fig. 1. The switch was used to induce capacitor discharge through the suspension in the cuvette. Since the resistance of the cuvette
129
Fig. 1. Experimental
setup. (A) Cuvette with platinum electrodes.
with the suspension was always 7 k0 and the capacitance of the discharge capacitor was 7 pF, the time constant for all pulses applied was 50 ms. The electrically induced changes in the surface charge of the cells were registered using the adsorption of electrically loaded indicators-9aminoacridine (9-AA) fluorescent cationic dye and two types of radioactively iodinated albumin: IBSA (containing predominantly negative charges) and IMBSA (polycation). During the fluorescent measurements 9-AA was added to the suspension in a final concentration of 5 PM, the cuvette with the electrodes was placed in a Perkin-Elmer Model 450 spectrophotometer with a fluorescent accessory, and the fluorescence was recorded at X,,, = 400 nm and X,, = 450 nm. Since the adsorbed dye is not fluorescent, the pulse-induced change in the fluorescence is a measure for the change of the adsorption of 9-AA and consequently a measure of the changes in the surface charge of the cells. In other experimental series IBSA or IMBSA Was added to the suspension. After the electrical pulse, aliquots of the suspension were centrifuged at 5000 g for 5 min and the radioactivities of the suspension and of the supernatant were determined using a liquid scintillation counter. The decrease in the radioactivity of the supernatant, compared with the activity of the suspension, is a criterion for the amount of adsorbed radioactive protein. All experiments were performed at room temperature. The viability of the yeasts after the application of the electrical pulses was determined on YEP-medium containing 3% agar and counting of the colonies.
RESULTS
AND
DISCUSSION
Figure 2 shows recordings of the fluorescence of a yeast suspension containing 9-AA after application of exponential pulses with a time constant of 50 ms and with different amplitudes. It can be seen that the pulse induces a decrease in the fluorescence, which develops for about 1 min and is long-lived. 9-AA is known to fluoresce when it is in solution, whereas adsorption of the dye results in quenching of the fluorescence. Since 9-AA is a cationic dye and does not pass through intact membranes, its fluorescence is used as a criterion for a surface charge [18,19]. In yeast suspensions, part of the dye is also adsorbed on the negative charges of the cell wall. The initial fluorescence (prior to the application of the pulse) is proportional to the amount of unadsorbed 9-M. The application of the pulse is followed by a decrease in the fluorescence, i.e. by an increase in the adsorption of the dye. The increased adsorption could also be due to an increase in the negative charges on the cell wall, to a higher surface charge of
130
100 v1
2 3
A3 8 ,' " %
h 3IL 2
h,
k
1000 Vlcm 1200Vlcm
i400vw
50
1600Vlcm
Pmin
time
Fig. 2. Changes in the fluorescence of 9-aminoacridine under the influence of pulses with a time constant of 50 ms and different amplitudes.
the membrane and to penetration of the dye into the cytoplasm and its adsorption on the intracellular components. The first possibility can be rejected. The cell wall is permeable to ions and its electrical resistance is approximately equal to the resistance of the medium. The intensity of the applied electrical field is the same as the average intensity for the entire suspension, i.e. l-l.6 kV/cm. It is highly unlikely that such a weak field induced a change in the number of electrical charges on the cell wall. The resistance of the membrane is much higher than that of the medium and of the cytoplasm. According to Laplace’s formula, if the cell radius is 2.5 pm, a pulse with an amplitude of 1 kV/cm at cos $I = 1 induces a transmembrane potential of 0.4 V. This voltage is applied to a membrane which is about 7 nm thick; therefore the field intensity in the membrane is about 500 kV/cm. Such a field causes poration of the membrane [4,5], resulting in penetration of the dye into the cytoplasm and its adsorption there, but it could cause other changes in the membrane as well, including a change in the surface charge. 9-AA is not suitable as an indicator for differentiating between these two possibilities. An indicator is needed for which the membrane remains impermeable even after the pulse. The pulses applied do not change the cell viability perceptibly (the experimental results are not demonstrated). Although porated, the yeasts do not lose essential cytoplasmic components such as enzymes or nucleic acids. Under experimental conditions similar to ours (pulses with an amplitude of 2.5 kV/cm and a time constant of 40 ms), Hashimoto et al. [20] obtained transfection with a frequency around 10e7. It follows from these results that single plasmids enter only single cells under these conditions. This suggests that sufficiently large protein molecules, such as e.g. bovine serum albumin with a molar mass of about 70 kD, do not pass through the electroporated cell membrane. The adsorption of two modifications of bovine serum albumin on yeast cells is demonstrated in Fig. 3. Prior to the pulse (at zero amplitude) IMBSA is adsorbed
131
I
14
I
500 Pulse
amplitude
I,
4
/V
1
1000
a,
J
1
cm-’
Fig. 3. Dependence of the amount of bound albumin on the pulse amplitude. values from 3-5 experiments. (- - -) Unmethylated albumin (IBSA); ( -) (IMBSA).
Data points are mean methylated albumin
more than IBSA. After a pulse the adsorption of IMBSA increases, while that of IBSA decreases. As in the case of 9-AA, the effects increase with the amplitude. At neutral pH values, there are both positive and negative charges in the IBSA molecule, with predominance of the negative charges. The adsorption of the summarily negatively charged protein molecules on the negatively charged cells is the result of the difference between the non-electrical attraction and electrical repulsion. The decrease in adsorption after a pulse suggests increased electrical repulsion and increased negative charge of the cells. IMBSA differs from IBSA only because all acid groups in it are methylated. The molecule is polycationic. The adsorption is due to unidirectional electrical and non-electrical attraction. The observed rise in the adsorption after a pulse confirms that an increase occurs in the negatively charged binding sites on the cell. The electrical pulse acts predominantly on the cell membrane and it is most probably there that the new binding sites are formed. Although the cell wall is impermeable to the protein molecules and separates them from the membrane, it is not an absolute obstacle to their adsorption. When the ionic concentration in the medium is 1 mM, the Debye length of 30 mn [21] is approximately equal to the thickness of the cell wall [22-241. This means that the electrical interaction is weakened about three times through the cell wall, but it is not eliminated. It leads to adsorption on the cell wall, induced by the surface charge of the membrane. There is a difference between the mechanisms of change of the adsorption of 9-AA and of IMBSA and IBSA. The electrical pulse induces a change in the surface charge and poration of the membrane. The fluorescence of 9-AA is quenched after a pulse because part of the dye is adsorbed on the intracellular sites that have become
132
accessible, whereas another part is adsorbed directly on the newly-formed negative charges of the membrane. The change in the adsorption of albumin is due only to a change in the surface charge of the membrane, which, however, acts at a distance through the cell wall. Although it is not possible to perform a quantitative comparison of the results obtained with different indicators, nevertheless all results confirm qualitatively the increase in the negative surface charge of the membrane after a pulse. The adsorption of 9-AA after a pulse increases quickly and preserves its high value for a long time (Fig. 2). The method does not permit a similar recording of the dynamics of adsorption of the protein indicators. Their adsorption is recorded after centrifugation of the suspension, between 5 and 10 min after the pulse. The results show that after that time the adsorption is still changed (Fig. 3). Hence it follows that the electrically induced change of the surface charge of the cell membranes is long-lived and that the effect is irreversible or at least slowly reversible. For a quantitative determination of the surface charge it is necessary to know the charge of the indicator molecules which are adsorbed and neutralize it. This is not possible with the methods used by us. The charge of the 9-AA ion is known, but-as we have shown-its adsorption does not depend on the surface charge only. The adsorption of IMBSA and IBSA depends both on the number of charges of the macromolecules (which is not known) and on steric factors. The experimental results presented do not provide information about the mechanism of electrical induction of a surface charge. Comparison with the results in refs. 4 and 5 shows that the electrical pulses cause parallel changes developing both in the transmembrane potential and in the membrane charge. One possible explanation is that the electrically induced transmembrane potential causes polarization of the cytoplasmic membrane, resulting in the appearance of additional electronegative groups on the outer side of the membrane. ACKNOWLEDGEMENT
This work was supported by the Bulgarian Academy of Sciences. REFERENCES 1 2 3 4 5 6 7 8
D. Gross, L.M. Loew and W.W. Webb, Biophys. J., 50 (1986) 339. K. Schwister and B. Deuticke, Biochim. Biophys. Acta, 816 (1985) 332. E.H. Serpersu, K. Kinosita and T.Y. Tsong, Biochim. Biophys. Acta, 812 (1985) 779. T. Tomov and I. Tsoneva, Bioelectrochem. Bioenerg., 19 (1988) 397. T. Tomov and I. Tsoneva, C.R. Acad. Bulg. Sci., 11 (1988) 127. U. Zimmermann, Biochim. Biophys. Acta, 694 (1982) 227. H. Berg, Stud. Biophys., 119 (1987) 17. E. Neumann and E. Boldt in H. Tschesche (Ed.), Modem Methods in Protein Chemistry, Vol. 3, Walter de Gruyter, Berlin, New York, 1988, p. 359. 9 K. Redmann, Stud. Biophys., 74 (1978) 21. 10 I. Tsoneva and T. Tomov, Bioelectrcchem. Bioenerg., 12 (1984) 253. 11 H. Weber, W. Farster, H. Berg and H.E. Jacob, Curr. Genet., 4 (1981) 165.
133 12 13 14 15 16 17 18 19 20 21 22 23 24
R. S&nettler, U. Zimmermann and C.C. Emeis, FEMS Microbial. Lett., 24 (1984) 81. R. S&nettler and U. Zimmermann. FEMS Microbial. Lett., 27 (1985) 195. I. Tsoneva, P. Doinov and D.S. Dimitrov, FEMS Microbial. Lett., 60 (1989) 61. F.C. Greenwood, W.M. Hunter and J.G. Glover, Biochem. J., 89 (1963) 114. J.D. Mandel and A.D. Hershey, Anal. Biochem., 1 (1960) 66. N. Sueoka and T.J. Cheng, J. Mol. Biol., 4 (1962) 161. G.F.W. Searle, J. Barber and J.D. Mills, B&him. Biophys. Acta, 461 (1977) 413. G.W.H. Bars&Pauwels, B&him. Biophys. Acta, 650 (1981) 88. H. Hasbimoto, H. Morikawa, Y. Yamada and A. Kimura, Appl. Microbial. Biotechnol., 21 (1985) 336. V.S. Markin and Yu.A. Chizmadzhev, Induced Ion Transport, Nauka, Moscow, 1974, p. 17 (in Russian). R.J. Morth, Nature (London), 190 (1961) 1215. M.L. Watson, J. B&hem. Cytol., 4 (1958) 475. E. Streiblova, J. Bacterial., 95 (1968) 700.
Bioelectrochemistry and Bioenergetics, 22 (1989) 127 -133 A section of J. Electroanal. Chem., and constituting Vol. 276 (1989) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
Changes in the surface charge of cells induced by electrical pulses * T.Ch. Tomov and I.Ch. Tsoneva Central Laboratory of Biophysics, Burg. Acad. Sci., G. Bonchev str. bl. 21, Sofia-1113 (Bulgaria) (Received 4 March 1989; in revised form 12 June 1989)
ABSTRACT The effect of electrical pulses with exponential shape on the adsorbing capacity of the cell surface of yeasts was studied experimentally. Pulses with amplitudes of l-l.6 kV/cm are shown to potentiate the adsorption of 9-aminoacridine (cationic dye) and of methylated serum albumin (polycation), reducing the adsorption of unmethylated serum albumin (with predominance of negative charges). The effects last for no less than 10 min. The change in the adsorption capacity is assumed to be due to a change in the surface charge of the cell membranes. The applicability of indicators having different molar mass for studying the surface charge when there is electroporation, is discussed.
INTRODUCTION
When an electric field is applied to a cell suspension, a transmembrane potential A@, is induced across the cell membrane. When the membrane resistance is much higher than the resistance of the medium and of the cytoplasm, according to Laplace’s formula A+,,,= 1 SEr cos cp where E is the field intensity, r is the cell radius and cp is the angle between the field vector and the membrane surface element. In accordance with the theory, the induction of A@,, has been demonstrated experimentally by Gross et al. [l]. Laplace’s transmembrane potential exists only during the inducing pulse (i.e. when E # 0), though under certain conditions (pulse amplitude and duration, composition of the medium, etc.), it can induce longer-lasting effects-ion transport l Presented at the IVth International A. Frumkin Symposium “Bioelectrochemistry row”, Suzdal, U.S.S.R., 24-28 October 1988.
(1302-4598/89/$03.50
0 1989 Elsevier Sequoia S.A.
Today and Tomor-
128
[2], reversible or irreversible poration [3], appearance of an electrically induced long-lived membrane potential [4,5], electrofusion [6,7] and electrotransfection [8]. The effectiveness of the fusion and transfection depends on the surface charge (on the electrostatic repulsion) of the membranes. On the other hand, there exists a correlation between the surface charge and the transmembrane potential [9,10]. It may be expected that electrical pulses inducing fusion of the cells and a long-lived membrane potential also induce changes in the surface charge of the cell membrane. The aim of the present work is to seek experimentally the influence of exogenous electrical pulses on the surface charge of free cells. The experimental object (yeasts) was chosen because: (1) the electrically induced membrane potential has been studied in yeasts [4,5]; (2) the yeasts (intact or in the form of protoplasts) are frequently used as objects for electrofusion [ll-141; (3) yeast suspensions are convenient for work from a methodological point of view.
MATERIAL AND METHODS
The yeast Saccharomyces cerevisae AH215, in an exponential growth phase, was investigated. The yeast was cultivated in YEP medium (1% yeast extract, 2% peptone, 2% glucose). Prior to the experiment the cells were washed thrice in a medium with low ionic strength: 250 mM sucrose, 1 mM Tris+ HCL, pH = 7.4. Bovine serum albumin (BSA) was labelled with 1311 by the method described in ref. 15. To 20 mg BSA dissolved in 1 ml phosphate buffer (20 mM, pH = 7.5) we added consecutively with stirring 10 MB9 K13*I without carrier, 0.05 ml 10% chloramine-T solution (oxidation of iodine and iodination of tyrosine groupes), 0.1 ml 10% sodium methabisulphite (reduction of the unreacted iodine) and 0.05 ml 1 M KI (desorption of the adsorbed unreacted 13rI). The radioisotope-labelled albumin (IBSA) was isolated from the reaction mixture by means of column gel chromatography on Sephadex G 25 in a medium of 20 mM Tris+ HCL, pH = 7.5. Methylated IBSA (IMBSA) was obtained by applying the method described in refs. 16 and 17 with modifications: 2 ml ether was added to 5 mg IBSA dissolved in 0.5 ml 20 mM Tris, pH = 7.5. The protein precipitate was sedimented by centrifugation for 5 min at 5000 g. The supematant was discarded and the pellet was washed thrice with methanol, after which it was resuspended in 3 ml methanol; 0.01 ml 10 M HCl was added and the suspension was stored at room temperature. Three days later the precipitate (i.e. IMBSA) was washed thrice with methanol and was dissolved in 0.5 ml 10 mM Tris+ HCl, pH = 7.6. A yeast suspension with a concentration of approximately lo8 cells/ml in a basic medium with composition 250 mM sucrose and 1 mM Tris, pH = 7.4, was subjected to electrical pulses with exponential shape (capacitor discharge). For this purpose the suspension was poured into a 1 X 1 cm glass fluorimetric cuvette, in which platinum foil electrodes were placed on two opposite walls. The electrodes were connected to the device shown in Fig. 1. The switch was used to induce capacitor discharge through the suspension in the cuvette. Since the resistance of the cuvette
129
Fig. 1. Experimental
setup. (A) Cuvette with platinum electrodes.
with the suspension was always 7 k0 and the capacitance of the discharge capacitor was 7 pF, the time constant for all pulses applied was 50 ms. The electrically induced changes in the surface charge of the cells were registered using the adsorption of electrically loaded indicators-9aminoacridine (9-AA) fluorescent cationic dye and two types of radioactively iodinated albumin: IBSA (containing predominantly negative charges) and IMBSA (polycation). During the fluorescent measurements 9-AA was added to the suspension in a final concentration of 5 PM, the cuvette with the electrodes was placed in a Perkin-Elmer Model 450 spectrophotometer with a fluorescent accessory, and the fluorescence was recorded at X,,, = 400 nm and X,, = 450 nm. Since the adsorbed dye is not fluorescent, the pulse-induced change in the fluorescence is a measure for the change of the adsorption of 9-AA and consequently a measure of the changes in the surface charge of the cells. In other experimental series IBSA or IMBSA Was added to the suspension. After the electrical pulse, aliquots of the suspension were centrifuged at 5000 g for 5 min and the radioactivities of the suspension and of the supernatant were determined using a liquid scintillation counter. The decrease in the radioactivity of the supernatant, compared with the activity of the suspension, is a criterion for the amount of adsorbed radioactive protein. All experiments were performed at room temperature. The viability of the yeasts after the application of the electrical pulses was determined on YEP-medium containing 3% agar and counting of the colonies.
RESULTS
AND
DISCUSSION
Figure 2 shows recordings of the fluorescence of a yeast suspension containing 9-AA after application of exponential pulses with a time constant of 50 ms and with different amplitudes. It can be seen that the pulse induces a decrease in the fluorescence, which develops for about 1 min and is long-lived. 9-AA is known to fluoresce when it is in solution, whereas adsorption of the dye results in quenching of the fluorescence. Since 9-AA is a cationic dye and does not pass through intact membranes, its fluorescence is used as a criterion for a surface charge [18,19]. In yeast suspensions, part of the dye is also adsorbed on the negative charges of the cell wall. The initial fluorescence (prior to the application of the pulse) is proportional to the amount of unadsorbed 9-M. The application of the pulse is followed by a decrease in the fluorescence, i.e. by an increase in the adsorption of the dye. The increased adsorption could also be due to an increase in the negative charges on the cell wall, to a higher surface charge of
130
100 v1
2 3
A3 8 ,' " %
h 3IL 2
h,
k
1000 Vlcm 1200Vlcm
i400vw
50
1600Vlcm
Pmin
time
Fig. 2. Changes in the fluorescence of 9-aminoacridine under the influence of pulses with a time constant of 50 ms and different amplitudes.
the membrane and to penetration of the dye into the cytoplasm and its adsorption on the intracellular components. The first possibility can be rejected. The cell wall is permeable to ions and its electrical resistance is approximately equal to the resistance of the medium. The intensity of the applied electrical field is the same as the average intensity for the entire suspension, i.e. l-l.6 kV/cm. It is highly unlikely that such a weak field induced a change in the number of electrical charges on the cell wall. The resistance of the membrane is much higher than that of the medium and of the cytoplasm. According to Laplace’s formula, if the cell radius is 2.5 pm, a pulse with an amplitude of 1 kV/cm at cos $I = 1 induces a transmembrane potential of 0.4 V. This voltage is applied to a membrane which is about 7 nm thick; therefore the field intensity in the membrane is about 500 kV/cm. Such a field causes poration of the membrane [4,5], resulting in penetration of the dye into the cytoplasm and its adsorption there, but it could cause other changes in the membrane as well, including a change in the surface charge. 9-AA is not suitable as an indicator for differentiating between these two possibilities. An indicator is needed for which the membrane remains impermeable even after the pulse. The pulses applied do not change the cell viability perceptibly (the experimental results are not demonstrated). Although porated, the yeasts do not lose essential cytoplasmic components such as enzymes or nucleic acids. Under experimental conditions similar to ours (pulses with an amplitude of 2.5 kV/cm and a time constant of 40 ms), Hashimoto et al. [20] obtained transfection with a frequency around 10e7. It follows from these results that single plasmids enter only single cells under these conditions. This suggests that sufficiently large protein molecules, such as e.g. bovine serum albumin with a molar mass of about 70 kD, do not pass through the electroporated cell membrane. The adsorption of two modifications of bovine serum albumin on yeast cells is demonstrated in Fig. 3. Prior to the pulse (at zero amplitude) IMBSA is adsorbed
131
I
14
I
500 Pulse
amplitude
I,
4
/V
1
1000
a,
J
1
cm-’
Fig. 3. Dependence of the amount of bound albumin on the pulse amplitude. values from 3-5 experiments. (- - -) Unmethylated albumin (IBSA); ( -) (IMBSA).
Data points are mean methylated albumin
more than IBSA. After a pulse the adsorption of IMBSA increases, while that of IBSA decreases. As in the case of 9-AA, the effects increase with the amplitude. At neutral pH values, there are both positive and negative charges in the IBSA molecule, with predominance of the negative charges. The adsorption of the summarily negatively charged protein molecules on the negatively charged cells is the result of the difference between the non-electrical attraction and electrical repulsion. The decrease in adsorption after a pulse suggests increased electrical repulsion and increased negative charge of the cells. IMBSA differs from IBSA only because all acid groups in it are methylated. The molecule is polycationic. The adsorption is due to unidirectional electrical and non-electrical attraction. The observed rise in the adsorption after a pulse confirms that an increase occurs in the negatively charged binding sites on the cell. The electrical pulse acts predominantly on the cell membrane and it is most probably there that the new binding sites are formed. Although the cell wall is impermeable to the protein molecules and separates them from the membrane, it is not an absolute obstacle to their adsorption. When the ionic concentration in the medium is 1 mM, the Debye length of 30 mn [21] is approximately equal to the thickness of the cell wall [22-241. This means that the electrical interaction is weakened about three times through the cell wall, but it is not eliminated. It leads to adsorption on the cell wall, induced by the surface charge of the membrane. There is a difference between the mechanisms of change of the adsorption of 9-AA and of IMBSA and IBSA. The electrical pulse induces a change in the surface charge and poration of the membrane. The fluorescence of 9-AA is quenched after a pulse because part of the dye is adsorbed on the intracellular sites that have become
132
accessible, whereas another part is adsorbed directly on the newly-formed negative charges of the membrane. The change in the adsorption of albumin is due only to a change in the surface charge of the membrane, which, however, acts at a distance through the cell wall. Although it is not possible to perform a quantitative comparison of the results obtained with different indicators, nevertheless all results confirm qualitatively the increase in the negative surface charge of the membrane after a pulse. The adsorption of 9-AA after a pulse increases quickly and preserves its high value for a long time (Fig. 2). The method does not permit a similar recording of the dynamics of adsorption of the protein indicators. Their adsorption is recorded after centrifugation of the suspension, between 5 and 10 min after the pulse. The results show that after that time the adsorption is still changed (Fig. 3). Hence it follows that the electrically induced change of the surface charge of the cell membranes is long-lived and that the effect is irreversible or at least slowly reversible. For a quantitative determination of the surface charge it is necessary to know the charge of the indicator molecules which are adsorbed and neutralize it. This is not possible with the methods used by us. The charge of the 9-AA ion is known, but-as we have shown-its adsorption does not depend on the surface charge only. The adsorption of IMBSA and IBSA depends both on the number of charges of the macromolecules (which is not known) and on steric factors. The experimental results presented do not provide information about the mechanism of electrical induction of a surface charge. Comparison with the results in refs. 4 and 5 shows that the electrical pulses cause parallel changes developing both in the transmembrane potential and in the membrane charge. One possible explanation is that the electrically induced transmembrane potential causes polarization of the cytoplasmic membrane, resulting in the appearance of additional electronegative groups on the outer side of the membrane. ACKNOWLEDGEMENT
This work was supported by the Bulgarian Academy of Sciences. REFERENCES 1 2 3 4 5 6 7 8
D. Gross, L.M. Loew and W.W. Webb, Biophys. J., 50 (1986) 339. K. Schwister and B. Deuticke, Biochim. Biophys. Acta, 816 (1985) 332. E.H. Serpersu, K. Kinosita and T.Y. Tsong, Biochim. Biophys. Acta, 812 (1985) 779. T. Tomov and I. Tsoneva, Bioelectrochem. Bioenerg., 19 (1988) 397. T. Tomov and I. Tsoneva, C.R. Acad. Bulg. Sci., 11 (1988) 127. U. Zimmermann, Biochim. Biophys. Acta, 694 (1982) 227. H. Berg, Stud. Biophys., 119 (1987) 17. E. Neumann and E. Boldt in H. Tschesche (Ed.), Modem Methods in Protein Chemistry, Vol. 3, Walter de Gruyter, Berlin, New York, 1988, p. 359. 9 K. Redmann, Stud. Biophys., 74 (1978) 21. 10 I. Tsoneva and T. Tomov, Bioelectrcchem. Bioenerg., 12 (1984) 253. 11 H. Weber, W. Farster, H. Berg and H.E. Jacob, Curr. Genet., 4 (1981) 165.
133 12 13 14 15 16 17 18 19 20 21 22 23 24
R. S&nettler, U. Zimmermann and C.C. Emeis, FEMS Microbial. Lett., 24 (1984) 81. R. S&nettler and U. Zimmermann. FEMS Microbial. Lett., 27 (1985) 195. I. Tsoneva, P. Doinov and D.S. Dimitrov, FEMS Microbial. Lett., 60 (1989) 61. F.C. Greenwood, W.M. Hunter and J.G. Glover, Biochem. J., 89 (1963) 114. J.D. Mandel and A.D. Hershey, Anal. Biochem., 1 (1960) 66. N. Sueoka and T.J. Cheng, J. Mol. Biol., 4 (1962) 161. G.F.W. Searle, J. Barber and J.D. Mills, B&him. Biophys. Acta, 461 (1977) 413. G.W.H. Bars&Pauwels, B&him. Biophys. Acta, 650 (1981) 88. H. Hasbimoto, H. Morikawa, Y. Yamada and A. Kimura, Appl. Microbial. Biotechnol., 21 (1985) 336. V.S. Markin and Yu.A. Chizmadzhev, Induced Ion Transport, Nauka, Moscow, 1974, p. 17 (in Russian). R.J. Morth, Nature (London), 190 (1961) 1215. M.L. Watson, J. B&hem. Cytol., 4 (1958) 475. E. Streiblova, J. Bacterial., 95 (1968) 700.