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Temperature dependence of electric field effects on Na,K-ATPase
291
Bioelectrociremistry qtld Bioenergehzs, 28 (1992) 29 l-299 A section of J. Elecrroanal. C/tern., and canstituting Vol. 343 (1992) Elsevier Sequoia S.A., Lausanne
JEC
BB 01519
Temperature d.ependence ‘on Na,K-ATPase *
of electric field effects
iviartir~ ISlant and Lily Soo Department of Pi~ysiology and Cehlar NY 10032 (USA) (Received
6 November
Biophysics,
Columbia
Uniwrsity,
630 West 168th Street, New York,
1991)
Abstract We have shown that the effect of alternating currents (a.~.) on Na,K-ATPase varies with temperature, and that the observations can be explained by assuming a simple theoretical model of changes in the ion binding to the activation sites due to an imposed alternating electric field. Previous observations showing that the effect of an electric field on the enzyme can lead to either an enhancement or a decrease in enzyme activity, depending upon the initial activity, also appears to be true when the enzyme activity is varied by changing the temperature. This suggests very strongly that the mechanism of action of a-c. is the result cf changing in ion binding and not changes in lipid phase structure. INTRODUCTION
Recent experiments on the effects of alternating currents (a.c.) on ATP-splitting by the membrane Na,K-ATPase [l-4] suggest that the mechanism involves changes in the activation of the ion-binding sites by the electric field. The model based on changes in ion activation (CIA) differs from the electroconformational coupling (ECC) model proposed on the basis of earlier studies of ion transport processes involving Na,K-ATPase [5-73. The ECC model suggests that there is a direct action of the electric field on the enzyme in the membrane (e.g. field-dipole interactions), leading to a change in its conformation, while the CIA model can be effective with interaction at the external binding site only. There is an overlap in the range of a.c. fields used in the two studies, and the work on ATP splitting by Na,ATPase in microsomes and on ion transport by Na,K-ATPase in erythrocytes agree on the observed enhanced ATP splitting and ion transport at low enzyme activity. There is also approximate agreement on the
* Dedicated collaboration
to Professor GiuIio Milazzo in grateful recognition in furthering the development of bioelectrochemistry.
0302-4598/92/$05.00
0
1992 - Elsevier
Sequoia
of our many years
S.A. All rights reserved
of friendship
and
292
lo2 Hz for ATP splitting and 10’ Hz for Rb observed optimal frequencies: accumulation_ The most important difference is the observed broad frequency optimum at IO” Hz for sodium efflux from erythrocytes, while the ATP splitting indicates no such effect in the range approaching this frequency. The two ionic fluxes (Rb in and Na out) are physioIogicalIy coupled, and a separation by three orders of magnitude in frequency is unlikely. The differences in observed frequency optima can I.2 explained by differences between intact erythrocytes with Na,K-ATPase molecules all oriented the same way in the membrane, and microsomes that have the Na,K-ATPase oriented in both directions. In addition, it appears that the microsomes are leaky, and ions as well as small molecules like ATP and ouabain have access to the binding sites on both sides of the membrane. In the erythrocyte experiments, K+ ion (and ouabain) binding occur on the outside surface of the cell, and one would expect Rb+ (a K+ analogue) to be transported under the low frequency conditions that affect the outer surface. To affect Na+ efflux, the a-c. signal must penetrate to the Na+ binding site on the inner surface of the membrane, an effect that is appreciable only at higher (megahertz range) frequencies. Another major difference between the two sets of experiments is that the effect of a.c. on ATP splitting by Na,K-ATPase suspensions shows decreases for the enzyme under normal conditions, and increases only when the enzyme activity is lowered to less than half its optimal value by changes in temperature, ouabain concentration and activating ion ratio. Ion transport by the enzyme in a.c. fields has been reported to increase only. The two sets of observations are compatible, since the reported ion transport experiments were done at low temperatures where one would expect enhanced ATP splitting and enhanced ion transport. It would be important to determine whether ion transport is decreased at normal temperatures, and, if so, how the ECC model would explain this. Since temperature appears to be a critical property in the response of this enzyme to a-c. fields, it is important to study the effect of this variable in some detail. In this paper we report on the effect of temperature on ATP-splitting by Na,K-ATPase and try to shed light on the mechanism of the interaction of the enzyme with electric fields. EXPERIMENTAL
Na,K-ATPase-containing vesicles were prepared from frozen rabbit kidneys by modification of the method of Kinsolving et al. 181, as described by Britten and Blank [9]. Details of the method are given in earlier papers by Blank and Soo [1,2]. The microsomal preparations were polydisperse with a range of diameters mainly between 0.1 and 1 pm, and concentrations of (5-7) x 10’. particles/ml. The protein .concentrations (about 1 mg/ml).and the enzymatic properties of different preparations were about the same. ATPase activity was estimated by incubating enzyme and the Na,ATP substrate at various temperatures for 15 min in 1 ml of solution normally containing the
cation
concentrations 81 mM Na’, 16 mM K+ and 5 mM Mg’+. In all cases, the major anion was 61-. Platinum electrodes in plastic tubing filled with salt-impregnated 1.5% agar gels were inserted into U-tubes containing the same amount of enzyme suspension as in the controls. The platinum electrodes wdrc connected to a Hewlett Packard HP 3312A function generator, and a-c. flowed through the U-tube (6-7 mm internal diameter) for 15 min. Sham controls were run for the same period of time. The distance between the pIatinum/agar salt bridge electrodes was kept at 3 cm, and because of the geometry of the system a 1.00 V (peak) output from the function generator was attenuated to 0.12 V (peak) across the enzyme suspension. The a.c. signals covered an amplitude range of 1 mV-1 V and a frequency range of 10 Hz-100 kHz. The current was approximately 0.05-50 mA/cm’ r.m.s. in the four voltage ranges studied. Reactions in both the stimulated and control solutions were terminated with 0.5 ml of 15% (w/v) trichloracetic acid, and the inorganic phosphate was assayed. 8ur measurements are reported as the effect of a.c. on enzyme activity E divided by the normal activity A, ix. the ratio E/A of cxpcrimcnt to control. RESULTS
Under normal conditions cf temperature and optimal ion ratio [1,2], an enzyme preparation shows a decreased ability to split ATP (E/A < 1) with imposed a-c.
1.6 i
I
ld-..- ,
._-c]---_-__---_._-__-
___-__-_._____-__-
I
.._.a...e__.._ 4. -.--.__ 1
.~
-
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--
_..*
-.--..
P
~
,,*j
._.
.
.
_
.
.,
._ .,.-.
_
P iii
1 l:‘--’
. .. .. . ____ _ _ . . 8. .
()*_C-_ ___ _-_.-._-.-.-A. 1 1 o&__ ____ _..__ ___ _. _
I
.._?..m .._.. . .I _ . .* _..I..-_ ,.. =I -mmI ._.______“___
- -a
R e -
._-_-.__ -... I. .I I- ._____._._.
I 0.4 i 0
f i
0.02
0.04
0.06
0.06 ACtiVQ
0.1
0.12
0.14
1 O.;S
Fig. 1. Na.K-ATPase temperature study. The ratio E/A of enzyme activity with a-c. to that without a.c. vs. the enzyme activity in pmol phosphate/mg protein/min. The a.c. frequency was 100 Hz and the peak voltage was 0.01 V. The points represent individual pairs of runs. Above an enzyme activity of 0.06, E/A < 1, while below this value E/A tend to be > 1. The low values of enzyme activity were obtained using the lower temperatures in the range 15-40°C. These results are similar to those obtained in experiments in which the enzyme activity was lowered by introducing 1O-4-lO-” M auabain [2].
294
._._---.-_
__. -.______._.___ _._- ._.._.__I
1 6 #
-
j
I
.._
._.
.
.
I
-.
.___._.__I
I.-._~.
-
..--._
o,~~________.__.
_-.._--.-.__xTz_. I
I
___-__. - -. .__. _ ._.__________._.___; _. ___.__._
o*$*-___
I
*.4! 10
0
20
25
30
35
40
45
TemperaturePC
Fig. 2. Na,K-ATPase temperature study. The ratio E/A of enzyme activity with a.c. to that without a.~. as a function of temperature. 6 was measured under an imposed current (at 100 Hz and IO mV). There appears to be an abrupt change of slope at 25°C where E/A = 1. Standard errors for the individual E and A points are shown in Fig. 3.
This can be seen in Fig. 1 for the data points above an enzyme activity of 0.06 for this preparation, which corresponds to temperatures between 25 and 40°C. The data points represent individual pairs of runs, where A is the activity under normal
I
_
0.14 r 0.1_9;___ _ ._ .-.
o!
10
-
__ . . ._._ .
._ . . .
.._.-..--_-.
.--.-
_ __. -‘z” /J .’ “- .-v --_. _..
I
15
20
25
35
30
Temperature
40
45
A
1°C
Fig. 3. The enzyme activity in pmol phosphate/mg protein/min (IB ) as a function of temperature. On the same temperature scale, we present the enzyme activity in the presence of a.~. at a frequency of 100 Hz and a peak voltage of 0.01 V. (+ 1. Standard error bars are shown except where they are smaller than the data points. At 25”C, the overlapping points have the following S.E.M. values: E = 0.0037; A, 0.0028.
conditions and E is under an imposed current (at 100 Hz and IO mV). At temperatures between 15 and 25”C, enzynle activity is lowered and there is an enhanced activity due to a-c. (E/A > 1). The enhanced effect of the a.c. at low enzyme activity is similar to the data obkined when reducing. the activity by introducing (lo- 4-1O-s M) ouabain 123. Figure 2 is a plot of E/A as a function of temperature. There appears to be an abrupt change of slope at 25°C where E/A = I. At lower temperatures, of a d(E/A)/dT is - 0.062 and above 25°C the slope is - 0.0086. The coincidence sharp change of slope with a change in response to an eIectric field suggests that a lipid phase’change may be involved in the mechanism. Figure 3 is a pIot of the dependence of the errzyme activity on the temperature. The full rectangles show the activity under normal conditions and the plus signs she;Y. the activity under an imposed current (at 100 Hz and 10 mV). Both curves vary smoothly over the range studied and they interseci at 25°C. The individual for the normal conditions and -4.9 x lo-” for the slopes are -3.6 x lo-’ electrically stimulated conditions in units of activity per degree Celsius. DISCUSSION
The effect of a.~.on iorz birtdiug to the Na,K-ATPasr The membrane enzyme Na,K-ATPase responsibk for establishing ion gradients across cell membranes extends throughout the membrane. The catalytic site is on the inside surface, but the enzyme is activated when lC+ ions interact with specific binding sites on the outside surface and Na + ion: interact with binding sites on the inside. When activated, the enzyme catalyzes the coupled transport of three Na+ ions from inside out and two Kf ions from outside in for each ATP split. It is clear that ion concentrations in particular regions are able to affect the activation of the enzyme, and electric fields that affect the ion binding would also change the enzyme activity. To explore the effects of electric fields on Na,K-ATPase, we have developed a simple theoretical model of ion binding at the activation sites on the enzyme surface and derived equations to describe the effects of imposed electric fields. The binding constant K of a cation at an anionic activation site is governed by and the the iocai electrical potential E (frequently calkd the surikce potential), form of the equation is usually written K =!c,
exp( -k,E)
with e the unit charge, k the? Boltzmann where k , is a constant and k, =e/kT, constant and T the absolute temperature. The value of k, at room temperature is about (25 mV)- ‘. If one imposes an external potential IJ, the new value of the binding constant becomes
K” =k,
exp[-k,(E+V)]
(2)
296
the effect of an imposed sine wave, let us assume an alternating To approximate square wave of the same frequency. (This approximation is obviously better at higher frequency.) The value of K, for a full cycle (of duration t) is the average of the plus and minus values in the two half-cycles: K, = f i”“k
! exp[--k,(h+V)ldr+f~~~~~
exp[k,(E-V)]dr I
-exp[--k,(E+V)]
++
exp[-k,(E-V)]
(4)
This is equal to K,=
k, exp( -k,E)
1 1
exp ( -k,V) + exp( k,V) 2
Therefore
(3)
1
K,, = K cosh( k,V)
(5) (6)
Since the cash function is equal to unity when V = 0 and increases for higher values of V, it appears that the imposed a-c. field atways increases on binding. The temperature dependence of both binding constants can be evaluated by taking the derivatives of eqns. (1) and (6): dK = k, exp( -k,E) dT dK -=-dT
dk,E (7)
dT
Ke E k
(8)
T2 d cosh( k,V)
d K” -=K dT
dT = K sinh(k,V)
d K” -=-dT
Ke E
dK, -=dT
dK
k dT
T2
- g
dK + cosh( k,V) dT
w2v/) dT
Ke E + cosh( k,V) k T-
sinh( k,V)
V - E sinh( k,V)
(9)
1 1
(10)
+ cosh( k,V)
+ cosh( k,V)
(12)
For small values of k2V, i.e. for V GX25 mV, and V less than or comparable with E, the value in square brackets is less than unity. Therefore the slope of the temperature curve for the imposed a.c. would be expected to be smaller. In Fig. 3, the ratio of the slopes is 0.73. Equation (12) does not lead to a unique solution but defines a line consisting of pairs of values of V and E for that ratio.
297
E.$fect cf temperature on the Na,K-ATPuse response to U.C. From Fig. 3, we see that the variation of the temperature leads to a smooth change in the enzyme activity which increases almost linearly in the range studied. At higher temperatures, the enzyme starts to inactivate (at shorter times when the enzyme is exposed to higher temperatures), but this is not a factor at the temperatures and durations studied here [9]. When the a.c. field is imposed, the variation of the effect is still smooth, but the slope of the temperature dependence curve decreases, in line with the predictions of the model developed’ above. The cross-over point of the two curves is at 2S”C, which is also the point where the effect of the a.c. field switches from enhancing to inhibiting enzyme activity. The abrupt change of slope shown in Fig. 2 has usually been interpreted as a phase transition. Thermotropic changes in the activity of Na,K-ATPase have been shown to occur in hepatocyte membranes at 26°C [lo], and may very well be linked to the properties of the lipids that form the matrix for the Na,K-ATPase vesicles in our preparation. Furthermore, it is well known that the activity of Na,K-ATPase depends upon the lipids that are present in the vesic!e membranes. Relipidation oC: delipidated enzyme 1113 causes both phosphatase and ATPase activity to return with increasing amounts of phospholipid, and the stoichiometry is different for different lipids, indicating specificity of interaction. These studies also suggest that the enzyme is a functional dimer with K+ ions favoring independence of the This would mean that a.c. fields subunits and Na+ ions favoring interaction. somehow affect the interaction between the subunits, a possibility that is in line with the changes in ion binding predicted by the theoreticai model. This interpretation is not without its problems. The effect on enzyme activity is the same for t.emperature variation as with the other experimental variables studied (e.g. ouabain concentration, Na/K ratio). The effect of *%.c:‘ on enzyme activity appears to va’ry with the activity regardless of how one brings about the change. The thermotropic effect as observed in our system does not seem to be a convincing explanation, despite the abrupt change in Fig. 2. The two lines in Fig. 3 suggest continuous rather than abrupt changes. Also, one would have to postulate changes in lipid organization owing to changes in ouabain concentration or Na/K ratio as well. Effect of frequency Turning again to thz alternating square wave, let us consider the time spent at the plus and minus values of the square wave potential in comparison with the time spent going between them. Since the switch between the two values of potential is not instantaneous, the finite switch time occupies a greater part of the total time as the frequency of the switching increases. (There shoilld also be a dependence on the amplitude of the potential, since the time required to switch should increase with the distance that has to be covered.) Consequently, the effect of a-c. should decrease at higher frequency, as has been observed [1,2].
298
0: -1
0
1
2 kg (Frequency/Hz)
3
4
5
Fig. 4. Standard ertor oi the mean (S.E.M.) as u function of the logarithm of the frequency in hertz. The results a1 different applied voltages arc plotted separately: I 1 mV; A 10 mV; Cl 100 mV; + I V.
We have also observed another effect of frequency. Figure 4 presents a summary of the standard errors of the enzyme activity when measured at different frequencies and amplitudes. The marked increase with frequency suggests that the a.c. is affecting a process that requites a certain length of time to be effective: for example, an ion must be bound for a particuIar period, At higher frequencies the probability that the period will be interrupted increases and leads to a greater variability in the measurements. Eoahat!on of proposed mechanisms
It has been shown that a.c. electric fields cause changes in ion transport and ATP splitting via the Na,K-ATPase, and that the effect on ATP splitting depends upon the level of enzyme activity_ Therefore any proposed mechanism must accoun5. for both observed dependences: that on the electric field and that on the level of enzyme activity. The CIA model appears to account for the basic observations. The effect of electric field and enzyme activity are mechanistically linked through changes in ion binding at the activation sites. The ECC model does not appear to have addressed the problem of the variation of the effect of the electric field with the degree of ion activation. The thermal effects described here add an additional constraint to the understanding of the mechanism. It appears possible to expIain the new observations in terms of. changes in ion binding at the activation sites, using the expressions derived for the binding constants and their temperature coefficients, under both normal conditions and a.c. The ECC model can probably accommodate these new data by choosing new values of the eight rate constants that can be set, but this kind of manipuIation wil1 not be very convincing until physio-chemical reasons can
be given for the choices. To resolve the problem arising from the difference between the optimal frequencies for cation influx (lo3 Hz) and efflux (lo6 Hz), the ECC model uses values for the adjustable parameters without any indication of their physical meaning [X2]. The CIA model shows that changes in the ionic concentrations across the cell membrane, which lead to changes in ion binding and enzyme activation, vary with the frequency and amplitude of the imposed a.c. signal, and that the optimal frequency is approximately 10’ Hz. The observed difference between influx and efflux depends on the two differem sois or”binding sites invoIved. ACKNOWLEDGEMENTS
We thank the Office of Naval Research search Institute (EPRI) for their support.
(ONR)
and the Electric
Power Re-
REFERENCES 1 2 3 4 5 6 7 8 9 10
M. Blank and L. Soo, Bioelectrochem. Bioenerg., 22 (1989) 313. M. Blank and L. Soo, Bioelectrochem. Bioenerg., 24 (1990) 51. M. Btank and L. Soo, Bioelectromagnetics, in press. M. Blank, FASEB J., 6 (1992) 2734. E.H. Serpersu and T.Y. Tsong, J. Biol. Chem., 259 (1984) 7155. T.Y. Tsong and R.D. Astumian, Bioelectrochem. Bioenerg., 15 (1986) 457. D.S. Liu, R-D. Astumian and T.Y. Tsong, J. Biol. Chem., 265 (1990) 7260. CR. Kinsolving, R.L. Post and D.L. Beaver, J. Cell. Comp. Physiol., 62 (1963) 85. J.S. Britten and M. Blank, Biochem. Biophys. Acta, 159 (2968) 150. D. Schachter, in Watts and De Pont (Eds.). Progress in Protein-Lipid Interactions, Amsterdam, 1985, Chapter 6. I I P. Ottolenghi, Eur. J. Biochem., 99 (1979) 113. 12 B. Robertson and R.D. Astumian, J. Chem. Phys., 94 (1991) 7414. 13 M. Blank, J. Electrochem. Sot., 134 !I9871 1112.
Elsevier,
Bioelectrociremistry qtld Bioenergehzs, 28 (1992) 29 l-299 A section of J. Elecrroanal. C/tern., and canstituting Vol. 343 (1992) Elsevier Sequoia S.A., Lausanne
JEC
BB 01519
Temperature d.ependence ‘on Na,K-ATPase *
of electric field effects
iviartir~ ISlant and Lily Soo Department of Pi~ysiology and Cehlar NY 10032 (USA) (Received
6 November
Biophysics,
Columbia
Uniwrsity,
630 West 168th Street, New York,
1991)
Abstract We have shown that the effect of alternating currents (a.~.) on Na,K-ATPase varies with temperature, and that the observations can be explained by assuming a simple theoretical model of changes in the ion binding to the activation sites due to an imposed alternating electric field. Previous observations showing that the effect of an electric field on the enzyme can lead to either an enhancement or a decrease in enzyme activity, depending upon the initial activity, also appears to be true when the enzyme activity is varied by changing the temperature. This suggests very strongly that the mechanism of action of a-c. is the result cf changing in ion binding and not changes in lipid phase structure. INTRODUCTION
Recent experiments on the effects of alternating currents (a.c.) on ATP-splitting by the membrane Na,K-ATPase [l-4] suggest that the mechanism involves changes in the activation of the ion-binding sites by the electric field. The model based on changes in ion activation (CIA) differs from the electroconformational coupling (ECC) model proposed on the basis of earlier studies of ion transport processes involving Na,K-ATPase [5-73. The ECC model suggests that there is a direct action of the electric field on the enzyme in the membrane (e.g. field-dipole interactions), leading to a change in its conformation, while the CIA model can be effective with interaction at the external binding site only. There is an overlap in the range of a.c. fields used in the two studies, and the work on ATP splitting by Na,ATPase in microsomes and on ion transport by Na,K-ATPase in erythrocytes agree on the observed enhanced ATP splitting and ion transport at low enzyme activity. There is also approximate agreement on the
* Dedicated collaboration
to Professor GiuIio Milazzo in grateful recognition in furthering the development of bioelectrochemistry.
0302-4598/92/$05.00
0
1992 - Elsevier
Sequoia
of our many years
S.A. All rights reserved
of friendship
and
292
lo2 Hz for ATP splitting and 10’ Hz for Rb observed optimal frequencies: accumulation_ The most important difference is the observed broad frequency optimum at IO” Hz for sodium efflux from erythrocytes, while the ATP splitting indicates no such effect in the range approaching this frequency. The two ionic fluxes (Rb in and Na out) are physioIogicalIy coupled, and a separation by three orders of magnitude in frequency is unlikely. The differences in observed frequency optima can I.2 explained by differences between intact erythrocytes with Na,K-ATPase molecules all oriented the same way in the membrane, and microsomes that have the Na,K-ATPase oriented in both directions. In addition, it appears that the microsomes are leaky, and ions as well as small molecules like ATP and ouabain have access to the binding sites on both sides of the membrane. In the erythrocyte experiments, K+ ion (and ouabain) binding occur on the outside surface of the cell, and one would expect Rb+ (a K+ analogue) to be transported under the low frequency conditions that affect the outer surface. To affect Na+ efflux, the a-c. signal must penetrate to the Na+ binding site on the inner surface of the membrane, an effect that is appreciable only at higher (megahertz range) frequencies. Another major difference between the two sets of experiments is that the effect of a.c. on ATP splitting by Na,K-ATPase suspensions shows decreases for the enzyme under normal conditions, and increases only when the enzyme activity is lowered to less than half its optimal value by changes in temperature, ouabain concentration and activating ion ratio. Ion transport by the enzyme in a.c. fields has been reported to increase only. The two sets of observations are compatible, since the reported ion transport experiments were done at low temperatures where one would expect enhanced ATP splitting and enhanced ion transport. It would be important to determine whether ion transport is decreased at normal temperatures, and, if so, how the ECC model would explain this. Since temperature appears to be a critical property in the response of this enzyme to a-c. fields, it is important to study the effect of this variable in some detail. In this paper we report on the effect of temperature on ATP-splitting by Na,K-ATPase and try to shed light on the mechanism of the interaction of the enzyme with electric fields. EXPERIMENTAL
Na,K-ATPase-containing vesicles were prepared from frozen rabbit kidneys by modification of the method of Kinsolving et al. 181, as described by Britten and Blank [9]. Details of the method are given in earlier papers by Blank and Soo [1,2]. The microsomal preparations were polydisperse with a range of diameters mainly between 0.1 and 1 pm, and concentrations of (5-7) x 10’. particles/ml. The protein .concentrations (about 1 mg/ml).and the enzymatic properties of different preparations were about the same. ATPase activity was estimated by incubating enzyme and the Na,ATP substrate at various temperatures for 15 min in 1 ml of solution normally containing the
cation
concentrations 81 mM Na’, 16 mM K+ and 5 mM Mg’+. In all cases, the major anion was 61-. Platinum electrodes in plastic tubing filled with salt-impregnated 1.5% agar gels were inserted into U-tubes containing the same amount of enzyme suspension as in the controls. The platinum electrodes wdrc connected to a Hewlett Packard HP 3312A function generator, and a-c. flowed through the U-tube (6-7 mm internal diameter) for 15 min. Sham controls were run for the same period of time. The distance between the pIatinum/agar salt bridge electrodes was kept at 3 cm, and because of the geometry of the system a 1.00 V (peak) output from the function generator was attenuated to 0.12 V (peak) across the enzyme suspension. The a.c. signals covered an amplitude range of 1 mV-1 V and a frequency range of 10 Hz-100 kHz. The current was approximately 0.05-50 mA/cm’ r.m.s. in the four voltage ranges studied. Reactions in both the stimulated and control solutions were terminated with 0.5 ml of 15% (w/v) trichloracetic acid, and the inorganic phosphate was assayed. 8ur measurements are reported as the effect of a.c. on enzyme activity E divided by the normal activity A, ix. the ratio E/A of cxpcrimcnt to control. RESULTS
Under normal conditions cf temperature and optimal ion ratio [1,2], an enzyme preparation shows a decreased ability to split ATP (E/A < 1) with imposed a-c.
1.6 i
I
ld-..- ,
._-c]---_-__---_._-__-
___-__-_._____-__-
I
.._.a...e__.._ 4. -.--.__ 1
.~
-
-r.
--
_..*
-.--..
P
~
,,*j
._.
.
.
_
.
.,
._ .,.-.
_
P iii
1 l:‘--’
. .. .. . ____ _ _ . . 8. .
()*_C-_ ___ _-_.-._-.-.-A. 1 1 o&__ ____ _..__ ___ _. _
I
.._?..m .._.. . .I _ . .* _..I..-_ ,.. =I -mmI ._.______“___
- -a
R e -
._-_-.__ -... I. .I I- ._____._._.
I 0.4 i 0
f i
0.02
0.04
0.06
0.06 ACtiVQ
0.1
0.12
0.14
1 O.;S
Fig. 1. Na.K-ATPase temperature study. The ratio E/A of enzyme activity with a-c. to that without a.c. vs. the enzyme activity in pmol phosphate/mg protein/min. The a.c. frequency was 100 Hz and the peak voltage was 0.01 V. The points represent individual pairs of runs. Above an enzyme activity of 0.06, E/A < 1, while below this value E/A tend to be > 1. The low values of enzyme activity were obtained using the lower temperatures in the range 15-40°C. These results are similar to those obtained in experiments in which the enzyme activity was lowered by introducing 1O-4-lO-” M auabain [2].
294
._._---.-_
__. -.______._.___ _._- ._.._.__I
1 6 #
-
j
I
.._
._.
.
.
I
-.
.___._.__I
I.-._~.
-
..--._
o,~~________.__.
_-.._--.-.__xTz_. I
I
___-__. - -. .__. _ ._.__________._.___; _. ___.__._
o*$*-___
I
*.4! 10
0
20
25
30
35
40
45
TemperaturePC
Fig. 2. Na,K-ATPase temperature study. The ratio E/A of enzyme activity with a.c. to that without a.~. as a function of temperature. 6 was measured under an imposed current (at 100 Hz and IO mV). There appears to be an abrupt change of slope at 25°C where E/A = 1. Standard errors for the individual E and A points are shown in Fig. 3.
This can be seen in Fig. 1 for the data points above an enzyme activity of 0.06 for this preparation, which corresponds to temperatures between 25 and 40°C. The data points represent individual pairs of runs, where A is the activity under normal
I
_
0.14 r 0.1_9;___ _ ._ .-.
o!
10
-
__ . . ._._ .
._ . . .
.._.-..--_-.
.--.-
_ __. -‘z” /J .’ “- .-v --_. _..
I
15
20
25
35
30
Temperature
40
45
A
1°C
Fig. 3. The enzyme activity in pmol phosphate/mg protein/min (IB ) as a function of temperature. On the same temperature scale, we present the enzyme activity in the presence of a.~. at a frequency of 100 Hz and a peak voltage of 0.01 V. (+ 1. Standard error bars are shown except where they are smaller than the data points. At 25”C, the overlapping points have the following S.E.M. values: E = 0.0037; A, 0.0028.
conditions and E is under an imposed current (at 100 Hz and IO mV). At temperatures between 15 and 25”C, enzynle activity is lowered and there is an enhanced activity due to a-c. (E/A > 1). The enhanced effect of the a.c. at low enzyme activity is similar to the data obkined when reducing. the activity by introducing (lo- 4-1O-s M) ouabain 123. Figure 2 is a plot of E/A as a function of temperature. There appears to be an abrupt change of slope at 25°C where E/A = I. At lower temperatures, of a d(E/A)/dT is - 0.062 and above 25°C the slope is - 0.0086. The coincidence sharp change of slope with a change in response to an eIectric field suggests that a lipid phase’change may be involved in the mechanism. Figure 3 is a pIot of the dependence of the errzyme activity on the temperature. The full rectangles show the activity under normal conditions and the plus signs she;Y. the activity under an imposed current (at 100 Hz and 10 mV). Both curves vary smoothly over the range studied and they interseci at 25°C. The individual for the normal conditions and -4.9 x lo-” for the slopes are -3.6 x lo-’ electrically stimulated conditions in units of activity per degree Celsius. DISCUSSION
The effect of a.~.on iorz birtdiug to the Na,K-ATPasr The membrane enzyme Na,K-ATPase responsibk for establishing ion gradients across cell membranes extends throughout the membrane. The catalytic site is on the inside surface, but the enzyme is activated when lC+ ions interact with specific binding sites on the outside surface and Na + ion: interact with binding sites on the inside. When activated, the enzyme catalyzes the coupled transport of three Na+ ions from inside out and two Kf ions from outside in for each ATP split. It is clear that ion concentrations in particular regions are able to affect the activation of the enzyme, and electric fields that affect the ion binding would also change the enzyme activity. To explore the effects of electric fields on Na,K-ATPase, we have developed a simple theoretical model of ion binding at the activation sites on the enzyme surface and derived equations to describe the effects of imposed electric fields. The binding constant K of a cation at an anionic activation site is governed by and the the iocai electrical potential E (frequently calkd the surikce potential), form of the equation is usually written K =!c,
exp( -k,E)
with e the unit charge, k the? Boltzmann where k , is a constant and k, =e/kT, constant and T the absolute temperature. The value of k, at room temperature is about (25 mV)- ‘. If one imposes an external potential IJ, the new value of the binding constant becomes
K” =k,
exp[-k,(E+V)]
(2)
296
the effect of an imposed sine wave, let us assume an alternating To approximate square wave of the same frequency. (This approximation is obviously better at higher frequency.) The value of K, for a full cycle (of duration t) is the average of the plus and minus values in the two half-cycles: K, = f i”“k
! exp[--k,(h+V)ldr+f~~~~~
exp[k,(E-V)]dr I
-exp[--k,(E+V)]
++
exp[-k,(E-V)]
(4)
This is equal to K,=
k, exp( -k,E)
1 1
exp ( -k,V) + exp( k,V) 2
Therefore
(3)
1
K,, = K cosh( k,V)
(5) (6)
Since the cash function is equal to unity when V = 0 and increases for higher values of V, it appears that the imposed a-c. field atways increases on binding. The temperature dependence of both binding constants can be evaluated by taking the derivatives of eqns. (1) and (6): dK = k, exp( -k,E) dT dK -=-dT
dk,E (7)
dT
Ke E k
(8)
T2 d cosh( k,V)
d K” -=K dT
dT = K sinh(k,V)
d K” -=-dT
Ke E
dK, -=dT
dK
k dT
T2
- g
dK + cosh( k,V) dT
w2v/) dT
Ke E + cosh( k,V) k T-
sinh( k,V)
V - E sinh( k,V)
(9)
1 1
(10)
+ cosh( k,V)
+ cosh( k,V)
(12)
For small values of k2V, i.e. for V GX25 mV, and V less than or comparable with E, the value in square brackets is less than unity. Therefore the slope of the temperature curve for the imposed a.c. would be expected to be smaller. In Fig. 3, the ratio of the slopes is 0.73. Equation (12) does not lead to a unique solution but defines a line consisting of pairs of values of V and E for that ratio.
297
E.$fect cf temperature on the Na,K-ATPuse response to U.C. From Fig. 3, we see that the variation of the temperature leads to a smooth change in the enzyme activity which increases almost linearly in the range studied. At higher temperatures, the enzyme starts to inactivate (at shorter times when the enzyme is exposed to higher temperatures), but this is not a factor at the temperatures and durations studied here [9]. When the a.c. field is imposed, the variation of the effect is still smooth, but the slope of the temperature dependence curve decreases, in line with the predictions of the model developed’ above. The cross-over point of the two curves is at 2S”C, which is also the point where the effect of the a.c. field switches from enhancing to inhibiting enzyme activity. The abrupt change of slope shown in Fig. 2 has usually been interpreted as a phase transition. Thermotropic changes in the activity of Na,K-ATPase have been shown to occur in hepatocyte membranes at 26°C [lo], and may very well be linked to the properties of the lipids that form the matrix for the Na,K-ATPase vesicles in our preparation. Furthermore, it is well known that the activity of Na,K-ATPase depends upon the lipids that are present in the vesic!e membranes. Relipidation oC: delipidated enzyme 1113 causes both phosphatase and ATPase activity to return with increasing amounts of phospholipid, and the stoichiometry is different for different lipids, indicating specificity of interaction. These studies also suggest that the enzyme is a functional dimer with K+ ions favoring independence of the This would mean that a.c. fields subunits and Na+ ions favoring interaction. somehow affect the interaction between the subunits, a possibility that is in line with the changes in ion binding predicted by the theoreticai model. This interpretation is not without its problems. The effect on enzyme activity is the same for t.emperature variation as with the other experimental variables studied (e.g. ouabain concentration, Na/K ratio). The effect of *%.c:‘ on enzyme activity appears to va’ry with the activity regardless of how one brings about the change. The thermotropic effect as observed in our system does not seem to be a convincing explanation, despite the abrupt change in Fig. 2. The two lines in Fig. 3 suggest continuous rather than abrupt changes. Also, one would have to postulate changes in lipid organization owing to changes in ouabain concentration or Na/K ratio as well. Effect of frequency Turning again to thz alternating square wave, let us consider the time spent at the plus and minus values of the square wave potential in comparison with the time spent going between them. Since the switch between the two values of potential is not instantaneous, the finite switch time occupies a greater part of the total time as the frequency of the switching increases. (There shoilld also be a dependence on the amplitude of the potential, since the time required to switch should increase with the distance that has to be covered.) Consequently, the effect of a-c. should decrease at higher frequency, as has been observed [1,2].
298
0: -1
0
1
2 kg (Frequency/Hz)
3
4
5
Fig. 4. Standard ertor oi the mean (S.E.M.) as u function of the logarithm of the frequency in hertz. The results a1 different applied voltages arc plotted separately: I 1 mV; A 10 mV; Cl 100 mV; + I V.
We have also observed another effect of frequency. Figure 4 presents a summary of the standard errors of the enzyme activity when measured at different frequencies and amplitudes. The marked increase with frequency suggests that the a.c. is affecting a process that requites a certain length of time to be effective: for example, an ion must be bound for a particuIar period, At higher frequencies the probability that the period will be interrupted increases and leads to a greater variability in the measurements. Eoahat!on of proposed mechanisms
It has been shown that a.c. electric fields cause changes in ion transport and ATP splitting via the Na,K-ATPase, and that the effect on ATP splitting depends upon the level of enzyme activity_ Therefore any proposed mechanism must accoun5. for both observed dependences: that on the electric field and that on the level of enzyme activity. The CIA model appears to account for the basic observations. The effect of electric field and enzyme activity are mechanistically linked through changes in ion binding at the activation sites. The ECC model does not appear to have addressed the problem of the variation of the effect of the electric field with the degree of ion activation. The thermal effects described here add an additional constraint to the understanding of the mechanism. It appears possible to expIain the new observations in terms of. changes in ion binding at the activation sites, using the expressions derived for the binding constants and their temperature coefficients, under both normal conditions and a.c. The ECC model can probably accommodate these new data by choosing new values of the eight rate constants that can be set, but this kind of manipuIation wil1 not be very convincing until physio-chemical reasons can
be given for the choices. To resolve the problem arising from the difference between the optimal frequencies for cation influx (lo3 Hz) and efflux (lo6 Hz), the ECC model uses values for the adjustable parameters without any indication of their physical meaning [X2]. The CIA model shows that changes in the ionic concentrations across the cell membrane, which lead to changes in ion binding and enzyme activation, vary with the frequency and amplitude of the imposed a.c. signal, and that the optimal frequency is approximately 10’ Hz. The observed difference between influx and efflux depends on the two differem sois or”binding sites invoIved. ACKNOWLEDGEMENTS
We thank the Office of Naval Research search Institute (EPRI) for their support.
(ONR)
and the Electric
Power Re-
REFERENCES 1 2 3 4 5 6 7 8 9 10
M. Blank and L. Soo, Bioelectrochem. Bioenerg., 22 (1989) 313. M. Blank and L. Soo, Bioelectrochem. Bioenerg., 24 (1990) 51. M. Btank and L. Soo, Bioelectromagnetics, in press. M. Blank, FASEB J., 6 (1992) 2734. E.H. Serpersu and T.Y. Tsong, J. Biol. Chem., 259 (1984) 7155. T.Y. Tsong and R.D. Astumian, Bioelectrochem. Bioenerg., 15 (1986) 457. D.S. Liu, R-D. Astumian and T.Y. Tsong, J. Biol. Chem., 265 (1990) 7260. CR. Kinsolving, R.L. Post and D.L. Beaver, J. Cell. Comp. Physiol., 62 (1963) 85. J.S. Britten and M. Blank, Biochem. Biophys. Acta, 159 (2968) 150. D. Schachter, in Watts and De Pont (Eds.). Progress in Protein-Lipid Interactions, Amsterdam, 1985, Chapter 6. I I P. Ottolenghi, Eur. J. Biochem., 99 (1979) 113. 12 B. Robertson and R.D. Astumian, J. Chem. Phys., 94 (1991) 7414. 13 M. Blank, J. Electrochem. Sot., 134 !I9871 1112.
Elsevier,