JWLABS ECC 1989

ECC Contents

Implications of Electro Conformational Coupling to Rife Therapy

Written by John Wright (1989)

The ECC 1989 materials were given to us by Andy Cowan of the Federal Technology Transfer Consortium. His view was that it represents exactly what we are doing with Rife technology. There are some clear similarities. However, the experiments, like those of Royal Rife in this area, must be seen as being fundamentally different in physics from the dynamics of therapeutic application of the same principals.

In our opinion, the synthesis of ATP is brought about during electron therapy, (Rife transdermal stimulus therapy) as described in detail by John Crane. Our observations seem to indicate that this process is rarely inhibited. However, of the many thousands of reports we have collected, a small percentage of these experience the reverse effect, and in most cases the effect resumes upon the reversal of relevant polarity, relative to the body.

ATP synthesis must be at least as copious as described in the experiment, although there are undoubtedly other factors that may account for the increase in energy that users report. Fluctuating, oscillating, and alternating electric fields were used in the experiments, mostly at very low temperatures. As reported, these effects seemed to stop well below 37 degrees Centigrade, but this is assumed to be necessary for the cyclic action to be realized and measurable invitro.

20 Hertz up to 300 Hertz were used, (middle C on a piano is 256Hz) and it was also observed that the effect was negligible at frequencies above 1500 Hertz. It is consistent with our observations that higher frequency, up to 1 MHz, although capable of the effect in theory, was not considered useful because of the dramatic fall off of amplitude in this range.

As discovered recently by experimenters with JWLABS Model B in Germany, the transductance dynamic of the living body is effectively impossible to duplicate. Invitro current conductivity of cells, bacterial or otherwise, is limited by the conductivity of its substrate. Thus, the amount of salts required to simulate the invivo environment, is deadly to cells in fluid suspension. In a specially formulated medium, the effects of bacterial electrocution can be demonstrated. This electric energy level is still too powerful a force to permit reliable demonstration of the effects of frequency alone for the same purpose. These are microbial electrocution frequencies.

The anion channel is considered in the experiment, however, in the application of dc fluctuating current, presumably, the cation channel is also present invivo. We believe ac current was necessary in order to achieve uniformity during these experiments. The polarity of ac current is demonstrated to be of little relevance in practical application. Obviously, they are of great importance in dc applications. Further, the cation channel is believed to be the driving force that induces the movement of heavy metals and other toxins, for egression. This is would be impossible or nonexistent in alternating current, since it has no continuous polarity force.

Diffusion of current, and relevant intensity over the substrate is another issue. We believe the experiment did not consider current diffusion, or assumed a predictable amount of diffusion, therefore primarily observing the effects of migration current. These are critical to practical application of these effects. The exact relationship of the signal source, and the subject were not disclosed. Depending on amplitude, the distance of travel required for complete diffusion is not less than 2 inches invivo, and may be much more at greater power. Bearing in mind that the decay distance of the signal and the diffusion of current electric fields used are unknown, the effective energy requirements of the endergonic reactions described cannot be determined for practical use, although we assume the radix is appropriate for their purposes. Columnar relationships in arbitrary transcutaneous invivo applications would be predictably variable.

For argument sake, the very weak (less than 1/250th of an amphere) electric fields needed for these effects to take place are well below the level and frequency required to induce “…structural isomerism, de-polarizing or …de-magnetizing of human cells and brain tissue.” It is believed that such damage is caused only during prolonged exposure to microwave frequencies, but are also present in radio frequencies and the harmonics thereof. (Even in devices of less than 250 watts) At the microwave range, (such as used in Rife Ray machines), “cancerous cells increase in human blood, causes loss of memory, concentration, emotional instability, and a decrease of intelligence,” according to Anthony Wayne and Lawrence Newell, The Proven Dangers of Microwaves. It goes without saying that ray machines, or broadcast, lacking electron flow and relevant electric fields, cannot bring about ATP synthesis, but can do considerable irreversible harm that is not immediately apparent to the casual observer. End of argument.

Current at audio frequency, (as opposed to carrier wave frequencies used in broadcast machines), induces a reaction sequence or cascade of beneficial effects. Some of these effects are easily observable in the living blood via darkfield, and phase-contrast microscopes.

It should be pointed out that the mortal spectrum for micro-organisms is a repeating phenomenon. Rife was first to observe that micro-organisms could be illuminated under certain colors, or frequencies of light. Color was not enough to kill pathogens. Down the spectrum, where the physical impact is more potent, the corresponding frequencies are effective. This is an almost identical effect as seen in the microwave range, the radio range, the audio and sub-audio range. The latter two of which may be better described as the electrocution range for bacteria, viruses and other infective agents. These ranges of mortal frequency also happen to coincide with the frequencies that are responsible for the removal of crystals, fibroblasts, calcification, etc. There is a profound immunostimulus, including the revitalization, redistribution and reactivation of leukocytes. Cell recovery, as in Gangrene, MS and degenerative disorders are also carried out by use of these audio numbers.

The ECC model incidentally demonstrates the inherant conductivity of cells and cell membranes. The lower audio frequencies require far less energy to accomplish the same work. Half the energy is needed at 20 Hz as would be needed at 2000 Hz, in practical application.

The amount of energy needed at 1 MHz, (one million cycles per sec.) is significant enough that there is real doubt as to its safety. In Rife Ray applications, or broadcast, the dangers and detrimental effects are further amplified by ambient frequencies from media transmissions of telephonic and other radio and microwave signals, or what we call overcast. This creates an unknown harmonic, generally in microwave, which increases the danger exponentially. Current at audio frequencies has no such danger potential.

Electro Conformational Coupling 1989

The following material was submitted to JWLABS by the Federal Technology Consortium in 1990 as declassified research performed originally for the United States Department of Defense. No permission is hereby given for the use or transmission of the material for any purpose. For perusal ONLY. Do not distribute.

ECC 1989

p. 319 Bioelectrochemistry and Bioenergetics, 21 (1989)

319-331 A section of J. Electroanal. Chem., and constituting Vol. 275 (1989) Elsevier Sequoia S.A., Lausanne – Printed in the Netherlands

Electroconformational coupling (ECC): an electric field induced enzyme oscillation for cellular energy and signal transductions* Tian Yow Tsong, Dao-Sheng Liu and Francoise Chauvin Department of Biochemistry, University of Minnesota College of Biological Sciences, 1479 Gortner Avenue, St. Paul, MN 55108 (U.S.A.)

Aldolfas Gaigalas and R. Dean Astumian, National Institutes of Standards and Technology, Chemical Process Metrology Division, Gaithersburg, MD 20899 (U.S.A.)

(Received 20 November 1988; in revised form 4 February 1989)

Electro Conformational Coupling 1989 Abstract

Previous work has shown that membrane ATPases can extract free energy from applied oscillating electric fields for doing chemical work, e.g. to synthesize ATP from ADP and P(i) or to transport Rb and Na ions against their respective electrochemical gradient. Data of these experiments are briefly reviewed. Electroconformational Coupling (ECC) is used to interpret these results. Computer analysis of a four state cyclic enzyme mechanism reproduces many experimental features. It is shown that a coulombic interaction between an enzyme and an alternating electric field (ac) can cause the enzyme to oscillate between different conformational states. If the frequency of the applied field matches the kinetic characteristics of the system and the amplitude matches the energy required for inducing productive catalytic cycling, a phenomenological resonance between catalytic reaction and the periodic field is generated. A condition necessary for achieving energy coupling is the kinetic bias arising from the binding energy of the ligand. Analysis indicates that only dynamic electric fields, i.e. oscillating or fluctuating fields, can propel the cyclic reaction of the enzyme catalysis, and thus be effective for transducing energy. A stationary transmembrane electric field must be modulated, e.g. by opening and closing of an ion channel, to become oscillatory in order to produce the same effect. We propose that ECC is a fundamental process of cellular energy and signal transductions. Here, many membrane associated events are reduced to Michaelis-Menten types of enzyme catalytic reactions and they are thus amenable to the quantitative analysis of chemical kinetics.

Introduction

Electrochemical potential of ions have been postulated to play a major role in free energy transductions and information transfer of cells. In neural transmission, Na and K currents are responsible for the generation and propagation of the action potential [1,2]. In mitochondrial ATP synthesis, the proton gradient across the inner membrane is the high energy intermediate, which, upon translocation of protons along the electrochemical gradient, transfers its potential energy to ATPase for the synthesis of ATP [3-8]. In photosynthetic processes, the energy of a photon is used to pump a proton into an energy reservoir and ATP synthase then uses the electrochemical potential energy of the proton for synthesis of ATP [7,9,10]. Notwithstanding, there is no compelling evidence which would exclude a direct energy transfer between the electric field and a protein, thus allowing a temporary storage of energy in the conformational states of the protein [11]. Previously, we proposed a mechanism, Electroconformational Coupling (ECC), to test the feasibility of direct energy transaction between a transmembrane electric field and an enzyme conformational equilibrium for driving ion pumps and ATP synthesis [12-15]. Here we will summarize new experimental evidence and analysis based on the concept of ECC. We will examine and compare the ECC model and the common enzyme catalytic process as exemplified by the Michaelis-Menten Mechanism.

The electric potential across cell membranes is of the order of 10 to 250 mV, which corresponds to a field intensity of 20 to 500 kV/cm. Under such a strong field, molecules will behave quite differently than they will under the zero field condition. Ion pairs will dissociate, dipoles will orient, molecules will be electronically and automatically polarized, equilibria between different conformers of a protein will be shifted, etc. [16,17]. There are two geometric situations under which these changes can take place. The first is where all molecules and ions freely diffuse. The second is when they are fixed relative to the field direction. The first situation is represented by a reaction in an homogeneous aqueous solution. The field effect on the rapidly tumbling molecule is generally small and has been discussed elsewhere [12,16,17]. Here we will focus on the second situation which is more relevant for dealing with effects of an electric field on a membrane protein. Let us start by considering a general enzyme catalytic reaction.

Cyclic Process of Enzyme Catalysis

An enzyme catalytic process is a cyclic reaction because the enzyme is recycled at each turnover. A cyclic process will respond to a periodic driving force with which the enzyme can interact. As a result of this interaction, the enzyme will oscillate between its different conformational states. This phenomenon has been shown to have an implication in cellular membrane processes. To examine the cyclic behavior of an enzyme, we will consider the simple Michaelis-Menten mechanism (scheme 1 of Fig. 1). The enzyme bonds to the substrate to form an enzyme-substrate complex.

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{Fig. 1. Cyclic enzyme catalytic process. Many membrane processes mediated by receptors and enzymes exhibit kinetic characteristics similar to the {?-missing} of a Michaelis-Menten enzyme. Such an enzyme is susceptible to periodic perturbation. This paper considers how oscillating electric fields can interact with membrane ATPases and in so doing induce enzyme conformational oscillations, thus allowing utilization of the binding energy of ligands for catalyzing endergonic reactions. See text for details.} 

The product is then released and the initial enzyme state is regenerated when the complex dissociates. The driving force of this reaction is the negative free energy of the S to P conversion. In fact, with a non-reversible step at the product releasing step, the reaction is implied to proceed to the left even if the free energy has a positive sign. Enzyme recycling has a specific rate, given by the turnover rate of the Michaelis-Menten mechanism. Generally, most investigators agree that there is another state preceding the formation of the product, namely the enzyme-product complex, as shown in scheme 2. If the two reversible steps are much faster than the dissociation of the enzyme- product complex, the kinetics of scheme 2 will be indistinguishable from that of scheme 1, and scheme 2 is in essence a Michaelis-Menten mechanism. In the third scheme, we simply rewrite the second scheme in a more consistent manner. It becomes a cyclic mechanism. Again, the reaction is driven by the negative free energy of the S to P conversion, although the description of the process is inherently unidirectional since it is shown to proceed only in the clockwise direction. To be more precise, the enzyme state which favors the binding substrate must be different from the state which favors the binding of the product state. A distinction between E(1) and E(2) is necessary. The Michaelis-Menten mechanism of scheme 1 is now more generally written as scheme 4. However, scheme 4 has an inconsistency. We all accept that without an additional

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free energy source an enzyme can only catalyze a reaction towards but not against the equilibrium. If the product dissociation step is thought irreversible, then whether the S to P conversion is energetically favorable or not, the reaction will proceed clockwise anyway. We know from our experience with hundreds of enzymes that this is not the case. The inconsistency can easily be be removed by writing a reversible product dissociation step. Now the reaction becomes scheme 5. Scheme 5 has been used to describe many membrane transport and energy transducing processes. The reaction of scheme 5 can proceed clockwise or counter-clockwise depending on the sign of the free energy of the S to P conversion, clockwise if (delta)G is negative and counter-clockwise if it is positive. More importantly, when a cell membrane is involved this scheme represents an efficient mechanism for energy and signal transductions as we shall see later; yet, the enzyme E is basically a Michaelis-Menten enzyme. An external energy source can be coupled to this scheme so that enzyme can catalyze a reaction against its chemical potential gradient. Scheme 5 has a characteristic frequency. Only an oscillating driving force the frequency of which matches this charateristic frequency will be effective in propelling the “catalytic wheel” of scheme 5, as will be discussed more explicitly in the following sections.

Electroconformational Coupling

One example of a periodic or oscillating driving force in a living cell is the transmembrane electric field. Although the transmembrane potential of a cell has been considered constant in the past, recent analyses suggest that locally it may exhibit large amplitude oscillations of fluctuations when time (is) resolved to ms or us, (microsecond), levels. Many membrane integral proteins have been shown to be electrically active. For examples, the VDAC (voltage-dependent anion channel) from the outer membrane of the mitochondria [18], the Na-channel/batrachotoxin complex [19], and the acetylcholine receptor [20] open in specific ranges of transmembrane electric potential and close in other ranges. These field dependent conformational changes may be coupled to ligand binding processes for energy and signal transductions [12-15]. Let us consider a simple two state conformational transition,

(formulae) (1)

The equilibrium constant of the reaction is K = k(1)/k(-1). The properties which make a protein responsive to an electric field are its electric moment (u) and polarizability (a). The molar electric moment of P(1) is M(1) = u(1) + a(1)E and of P(2) , M(2) = u(2) + a(2)E. The change in molar electric moment for the conformational transition is (delta)M = M(2) – M(1). An electric field of strength E will shift the equilibrium according to the generalized van’t Hoff equation,

(formulae)(2)

The energy involved in this transaction is (delta)M*E. This energy can be utilized to {missing}

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{Fig. 2. Computer analysis of the cyclic kinetic scheme of eqn. 3). (A) Numerical integration of an ac field stimulated pumping of a neural substrate is shown. The MLAB computer program of National Institutes of Health was used. In the computation an interaction energy (delta)M*E of 12.6kJ/mol was used. Changes in the concentration of S(in) are plotted against stimulation time. The frequency and the strength of the ac field were adjusted so that there is an apparent resonance of the enzyme conformational transitions and the oscillating field. A fine feature of oscillation can be seen on top of the steady rise in the concentration of S(in). This rise would level off when the concentration gradient balanced the interaction energy. For the set of rate constants and other parameters used here, the resonance occurred around 300 Hz. (B) When an ac field of 1500 Hz was used, the resonance was broken and there was a fluctuation of the concentration of S(in) but no net accumulation of S(in) in the cytoplasmic side was detected. See refs. 12-15 for details.} drive an endergonic reaction. Equation (3) shows one such design for a membrane transport protein or enzyme.

(formulae) (3)

Here the protein is assumed to be a membrane integral protein and the activity of the protein is to transport the substrate S from the extracellular medium into the cytoplasm of the cell. For convenience only, two conditions will be introduced in our discussion. First, (delta)M of the P(1) to P(2) transition has a positive value. In other words, a positive electric field will favor the P(2) state and a negative electric field the P(1) state. Second, the affinity of S(out) for P(2) is much greater than that of S(in) for P(1). When these two conditions are imposed, a periodic perturbation by an oscillating electric field (ac field) will drive a clockwise flux of the enzyme and hence the transport of S(out) from the extracellular space into S(in) of the cytoplasm. Figure 2 gives some results of the computer analysis for eqn. (3). These computations and p. 324

{Fig. 3. Summary of the behavior of the cyclic kinetic scheme of eqn. (3) in response to an oscillating electric field. (A) In the scheme the affinity of S(out) for P(2) is assumed to be much greater than the affinity of S(in) for P(1). The substrate binding and dissociation steps are fast compared to the conformational transition steps. With these conditions, [P(1)] > [P(e)2] and [P(2)S] > [P(1)S] under zero field. (B) When the ac field is in its positive phase, it induces a large flux of P(1) – P(2) and a small flux of P(1)S – P(2)S. The catalytic wheel turns clockwise. (C) When the ac field is in its negative phase, it induces a large flux of P(2)S – P(1)S and a small flux of P(2) – P(1). The catalytic wheel again turns clockwise. These results indicate that the wheel turns only in one direction regardless of the polarity of the stimulating ac field. This means that the system can capture energy from the oscillating field for driving an endergonic reaction such as energy transduction or signal transduction. The direction of the revolving wheel is determined by the affinity of S to P(1) and P(2). If the affinity is greater for P(1) than for P(2), the wheel will turn counterclockwise.} analyses also reveal many interesting properties of eqn. (3) which are summarized in Fig. 3. Figure 3 indicates that, regardless of the polarity of the induced membrane potential during the experiment, the fluxes are directed towards the cytoplasm. This means that eqn. (3) is a molecular pump which is driven by an oscillating electric field by direct coulombic coupling to the enzyme conformational equilibrium. No interaction between the substrate and the electric field need be presumed. Several remarkable points are listed below.

(1) The scheme would transduce energy only if the four state scheme has inherent asymmetries. We have already mentioned the difference in the affinity of S for P(1) and P(2). This equivalent to the interaction energy discussed by Jencks [21]. Other asymmetry requirements have been discussed elsewhere [13,16,22].

(2) The frequency of the applied field must match the kinetic attributes of the system. When the ligand binding and dissociation steps are fast, the optimum frequency of the field for driving the reaction is determined by the conformational transition steps.

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(3) There is also an optimum field strength for the energy coupling. A field of strength exceeding this value tends to lock the enzyme into certain states, thus preventing its effective turnover [16].

Activation of Membrane ATPASE by Oscillating Electric Fields

Experiments on the activation of [Na,K]-ATPase of human erythrocytes and mitochondrial F(0)F(1)ATPase of beef heart by oscillating electric fields are good examples of how membrane proteins can respond to oscillating electric fields, capture energy and couple that energy to drive endergonic reactions.

[Na,K]-ATPase of Human Erythrocytes

Previous experiments from this laboratory indicated that [Na,K]-ATPase of human erythrocytes might be susceptible to electric perturbation and might be one of the sites of electroporation when an electric field of the order of kV/cm is used to induce pores in human red cells [23]. A membrane conductance which could be blocked by ouabain was detected. We then attempted to active this enzyme using a field which would generate a transmembrane potential of the order of 10 mV. Human red cells in an isotonic suspension were exposed to applied alternating electric fields of strengths up to 50 V/cm (peak-to-peak) and of frequencies between 1 Hz and 20 MHz. The transport of Na+ and Rb+ (or K+) into and out of the cytoplasm was measured by radioactive tracers and compared with control samples in which no ac field was imposed. The activity which was inhibited by ouabain was taken to be due to the catalytic action of the [Na,K]-ATPase. Other inhibitors of the enzyme such as oligomycin, vanadate and oubagenin also inhibited the electric field provoked activity. In contrast, inhibitors to anion transport and Na+/Na+ exchange have no appreciable effects except at concentrations 10-1000-fold higher than that required to inhibit these activities. The results of these experiments are summarized below [24-27].

(1) At 4 (degrees) C, the maximum ac stimulated activity (i.e. at optimum field strength and optimum frequency) was 15-20 ions/pump for the Rb+ uptake and 20-30 ions/pump for the Na+ efflux. The ratio of Na+ pump activity and Rb+ pump activity was roughly 3/2, although it was not strictly maintained for any one red cell sample.

(2) There was an optimum field strength of 20 V/cm (peak-to-peak) for activating both the Rb+ and the Na+ pumps. The maximum perturbation to the red cell transmembrane electric potential induced by this field is estimated to be 12 mV (i.e. +/-6 mV) by using the Maxwell relation. This value corresponds to a peak-to-peak electric field of 24 kV/cm if the thickness of the hydrophobic layer of the red cell membrane is assumed to be 5 nm.

(3) The optimum frequency for the Rb+ pump was 1.0 kHz and for the Na+ pump 1.0 MHz when a 20 V/cm ac field was used. The reason for this wide separation of optimum frequencies is not clear. It is, however, recognized that in the

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action potential of neurons, the rise of the Na+ current is 100 times faster than the rise of the K+ current.

(4) The Rb+ uptake and the Na+ efflux activities detected here were active transport against the respective ion concentration gradients. In the case of the Rb+ uptake, the cytoplasmic [Rb+] was 27 mM and the extracellular [Rb+] was 10-12.5 mM. If the electric field stimulated a passive permeation, it would also have stimulated the efflux of Rb+. The efflux was not effected by the electric field up to 60 V/cm. In the case of the Na+ efflux, the cytoplasmic [Na+] was 6-10 mM amd the extracellular [Na+] was 140-150 mM, and the electric field stimulated only the efflux but not the uptake.

(5) The Michaelis-Menten constant, K(m), of internal Na+ for Rb+ uptake and Na+ efflux was 8 mM and for the external Rb+, 1.5 mM, consistent with an ATP dependent pumping activity of the [Na,K]-ATPase.

(6) ATP depleted red cells (down to 10 uM) still retained nearly the same level of voltage stimulated activity, suggesting that ATP hydrolysis was not required. However, it is not known whether other nucleotide or phosphate ligands are essential.

(7) Although most experiments were carried out around 4 (degrees) C, where the basal activity (i.e. ATP hydrolysis activity) was negligible, experiments done at 25 (degrees)

{Fig. 4. Frequency windows for the electric activation of the Na+ pump and the Rb+ pump (or K+ pump) of human [Na,K]-ATPase. (A) Human erythrocytes in an isotonic saline were exposed to an ac field of 20 V/cm and 4 (degrees) C. The ouabain sensitive Na+ efflux (O) and Rb+ uptake (delta) are plotted against the frequency of the applied field. In the same experiment, no ouabain sensitive Na+ uptake and Rb+ efflux were induced. The cytoplasmic concentration of Na+ was 6 mM and that of Rb+, 27 mM (pre-loaded), and the external concentration of Na+ was 140 mM and of Rb+, 10 mM. Yet the ac field stimulated transport of both ions against there respective concentration gradient. No consumption of ATP was detected. (B) The Electroconformational Coupling (ECC) model was used to simulate the above results. By adjusting the rate constants, asymmetry factor and the gating charge, it is possible to reproduce the main feature of the frequency dependence curves of (A). No attempt was made to fit the experimental results numerically. All rate constants used in the computation were below the diffusion controlled limit. Details of the computations and adjustment of parameters will be presented.}

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showed enhanced pump activity due to the ac stimulation. However, at 37 (deg) C, where the basal activity was at its maximum, no further stimulation by the ac field was detected. (98.6F)

{Fig. 5. ATP synthesis in beef heart submitochondrial particles stimulated by pulsed electric fields. (A) Submitochondrial particles from beef heart were suspended in a medium containing 5 mM HEPES, 2.5 mM potassium phosphate, (32)P(i) tracer, 2.5 mM ADP, 1 mM MgCl(2), 1 mg/ml BSA, 2 mM NaCN, 0.25 M sucrose, 20 mM glucose, pH 7.0, with a varying concentration of dithiothretol (DTT). The sample was exposed to five pulses of a 25 kV/cm exponentially decaying electric field (time constant of 100 us). The ATP formed was trapped with a hexokinase system (each sample contained also 71.4 U/ml of hexokinase), and analyzed for yield. At the peak, the ATP yield was approximately 5 molecules of ATP per enzyme per pulse. The initial temperature of the sample was 15 (deg) C and at no time did the temperature exceed 25 (deg) C. A control sample was kept at 25 (deg) C and the count was subtracted from the data. (B) Submitochondrial particles were preincubated for the time shown on the abscissa in a 0.25 M sucrose, 50 mM potassium phosphate buffer before transferring to the medium given in (A) for a low amplitude ac stimulation (60 V/cm at 30 Hz). Incubation caused the dissociation of the natural inhibitor peptide and an increase in ATP yield (). A control sample kept at 15 (deg) C showed no ATP synthesis (o). See text for details.}

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These results established firmly that the electric field stimulated active transports of Na+, K+ and Rb+ were due to the activation of [Na,K]-ATPase, and that the energy required for driving the endergonic reaction was derived from the applied ac field. Figure 4A shows the frequency dependence of the Na+ pump and the Rb+ pump using a 20 V/cm ac field.

Mitochondrial ATPASE From Beef Heart

Witt and co-workers [28] were the first to report activation of ATP synthesis of chloroplasts by pulsed electric fields. The yield of ATP was less than one per enzyme per electric pulse and it was assumed that the electric field triggered the release of tightly bound nucleotides from the enzyme surface. Our experiments were performed with beef heart mitochondrial ATPase and our goal was to demonstrate that a single pulse of electric field can induce enzyme turnover [29-33]. The electron transport activity of submitchondrial particles was inhibited by rotenone or cyanide or both. Thus, the energy source for ATP synthesis, about 42 kJ/mol, must be derived from the applied electric field. Using the capacitor discharge technique it was shown that ATP was formed from ADP and P(i) and this synthesis was inhibited completely by oligomycin and FCCP (carbonyl cyanide p-triflouromethoxyphenol- hydrazone), the former being a potent inhibitor and the latter an ionophore of proton. When dithiothreitol was present in the medium, as much as 5 ATP molecules per enzyme were generated with a single pulse of 30 kV/cm (300 mV of transmembrane potential) with a decay constant of 100 us. We interpret these results as due to the oxidation-reduction cycle of certain SH groups in the ATP synthetic process. Firgure 5A shows some results of these experiments using the exponentially decaying electric field.

Alternating electric fields have also been used to demonstrate ATP synthesis. In most experiments, we used an electric field of 60 V/cm at various frequencies. The ATP yield per ac cycle is low (approximately 10^-4) because of the low energy level of the applied field. However, the experiment is simple and the results are essential for clarifying certain concepts of the ECC model, as discussed below.

Another interesting observation is that the natural inhibitor peptide of the ATPase has a profound effect on the electric field induced ATP synthesis. Synthesis was effective only if the peptide was removed. Figure 5B gives some of the results which show an increase in ATP yield as the natural inhibitor peptide dissociates with time from the enzyme.

Interpretation Using the ECC Model

The main features of the above results on [Na,K]-ATPase have been predicted from computer analyses of the ECC model using eqn. (3) [12-17]. These include the ability of eqn. (3) to absorb energy from an applied field for actively pumping a neutral substrate or an ion, an optimum field strength and an optimum frequency for this effect. Computer analysis, shown in Fig. 4B, reproduces the frequency dependence of the Na+ pump and the Rb+ pump. No quantitative fit of the data

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points was attempted. The difference in the frequency for effective pumping for Na+ and Rb+ was produced by adjusting the number of gating charges (2 for Rb+ pump and 3 for Na+ pump) and the rate constants of eqn. (3). Details of the simulation will be presented elsewhere.

The results on the ATP synthesis experiment need further elaboration. According to the ECC model, only a dynamic electric field (either oscillating or fluctuating) can induce a phenomenological resonance with the enzyme cyclic reation and be effective for energy coupling. An exponentially decaying electric field can be viewed as a fourier sum of many components, each with a characteristic frequency. Among these components are high frequency ones which would be able to induce enzyme turnover within a single electric discharge. This is judged unlikely to be the reason for enzyme turnover as the amplitude of these higher frequency components is bound to be small and the interaction energy (delta M*E) would be insufficient for ATP synthesis.

The above consideration led us to suggest that within the mitochondrial ATPase a mechanism exists which can modulate a stationary transmembrane electric field to become locally oscillatory [12-17]. We suggested that the F(o) subunit could fill this role by translocating protons at a defined time interval. When a proton approaches from one side of the membrane and then translocates to the other side, the electric potential experienced by the enzyme would temporarily change its sign, making it oscillatory. There are many mechanisms for modulating a stationary electric potential. An ion channel, a redox protein, a charge motion, etc. in the vicinity of an energy transducing enzyme can effectively produce an oscillating field under an energy sustaining constant potential. We mentioned that oxidation and reduction of certain SH groups may be involved in the turnover of the mitochondrial ATPase. The mechanisms and details of its involvement remain to be investigated.

Relation of Other Cellular Energy and Signal Transductions

We propose that the experiments and analyses presented above are reminiscent of how a cell transduces energy and signals [15,33,34]. A crucial question is whether the transmembrane potential of a cell is stationary or oscillatory. If it is oscillatory, the ECC model would be applicable for understanding the function of many membrane enzymes and receptors. If it is stationary, one would expect that such a potential be modulated by certain mechanisms before it can play a role in the cellular energy and signal transductions. Several methods of modulation may be conceived: (1) opening/closing of a channel protein, as in F(o)F(1)ATPase and the generation and propogation of an action potential, (2) altering the charge density of a protein, as in phosphorylation /dephosphorylation, (3) a redox reaction that transports electrons through a specified path, as in the electron transport chain, (4) binding/dissociation of ions or charged ligands, etc.

These ideas have been discussed previously. Most cellular events mediated by membrane receptors or enzymes basically have the character of a Michaelis- Menten enzyme, as depicted in scheme 5 of Fig. 1. Such a system is able to receive, decipher

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and respond to a periodic perturbation, i.e. an energy source or a signal, insofar as the receptor or enzyme is capable of interacting effectively with that periodic driving force. A reverse reaction will, in turn, allow the receptor or the enzyme to transmit energy or a signal. If the concept discussed here is near the truth, most useful information of the transmembrane electric activity of a cell is not contained in the measured stationary potential but rather in the local and time dependent fluctuations of the electric field.

Very weak electric fields (mV/cm) of various frequencies have been shown to trigger gene expression, stimulate RNA and protein biosynthesis, and influence cell differentiation, proliferation, bone healing, etc. (see e.g. refs. 35- 37). These phenomena may be understood using the concept of the ECC model, as well. Our task is then to identify cellular components that respond to these weak electric fields. In our previous work, we have examined how a very weak periodic field is amplified at the cite of the cell membrane [11,12,38,39]. With such mechanisms, organisms can communicate and navigate with extremely weak oscillatory electric, magnetic, acoustic or chemical potentials.

Acknowledgements

The work of T.Y.T., D.-S.L. AND F.C. has been supported by the United States Office of Naval Research and a National Science Foundation grant to T.Y.T. Contributions of our former and present associates and collaborators, Drs. K. Kinosita, E.H. Serpersu, J. Teissie, H.V. Westerhoff, P.B. Chock, Y.-D. Chen, B.E. Knox and F. Kamp, are greatly appreciated.

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