Bioelectrorheological model of the cell. 4. Analysis of the extensil deformation of cellular membrane in alternating electric field.

Analysis of the angular distribution of extensil mechanical stress, sigma e, generated in cytoplasmic membranes by an external oscillating electric field, is presented. Theoretical considerations show that sigma e is directly proportional to the local relative increase in membrane area and/or to the local relative decrease in its thickness. The magnitude of this stress depends on the position of the analyzed point of the membrane in relation to field direction. The maximal value, sigma eo, is reached at the cell "poles." The magnitude of sigma eo depends on electric and geometric parameters (in particular on field frequency) of the system studied. The foregoing analysis can be applied to quantitatively describe the destabilizing effects of the electric field on the cellular membrane, leading to its poration, fusion, and destruction.     

Bioelectrorheological model of the cell. 2. Analysis of creep and its experimental verification

The electrorheological model of the cell proposed in Part 1 of this work was used to analyze changes in time of the shape of a cell acted on by a constant-amplitude external alternating electric field, with lossiness of the media taken into account. Shear stress in the cell membrane was determined. This model was then subjected to preliminary experimental verification using Neurospora crassa (slime) spheroplasts subjected to an external alternating electric field of constant frequency (3 MHz) and varying magnitude for different periods of time. Reversible viscoelastic cell deformation and fatigue (stiffening) of the material were observed. A satisfactory fit of the experimental data to Burgers' rheological model was found, and the values of the elastic moduli E1 = 9 X 10(2) N/m2, E2 = 2 X 10(2) N/m2 and viscosities eta 1 = 8 X 10(4) Ns/m2; eta 2 = 7 X 10(3) Ns/m2 were evaluated.     

Deformability and stability of erythrocytes in high-frequency electric fields down to subzero temperatures.

High-frequency electric fields can be used to induce deformation of red blood cells. In the temperature domain T = 0 degrees to -15 degrees C (supercooled suspension) and for 25 degrees C this paper examines for human erythrocytes (discocytes, young cell population suspended in a low ionic strength solution with conductivity sigma(25 degrees) = 154 microS/cm) in a sinusoidal electric field (nu = 1 MHz, E0 = 0-18 kV/cm) the following properties and effects as a function of field strength and temperature: 1) viscoelastic response, 2) (shear) deformation (steady-state value obtained from the viscoelastic response time), 3) stability (by experimentally observed breakdown of cell polarization and hemolysis), 4) electrical membrane breakdown and field-induced hemolysis (theoretical calculations for ellipsoidal particles), and 5) mechanical hemolysis. The items 2-4 were also examined for the frequency nu = 100 kHz and for a nonionic solution of very low conductivity (sigma(25 degrees) = 10 microS/cm) to support our interpretations of the results for 1 MHz. Below 0 degrees C with decreasing temperature the viscoelastic response time tau(res)(T) for the cells to reach steady-state deformation values d(infinity,E) increases and the deformation d(infinity,E)(T) decreases strongly. Both effects are especially high for low field strengths. The longest response time of approximately 30 s was obtained for -15 degrees C and small deformations. For 1 MHz the cells can be highly elongated up to 2.3 times their initial diameter a0 for 25 degrees and 0 degrees C, 2.1a0 for -10 degrees C and still 1.95a0 for -15 degrees C. For T > or = 0 degrees C the deformation is limited by hemolysis of the cells, which sets in for E0(lysis)(25 degrees) approximately 8 kV/cm and E0(lysis)(0 degrees) approximately 14 kV/cm. These values are approximately three times higher than the corresponding calculated critical field strengths for electrically induced pore formation. Nevertheless, the observed depolarization and hemolysis of the cells is provoked by electrical membrane breakdown rather than by mechanical forces due to the high deformation. For the nonionic solution, where no electrical breakdown is expected in the whole range for E0, the cells can indeed be deformed to even higher values with a low hemolytic rate. Below 0 degrees C we observe no hemolysis at all, not even for the frequency 100 kHz, where the cells hemolyze at 25 degrees C for the much lower field strength E0(lysis) approximately 2.5 kV/cm. Obviously, pore formation and growth are weak for subzero temperatures.     

Bioelectrorheological model of the cell. 1. Analysis of stresses and deformations

An electrorheological model of a cell in alternating electric field is proposed. The model relates changes in the spherical cell's shape to the field conditions, electric parameters of cytoplasm, cell membrane and external medium, and to the rheological parameters of the membrane. Stresses were determined using Maxwell's stress tensor for isotropic media. Shear stresses in the cell membrane were analyzed. Predictions of the model for variations of shear stress in cellular membranes subjected to an external periodic electric field are presented and related to the conditions prevailing in electrobiological research.     

Study of mechanisms of electric field-induced DNA transfection. II. Transfection by low-amplitude, low-frequency alternating electric fields.

Electroporation for DNA transfection generally uses short intense electric pulses (direct current of kilovolts per centimeter, microseconds to milliseconds), or intense dc shifted radio-frequency oscillating fields. These methods, while remarkably effective, often cause death of certain cell populations. Previously it was shown that a completely reversible, high ionic permeation state of membranes could be induced by a low-frequency alternating electric field (ac) with a strength one-tenth, or less, of the critical breakdown voltage of the cell membrane (Teissie, J., and T. Y. Tsong. 1981. J. Physiol. (Paris). 77:1043-1053). We report the transfection of E. coli (JM105) by plasmid PUC18 DNA, which carries an ampicillin-resistance gene, using low-amplitude, low-frequency ac fields. E. coli transformants confer the ampicillin resistance and the efficiency of the transfection can be conveniently assayed by counting colonies in a selection medium containing ampicillin. For the range of ac fields employed (peak-to-peak amplitude 50-200 V/cm, frequency 0.1 Hz-1 MHz, duration 1-100 s), 100% of the E. coli survived the electric field treatment. Transfection efficiencies varied with field strength and frequency, and as high as 1 x 10(5)/micrograms DNA was obtained with a 200 V/cm square wave, 1 Hz ac field, 30 s exposure time, when the DNA/cell ratio was 50-75. Control samples gave a background transfection of much less than 10/micrograms DNA. With a square wave ac field, the transfection efficiency showed a frequency window: the optimal frequency was 1 Hz with a 200 V/cm field, and was approximately 0.1 Hz with a 50 V/cm field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioelectrorheological model of the cell. 3. Viscoelastic shear deformation of the membrane.

An analytical electromechanical model of a spherical cell exposed to an alternating electric field was used to calculate shear stress generated in the cellular membrane. Shape deformation of Neurospora crassa (slime) spheroplasts was measured. Statistical analysis permitted empirical evaluation of creep of the cellular membrane within the range of infinitesimal stress. Final results were discussed in terms of various rheological models.     

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.     

Rotation of cells in an alternating electric field theory and experimental proof

Summary Protoplasts ofAvena sativa rotate in an alternating electric field provided that at least two cells are located close to each other. An optimum frequency range (20 to 30 kHz) exists where rotation of all cells exposed to the field is observed. Below and above this frequency range, rotation of some cells is only occasionally observed. The angular velocity of rotation depends on the square of the electric field strength. At field strengths above the value leading to electrical breakdown of the cell membrane, rotation is no longer observed due to deterioration of the cells. The absolute value of the angular velocity of rotation at a given field strength depends on the arrangement of the cells in the electric field. A maximum value is obtained if the angle between the field direction and the line connecting the two cells is 45^o. With increasing distance between the two cells the rotation speed decreases. Furthermore, if two cells of different radii are positioned close to each other the cell with the smaller radius will rotate with a higher speed than the larger one. Rotation of cells in an alternating electric field is described theoretically by interaction between induced dipoles is adjacent cells. The optimum frequency range for rotation is related to the relaxation of the polarization process in the cell. The quadratic dependence of the angular velocity of rotation on the field strength results from the fact that the torque is the product of the external field and the induced dipole moment which is itself proportional to the external field. The theoretical and experimental results may be relevant for cyclosis (rotational streaming of cytoplasm) in living cells.     

Conformational flexibility of membrane proteins in electric fields. I. Ultraviolet absorbance and light scattering of bacteriorhodopsin in purple membranes

Bacteriorhodopsin of halobacterial purple membranes exhibits conformational flexibility in high electric field pulses (1-30 x 10(5) V m(-1), 1-100 micros). High-field electric dichroism data of purple membrane suspensions indicate two kinetically different structural transitions within the protein; involving a rapid (approximately 1 micros) concerted change in the orientation of both retinal and tyrosine and/or tryptophan side chains concomitant with alterations in the local protein environment of these chromophores. as well as slower changes (approximately 100 micros) of the microenvironment of aromatic amino acid residues concomitant with pK changes in at least two types of proton-binding sites. Light scattering data are consistent with the maintenance of the random distribution of the membrane discs within the short duration of the applied electric fields. The kinetics of the electro-optic signals and the steep dependence of the relaxation amplitudes on the electric field strength suggest a saturable induced-dipole mechanism and a rather large reaction dipole moment of 1.1 x 10(-25) C m ( = 3.3 x 10(4) debye) per cooperative unit at E = 1.3 x 10(5) V m(-1), which is indicative of appreciable cooperativity in the probably unidirectional transversal displacement of ionic groups on the surfaces of and within the bacteriorhodopsin proteins of the membrane lattice. The electro-optic data of bacteriorhodopsin are suggestive of a possibly general, induced-dipole mechanism for electric field-dependent structural changes in membrane transport proteins such as the gating proteins in excitable membranes or the ATP synthetases.     

Modification of electrokinetic properties of nuclei in human buccal epithelial cells by electric fields

The influence of an alternating (50 Hz) electric field (5--110 V/cm) on the state of human buccal epithelium cells was studied by the methods of intracellular microelectrophoresis, heterochromatin staining with orcein, and indigo carmine staining for viability and membrane integrity evaluations. Electric field exposure induced an increase in electrophoretic mobility of cell nuclei, decreased numbers of heterochromatin granules near the inner membrane of cell nucleus, and induced cell membrane damage; but cell viability was conserved. Nuclear and cell membrane properties varied with electric field strength and age of the donors. The data obtained are interpreted as evidence of electric field induced activation of the functional state of nuclei.     

On the measurement of shear elastic moduli and viscosities of erythrocyte plasma membranes by transient deformation in high frequency electric fields.

We present a new method to measure the shear elastic moduli and viscosities of erythrocyte membranes which is based on the fixation and transient deformation of cells in a high-frequency electric field. A frequency domain of constant force (arising by Maxwell Wagner polarization) is selected to minimize dissipative effects. The electric force is thus calculated by electrostatic principles by considering the cell as a conducting body in a dielectric fluid and neglecting membrane polarization effects. The elongation A of the cells perpendicular to their rotational axis exhibits a linear regime (A proportional to Maxwell tension or to square of the electric field E2) at small, and a nonlinear regime (A proportional to square root of Maxwell tension or to the electric field E) at large extensions with a cross-over at A approximately 0.5 micron. The nonlinearity leads to amplitude-dependent response times and to differences of the viscoelastic response and relaxation functions. The cells exhibit pronounced yet completely reversible tip formations at large extensions. Absolute values of the shear elastic modulus, mu, and membrane viscosity, eta, are determined by assuming that field-induced stretching of the biconcave cell may be approximately described in terms of a sphere to ellipsoid deformation. The (nonlinear) elongation-vs.-force relationship calculated by the elastic theory of shells agress well with the experimentally observed curves and the values of mu = 6.1 x 10(-6) N/m and eta = 3.4 x 10(-7) Ns/m are in good agreement with the micropipette results of Evans and co-workers. The effect of physical, biochemical, and disease-induced structural changes on the viscoelastic parameters is studied. The variability of mu and eta of a cell population of a healthy donor is +/- 45%, which is mainly due to differences in the cell age. The average mu value of cells of different healthy donors scatters by +/- 18%. Osmotic deflation of the cells leads to a fivefold increase of mu and 10-fold increase of eta at 500 mosm. The shear modulus mu increases with temperature showing that the cytoskeleton does not behave as a network of entropy elastic springs. Elliptic cells of patients suffering from elliptocytosis of the Leach phenotype exhibit a threefold larger value of mu than normal discocytes of control donors. Cross-linking of the spectrin by the divalent S-H agents diamide (1 mM, 15 min incubation) leads to an eightfold increase of mu whereas eta is essentially constant. The effect of diamide is reversed after treatment with S-S bond splitting agents.     

Cell shape-dependent rectification of surface receptor transport in a sinusoidal electric field.

In the presence of an extracellular electric field, transport dynamics of cell surface receptors represent a balance between electromigration and mutual diffusion. Because mutual diffusion is highly dependent on surface geometry, certain asymmetrical cell shapes effectively create an anisotropic resistance to receptor electromigration. If the resistance to receptor transport along a single axis is anisotropic, then an applied sinusoidal electric field will drive a net time-average receptor displacement, effectively rectifying receptor transport. To quantify the importance of this effect, a finite difference mathematical model was formulated and used to describe charged receptor transport in the plane of a plasma membrane. Representative values for receptor electromigration mobility and diffusivity were used. Model responses were examined for low frequency (10(-4)-10 Hz) 10-V/cm fields and compared with experimental measurements of receptor back-diffusion in human fibroblasts. It was found that receptor transport rectification behaved as a low-pass filter; at the tapered ends of cells, sinusoidal electric fields in the 10(-3) Hz frequency range caused a time-averaged accumulation of receptors as great as 2.5 times the initial uniform concentration. The extent of effective rectification of receptor transport was dependent on the rate of geometrical taper. Model studies also demonstrated that receptor crowding could alter transmembrane potential by an order of magnitude more than the transmembrane potential directly induced by the field. These studies suggest that cell shape is important in governing interactions between alternating current (ac) electric fields and cell surface receptors.     

Effects of Electromagnetic Fields on the Bioactivities of an Osteoblast-Like Cell Line (UMR-106)

In the present study, we first investigated the effects of various types of low-energy, low-frequency electromagnetic fields (EMFs) on DNA synthesis in UMR-106 osteoblast-like cells. The experimental groups were exposed to EMFs for 2 days (twice/day, 30 min/time), and DNA synthesis was measured. The results showed that the cells responded most sensitively to EMFs of some specific combinations of the parameters by an increase in DNA synthesis, implying that EMFs with a specific waveform rather than a complex one can be used in clinical electrotherapy. The parameters were as follows: pulsed electric field (PEF) with pulse width 0.2 ms, field strength 10 V/cm, frequency 125 Hz; sinusoidal electric field (SEF) with field strength 1 V/cm, frequency 10 Hz; and alternating magnetic field (AMF) with field density 0.5 mT, frequency 5 Hz. In addition to frequency, the field strength or field density within a suitable intensity scale played a dominant role in causing the DNA synthesis response. We then compared the effects of two kinds of fields, PEF and AMF, with the optimum parameters identified by the experiments, on alkaline phosphatase (ALP) activity, protein and collagen synthesis, and intracellular levels of cyclic adenosine monophosphate (cAMP). The results indicated that both fields could not only affect UMR-106 cells proliferation but could particularly affect a series of characteristic bioactivities of UMR-106 such as ALP activity and collagen synthesis. The intracellular cAMP levels were increased rapidly and greatly with exposure to both PEF and AMF, implying that the action of low-frequency EMFs proceeds via second messenger-dependent processes originating from signals at the cell membrane. The difference in action between PEF and AMF suggests that they may couple to the cell membrane in a partially different way.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field induced conformational changes of bacteriorhodopsin were studied in six types of dried film (randomly and electrically oriented membranes of purple as well as cation-depleted blue bacteriorhodopsin) by measuring the frequency dependence of the optical absorbance change and the dielectric dispersion and absorption. For the purple bacteriorhodopsin the optical absorbance change induced by alternating rectangular electric fields of ±300 kV/cm altered the sign twice in the frequency range from 0.001 Hz to 100 kHz (around 0.03 Hz and 100 kHz), indicating that the electric field induced conformational change in these samples consists of, at least, three steps. Similarly, it was found for the blue bacteriorhodopsin that at least two steps are involved. In accord with optical measurements, the dielectric behaviour due to alternating sinusoidal electric fields of±6kV/cm in the frequency range from 10 Hz to 10 MHz showed two broad dispersion/absorption regions, one below 1 kHz and the other around 10–100 kHz. This suggests that the conformational change of bacteriorhodopsin is also reflected by its dielectrical properties and that it is partially induced at 6 kV/cm. Including previous results obtained by analysis of the action of DC fields on purple membrane films, a model for a field-induced cyclic reaction for purple as well as blue bacteriorhodopsin is proposed. In addition it was found that there are electrical interactions among purple membrane fragments in dried films.     

Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions.

The observed equilibrium constants (Kobs) of the creatine kinase (EC 2.7.3.2), myokinase (EC 2.7.4.3), glucose-6-phosphatase (EC 3.1.3.9), and fructose-1,6-diphosphatase (EC 3.1.3.11) reactions have been determined at 38 degrees C, pH 7.0, ionic strength 0.25, and varying free magnesium concentrations. The equilibrium constant (KCK) for the creatine kinase reaction defined as: KCK = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] [H+]) was measured at 0.25 ionic strength and 38 degrees C and was shown to vary with free [Mg2+]. The value was found to be 3.78 x 10(8) M-1 at free [Mg2+] = 0 and 1.66 x 10(9) M-1 at free [Mg2+] = 10(-3) M. Therefore, at pH 7.0, the value of Kobs, defined as Kobs = KCK[H+] = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] was 37.8 at free [Mg2+] = 0 and 166 at free [Mg2+] = 10(-3) M. The Kobs value for the myokinase reaction, 2 sigma ADP equilibrium sigma AMP + sigma ATP, was found to vary with free [Mg2+], being 0.391 at free [Mg2+] = 0 and 1.05 at free [Mg2+] = 10(-3) M. Taking the standard state of water to have activity equal to 1, the Kobs of glucose-6-P hydrolysis, sigma glucose-6-P + H2O equilibrium sigma glucose + sigma Pi, was found not to vary with free [Mg2+], being 110 M at both free [Mg2+] = 0 and free [Mg2+] = 10(-3) M. The Kobs of fructose-1,6-P2 hydrolysis, sigma fructose-1,6-P2 equilibrium sigma fructose-6-P + sigma Pi, was found to vary with free [Mg2+], being 272 M at free [Mg2+] = 0 and 174 M at free [Mg2+] = 0.89 x 10(-3) M.     

Dipole interactions in electrofusion. Contributions of membrane potential and effective dipole interaction pressures.

The contributions of pulse-induced dipole-dipole interaction to the total pressure acting normal to the membranes of closely positioned pronase treated human erythrocytes during electrofusion was calculated. The total pressure was modeled as the sum of pressures arising from membrane potential and dipole-dipole attraction opposed by interbilayer repulsion. The dipole-dipole interaction was derived from the experimentally obtained cell polarizability. The threshold electric field amplitude necessary for fusion of pronase-treated human erythrocytes was experimentally obtained at various combinations of pulse duration, frequency, and the conductivity of the external medium. The theoretical values of the critical electric field amplitude compared favorably to the experimentally obtained threshold field amplitudes. Fusion by dc pulses may be primarily attributed to attainment of sufficiently high membrane potentials. However, with decreasing external conductivity and increasing sinusoidal pulse frequency (100 kHz-2.5 MHz), the induced dipole-dipole interactions provide the principal driving force for membrane failure leading to fusion.     

Recognition and processing of randomly fluctuating electric signals by Na,K-ATPase.

Previous work has shown that Na,K-ATPase of human erythrocytes can extract free energy from sinusoidal electric fields to pump cations up their respective concentration gradients. Because regularly oscillating waveform is not a feature of the transmembrane electric potential of cells, questions have been raised whether these observed effects are biologically relevant. Here we show that a random-telegraph fluctuating electric field (RTF) consisting of alternating square electric pulses with random lifetimes can also stimulate the Rb(+)-pumping mode of the Na,K-ATPase. The net RTF-stimulated, ouabain-sensitive Rb+ pumping was monitored with 86Rb+. The tracer-measured, Rb+ influx exhibited frequency and amplitude dependencies that peaked at the mean frequency of 1.0 kHz and amplitude of 20 V/cm. At 4 degrees C, the maximal pumping activity under these optimal conditions was 28 Rb+/RBC-hr, which is approximately 50% higher than that obtained with the sinusoidal electric field. These findings indicate that Na,K-ATPase can recognize an electric signal, either regularly oscillatory or randomly fluctuating, for energy coupling, with high fidelity. The use of RTF for activation also allowed a quantitative theoretical analysis of kinetics of a membrane transport model of any complexity according to the theory of electroconformational coupling (ECC) by the diagram methods. A four-state ECC model was shown to produce the amplitude and the frequency windows of the Rb(+)-pumping if the free energy of interaction of the transporter with the membrane potential was to include a nonlinear quadratic term. Kinetic constants for the ECC model have been derived.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycoproteins bound to ion channels mediate detection of electric fields: a proposed mechanism and supporting evidence

The mechanism by which animals detect weak electric and magnetic fields has not yet been elucidated. We propose that transduction of an electric field (E) occurs at the apical membrane of a specialized cell as a consequence of an interaction between the field and glycoproteins bound to the gates of ion channels. According to the model, a glycoprotein mass (M) could control the gates of ion channels, where M > 1.4 x 10(-18)/E, resulting in a signal of sufficient strength to overcome thermal noise. Using the electroreceptor organ of Kryptopterus as a mathematical and experimental model, we showed that at the frequency of maximum sensitivity (10 Hz), fields as low as 2 microV/m could be detected, and that the observation could be explained if a glycoprotein mass of 0.7 x 10(-12) kg (a sphere 11 microm in diameter) were bound to channel gates. Antibodies against apical membrane structures in Kryptopterus blocked field transduction, which was consistent with the proposal that it occurred at the membrane surface. Although the target of the field was hypothesized to be an ion channel, the proposed mechanism can easily be extended to include other kinds of membrane proteins.     

Annexin proteins PP4 and PP4-X. Comparative characterization of biological activities of placental and recombinant proteins.

Several G-proteins (GTP-binding proteins) were identified by SDS/PAGE in the cytosol (105,000 g supernatant) and membrane fractions of the oestrogen-dependent human mammary-tumour cell line ZR-75-1. These proteins, with molecular masses in the range 18-29 kDa, specifically bind [alpha-32P]GTP, which can be displaced by unlabelled GTP, GDP and their non-hydrolysable analogues guanosine 5'-[delta-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-thio]diphosphate (GDP[S]), but not by GMP, ATP, ADP, AMP and other unrelated nucleotides. The apparent dissociation constant for GTP was approx. 2 x 10(-8)M. Homogenization of ZR-75-1 cells in high-salt buffer (1 M-KCl), and successive washing of the membrane fraction, suggested that, among the major G-proteins found, the 18 kDa protein is predominantly soluble, whereas the 27-29 kDa complex is primarily bound to the membrane fraction under the experimental conditions employed. Possible translocation of these G-proteins between membrane and cytosol was analysed. No redistribution of the 27-29 kDa complex was observed, whereas GTP[S] in the presence of Mg2+ caused apparent translocation of the 18 kDa protein to the membrane fraction. This effect was specific for GTP and stable GTP analogues, whereas GDP, GMP, ATP, ADP, AMP and other unrelated nucleotides were ineffective. GTP[S] and guanosine 5'-[beta gamma-imido]-triphosphate (p[NH]ppG) were equally potent (apparent Kd approximately 5 x 10(-6)M), whereas GTP was rather weak. The nucleotide effect is temperature-, time- and concentration-dependent. The translocation process was reversible, slow, and reached its maximum between 30 and 60 min at 37 degrees C. The apparent translocation of this small G-protein from the cytosol to the membrane fraction, and the specific effect of GTP analogues, suggest that this process may have functional significance in mammary-tumour cells.