Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Giant protoplasts of Saccharomyces cerevisiae of 10-35 microm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and "patchability" of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66+/-0.07 microF/cm(2)) and conductance (23-44 microS/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.     

Electric Field Effects with Alcohol Dehydrogenase

Extending previous work with electrostimulation of yeast dehydrogenases in cell suspension, we attempted to influence the alcohol dehydrogenase system in vitro using methylene blue as the acceptor. According to the conformational coupling model and the free radical hypothesis, the possibility of capacitive current treatment was tested by short applications of E = 10, 20, and 30 V/cm at a frequency of 50 Hz. Besides a temperature rise, no significant kinetic data from spectrophotometry or polarography could be measured, nor could inductive coupling using magnetic fluxes of 1, 5, and 10 mT. On the other hand, marked changes of relative electrofusion yield were found after interaction of dehydrogenases with membrane surfaces of barley protoplasts. It was confirmed that in unbuffered solution containing 0.5 M mannitol, the isoelectric point (PI) of enzymes determines the relative fusion yield: Fr < 1 for pI < 7 and Fr > 1 for pI > 7. We must conclude that only the position of dehydrogenases at or within cell membranes shows responses to weak ac or stronger dc treatments influencing additionally electric moments and conformations by surface potential and adsorption energy.     

The Effect of PS Content on the Ability of Natural Membranes to Fuse with Positively Charged Liposomes and Lipoplexes

Supramolecular aggregates containing cationic lipids have been widely used as transfection mediators due to their ability to interact with negatively charged DNA molecules and biological membranes. First steps of the process leading to transfection are partly electrostatic, partly hydrophobic interactions of liposomes/lipoplexes with cell and/or endosomal membrane. Negatively charged compounds of biological membranes, namely glycolipids, glycoproteins and phosphatidylserine (PS), are responsible for such events as adsorption, hemifusion, fusion, poration and destabilization of natural membranes upon contact with cationic liposomes/lipoplexes. The present communication describes the dependence of interaction of cationic liposomes with natural and artificial membranes on the negative charge of the target membrane, charges which in most cases were generated by charging the PS content or its exposure. The model for the target membranes were liposomes of variable content of PS or PG (phosphatidylglycerol) and erythrocyte membranes in which the PS and other anionic compound content/exposure was modified in several ways. Membranes of increased anionic phospholipid content displayed increased fusion with DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) liposomes, while erythrocyte membranes partly depleted of glycocalix, its sialic acid, in particular, showed a decreased fusion ability. The role of the anionic component is also supported by the fact that erythrocyte membrane inside-out vesicles fused easily with cationic liposomes. The data obtained on erythrocyte ghosts of normal and disrupted asymmetry, in particular, those obtained in the presence of Ca²⁺, indicate the role of lipid flip-flop movement catalyzed by scramblase. The ATP-depletion of erythrocytes also induced an increased sensitivity to hemoglobin leakage upon interactions with DOTAP liposomes. Calcein leakage from anionic liposomes incubated with DOTAP liposomes was also dependent on surface charge of the target membranes. In all experiments with the asymmetric membranes the fusion level markedly increased with an increase of temperature, which supports the role of membrane lipid mobility. The decrease in positive charge by binding of plasmid DNA and the increase in ionic strength decreased the ability of DOTAP liposomes/lipoplexes to fuse with erythrocyte ghosts. Lower pH promotes fusion between erythrocyte ghosts and DOTAP liposomes and lipoplexes. The obtained results indicate that electrostatic interactions together with increased mobility of membrane lipids and susceptibility to form structures of negative curvature play a major role in the fusion of DOTAP liposomes with natural and artificial membranes.     

Effect of membrane surface charge on nickel uptake by purified mung bean root protoplasts

The influence of membrane surface charge on cation uptake was investigated in protoplasts prepared from roots of mung bean (Vigna radiata L.). Confocal laser scanning microscopy showed that a fluorescent trivalent cation accumulated to very high concentrations at the surface of the protoplasts when they were incubated in medium containing low concentrations of Ca or other cations, but that this accumulation could be completely reversed by suppression of membrane surface negativity by high cation concentrations. Influx of 63Ni was strongly reduced by a range of divalent cations. Increasing the Ca concentration in the medium from 25 microM to 10 mM inhibited 63Ni influx by more than 85%. 63Ni influx was also inhibited by 85% by reducing the pH from 7 to 4. Computation of the activity of Ni at the membrane surface under the various treatment conditions showed that Ni uptake was closely correlated with its activity at the membrane surface but not with its concentration in the bulk medium. It was concluded that the effects on Ni uptake of addition of monovalent, divalent and trivalent cations, and of variations in pH are all consistent with the proposition that the activity of Ni at the membrane surface is the major determinant of the rate of Ni influx into mung bean protoplasts. It is proposed that the surface charge on the plasma membrane will influence the membrane transport of most charged molecules into cells.     

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.     

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Giant protoplasts of Saccharomyces cerevisiae of 10-35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 ± 0.07 µF/cm²) and conductance (23-44 µS/cm²) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.     

Flow cytometric quantification of electroporation-mediated uptake of macromolecules into plant protoplasts

Summary Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplasts. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasma membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to the presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast populations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and transient gene expression following plasmid uptake. Thus, simultaneous electroporation of protoplasts with foreign DNA and FITC-dextran followed by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.Abbreviations ADC analogue to digital converter - CAT chloramphenicol acetyl transferase (enzyme) - cat chloramphenicol acetyl transferase (gene) - CPW solution cell and protoplast wash solution - DC direct current - EF electrofusion - FALS forward angle light scatter - FITC fluorescein isothiocyanate - FITC-dextran fluorescein isothiocyanate conjugated dextran - PI propidium iodide - PMT photomultipliertube - TLC thin layer chromatography     

Surface Charge Density of Hetero-Fused Plant Protoplasts: an Electrophoretic Study

Electrophoretic mobilities of hetero-fused plant protoplasts,which were obtained by electrofusion of barley mesophyll cellprotoplasts and Rauwolfia serpentina cultured cell protoplasts,and those of the unfused parent protoplasts were measured invarious media of different pH values. At pH 5.2, the zeta potentialof the fused protoplasts was intermediate between those of thebarley and R. serpentina protoplasts and the average surfacecharge density of the fused protoplasts was closer to that ofR. serpentina than to that of barley. The distribution of thesurface charge density of fused protoplast obtained at pH 5.2is discussed in terms of the surface charge densities and thesizes of parent protoplasts. These results revealed that thesurface charge density of fused protoplasts was determined bythe surface charge densities and the ratio of the surface areasof the respective parent protoplasts. (Received December 28, 1989; Accepted August 10, 1990)     

Dextran sulfate-dependent fusion of liposomes containing cationic stearylamine.

The incorporation of the positively charged stearylamine into phosphatidylcholine liposomes was studied by measuring electrophoretic mobilities. Up to a molar ratio SA/PC = 0.5 an increase of the positive zeta potential can be observed. Addition of the negatively charged macromolecule dextran sulfate leads to a change of the sign of the surface potential of the PC/SA liposomes indicating binding of the macromolecule to the surface. This process is accompanied by an increase in turbidity, which is dependent on the molecular weight of the dextran sulfate and the SA concentration (measured by turbidimetry). Using the NBD/Rh and Pyr-PC fluorescence assays the fusion of SA containing liposomes was investigated. A strong influence of the SA content and molecular weight of dextran sulfate on the fusion extent was observed. The fusion extent is proportional to the SA content in the PC membrane and the molecular weight of dextran sulfate. PC/SA/PE liposomes exhibit a higher fusion extent after addition of dextran sulfate compared to PC/SA liposomes indicating that PE additionally destabilizes the bilayer. Freeze-fracture electron microscopy reveals that the reaction products are large complexes composed of multilamellar stacks of tightly packed, straight membranes and aggregated vesicles. The tight packing of the membranes in the stacks (and the narrow contact of the aggregated vesicles) indicates a strong adherence of opposite membrane surfaces induced by dextran sulfate.     

ON THE CAUSE OF THE INFLUENCE OF IONS ON THE RATE OF DIFFUSION OF WATER THROUGH COLLODION MEMBRANES. II

1. It had been shown in previous publications that when pure water is separated from a solution of an electrolyte by a collodion membrane the ion with the same sign of charge as the membrane increases and the ion with the opposite sign of charge as the membrane diminishes the rate of diffusion of water into the solution; but that the relative influence of the oppositely charged ions upon the rate of diffusion of water through the membrane is not the same for different concentrations. Beginning with the lowest concentrations of electrolytes the attractive influence of that ion which has the same sign of charge as the collodion membrane upon the oppositely charged water increases more rapidly with increasing concentration of the electrolyte than the repelling effect of the ion possessing the opposite sign of charge as the membrane. When the concentration exceeds a certain critical value the repelling influence of the latter ion upon the water increases more rapidly with a further increase in the concentration of the electrolyte than the attractive influence of the ion having the same sign of charge as the membrane. 2. It is shown in this paper that the influence of the concentration of electrolytes on the rate of transport of water through collodion membranes in electrical endosmose is similar to that in the case of free osmosis. 3. On the basis of the Helmholtz theory of electrical double layers this seems to indicate that the influence of an electrolyte on the rate of diffusion of water through a collodion membrane in the case of free osmosis is due to the fact that the ion possessing the same sign of charge as the membrane increases the density of charge of the latter while the ion with the opposite sign diminishes the density of charge of the membrane. The relative influence of the oppositely charged ions on the density of charge of the membrane is not the same in all concentrations. The influence of the ion with the same sign of charge increases in the lowest concentrations more rapidly with increasing concentration than the influence of the ion with the opposite sign of charge, while for somewhat higher concentrations the reverse is true.     

Effects of chemical sanitization using NaOH on the properties of polysulfone and polyethersulfone ultrafiltration membranes

Membranes used in bioprocessing applications are typically sanitized before use to insure aseptic operation. However, there is almost no information in the literature on the effects of this preuse sanitization step on the properties of the membrane. Experiments were performed with commercially available hollow fiber polysulfone (PSf) and polyethersulfone (PES) membranes with different nominal molecular weight cutoffs. Data were obtained for the membrane hydraulic permeability, dextran retention coefficients, zeta potential (surface charge), and extent of protein adsorption both before and after sanitization with 0.5 N NaOH at 45°C for 30 min. Changes in chemical composition were examined using ATR‐FT‐IR and XPS. Sanitization caused a large increase in the net negative charge for all membranes. There was a small reduction in hydraulic permeability and a significant increase in dextran retention for the polyethersulfone membranes, consistent with a reduction in the effective pore size. Spectroscopic analyses suggest that this change is likely due to the base‐catalyzed hydrolysis of the lactam ring in polyvinylpyrrolidone (PVP) that is typically is used as a wetting/pore‐forming agent in PSf and PES membranes. Preuse sanitization also appeared to have a small effect on protein adsorption, although the extent of adsorption was quite low for both the virgin and sanitized membranes. The observed changes in membrane properties could have a significant impact on the ultrafiltration performance, demonstrating the importance of standardizing the sanitization procedures even in process development and scale‐down validation studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:90–96, 2015     

The influence of phytohormones on zeta potential and electrokinetic charges of winter wheat cells

The zeta potential measurements of protoplasts obtained from winter wheat cell culture and phospholipid liposomes were performed to determine the electrokinetic charge in a medium containing various phytohormones (kinetin, 2,4-D and zearalenone) in absence and in presence of 2 x 10(-5) MCa2+. Calli were induced from immature inflorescences (inf) and embryos (emb) and cultured to obtain non-embryogenic (NE) and embryogenic (E) cell tissues. All investigated phytohormones indicate ability to adsorb to the negatively charged surfaces (latex, L88 - model negative adsorption site) both in water solutions and at the presence of mannitol and buffer (MES). In biological systems (protoplasts and liposomes - prepared from phospholipids of protoplasts) the electrokinetic charges were dependent on the phospholipid and protein composition of cells. The influence of protein groups on electrokinetic charge was calculated from charge values of protoplasts and liposomes, assuming additivity of surface charges. The comparison of calculated charges for protoplasts and liposomes indicate that 2,4-D is better adsorbed to the phospholipid and proteins of NE cells whereas kinetin is bound to the phospholipid and protein sites of E calli. This effect may be connected with embryogenesis process, where non-embryogenic culture of wheat requires 2,4-D in the medium, and embryogenic culture requires cytokinin rather. Zearalenone binding is especially dependent on the kind of explant.     

A test of discreteness-of-charge effects in phospholipid vesicles: measurements using paramagnetic amphiphiles

A new series of negatively charged, paramagnetic alkylsulfonate probes was synthesized and can be used to measure both the internal and the external surface potentials of model membrane systems. We tested for discreteness-of-charge effects in lipid membranes by comparing the surface potentials, estimated by use of these negatively charged amphiphiles, with that of a series of positively charged alkylammonium nitroxides in charged membranes. From the partitioning of these probes, the membrane surface potential was estimated in phosphatidylcholine membranes containing either phosphatidylserine or didodecyldimethylammonium bromide. The surface potentials, estimated with either positive or negative probes, were identical, within experimental error, in either positive or negative membranes, and they were well accounted for by a simple Gouy-Chapman-Stern theory. This symmetry, with respect to the sign of the charge, indicates that discreteness-of-charge effects are not significant in determining the potential-sensitive phase partitioning of these probes in model membranes. Thus, despite the fact that charge on membranes is discrete, models that assume a uniform density of charge in the plane of the membrane adequately account for the potentials measured by these amphiphilic probes.     

Poisson-Boltzmann Theory for Membranes with Mobile Charged Lipids and the pH-Dependent Interaction of a DNA Molecule with a Membrane

We consider a planar stiff model membrane consisting of mobile surface groups whose state of charge depends on the pH and the ionic composition of the adjacent electrolyte solution. To calculate the mean-field interaction potential between a charged object and such a model membrane, one needs to solve a Poisson-Boltzmann boundary value problem. We here derive and discuss the boundary condition at the membrane surface, a condition that is generally appropriate for biological membranes where two charge-regulating mechanisms are present at the same time: the pH-dependent chemical charge regulation and a regulation through the in-plane mobility of the surface groups. As an application of this general formalism, we consider the specific example of a single DNA molecule, approximated by a cylinder with smeared-out surface charges, interacting with such a model membrane. We study the effect that the two competing charge-regulating mechanisms have on the DNA/membrane interaction and the distribution of surface ions in the plane of the membrane. We find that, at short DNA-membrane distances, membrane fluidity can have a considerable impact on the DNA adsorption behavior and can lead to such counterintuitive phenomena as the adsorption of a negatively charged DNA onto a (on average) negatively charged membrane.     

Influence of the conformational state on the isoelectric points of rat brain synaptosomes, mitochondria and mitoplasts

The isoelectric points of rat brain synaptosomes, mitochondria and mitoplasts have been determined by using different charged two-phase systems containing dextran and poly(ethylene glycol). The cross-partition diagrams of these organelles show isoelectric points at pH 4.1, 4.5 and 4.7, respectively. The influence of the conformational state of mitochondrial membranes upon their partition in two-phase systems has been studied. Shrunk mitoplasts showed a large change in their partition behavior as reflected by an increased affinity for the lower dextran phase, while shrinkage of mitochondria did not affect their partition. Shrunk mitoplasts showed the same isoelectric point of pH 4.7 as swollen mitoplasts, which indicates that no charge changes occurred on the outer side of the inner mitochondrial membrane during shrinkage of mitoplasts.     

Electrostatic effects upon adsorption and desorption of polylysines on the surface of lipid membranes of different composition

Electrokinetic measurements are carried out in suspensions of liposomes made from mixtures of charged (cardiolipin, CL) and neutral (phosphatidylcholine, PC) lipids in the presence of lysine and lysine-based polypeptides. Neither monolysine nor polylysines adsorbed on neutral (PC) membranes. In the case of negatively charged membranes (CL/PC) all polypeptides showed a sharp dependence of liposome electrophoretic mobility on the amount of polymer added to the cell. In suspension of cardiolipin liposomes the position of zero charge point coincided for all high-molecular polylysines; thus, pentalysine neutralizes the membrane surface, whereas polycations with a higher polymerization degree change a sign of the surface charge. Electrophoretic mobility of liposomes in plateau range depended on the molecular weight of polylysines and composition of liposomes; for large macromolecules the absolute value came close to its value for the initial liposomes. Adsorption of polycations on planar bilayer lipid membranes (BLM) resulted in alteration of the boundary potential measured by the method of intramembranous field compensation (IFC). The electrokinetic measurements and IFC method gave close results in the case of lysine monomers; their surface concentration could be fitted by an isotherm of the molecule distribution between the membrane surface and solution. Considerable differences of the surface and boundary potentials found in the case of pentalysine, correspond to changes in the dipole component of boundary potential induced by the adsorbed molecules. Using the IFC method, the kinetics of the adsorption process before saturation was studied. The adsorption of polylysines was markedly slower (more than hour) than that of pentalysine (tens of min) or monolysine (minutes). Washout experiments showed that adsorption of penta-and monolysine on planar BLM was reversible, while that of high-molecular polylysines was practically irreversible.     

Effects of la on surface charges, dielectrophoresis, and electrofusion of barley protoplasts

When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 millimolar lanthanum ion (La³⁺) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N, N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl₂), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La³⁺ -treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La³⁺ -treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La³⁺ after washing without EDTA and released most of the bound La³⁺ by washing with 1 millimolar EDTA. This tightly bound La³⁺ seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La³⁺ -treated protoplasts after washing. ζ-potentials of protoplasts were -39.0±3.2 millivolts, -16.7 ± 2.6 millivolts, and virtually zero in media containing 0, 0.1, and 0.3 millimolar La³⁺ (I = 7.2 millimolar), respectively, and had a positive value (+ 14.2 ± 2.2 millivolts) in the presence of 1 millimolar La³⁺. These effects of La³⁺ on ζ-potentials were easily abolished by washing without EDTA. This indicates that charged species located at the surface of plasma membrane of protoplasts cannot account for the sites at which La³⁺ exerts its inhibition of dielectrophoresis and electrofusion. In contrast, the promotion of spherical fusion and the reduction of broken fusion products observed in the presence of La³⁺ were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently.     

Insulin adsorption into porous charged membranes: Effect of the electrostatic interaction

Insulin adsorption into a series of porous charged membranes was investigated by batch adsorption experiments, and the experimental results were analyzed by the homogeneous diffusion model. The membranes used in this study were prepared by pore‐surface modification of porous poly(acrylonitrile) (PAN) membranes by grafting with weak acidic and basic functional groups. The amount of insulin adsorbed into the membrane was determined from the material balance of insulin. The insulin partition coefficient K between the membrane and solution was estimated from the equilibrium adsorption amount, and the effective diffusion coefficient D was estimated by matching the model with the experimental data as a fitting parameter. The dependence of K and D on the charge properties of the insulin and membrane is observed and discussed. The partition coefficient K increased when the insulin and the membrane carried opposite charges, on the other hand, the effective diffusion coefficient D was reduced. These results indicate that the electrostatic interaction between the insulin and the membranes played an important role in the insulin adsorption. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

Differential effects of electrofusion and electropermeabilization parameters on the membrane integrity of plant protoplasts

Ethane was used as an indicator of lipid peroxidation in order to characterize the membrane damage induced by electrical pulses during the processes of electrofusion and electropermeabilization. The increase of ethane in fused protoplasts ofVicia faba L. was found to be correlated with the intensity of field strength and pulse number, which also affected the yield of hybrids. The degree of membrane damage is postulated to depend on the accumulation of lipid free-radicals, which can be increased by light, by longer storage time of protoplasts and by higher field strength and pulse number. As a result, the conditions for electropermeabilization lead to greater membrane damage compared with those for electrofusion. The measurement of ethane production may prove to be useful for characterization of the membrane integrity, viability and regeneration ability of protoplasts.     

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3-pyrene)maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.