Influence of obstacles on lipid lateral diffusion: computer simulation of FRAP experiments and application to proteoliposomes and biomembranes

Fluorescence Recovery After Photobleaching experiments were simulated using a computer approach in which a membrane lipid leaflet was mimicked using a triangular lattice obstructed with randomly distributed immobile and non-overlapping circular obstacles. Influence of the radius r and area fraction c of these obstacles and of the radius R of the observation area on the relative diffusion coefficient D ^* (Eq. (1)) and mobile fraction M was analyzed. A phenomenological equation relating D ^* to r and c was established. Fitting this equation to the FRAP data we obtained with the probe NBD-PC embedded in bacteriorhodopsin/egg-PC multilayers suggests that this transmembrane protein rigidifies the surrounding lipid phase over a distance of about 18 Å (two lipid layers) from the protein surface. In contrast, analysis of published diffusion constants obtained for lipids in the presence of gramicidin suggests that in terms of lateral diffusion, this relatively small polypeptide does not significantly affect the surrounding lipid phase. With respect to the mobile fraction M, and for point obstacles above the percolation threshold, an increase in R led to a decrease in M which can be associated with the existence of closed domains whose average size and diffusion properties can be determined. Adaptation of this model to the re-interpretation of the FRAP data obtained by Yechiel and Edidin (J Cell Biol (1987) 115:755–760) for the plasma membrane of human fibroblasts consistently leads to the suggestion that the lateral organization of this membrane would be of the confined type, with closed lipid domains of 0.5 µm² in area.Abbreviations and notations used BR bacteriorhodopsin - DMPC dimyristoylphosphatidylcholine - diOC18 dioctadecyloxatricarbocyanine - egf-PC egg-yolk phosphatidylcholine - NBD-PC 1-acyl2-[t2-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine - MOPS 3-[N-morpholino]propane sulfonic acid - FRAP Fluoresence Recovery After photobleaching - D observed diffusion coefficient - D0 diffusion coefficient in the absence of obstacles - D ^* relative diffusion constant (Eq. 1) - M mobile fraction - c obstacle area fraction - r obstacle radius - R observation area radius - r _d diffusion area radius Correspondence to: A. Lopez     

Electrostatic interactions in proteins: Calculations of the electrostatic term of free energy and the electrostatic potential field

The pH-dependence of the electrostatic energy of interactions between titratable groups is calculated for some well studied globular proteins: basic pancreatic trypsin inhibitor, sperm whale myoglobin and tuna cytochrome c. The calculations are carried out using a semi-empirical appraach in terms of the macroscopic model based on the Kirkwood-Tanford theory. The results are discussed in the light of their physicochemical and biological properties. It was found that the pH-dependence of the electrostatic energy correlates with the III–IV transition of cytochrome c. The electrostatic field of the cysteine proteinase inhibitor, cystatin, was calculated in two ways. In the first one, the electrostatic field created by the pH dependent charges of the ionizable groups and peptide dipoles was calculated using the approach proposed. In the second one, the finite-difference method was used. The results obtained by the two methods are in overall agreement. The calculated field was discussed in terms of the binding of cystatin to papain.     

Effects of ceramide and other simple sphingolipids on membrane lateral structure

The available data concerning the ability of ceramide and other simple sphingolipids to segregate laterally into rigid, gel-like domains in a fluid bilayer has been reviewed. Ceramides give rise to rigid ceramide-enriched domains when their N-acyl chain is longer than C12. The high melting temperature of hydrated ceramides, revealing a tight intermolecular interaction, is probably responsible for their lateral segregation. Ceramides compete with cholesterol for the formation of domains with lipids such as sphingomyelin or saturated phosphatidylcholines; under these conditions displacement of cholesterol by ceramide involves a transition from a liquid-ordered to a gel-like phase in the domains involved. When ceramide is generated in situ by a sphingomyelinase, instead of being premixed with the other lipids, gel-like domain formation occurs as well, although the topology of the domains may not be the same, the enzyme causing clustering of domains that is not detected with premixed ceramide. Ceramide-1-phosphate is not likely to form domains in fluid bilayers, and the same is true of sphingosine and of sphingosine-1-phosphate. However, sphingosine does rigidify pre-existing gel domains in mixed bilayers.     

Effect of a 2-hydroxylated fatty acid on Cholesterol-rich membrane domains

Abstract

2-Hydroxyoleic acid (2OHOA) is a synthetic fatty acid with antihypertensive properties that is able to alter structural membranes properties. The main purpose of this study was to analyze the effect of 2OHOA on the membrane architecture in cholesterol (Cho)-rich domains. For this purpose, model membranes mimicking the composition of lipid rafts and PC- or PE-Cho-rich domains were examined in the absence and presence of 2OHOA by synchrotron X-ray diffraction, atomic force microscopy (AFM) and microcalorimetry (DSC) techniques. Our results demonstrate that 2OHOA phase separates from lipid raft domains and affects the lateral organization of lipids in the membrane. In model raft membranes, 2OHOA interacted with the sphingomyelin (SM) gel phase increasing the thickness of the water layer, which should lead to increased bilayer fluidity. The hydrogen binding competition between 2OHOA and Cho could favour the enrichment of 2OHOA in SM domains separated from the SM-Cho domains, resulting in an enhanced phase separation into SM-2OHOA-rich liquid-disordered (non-raft) and SM-Cho-rich liquid-ordered (raft) domains. The segregation into 2OHOA-rich/Cho-poor and 2OHOA-poor/Cho-rich domains was also observed in PC bilayers.     

Lipid lateral diffusion and membrane heterogeneity

The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using ²H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (l_d) and liquid ordered (l_o) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of l_d and l_o phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the l_dand l_o phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-T_m and one low-T_m lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid.     

Lateral diffusion of proteins and lipids in the plasma membrane of rose protoplast

Summary Lateral diffusion measurements have been made on lipids and proteins in the plasma membrane of live protoplasts derived from rose (Rosa sp. Paul's Scarlet) suspension-cultured cells. Two different fluorescent lipid probes exhibited markedly different diffusion rates, indicating possible heterogeneity in the lipid domain of the membrane. Membrane proteins were labeled directly with covalently-reactive fluorophores, and factors that might perturb the lateral diffusion of these labeled proteins were investigated. Treatment of the protoplasts with various cytoskeleton-disrupting drugs generally had little effect on protein diffusion, although treatment with oryzalin, a microtubule-disrupting drug, did slightly reduce the mobile fraction of membrane proteins. Elevation of the CaCl₂ concentration in the medium from 1 mM to 10 mM significantly reduced the mobile fraction of membrane proteins and also increased the fraction of protoplasts that were able to regenerate cell walls and divide in culture. These results are discussed in relation to reported evidence of lipid domains in the plasma membranes of other cells and protoplasts. The relative importance of lipid domains and membrane-cytoskeleton interaction in governing protein diffusion is considered.Abbreviations D lateral diffusion coefficient - RCA Ricinus communis agglutinin - BPA Bauhinia purpurea agglutinin - DTAF dichlorotriazinylaminofluorescein - FTSC fluorescein-5-thiosemicarbazide - C₁₈-Fl 5-(N-octadecanoyl)aminofluorescein - LY-Chol Lucifer yellow conjugate of cholesterol, i.e., dilithium 4-amino-N-[(-(carbo(5-cho-lesten-3-yl)oxy)hydrazinocarbonyl)amino]-1,8-naphthalimide-3,6-disulfonate - APM amiprophosmethyl - DMSO dimethylsulfoxide - FPR fluorescence photobleaching recovery - sd standard deviation - FRAF fluorescence redistribution after fusion - M mobile fraction     

Effect of NaCl and CaCl2 on the lateral diffusion of zwitterionic and anionic lipids in bilayers

The influence of addition of NaCl or CaCl₂ (0.3 and 0.1 M, respectively) on the lateral diffusion coefficient (D_L) of dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylglycerol (DOPG) was measured by the pulsed field gradient NMR technique. D_L of DOPC was unaffected, whereas the DOPG diffusion decreased with salt concentration. ²³Na NMR quadrupole splittings of DOPG between 20 and 60 °C and added NaCl between 0 and 15 wt% decreased only slightly with salt content, but increased with increasing temperature. Similar results were obtained for palmitoyloleoylphosphatidylglycerol, in which the palmitoyl chain order parameter increased slightly with salt. A model with free and “bound” ions was used to interpret the splitting data.With increasing salt content a decrease in the water permeability for DOPG was observed, but not for DOPC, as measured by water diffusion perpendicular to the oriented lipid bilayers.It was concluded that calcium and sodium ions interacted with the DOPG head-groups resulting in a decrease in the “free area” per lipid molecule due to a screening of the charged lipid head-groups. Thus, there was a closer packing of DOPG, leading to a decrease in D_L and water permeability. DOPC did not show any changes in the bilayer properties upon the addition of ions.     

Photosynthesis can be enhanced by lateral CO2 diffusion inside leaves over distances of several millimeters

This study examines the extent to which lateral gas diffusion can influence intercellular CO(2) concentrations (c(i)) and thus photosynthesis in leaf areas with closed stomata. Leaves were partly greased to close stomata artificially, and effects of laterally diffusing CO(2) into the greased areas were studied by gas-exchange measurement and chlorophyll fluorescence imaging. Effective quantum yields (Delta F/F(m)') across the greased areas were analysed with an image-processing tool and transposed into c(i) profiles, and lateral CO(2) diffusion coefficients (D(C'lat)), directly proportional to lateral conductivities (), were estimated using a one-dimensional (1D) diffusion model. Effective CO(2) diffusion distances in Vicia faba (homobaric), Commelina vulgaris (homobaric) and Phaseolus vulgaris (heterobaric) leaves clearly differed, and were dependent on D(C'lat), light intensity, [CO(2)], and [O(2)]: largest distances were approx. 7.0 mm for homobaric leaves (with high D(C'lat)) and approx. 1.9 mm for heterobaric leaves (low D(C'lat)). Modeled lateral CO(2) fluxes indicate large support of photosynthesis over submillimeter distances for leaves with low D(C'lat), whereas in leaves with large D(C'lat), photosynthesis can be stimulated over distances of several millimeters. For the plant species investigated, the surplus CO(2) assimilation rates of the greased leaf areas (A(gr)) differed clearly, depending on lateral conductivities of the respective leaves.     

Electrostatic effects in a large enzyme complex: subunit interactions and electrostatic potential field of the icosahedral beta 60 capsid of heavy riboflavin synthase

The role of the electrostatic interactions in the stability of the icosahedral beta 60 capsid of heavy riboflavin synthase from Bacillus subtilis has been investigated using an approach based on the theory of Kirkwood and Tanford. The pH dependence of the electrostatic subunit interactions agrees well with experimental data. The electrostatic subunit interaction energy has a pronounced minimum at pH 8.2 for both the ligated and ligand-free capsid. The latter is characterized by a reduction of the magnitude and the pH range of the electrostatic attraction. It is found that only 8 charged groups, which form one cluster and two ion pairs, provide a significant contribution to the capsid stability. The analysis has shown that the aggregation/disaggregation equilibrium seems to be regulated by electrostatic interactions between beta-subunits forming dimers, which connect the relatively stable pentamers in the beta-60 capsid. The release of the ligand causes a reduction of the electrostatic attraction of the dimers, which may induce disaggregation of the capsid. The electrostatic potential field due to the titratable groups and alpha-helix macrodipoles has been calculated on the basis of the Coulomb relation. Two different values of the dielectric constant have been used for the protein and the surrounding solvent, respectively. The electrostatic potential shows a radially polar distribution with a positive pole at the inner capsid wall and a negative pole outside the capsid. An interesting feature of the electrostatic field is the formation of positive potential "channels" that coincide with the channels constituted by the pentameric and trimeric beta-subunit aggregates. It is supposed that the electrostatic potential field plays a role in enzyme-substrate recognition.     

Predicting membrane protein type by functional domain composition and pseudo-amino acid composition

Given the sequence of a protein, how can we predict whether it is a membrane protein or non-membrane protein? If it is, what membrane protein type it belongs to? Since these questions are closely relevant to the function of an uncharacterized protein, their importance is self-evident. Particularly, with the explosion of protein sequences entering into databanks and the relatively much slower progress in using biochemical experiments to determine their functions, it is highly desired to develop an automated method that can be used to give a fast answers to these questions. By hybridizing the functional domain (FunD) and pseudo-amino acid composition (PseAA), a new strategy called FunD-PseAA predictor was introduced. To test the power of the predictor, a highly non-homologous data set was constructed where none of proteins has 25% sequence identity to any other. The overall success rates obtained with the FunD-PseAA predictor on such a data set by the jackknife cross-validation test was 85% for the case in identifying membrane protein and non-membrane protein, and 91% in identifying the membrane protein type among the following 5 categories: (1) type-1 membrane protein, (2) type-2 membrane protein, (3) multipass transmembrane protein, (4) lipid chain-anchored membrane protein, and (5) GPI-anchored membrane protein. These rates are much higher than those obtained by the other methods on the same stringent data set, indicating that the FunD-PseAA predictor may become a useful high throughput tool in bioinformatics and proteomics.     

Lateral diffusion of gramicidin S,M-13 coat protein and glycophorin in bilayers of saturated phospholipids: Mean field and Monte Carlo studies

We have developed a general model that relates the lateral diffusion coefficient of one isolated large intrinsic molecule (mol. wt. ?1000) in a phosphatidylcholine bilayer to the static lipid hydrocarbon chain order. We have studied how protein lateral diffusion can depend upon protein-lipid interactions but have not investigated possible non-specific contributions from gel-state lattice defects. The model has been used in Monte Carlo simulations or in mean-field approximations to study the lateral diffusion coefficients of Gramicidin S, the M-13 coat protein and glycophorin in dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC) bilayers as functions of temperature. Our calculated lateral diffusion coefficients for Gramicidin S and the M-13 coat protein are in good agreement with what has been observed and suggest that Gramicidin S is in a dimeric form in DMPC bilayers. In the case of glycophorin we find that the ‘ice breaker’ effect can be understood as a consequence of perturbation of the lipid polar region around the protein. In order to understand this effect is is necessary that the protein hydrophilic section perturb the polar regions of at least approx. 24 lipid molecules, in good agreement with the numbers of 29–30 measured using ³¹P-NMR. Because of lipid-lipid interactions this effect extends itself out to four or five lipid layers away from the protein so that the hydrocarbon chains of between approx. 74 and approx. 108 lipid molecules are more disordered in the gel phase, so contributing less to the transition enthalpy, in agreement with the numbers of 80–100 deduced from differential scanning calorimetry (DSC). An understanding of the abrupt change in the diffusion coefficient at a temperature below the main bilayer transition temperature requires an additional mechanism. We propose that this change may be a consequence of a ‘coupling-uncoupling’ transition involving the protein hydrophilic section and the lipid polar regions, which may be triggered by the lipid bilayer pretransition. Our calculation of the average number of gauche bonds per lipid chain as a function of temperature and distance away from an isolated polypeptide or integral protein shows the extent of statically disordered lipid around such molecules. The range of this disorder depends upon temperature, particularly near the main transition.     

Modelling mechanosensitivity in membranes: effects of lateral tension on ionic pores in a microcystin toxin-containing membrane

Lipid bilayers of diphytanoyl lecithin (DPhL) in which a cyanobacterial toxin, microcystin-LR (MC-LR) was incorporated, were found to be a convenient model of natural mechanosensitive membranes. The effects of pressure difference, leading to lateral membrane tension, on artificial membranes formed on the tips of glass micropipettes were investigated using patch-clamp methodology. Emplacement of MC-LR from the bathing solution was enhanced by transmembrane voltage and/or pressure difference. MC-LR pores could be recorded over a wide voltage range, their opening probability being first increased and then reduced at high membrane potential. The pores exhibited several open pore conductance levels, the higher conductance states being more probable at greater lateral tensions. Ion gradient experiments established that the MC-LR pores are cation selective, but discriminate only weakly between K and Na. These results suggest that a lipid liquid crystal matrix containing monomers of multimeric pore-forming molecules could be used as a mechanical sensor and molecular switch.Offprint requests to: P. N. R. Usherwood     

The study of the conformation and interaction of two tachykinin peptides in membrane mimicking systems by NMR spectroscopy and pulsed field gradient diffusion

Pulsed‐field gradient diffusion has been used to study the binding of two tachykinin peptides, [Tyr⁸]‐substance P (SP) and [Tyr⁰]‐neurokinin A (NKA) to two membrane‐mimicking micelles, dodecylphosphocholine, and sodium dodecylsulfate. The structure of these peptides bound to the micelles have also been studied by using two‐dimensional nmr and restrained simulated annealing calculations. No major difference in the structures of each peptide in the two micellar media was found. The difference between the micelle‐bound structure of [Tyr⁸]SP and that of SP was also minor. The longer helical conformation on the C‐terminus for [Tyr⁰]NKA was observed, compared with that for NKA. The relationship between the difference in the biological potencies of [Tyr⁸]SP and SP and the differences in their structure, especially the interaction of the side chains of the two aromatic residues, and the difference in their binding affinities to membrane was discussed. In addition, differences between the result of restrained molecular dynamics simulations of [Tyr⁸]SP in the presence of an explicit micelle and the present results were observed and discussed. © 1999 John Wiley & Sons, Inc. Biopoly 50: 555–568, 1999     

A simple theory of peptide interactions on a membrane surface: excluded volume and entropic order

A simple theory of the interactions of peptides bound onto a lipid membrane is developed, modeling the peptides as rods on a surface. At low peptide surface-concentration, excluded volume dominates the peptide-peptide interactions and the orientation of the peptides is random, resulting in an isotropic configuration. However, at high peptide density on the membrane, the peptides become orientationally ordered, resulting in an anisotropic configuration. This effect is entirely entropic in origin, and simply reflects the fact that peptides can be exchanged more easily on the surface if they are equally aligned, resulting in a larger number of possible configurations. In three dimensions, this phenomenon corresponds to the well-known isotropic-nematic phase transition. In two dimensions, the problem is not as well understood. The theoretical treatment presented here yields a simple, manageable expression which can be compared with experimental data. Two-dimensional ordering results in an increase in the apparent binding constant of peptides to membranes at high concentration of peptides relative to what is expected from the effect of excluded volume alone. The possible implications of side-by-side alignment for several biological processes, such as peptide translocation across membranes and plaque formation in Alzheimer's disease, are discussed.     

Low frequency and low intensity pulsed electromagnetic field exerts its antiinflammatory effect through restoration of plasma membrane calcium ATPase activity

Rheumatoid arthritis (RA) is a chronic inflammatory disorder affecting 1% of the population worldwide. Pulsed electromagnetic field (PEMF) has a number of well-documented physiological effects on cells and tissues including antiinflammatory effect. This study aims to explore the antiinflammatory effect of PEMF and its possible mechanism of action in amelioration of adjuvant induced arthritis (AIA). Arthritis was induced by a single intradermal injection of heat killed Mycobacterium tuberculosis at a concentration of 500 μg in 0.1 ml of paraffin oil into the right hind paw of rats. The arthritic animals showed a biphasic response regarding changes in the paw edema volume. During the chronic phase of the disease, arthritic animals showed an elevated level of lipid peroxides and depletion of antioxidant enzymes with significant radiological and histological changes. Besides, plasma membrane Ca²⁺ ATPase (PMCA) activity was inhibited while intracellular Ca²⁺ level as well as prostaglandin E₂ levels was noticed to be elevated in blood lymphocytes of arthritic rats. Exposure of arthritic rats to PEMF at 5 Hz × 4 μT × 90 min, produced significant antiexudative effect resulting in the restoration of the altered parameters. The antiinflammatory effect could be partially mediated through the stabilizing action of PEMF on membranes as reflected by the restoration of PMCA and intracellular Ca²⁺ levels in blood lymphocytes subsequently inhibiting PGE₂ biosynthesis. The results of this study indicated that PEMF could be developed as a potential therapy for RA in human beings.     

The effect of natural and synthetic fatty acids on membrane structure,microdomain organization,cellular functions and human health

This review deals with the effects of synthetic and natural fatty acids on the biophysical properties of membranes, and on their implication on cell function. Natural fatty acids are constituents of more complex lipids, like triacylglycerides or phospholipids, which are used by cells to store and obtain energy, as well as for structural purposes. Accordingly, natural and synthetic fatty acids may modify the structure of the lipid membrane, altering its microdomain organization and other physical properties, and provoking changes in cell signaling. Therefore, by modulating fatty acids it is possible to regulate the structure of the membrane, influencing the cell processes that are reliant on this structure and potentially reverting pathological cell dysfunctions that may provoke cancer, diabetes, hypertension, Alzheimer's and Parkinson's disease. The so-called Membrane Lipid Therapy offers a strategy to regulate the membrane composition through drug administration, potentially reverting pathological processes by re-adapting cell membrane structure. Certain fatty acids and their synthetic derivatives are described here that may potentially be used in such therapies, where the cell membrane itself can be considered as a target to combat disease. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Cholesterol and phosphatidylethanolamine lipids exert opposite effects on membrane modulations caused by the M2 amphipathic helix

Membrane curvature remodeling induced by amphipathic helices (AHs) is essential in many biological processes. Here we studied a model amphipathic peptide, M2AH, derived from influenza A M2. We are interested in how M2AH may promote membrane curvature by altering membrane physical properties. We used atomic force microscopy (AFM) to examine changes in membrane topographic and mechanical properties. We used electron paramagnetic resonance (EPR) spectroscopy to explore changes in lipid chain mobility and chain orientational order. We found that M2AH perturbed lipid bilayers by generating nanoscale pits. The structural data are consistent with lateral expansion of lipid chain packing, resulting in a mechanically weaker bilayer. Our EPR spectroscopy showed that M2AH reduced lipid chain mobility and had a minimal effect on lipid chain orientational order. The EPR data are consistent with the surface-bound state of M2AH that acts as a chain mobility inhibitor. By comparing results from different lipid bilayers, we found that cholesterol enhanced the activity of M2AH in inducing bilayer pits and altering lipid chain mobility. The results were explained by considering specific M2AH-cholesterol recognition and/or cholesterol-induced expansion of interlipid distance. Both AFM and EPR experiments revealed a modest effect of anionic lipids. This highlights that membrane interaction of M2AH is mainly driven by hydrophobic forces. Lastly, we found that phosphatidylethanolamine (PE) lipids inhibited the activity of M2AH. We explained our data by considering interlipid hydrogen-bonding that can stabilize bilayer organization. Our results of lipid-dependent membrane modulations are likely relevant to M2AH-induced membrane restructuring.     

Electrostatic control of chloroplast coupling factor binding to thylakoid membranes as indicated by cation effects on electron transport and reconstitution of photophosphorylation

1. Increase in electron transport rate and the decay rate of the 518 nm absorption change, induced by EDTA treatment, is prevented by cations. The order of effectiveness is C³⁺ > C²⁺ > C⁺.2. In this respect methyl viologen is an effective divalent cation in addition to its action as an electron acceptor.3. Complete cation irreversible EDTA-induced uncoupling occurs in the dark in 2 min. Light greatly stimulates the rate of uncoupling by EDTA. It is concluded that the uncoupling is due to release of coupling factor I from the thylakoid membrane.4. Binding of purified coupling factor I to coupling factor I-depleted thylakoids can be achieved with any cation. The order of effectiveness is C³⁺ > C²⁺ > C⁺, reconstituted thylakoids are active in photophosphorylation regardless of the cation used for coupling factor I binding.5. The marked difference in the concentration requirements for cation effects on 9-aminoacridine fluorescence yield and for prevention of uncoupling by EDTA indicate that coupling factor I and its binding site have a lower surface charge density than the net surface charge density of the thylakoid membrane.6. It is concluded that coupling factor I binding only occurs when negative charges on coupling factor I and its binding site are electrostatically screened by cations.7. Previously reported examples of uncoupling by low ionic conditions are discussed in relation to the basic concepts of diffuse electrical layer theory.     

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.