Electrophotoluminescence and the electrical properties of the photosynthetic membrane. I. Initial kinetics and the charging capacitance of the membrane

Preilluminated chloroplast membranes, and particularly hypotonically swollen vesicles (blebs), give rise to a strong characteristic luminescence (electrophotoluminescence, EPL; Ellenson and Sauer, 1976, Photochem. Photobiol., 23:113-123; Arnold and Azzi, 1971, Photochem. Photobiol., 14:233-240) during the application of a strong external electric field. A detailed kinetic study of EPL was carried out and the initial kinetics from the field onset are reported here. The fast rise time (less than 0.2 mus) of the applied external electric field together with a high instrumental time resolution allowed the observation of a characteristic delay (lag time) between the field onset and the appearance of the induced emission. The lag time decreased with increase in the applied field strength and/or the conductivity of the suspension and is interpreted to be a consequence of (a) the necessity to reach a threshold electrical potential difference in the bleb membrane, below which no emission can be triggered, and (b) the finite time required to attain such a transmembranal field during the charging process of the membrane. A quantitative analysis, connecting the lag time, the controllable experimental parameters, and the membrane electrical characteristics is presented. Its verification was carried out in both size-selected and heterogeneous bleb populations. In the latter, experiments were consistent with the assumption that the lag time reflects the charging of the largest blebs. The results indicate (a) the possibility of directly measuring the specific membrane capacitance, yielding an estimate of Cm = 1.2 +/- 0.3 microF/cm2 (the precision being particle size-homogeneity dependent); (b) A minimal transmembranal potential difference (of approximately 240 mV) is necessary to induce electrophotoluminescence; and (c) the lag duration depends on the time elapsed between the preillumination and the external field application. Correlated with the study of ionophore effects on the lag time, this suggests additivity of the light- and field-induced transmembrane potentials in attaining the threshold for emission.     

Breakdown of lipid bilayer membranes in an electric field

The behaviour of lipid bilayer membranes, made of oxidized cholesterol, and UO₂²⁺-modified azolectin membranes in a high electric field has been investigated using the voltage clamp method. When a voltage pulse is applied to the membrane of these compositions, the mechanical rupture of the membranes is preceded by a gradual conductance increase which remains quite reversible till a certain moment. The voltage drop at this reversible stage of breakdown leads to a very rapid (characteristic time of less than 5 μs) decrease in the membrane conductance. At repeated voltage pulses of the same amplitude with sufficient intervals between them (approx. 10 s), the current oscillograms reflecting the reversible resistance decrease are well reproduced on the same membrane. The time of attainment of the predetermined level of the membrane conductance is strongly dependent on voltage. At different stages of breakdown we have investigated changes in the conductance of UO₂²⁺-modified membrane after the application of two-step voltage pulses, the kinetics of development of the reversible decrease in the membrane resistance in solutions of univalent and divalent ions, and also the influence of sucrose and hemoglobin on the current evolution. The relationship between the reversible conductance increase, the reversible electrical breakdown [15] and the rupture of membrane in an electric field is discussed. We propose the general interpretation of these phenomena, based on the representation of the potential-dependent appearance in the membrane of pores, the development of which is promoted by an electric field.     

An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization.

When applied on intact cell suspension, electric field pulses are known to induce membrane permeabilization (electropermeabilization) and fusion (electrofusion). These effects are triggered through a modulation of the membrane potential difference. Due to the vectorial character of the electric field effects, this modulation, which is superimposed on the resting membrane potential difference, is position-dependent on the cell surface. This explains the difference between the experimentally observed critical field strengths requested to trigger the processes of permeabilization and fusion. The critical membrane potential difference which induces membrane permeabilization can be calculated from these experimental observations. It is observed that its value is always about 200 mV for many different cell systems as we previously reported in the case of pure lipid vesicles. This is much less than assumed in most previous studies.     

A model for continuous enzymatic saccharification of cellulose with simultaneous removal of glucose syrup

Cellulase of Trichoderma viride was concentrated in various molecular cutoff membranes, and flux rates and retention of activity were studied under ultra-filtration conditions. Little or no Cellulase was discharged through the membranes tested. The concentrated (5–8-fold) enzymes were used to saccharify finely ground substrate (Solka Floe) in stirred tank (STR) and membrane reactors (MR). A pressure filtration vessel provided with a membrane for simultaneous removal of low molecular weight products (glucose) from the reacting system (Cellulose-Cellulase) is designated as a membrane reactor. Continuous digestion of dense cellulose suspension in the membrane reactor was achieved. Using PM-30 (Amicon) membrane reasonably high mass flux values (9.7–23.3 gals/ft²—day) were obtained in separating glucose from a digest of 30% cellulose suspension. Abcor membrane (HFA 300) was equally effective and necessitated less care in handling. Nearly 14% glucose concentration has been achieved in less than 50 hrs in STR by digesting a 30% cellulose suspension. Based on experimental data a model system is proposed for the continuous steady state Saccharification of ground substrate in which there is continuous removal of concentrated glucose syrup, and a feedback of enzyme.     

Effect of cell density on energy-dependent calcium uptake by Balb/c 3T3 membranes is independent of protein synthesis and attachment to substratum.

Membranes isolated from subconfluent cultures of Balb/c 3T3 cells have low energy-dependent calcium uptake activity. Replating confluent cells at low density results in a prompt fall of energy-dependent calcium uptake by membrane fractions. The level to which uptake activity falls is a function of the density at which the cells are plated (Moore and Pastan, '77b). To determine if regulation of energy-dependent uptake of calcium by membrane fractions is dependent upon attachment to a substrate and to further characterize conditions that regulate the process, we examined calcium uptake activity of membranes isolated from cells in suspension. With cells in suspension energy-dependent calcium uptake activity of isolated membranes falls promptly if cells are diluted to a low density (less than 10(5) cells/ml) and is a function of cell density. When cells in suspension at low cell densities are concentrated to high cell densities (greater than 2 x 10(6) cells/ml), calcium uptake activity of the isolated membrane fraction is increased as a function of cell density. These changes of membrane calcium uptake activity occur promptly and do not require protein synthesis.     

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.     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G₁ protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

Molecular Dynamics Simulations of Lipid Membrane Electroporation

The permeability of cell membranes can be transiently increased following the application of external electric fields. Theoretical approaches such as molecular modeling provide a significant insight into the processes affecting, at the molecular level, the integrity of lipid cell membranes when these are subject to voltage gradients under similar conditions as those used in experiments. This article reports on the progress made so far using such simulations to model membrane—lipid bilayer—electroporation. We first describe the methods devised to perform in silico experiments of membranes subject to nanosecond, megavolt-per-meter pulsed electric fields and of membranes subject to charge imbalance, mimicking therefore the application of low-voltage, long-duration pulses. We show then that, at the molecular level, the two types of pulses produce similar effects: provided the TM voltage these pulses create are higher than a certain threshold, hydrophilic pores stabilized by the membrane lipid headgroups form within the nanosecond time scale across the lipid core. Similarly, when the pulses are switched off, the pores collapse (close) within similar time scales. It is shown that for similar TM voltages applied, both methods induce similar electric field distributions within the membrane core. The cascade of events following the application of the pulses, and taking place at the membrane, is a direct consequence of such an electric field distribution.     

Classifying surfactants with respect to their effect on lipid membrane order

We propose classifying surfactants with respect to their effect on membrane order, which is derived from the time-resolved fluorescence anisotropy of DPH. This may help in understanding why certain surfactants, including biosurfactants such as antimicrobial lipopeptides and saponins, often show a superior performance to permeabilize and lyse membranes and/or a better suitability for membrane protein solubilization. Micelle-forming surfactants induce curvature stress in membranes that causes disordering and, finally, lysis. Typical detergents such as C(12)EO(8), octyl glucoside, SDS, and lauryl maltoside initiate membrane lysis after reaching a substantial, apparently critical extent of disordering. In contrast, the fungicidal lipopeptides surfactin, fengycin, and iturin from Bacillus subtilis QST713 as well as digitonin, CHAPS, and lysophosphatidylcholine solubilize membranes without substantial, overall disordering. We hypothesize they disrupt the membrane locally due to a spontaneous segregation from the lipid and/or packing defects and refer to them as heterogeneously perturbing. This may account for enhanced activity, selectivity, and mutual synergism of antimicrobial biosurfactants and reduced destabilization of membrane proteins by CHAPS or digitonin. Triton shows the pattern of a segregating surfactant in the presence of cholesterol.     

Mechanical and functional aspects of membrane skeletons

Membrane skeletons can be characterized as cytoskeletal structures lying parallel to the bilayer part of cellular and organelle membranes. Typical examples are spectrin network and actin-myosin cortex. We approach the problem of elucidating the function of membrane skeletons by theoretically analyzing mechanical models of the cellular behavior. Membranes of different physical and chemical properties are considered. In erythrocytes and some organelles membrane bilayers are smooth and simply underlaid or overlaid by membrane skeletons. It is argued that there the role of a membrane skeleton is, either, to keep the membrane composition laterally homogeneous as it is in the case of the erythrocyte, or, that it is involved in the processes of the lateral separation of integral membrane proteins as it is happening in the case of some intermediate steps of the vesicular membrane trafficking. In the second type of membranes the bilayer part is ruffled and folded, and there the membrane skeletons play a role in the determination of the cortical tension. Here we explore in more detail the mechanical behavior of a cell with such properties of its boundary. The shape transformations are described which occur under the influence (i) of different external forces, i.e., when an originally spherical cell is aspirated into the micropipette or when such a cell is adsorbed on a flat surface, and (ii) of different internal forces on the cell boundary exerted by the cytoskeletal elements.     

Cytotoxic and cytolytic activity of nonadecafluoro-n-decanoic acid on Acholeplasma laidlawii.

We studied the interactions between the perfluorinated fatty acid nonadecafluoro-n-decanoic acid (NDFDA) and the cell wall-less procaryote Acholeplasma laidlawii, which were cultured in an identical medium base but with different serum supplements. When grown in mycoplasma media supplemented with PPLO serum fraction (Difco Laboratories, Detroit, Mich.), A. laidlawii was rapidly killed by low concentrations of toxicant (less than 1.0 mM). At higher concentrations (greater than 10 mM), NDFDA treatment appeared to lyse cells. A. laidlawii cells grown in horse serum-supplemented mycoplasma media were both killed and lysed at the same NDFDA concentration (greater than 10 mM). These data suggest that this perfluorinated fatty acid can be cytotoxic and cytolytic to mycoplasmas. Changes in active concentrations occurred in parallel with changes in growth medium serum supplementation, which is known to alter mycoplasma membrane composition. We propose that NDFDA interacts with the membranes of A. laidlawii cells, resulting in cell death or cell lysis or both.     

Cytotoxic and cytolytic activity of nonadecafluoro-n-decanoic acid on Acholeplasma laidlawii

We studied the interactions between the perfluorinated fatty acid nonadecafluoro-n-decanoic acid (NDFDA) and the cell wall-less procaryote Acholeplasma laidlawii, which were cultured in an identical medium base but with different serum supplements. When grown in mycoplasma media supplemented with PPLO serum fraction (Difco Laboratories, Detroit, Mich.), A. laidlawii was rapidly killed by low concentrations of toxicant (less than 1.0 mM). At higher concentrations (greater than 10 mM), NDFDA treatment appeared to lyse cells. A. laidlawii cells grown in horse serum-supplemented mycoplasma media were both killed and lysed at the same NDFDA concentration (greater than 10 mM). These data suggest that this perfluorinated fatty acid can be cytotoxic and cytolytic to mycoplasmas. Changes in active concentrations occurred in parallel with changes in growth medium serum supplementation, which is known to alter mycoplasma membrane composition. We propose that NDFDA interacts with the membranes of A. laidlawii cells, resulting in cell death or cell lysis or both.     

Electrically Stimulated Fusion of Different Plant Cell Protoplasts : MESOPHYLL CELL AND GUARD CELL PROTOPLASTS OF VICIA FABA

Cell fusion is induced between guard cell and mesophyll cell protoplasts of Vicia faba by electrical field application. The process of fusion is initiated by electrical breakdown of the cell membrane. Prior to the application of an external electrical field pulse which brings about reversible breakdown of the membrane, the cells (suspended in a low-conducting medium) are brought into close contact with one another by exposing them to an external alternating, nonuniform field (5 volts, electrode distance, 200 micrometers; 500 kiloHertz). During this process, they form “pearl chains” which may become sufficiently long to form bridges between the electrodes. The process is reversible as long as this voltage is not exceeded. Cell fusion is initiated as a result of an electrical field pulse of 50 microseconds duration and of sufficiently high intensity to induce reversible electrical breakdown of the membranes. The process of fusion is completed within 40 minutes or less in the case of guard cell protoplasts, as well as in the case of fusion between guard cell and mesophyll cell protoplasts. The fused cells are spherical in shape, if the fusion product consists only of two or three cells.     

Kinetics of cytolytic T lymphocyte binding to target cells in suspension

Cytolytic T lymphocytes (CTL) were able to specifically bind and lyse allogeneic P815 tumor cells and LPS blast cells in suspension. An assay was developed to measure the rate of target cell binding in suspension independent of the rate of lysis. Target cell binding was found to plateau within 3 hr in suspension. The presence of free, functional CTL and targets at these plateaus was demonstrated, indicating that target cell binding was an equilibrium process. Scatchard plots were used to derive values for Kd (apparent affinity) and bmax (maximum binding). Target cell binding in suspension could not be blocked by purified plasma membranes. Target cell binding was compared for CTL generated by secondary in vitro stimulation with intact cells or with purified membranes. These 2 CTL populations yielded distinct values for Kd and bmax. Implications of this kinetic difference for CTL recognition of purified plasma membranes are discussed.     

A novel method for measuring membrane conductance changes by a voltage-sensitive optical probe.

This study presents a method whose principles enable using a voltage-sensitive optical probe, to quantitatively measure conductivity changes elicited in membrane vesicles and cells. The procedure is based on the fact that the amplitude of the transmembrane potential difference, established across a membrane by an external electric field, is decreased when membrane conductivity is increased upon incorporation of ionophores into the membrane. The method was applied to osmotically swollen thylakoid membranes whose membrane conductivity was changed by the addition of gramicidin or ionomycin. The electric field induced stimulated luminescence from photosystem I (electrophotoluminescence-EPL) was used as a voltage-sensitive optical probe. We calculated the induced conductance changes by using a calibrated EPL vs external electric field response curve and measuring the ionophore-mediated attenuation of the EPL signal. The calculated ionophore-unmodified conductance of the thylakoid membrane yields a value of 171 +/- 56 nS/cm. The value of the membrane conductance, modified by 10 nM gramicidin was found to be 190 +/- 56 nS/cm. The modified membrane conductance and the membrane conductance changes induced by 1 microM ionomycin in the presence of CaCl2 were found to be 186 +/- 3 nS/cm and 15 +/- 3 nS/cm, respectively.     

A long-lived fusogenic state is induced in erythrocyte ghosts by electric pulses

Treatment of erythrocyte ghosts in random positions in a suspension with membrane fusion-inducing direct current electric field pulses causes the membranes to become fusogenic. Significant fusion yields are observed if the membranes are dielectrophoretically aligned into membrane-membrane contact with a weak alternating electric field as much as 5 min after the application of the pulses. This demonstrates that a long-lived membrane structural alteration is involved in this fusion mechanism. Other experiments indicate that the areas on the membrane which become fusogenic after treatment with the pulses may be very highly localized. The locations of these fusogenic areas coincide with where the trans-membrane electric field strength was greatest during the pulse. The fusogenic membrane alteration, or components thereof, in these areas laterally diffuses very slowly or not at all, or, to be fusogenic, must be present at concentrations in the membrane above a certain threshold. The loss of soluble 0.9-3-nm-diameter fluorescent probes from resealed cytoplasmic compartments of randomly positioned erythrocyte ghosts occurs through electric field pulse-induced pores only during a pulse but not between pulses or after a train of pulses if the probe diameter is 1.2 nm or greater. For a given pulse treatment of membranes in random positions in suspensions, an increase in ionic strength of the medium results in (a) a decrease in loss during the pulse, (b) no difference in loss between pulses, and (c) an increase in fusion yield when membrane-membrane contact is established. The latter two results (b and c) are incompatible with a fusion mechanism that proposes a simple relationship between electric field-induced pores and fusion.     

Cellular membrane potentials induced by alternating fields

Membrane potentials induced by external alternating fields are usually derived assuming that the membrane is insulating, that the cell has no surface conductance, and that the potentials are everywhere solutions of the Laplace equation. This traditional approach is reexamined taking into account membrane conductance, surface admittance, and space charge effects. We find that whenever the conductivity of the medium outside the cell is low, large corrections are needed. Thus, in most of the cases where cells are manipulated by external fields (pore formation, cell fusion, cell rotation, dielectrophoresis) the field applied to the cell membrane is significantly reduced, sometimes practically abolished. This could have a strong bearing on present theories of pore formation, and of the influence of weak electric fields on membranes.     

Na, k, and nonspecific solute requirements for induction and function of galactose active transport in an antarctic psychrophilic marine bacterium

Strong adherence of bacteria, yeast, erythrocytes, leukocytes, platelets, spores, and polystyrene spheres to membrane filter materials was noted during filtration through membranes with pore size diameters much larger than the particles themselves. Quantitative recovery on the membrane filters of these particles from low-concentration suspensions was achieved during gravity- or vacuum-assisted filtration through membranes with pore diameters as much as 30 times that of the filtered particles. Mechanical sieving was not responsible. The phenomenon was judged to be electrostatic. It could be partially blocked by pretreating the filter with a nonionic surfactant (Tween 20), and elution of adherent particles was achieved with 0.05% Tween 20. Gram-positive cocci were removed from suspension more efficiently than gram-negative rods. The commonly used cellulose membranes adsorbed more bacteria, blood cells, and other particles than did polycarbonate filters. Of lesser adsorptive capacity were vinyl acetate, nylon, acrylic, and Teflon membranes. Backwashing with saline, serum, 6% NaCl, dextran solutions, or phosphate buffers of varying molality and pH removed only a fraction of adherent particles. Tween 20 (0.05%) eluted up to 45% of adherent particles in a single back-filtration. Selected filters quantitatively removed the particles tested, which then could be washed and subjected to reagents for a variety of purposes. It is important to anticipate the removal of particles during membrane filtration, since it is not a simple mechanical event.     

The gene expression and deficiency phenotypes of Cockayne syndrome B protein in Caenorhabditis elegans

Maculatin 1.1 is an antimicrobial peptide isolated from the Australian tree frog Litoria genimaculata that adopts an amphipathic, alpha-helical structure in solution. Its orientation and conformation when incorporated to pre-formed DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) vesicles was determined using polarised Fourier transform infrared-attenuated total reflection infrared and deuterium exchange experiments. For DMPG membranes, our results show insertion of 70% of the maculatin 1.1 molecules, with an angle of insertion of approximately 35 degrees to the membrane normal and with a predominant alpha-helical structure. These results suggest that maculatin 1.1 acts through a pore-forming mechanism to lyse bacterial membranes. A similar degree of insertion in DMPG (65%) and alpha-helical structure was observed for a biologically inactive, less amphipathic maculatin 1.1 analogue, P15A, although the helix tilt was found to be greater (46 degrees) than for maculatin 1.1. Similar experiments performed using DMPC liposomes showed poor insertion, less than 5%, for both maculatin 1.1 and its analogue. In addition, the shape of the amide I band in these samples is consistent with alpha-helix, beta-structure and disordered structures being present in similar proportion. These results clearly show that maculatin 1.1 inserts preferentially in negatively charged membranes (DMPG) which mimic the negatively charged membrane of Gram-positive bacteria. We attribute the high percentage of insertion of the biologically inactive analogue in DMPG to the fact that its concentration on the membrane surface in our experiments is likely to be much higher than that found in physiological conditions.