Electrorotation of single yeast cells at frequencies between 100 Hz and 1.6 GHz.

The determination of complete electrorotation spectra of living cells has been made possible by the development of a quadrature generator and an electrode assembly that span the frequency range between 100 Hz and 1.6 GHz. Multiple spectra of single cells of the yeast Saccharomyces cerevisiae have been measured at different medium conductivities ranging from 0.7 to 550 microS cm-1. A spherical four-shell model was applied that simulated the experimental data well and disclosed the four-layer structure of the cell envelope attributed to the plasma membrane, the periplasmic space, and a thick inner and a thin outer wall region. Below 10 kHz an additional rotation effect was found, which changed its direction depending on the ionic strength of the medium. This is supposed to be connected with properties of the cell surface and its close vicinity. From the four-shell simulation the following physical properties of cell compartments could be derived: specific capacitance of plasma membrane (0.76 microF cm-2), periplasmic space (0.5 microF cm-2), and outer wall region (0.1 microF cm-2). The conductivity of cytoplasm, plasma membrane, and inner wall region were found to vary with medium ionic strength from 9 to 12 mS cm-1, 5.8 nS cm-1 to approximately 50 nS cm-1, and 6 microS cm-1 to 240 microS cm-1, respectively.     

Dielectric properties of yeast cells as determined by electrorotation.

Electrorotational spectra of yeast cells, Saccharomyces cerevisiae strain R XII, were measured over a frequency range of nearly 7 decades. The physical properties of distinct cell parts were simultaneously determined for individual cells by comparison with an electrical two-shell model: The conductivity of the cytoplasm, cell wall and cytoplasmic membrane of living cells were found to be 5.5 mS/cm, 0.1 to more than 0.5 mS/cm and less than 0.25 nS/cm to 4.5 microS/cm, respectively. The conductivity of the cytoplasmic membrane was dependent on the conductivity of the medium. Membrane behaviour is interpreted as an opening of membrane channels when the environment becomes more physiological. The specific membrane capacitance was determined to be 1.1 microF/cm2 and the thickness of the cell wall was calculated as 0.11 micron. Heat treated cells showed an increased membrane conductivity of more than 0.1 microS/cm (at 25 microS/cm medium conductivity) and a drop in cytoplasmic conductivity to between 0.1 and 0.8 mS/cm, depending on the length of time the cells were suspended in low conductivity water (25 microS/cm), indicating a perforation of the membrane. A slightly decreased spinning speed scaling factor for dead cells suggests a modification to the cellular surface, while the principal structure of the cell wall appears to be uneffected. It can be demonstrated by these observations, that cellular electrorotation permits the simultaneous investigation of the different cellular compartments of individual cells in vivo under various environmental conditions.     

Electrorotation studies of baby hamster kidney fibroblasts infected with herpes simplex virus type 1

The dielectric properties of baby hamster kidney fibroblast (BHK(C-13)) cells have been measured using electrorotation before and after infection with herpes simplex virus type 1 (HSV-1). The dielectric properties and morphology of the cells were investigated as a function of time after infection. The mean specific capacitance of the uninfected cells was 2.0 microF/cm2, reducing to a value of 1. 5 microF/cm2 at 12 h after infection. This change was interpreted as arising from changes in the cell membrane morphology coupled with alterations in the composition of the cell membrane as infection progressed. The measured changes in the cell capacitance were correlated with alterations in cellular morphology determined from scanning electron microscope (SEM) images. Between 9 and 12 h after infection the internal permittivity of the cell exhibited a rapid change, reducing in value from 75epsilono to 58epsilono, which can be correlated with the generation of large numbers of Golgi-derived membrane vesicles and enveloped viral capsids. The data are discussed in relation to the known life cycle of HSV-1 and indicate that electrorotation can be used to observe dynamic changes in both the dielectric and morphological properties of virus-infected cells. Calculations of the dielectrophoretic spectrum of uninfected and infected cells have been performed, and the results show that cells in the two states could be separated using appropriate frequencies and electrode arrays.     

Differences in membrane properties between unfertilised and fertilised single rabbit oocytes demonstrated by electro-rotation. Comparison with cells from early embryos

The apparent membrane capacity of tubular rabbit oocytes increases from 1.7-2.0 microF/cm2 before fertilisation to 3.7-4.0 microF/cm2 after fertilisation. The membrane conductivity measured on single cells was also increased by fertilisation from less than 1 mS/cm2 to 14 mS/cm2. Cells obtained from 2-, 4- or 8-cell embryos exhibited intermediate values of membrane capacity (2.3-2.8 microF/cm2) and conductivity (5-22 mS/cm2). The values quoted are those effective between 1 and 10 kHz, the frequency of the rotating field used. The large apparent capacities are probably due to the presence of structures such as microvilli which cause the actual membrane area to exceed the smooth sphere area. It must be assumed that these structures change in form or number on fertilisation, and that they persist in embryos, at least up to the 8-cell stage. No difference was apparent between cells fertilised in vitro or in vivo. Comparison of the above zona-free data with measurements on zona-complete oocytes indicate how fertilised and unfertilised rabbit eggs may be distinguished from one another, even in the presence of the zona pellucida.     

Electro-rotation of mouse oocytes: single-cell measurements of zona-intact and zona-free cells and of the isolated zona pellucida

Passive electrical properties of oocytes and of zonae pellucidae, and the mechanical coupling between them, can be elucidated by means of rotating-field-induced rotation. In low-conductivity media (25-100 microS/cm) rotation of mouse oocytes (with or without their zonae) requires fields in the 1-100 kHz frequency range. However, an isolated zona shows weak rotation in the opposite direction to that of a cell, and in response to much higher field frequencies (approx. 1 MHz). In zona-intact mouse oocytes, the rotation of cell and zona are not rigidly coupled: thus rotation of the cell can still be induced when the zona is held stationary. However, rotation of freely suspended zona-intact cells is much slower than that of zona-free cells and requires an optimum field frequency that is approximately 1.5 kHz higher. These observations show that the electrical properties of the oocyte that are measured by rotation are altered by the presence of the zona pellucida, even though no such influence has been detected using micro-electrodes. The data are consistent with the zona acting as a porous shell with a conductivity of 40 microS/cm (preliminary estimate made at a single medium conductivity of 26 microS/cm). Measurements on cells from which the zonae had been removed gave values for the membrane capacity and resistivity of 1.2-1.3 microF/cm2 and 400 omega.cm2, respectively. These values may reflect the presence of plasmalemma microvilli. The results strongly suggest that the technique may be useful for studies of cell maturation and for in vitro fertilization, because the cells may be further cultured after measurement.     

Dielectric spectroscopy of single human erythrocytes at physiological ionic strength: dispersion of the cytoplasm.

Usually dielectrophoretic and electrorotation measurements are carried out at low ionic strength to reduce electrolysis and heat production. Such problems are minimized in microelectrode chambers. In a planar ultramicroelectrode chamber fabricated by semiconductor technology, we were able to measure the dielectric properties of human red blood cells in the frequency range from 2 kHz to 200 MHz up to physiological ion concentrations. At low ionic strength, red cells exhibit a typical electrorotation spectrum with an antifield rotation peak at low frequencies and a cofield rotation peak at higher ones. With increasing medium conductivity, both electrorotational peaks shift toward higher frequencies. The cofield peak becomes antifield for conductivities higher than 0.5 S/m. Because the polarizability of the external medium at these ionic strengths becomes similar to that of the cytoplasm, properties can be measured more sensitively. The critical dielectrophoretic frequencies were also determined. From our measurements, in the wide conductivity range from 2 mS/m to 1.5 S/m we propose a single-shell erythrocyte model. This pictures the cell as an oblate spheroid with a long semiaxis of 3.3 microns and an axial ratio of 1:2. Its membrane exhibits a capacitance of 0.997 x 10(-2) F/m2 and a specific conductance of 480 S/m2. The cytoplasmic parameters, a conductivity of 0.4 S/m at a dielectric constant of 212, disperse around 15 MHz to become 0.535 S/m and 50, respectively. We attribute this cytoplasmic dispersion to hemoglobin and cytoplasmic ion properties. In electrorotation measurements at about 60 MHz, an unexpectedly low rotation speed was observed. Around 180 MHz, the speed increased dramatically. By analysis of the electric chamber circuit properties, we were able to show that these effects are not due to cell polarization but are instead caused by a dramatic increase in the chamber field strength around 180 MHz. Although the chamber exhibits a resonance around 180 MHz, the harmonic content of the square-topped driving signals generates distortions of electrorotational spectra at far lower frequencies. Possible technological applications of chamber resonances are mentioned.     

Dielectric properties of E. coli cell as simulated by the three-shell spheroidal model

Dielectric properties of E. coli cell have been re-studied by means of the three-shell spheroidal model, where the three shells correspond to the outer membrane, the periplasmic space and the inner membrane, respectively. With the model, a curve-fitting procedure has been developed to analyze the dielectric spectra. Although E. coli cell has been studied before, its special morphological structure was taken into account more comprehensively than any previous model in the present work. Dielectric properties of various cell components have been estimated from the observed dielectric spectra, especially the permittivity of the outer membrane, which was evaluated quantitatively for the first time. The values of epsilon(om) were 12 for kappa(om) of 0 to 10(-4) S/m and 34 for kappa(om) of 10(-3) S/m. The specific capacitance of the inner membrane was 0.6-0.70 microF/cm(2). The relative permittivity and the conductivity of the cytoplasm were about 100 and 0.22 S/m, respectively, and the conductivity of the periplasmic space was 2.2-3.2 S/m.     

Na+ transport and impedance properties of cultured renal (A6 and 2F3) epithelia

Previous impedance analysis studies of intact epithelia have been complicated by the presence of connective tissue or smooth muscle. We now report the first application of this method to cultured epithelial monolayers. Impedance analysis was used as a nondestructive method for deducing quantitative morphometric parameters for epithelia grown from the renal cell line A6, and its subclonal cell line 2F3. The subclonal 2F3 cell line was chosen for comparison to A6 because of its inherently higher Na+ transport rate. In agreement with previous results, 2F3 epithelia showed significantly higher amiloride-sensitive short-circuit currents (Isc) than A6 epithelia (44 +/- 2 and 27 +/- 2 microA/cm2, respectively). However, transepithelial conductances (GT) were similar for the two epithelia (0.62 +/- 0.04 mS/cm2 for 2F3 and 0.57 +/- 0.04 mS/cm2 for A6) because of reciprocal differences in cellular (Gc) and paracellular (Gj) conductances. Significantly lower Gj and higher Gc values were observed for 2F3 epithelia than A6 (Gj = 0.23 +/- 0.02 and 0.33 +/- 0.04 mS/cm2 and Gc = 0.39 +/- 0.16 and 0.26 +/- 0.10 mS/cm2, respectively). Nonetheless, the cellular driving force for Na+ transport (Ec) and the amount of transcellular Na+ current under open-circuit conditions (Ic) were similar for the two epithelia. Three different morphologically-based equivalent circuit models were derived to assess epithelial impedance properties: a distributed model which takes into account the resistance of the lateral intercellular space and two models (the "dual-layer" and "access resistance" models), which corrected for impedance of small fluid-filled projections of the basal membrane into the underlying filter support. Although the data could be fitted by the distributed model, the estimated value for the ratio of apical to basolateral membrane resistances was unreasonably large. In contrast, the other models provided statistically superior fits and reasonable estimates of the membrane resistance ratio. The dual-layer model and access resistance models also provided similar estimates of apical and basolateral membrane conductances and capacitances. In addition, both models provided new information concerning the conductance and area of the basolateral protrusions. Estimates of the apical membrane conductance were significantly higher for 2F3 (0.79 +/- 0.23 mS/cm2) than A6 epithelia (0.37 +/- 0.07 mS/cm2), but no significant difference could be detected for apical membrane capacitances (1.4 +/- 0.04 and 1.2 +/- 0.1 microF/cm2 for 2F3 and A6, respectively) or basolateral membrane conductances (3.48 +/- 1.67 and 2.95 +/- 0.40 mS/cm2). The similar basolateral membrane properties for the two epithelia may be explained by their comparable transcellular Na+ currents under open-circuit conditions.     

Electrorotation of Erythrocytes Treated with Dipicrylamine: Mobile Charges within the Membrane Show their ``Signature' in Rotational Spectra

In this study, electrorotation spectra of individual cells (that is, frequency dependence of cell rotation speed) have been proved to yield information not only about the passive electric properties of cell constituents, but also about the presence of mobile charges within the plasma membrane being part of ion carrier transport systems. Experiments on human erythrocytes pretreated with the lipophilic anion dipicrylamine (DPA) gave convincing evidence that these artificial mobile charges adsorbed to the plasma membrane contributed significantly to the rotational spectrum at relatively low conductivity of the external medium (2–5 mS m⁻¹). Theoretical integration of the mobile charge concept into the single-shell model (viewing the cell as a homogenous sphere surrounded by a membrane) led to a set of equations which predicted electrorotational behavior of DPA-treated cells in dependence on medium conductivity. The quantitative data on the partition and the transmembrane translocation rate of the DPA anion extracted from the experimental rotational spectra agreed well with the corresponding literature values. Received: 14 February 1996/Revised: 29 May 1996     

Frequency domain analysis of membrane capacitance of cultured cells (HeLa and myeloma) using the micropipette technique.

The membrane capacitance and conductance of cultured cells (HeLa and mouse myeloma) are investigated using the micropipette method. Mean values of the membrane capacities were found to be 1.9 microF/cm2 for HeLa cells and 1.0 microF/cm2 for myeloma cells. These values are in agreement with those obtained using the suspension method. Whereas the suspension method is unable to provide the information on membrane conductance, the micropipette method is able to measure even an extremely small membrane conductance if leakage current is negligibly small. The membrane conductances were found, using this technique, to be approximately 90-100 microS/cm2 for both HeLa and myeloma cells. One of the purposes of this study is to establish the frequency profile of membrane capacitance. It was found, however, that membrane capacitances of these cells are independent of frequency between 1 Hz and 1 KHz within the resolution of this technique.     

Electrorotation of Isolated Generative and Vegetative Cells, and of Intact Pollen Grains of Lilium longiflorum

The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m⁻¹. The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques. Received: 9 June 1997/Revised: 4 August 1997     

Dielectric properties of mouse lymphocytes and erythrocytes

In order to study the effect of the nucleus on dielectric behavior of the whole cell, permittivity (dielectric constant) and conductivity of mouse lymphocytes and erythrocytes were measured over a frequency range from 0.1 to 250 MHz. Erythrocytes (spherocytes) showed a single dielectric dispersion, which was explained by a single-shell model that is a conducting sphere covered with a thin insulating shell. On the other hand, lymphocytes showed a broad dielectric dispersion curve which was composed of two subdispersions. The high-frequency subdispersion, which was not found for erythrocytes, was assigned to the Maxwell-Wagner dispersion of the nucleus occupying about 65% of the total cell volume. Analysis of the lymphocyte dispersion was carried out by a double-shell model, in which a shelled sphere, i.e., nucleus, is incorporated into the single-shell model. The following electrical parameters were consequently estimated; the capacitance of the plasma membrane, 0.86 microF.cm-2; the conductivity of the cytoplasm, 3.2 mS.cm-1; the capacitance and conductance of the nuclear envelope are, respectively, 0.62 microF.cm-2 and 15 S.cm-2, and the permittivity and conductivity of the nucleoplasm are 52 and 13.5 mS.cm-1.     

A new microsystem for automated electrorotation measurements using laser tweezers

We have developed a new microsystem for fast, automated studies of reactions and kinetics of single cells with biochemical or pharmacological agents. A cell spins in an external rotating electric field and the frequency dependence characterises the passive dielectric properties of membrane and cytoplasm. We use a planar microelectrode chip with microchannel (easily covered with a removable slip) for the application of frequencies exceeding 250 MHz to determine cytoplasmic properties in low and high conductivity electrolyte solutions. The laser tweezers serve as a bearing system, rotation is induced by microelectrodes and rotation speed is recorded automatically. This opens up new possibilities in biotechnology, e.g. for drug screening as demonstrated by measuring the influence of ionomycin on the passive dielectric properties of T-lymphoma cells. Additionally, a possible infrared-induced long-term cell damage could be observed by electrorotation and is discussed.     

Frequency domain studies of impedance characteristics of biological cells using micropipet technique. I. Erythrocyte.

This study aims at precise measurement of the membrane capacity and its frequency dependence of small biological cells using the micropipet technique. The use of AC fields as an input signal enables the magnitude and phase angle of membrane impedance to be measured at various frequencies. The micropipet technique was applied to human erythrocyte, and passive membrane capacity and conductivity were determined between 4 Hz and 10 KHz. Membrane capacity thus determined changed from 1.05 to 0.73 microF/cm2 between 4 Hz and 10 KHz. In addition to the micropipet technique, we used suspension method between 50 KHz and 10 MHz for the purpose of supplementing the new method with the one which has been in use for many years. We obtained a membrane capacity of 0.65-0.8 microF/cm2 using this technique. These values agree with the capacitance obtained with the micropipet method. Although this paper discusses only human erythrocytes, the study has been performed with lymphocytes and various forms of cancer cells. This paper is the first of the series of reports on frequency domain studies of the impedance characteristics of various biological cells.     

Dielectrophoresis and electrorotation of neurospora slime and murine myeloma cells.

Dielectrophoresis and electrorotation are commonly used to measure dielectric properties and membrane electrical parameters of biological cells. We have derived quantitative relationships for several critical points, defined in Fig. A 1, which characterize the dielectrophoretic spectrum and the electrorotational spectrum of a cell, based on the single-shell model (Pauly, H., and H.P. Schwan, 1959. Z. Naturforsch. 14b:125-131; Sauer, F.A. 1985. Interactions between Electromagnetic Field and Cells. A. Chiabrera, C. Nicolini, and H.P. Schwan, editors. Plenum Publishing Corp., New York. 181-202). To test these equations and to obtain membrane electrical parameters, a technique which allowed simultaneous measurements of the dielectrophoresis and the electrorotation of single cells in the same chamber, was developed and applied to the study of Neurospora slime and the Myeloma Tib9 cell line. Membrane electrical parameters were determined by the dependence of the first critical frequency of dielectrophoresis (fct1) and the first characteristic frequency of electrorotation (fc1) on the conductivity of the suspending medium. Membrane conductances of Neurospora slime and Myeloma also were found to be 500 and 380 S m-2, respectively. Several observations indicate that these cells are more complex than that described by the single-shell model. First, the membrane capacities from fct1 (0.81 x 10(-2) and 1.55 x 10(-2) F m-2 for neurospora slime and Myeloma, respectively) were at least twice those derived from fc1. Second, the electrorotation spectrum of Myeloma cells deviated from the single-shell like behavior. These discrepancies could be eliminated by adapting a three-shell model (Furhr, G., J. Gimsa, and R. Glaser. 1985. Stud. Biophys. 108:149-164). Apparently, there was more than one membrane relaxation process which could influence the lower frequency region of the beta-dispersion. fct1 of Myeloma in a medium of given external conductivity were found to be similar for most cells, but for some a dramatically increased fct1 was recorded. Model analysis suggested that a decrease in the cytoplasmatic conductivity due to a drastic ion loss in a cell could cause this increase in fct1. Model analysis also suggested that the electrorotation spectrum in the counter-field rotation range and fc1 would be more sensitive to conductivity changes of the cytoplasmic fluid and to the influence of internal membranes than would fct1, although the latter would be sensitive to changes in capacitance of the cytoplasmic membranes.     

Low frequency electrorotation of fixed red blood cells.

Electrorotation of fixed red blood cells has been investigated in the frequency range between 16 Hz and 30 MHz. The rotation was studied as a function of electrolyte conductivity and surface charge density. Between 16 Hz and 1 kHz, fixed red blood cells undergo cofield rotation. The maximum of cofield rotation occurs between 30 and 70 Hz. The position of the maximum depends weakly on the bulk electrolyte conductivity and surface charge density. Below 3.5 mS/m, the cofield rotation peak is broadened and shifted to higher frequencies accompanied by a decrease of the rotation speed. Surface charge reduction leads to a decrease of the rotation speed in the low frequency range. These observations are consistent with the recently developed electroosmotic theory of low frequency electrorotation.     

Capacitance and resistance of the bilayer lipid membrane formed of phosphatidylcholine and cholesterol

Capacity and electric resistance of lipid membranes composed of lecithin and cholesterol were determined. The components were chosen for the study because they were present in biological membranes. Capacitance of the lecithin and cholesterol membranes amounts to 0.38 and 0.61 microF/cm(2), and resistance to 1.44(10(4)and 2.12(10(6)Omega cm(2), respectively. A 1:1 complex appears as a result of lecithin-cholesterol membrane formation. Parameters of the membrane formed of the lecithin-cholesterol complex were determined: surface concentration (Gamma(3)), capacitance (C(3)), and conductance (R;(3)(-1), as well as the stability constant (K) of the complex. The mean values of those magnitudes are as follows: 4.265(10(-6)mol/m(2), 0.54 microF/cm(2), 1.381(10(-6)Omega(-1)cm(-2)and 3.748(10(7), respectively.     

Dielectric properties of alginate beads and bound water relaxation studied by electrorotation

The electrical and dielectric properties of Ba²⁺ and Ca²⁺ cross‐linked alginate hydrogel beads were studied by means of single‐particle electrorotation. The use of microstructured electrodes allowed the measurements to be performed over a wide range of medium conductivity from about 5 mS/m to 1 S/m. Within a conductivity range, the beads exhibited measurable electrorotation response at frequencies above 0.2 MHz with two well‐resolved co‐ and antifield peaks. With increasing medium conductivity, both peaks shifted toward higher frequency and their magnitudes decreased greatly. The results were analyzed using various dielectric models that consider the beads as homogeneous spheres with conductive loss and allow the complex rotational behavior of beads to be explained in terms of conductivity and permittivity of the hydrogel. The rotation spectra could be fitted very accurately by assuming (a) a linear relationship between the internal hydrogel conductivity and the medium conductivity, and (b) a broad internal dispersion of the hydrogel centered between 20 and 40 MHz. We attribute this dispersion to the relaxation of water bound to the polysaccharide matrix of the beads. The dielectric characterization of alginate hydrogels is of enormous interest for biotechnology and medicine, where alginate beads are widely used for immobilization of cells and enzymes, for drug delivery, and as microcarriers for cell cultivation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 227–237, 1999     

Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation.

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.