Thermoosmosis through charged membranes

Thermoosmosis through oxidized collodion and collodion-sulfonated polystyrene interpolymer membranes has been observed in KCl solutions of various concentrations. The effective temperature difference acting for thermoosmosis was determined by measuring the thermal membrane potential appearing on both sides of membrane. It was found that the velocity of thermoosmosis is proportional to the effective temperature difference and the proportionality constant (thermoosmotic coefficient) is a function of electrolyte concentration. The dependence of the thermoosmotic coefficient of charged membranes on the electrolyte concentration is found to have a characteristic feature.     

Thermal membrane potential through charged membranes in electrolyte solutions.

Measurements of the thermal membrane potential across cation and anion exchange membranes were carried out by using the same solution of various 1-1 electrolytes on both sides of the membrane. In all cases a good linear relationship was observed between the thermal membrane potential increment psi and the temperature difference increment T. The slope of the linear plot varied with the concentration of the electrolyte. The value of increment psi/increment T versus logarithmic activity of the electrolyte plot was linear with a slope of +/- R/F if the transport number of counterion was unity. The magnitude of increment psi/increment T was independent of coion species but dependent on counterions. These experimental results are in agreement with a theory presented previously. The thermal membrane potential caused by the direct effect of temperature differences and that by the indirect effect arising from the changes in ionic and water chemical potentials due to the temperature difference are separately discussed.     

Phase transition in charged lipid membranes.

Experimental results on the effect of electrostatics on bilayer phase transitions are compared with corresponding data for monolayers and the predictions of electrical double layer theory. The two substantial conclusions which emerge are that: (i) double layer theory based on a continuous surface charge distribution cannot explain all the relevant data, a situation which may be improved by taking into account the discrete nature of the surface charge distribution; (ii) the crystal - liquid crystal phase transition of charged bilayer membranes is always a continuous one which takes place through an intermediate state consisting of both fluid and frozen domains.     

A theoretical model for electron transport through chlorophyll-containing bileaflet membranes

Summary A model of electron transport through bileaflet membranes comprised of chlorophylls and carotenoids is proposed and can account for the photoresponses observed in chloroplast-extract membranes. Some features of the model are as follows: In the absence of any specific additive, such as phycocyanin, the energy barrier for electron transfer between chromophores in the membrane and a redox pair in the aqueous solution is significantly high, so that the only realistic mechanism for electron exchange through the interface is by quantum-mechanical tunneling. In an analogy to theories developed for the explanation of semiconductor electrode behavior, the essential requirement of energy overlap between occupied and unoccupied electronic states on either side of the membrane-water interface is explored. The extent of overlap can be controlled by the potential difference developed across the interface. The presence of the carotenoids can reduce the energy barrier for electron movement within she membrane so that excited electrons can pass from the chlorophyll chromophores on one tide of the membrane interface to those on the other side. It is suggested that electron tunneling is the mechanism by which reducing substances belonging to a redox pair of high oxidation-reduction potential deliver their electrons to vacant orbitals in the photosynthetic membranes.     

Electrical measurement of electroneutral fluxes of divalent cations through charged planar phospholipid membranes

The voltage-sensitive channel-former monazomycin is used as a conductance probe to monitor changes in the trans electrostatic surface potentials of negatively-charged planar phospholipid bilayers. Cis-to-trans electroneutral fluxes of divalent cations mediated by ionophores A23187 and X537A are sensed via the effect of transported divalent cations on the trans surface potentials. Quantitative determinations of neutral Ca2+ and Mg2+ fluxes are made and related to ionophore function.     

A theoretical analysis of pH dependence of sodium channel conductance

A new theory termed tunnel-acid-group-potential (TAGPT), which explains the effects of pHo and pHi on the ion conductance through different membrane channels, is presented. It is suggested that shifts in pHo and pHi change the values of negative charges generated by acid groups of side chains of some polar (Glu, Asp) amino acid residues lining the tunnel part of the channel. The resulting electrostatic field modification affects the heights of rate-limiting energy barriers (for ion transport) in the transition zones between the tunnel and the vestibules, which changes the channel conductance.     

Polymer concentration dependence of the helix to random coil transition of a charged polypeptide in aqueous salt solution.

The helix to coil transition of poly(L-glutamic acid) was investigated in 0.05 and 0.005 M aqueous potassium chloride solutions by use of potentiometric titration and circular dichroism measurement. Polymer concentration dependence of the transition was observed in the range from 0.006 to 0.04 monomol/e in 0.005 M KG1 solution. The polymer concentration dependence can be interpreted by current theories of the transition of charged polypeptides and of titration curves of linear weak polyelectrolytes taking the effect of polymer concentration into consideration.     

An extended kinetic analysis of valinomycin-induced Rb-transport through monoglyceride membranes.

The time course of the current following a voltage jump, which is applied to monoglyceride bilayers in the presence of valinomycin, shows two relaxation times. This is basically in agreement with a simple carrier model which has been described in full detail formerly. Relaxation times and amplitudes allow a calculation of the rate constants of the transport model. The presented data supplement an analysis which was hitherto based only on the slower relaxation process and on information derived from the nonlinearity of current-voltage characteristics. The additional resolution of the faster relaxation time allowed an approximate determination of the voltage dependence of the translocation rate constant for carrier-ion-complex and provided evidence for a small voltage dependence of the interfacial reaction. The dependence of the relaxation parameters on the ion concentration in the aqueous phase was interpreted assuming a saturation of the ion concentration at the reaction plane at high bulk concentrations.     

pH dependence of penicillin amidase enantioselectivity for charged substrates.

The pH dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) was studied for penicillin amidase catalysed hydrolysis of charged enantiomeric substrates. Theoretical analysis shows that a pH dependence can only be observed around the pK values of groups in the active site whose ionisation control the enzyme activity. For charged substrates that may perturb these pK values, a pH dependence of E is also expected. This was experimentally verified around these pK values. The S'(1)-stereospecificity of penicillin amidase was studied for the hydrolysis of the enantiomeric phenylacetyl-S/R-Phe and for the racemic phenylacetyl-S,R-PhG. The S(1)-stereospecificity was investigated for the hydrolysis of the enantiomeric S/R-PhG-NH(2). The observed pH modulation of E (more than 3-fold for the studied substrates in the pH range 4.5-9) was found to be a result of compensatory effects for binding and catalysis. The ratios k(cat, S)/k(cat,R) and K(m,S)/K(m,R) for the hydrolysis of the enantiomeric phenylacetyl-Phe were found to decrease from 1000 to 10 and from 0.1 to 0.01, respectively in the pH range 5-8. The dependence was stronger for the S'(1)- than for the S(1)-subsite. This is probably due to the stronger influence of the substrate carboxyl group in the S'(1)-subsite than that of the substrate amino group in the S(1)-subsite on the pK of the N-terminal Ser B1 that is essential for the activity. The observed pH dependence of E was used to discuss the importance of ground-state interactions for discrimination between enantiomers and for enzyme catalysis in general. The experimental results conform to the split site model according to which a better binding must not be fundamentally inhibitory.     

Electrostatic interactions at charged lipid membranes. Calcium binding

A quantitative study of calcium-ion binding by the negatively-charged phospholipid methylphosphatidic acid is presented. Experimental results are compared with the predictions of the Gouy-Chapman theory, taking into account both the ions bound at the membrane surface and the ions held in the diffuse layer. This theory suffices to explain the titration of the calcium/lipid system, but fails to explain completely the behaviour of the ordered-fluid transition temperature, which shows a splitting that according to electrostatic theory alone should not occur. The dependence of the calcium-lipid binding constant. upon 1: 1 electrolyte concentration is correctly predicted by the theory; the latter however gives equations which can only be solved numerically. A simple, approximate equation is therefore given (in the text, eq. 34) for the prediction of the degree of calcium binding to a negatively-charged lipid membrane.     

An experimental and theoretical analysis of ultrasound-induced permeabilization of cell membranes

Application of ultrasound transiently permeabilizes cell membranes and offers a nonchemical, nonviral, and noninvasive method for cellular drug delivery. Although the ability of ultrasound to increase transmembrane transport has been well demonstrated, a systematic dependence of transport on ultrasound parameters is not known. This study examined cell viability and cellular uptake of calcein using 3T3 mouse cell suspension as a model system. Cells were exposed to varying acoustic energy doses at four different frequencies in the low frequency regime (20-100 kHz). At all frequencies, cell viability decreased with increasing acoustic energy dose, while the fraction of cells exhibiting uptake of calcein showed a maximum at an intermediate energy dose. Acoustic spectra under various ultrasound conditions were also collected and assessed for the magnitude of broadband noise and subharmonic peaks. While the cell viability and transport data did not show any correlation with subharmonic (f/2) emission, they correlated with the broadband noise, suggesting a dominant contribution of transient cavitation. A theoretical model was developed to relate reversible and irreversible membrane permeabilization to the number of transient cavitation events. The model showed that nearly every stage of transient cavitation, including bubble expansion, collapse, and subsequent shock waves may contribute to membrane permeabilization. For each mechanism, the volume around the bubble within which bubbles induce reversible and irreversible membrane permeabilization was determined. Predictions of the model are consistent with experimental data.     

Defensins promote fusion and lysis of negatively charged membranes.

Defensins, a family of cationic peptides isolated from mammalian granulocytes and believed to permeabilize membranes, were tested for their ability to cause fusion and lysis of liposomes. Unlike alpha-helical peptides whose lytic effects have been extensively studied, the defensins consist primarily of beta-sheet. Defensins fuse and lyse negatively charged liposomes but display reduced activity with neutral liposomes. These and other experiments suggest that fusion and lysis is mediated primarily by electrostatic forces and to a lesser extent, by hydrophobic interactions. Circular dichroism and fluorescence spectroscopy of native defensins indicate that the amphiphilic beta-sheet structure is maintained throughout the fusion process. Taken together, these results support the idea that protein-mediated membrane fusion depends not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form a three-dimensional amphiphilic structure, which promotes the efficient mixing of the lipids between membranes. A molecular model for membrane fusion by defensins is presented, which takes into account the contributions of electrostatic forces, hydrophobic interactions, and structural amphiphilicity.     

Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes. Evidence of affinity for negatively charged membranes.

The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed of l-alpha-1,2-dimyristoylphosphatidylcholine, l-alpha-1,2-dimyristoylphosphatidylglycerol and l-alpha-1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing l-alpha-1,2-dimyristoylphosphatidylglycerol; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane. PEBP affinity for negatively charged membranes is puzzling considering the previous identification of the protein as a phosphatidylethanolamine-binding protein, and suggests that the association of PEBP with phospholipid membranes is driven by a mechanism other than its binding to solubilized phosphatidylethanolamine. An explanation was suggested by its three-dimensional structure: a small cavity at the protein surface has been reported to be the binding site of the polar head of phosphatidylethanolamine, while the N-terminal and C-terminal parts of PEBP, exposed at the protein surface, appear to be involved in the interaction with membranes. To test this hypothesis, we synthesized the two PEBP terminal regions and tested them with model membranes in parallel with the whole protein. Both peptides displayed the same behaviour as whole PEBP, indicating that they could participate in the binding of PEBP to membranes. Our results strongly suggest that PEBP directly interacts with negatively charged membrane microdomains in living cells.     

Effect of profilin on actin critical concentration: a theoretical analysis

To explain the effect of profilin on actin critical concentration in a manner consistent with thermodynamic constraints and available experimental data, we built a thermodynamically rigorous model of actin steady-state dynamics in the presence of profilin. We analyzed previously published mechanisms theoretically and experimentally and, based on our analysis, suggest a new explanation for the effect of profilin. It is based on a general principle of indirect energy coupling. The fluctuation-based process of exchange diffusion indirectly couples the energy of ATP hydrolysis to actin polymerization. Profilin modulates this coupling, producing two basic effects. The first is based on the acceleration of exchange diffusion by profilin, which indicates, paradoxically, that a faster rate of actin depolymerization promotes net polymerization. The second is an affinity-based mechanism similar to the one suggested in 1993 by Pantaloni and Carlier although based on indirect rather than direct energy coupling. In the model by Pantaloni and Carlier, transformation of chemical energy of ATP hydrolysis into polymerization energy is regulated by direct association of each step in the hydrolysis reaction with a corresponding step in polymerization. Thus, hydrolysis becomes a time-limiting step in actin polymerization. In contrast, indirect coupling allows ATP hydrolysis to lag behind actin polymerization, consistent with experimental results.     

Scaling analysis of the concentration dependence on elasticity of agarose gel

Since the critical exponent of the elastic modulus is related to the spatial dimension and the critical exponent of the correlation length, depending on the characteristics of elasticity, we experimentally evaluated both the elastic modulus of a sol-gel transition system and also the correlation length. We could determine the correlation length of agarose gel by the dynamic light scattering method; it was well described by the power law as a function of the deviation from the sol-gel transition point. Three scaling laws between the critical exponent of the correlation length (v) and that of the elastic shear modulus (t) were compared, and the critical exponent of the elastic modulus was described by the equation of de Gennes expression (t=1+v(d-2), where d is the spatial dimension). This result suggests that agarose fibers are stiff enough to show scalar elasticity.     

An ion switch regulates fusion of charged membranes

Here we identify the recruitment of solvent ions to lipid membranes as the dominant regulator of lipid phase behavior. Our data demonstrate that binding of counterions to charged lipids promotes the formation of lamellar membranes, whereas their absence can induce fusion. The mechanism applies to anionic and cationic liposomes, as well as the recently introduced amphoteric liposomes. In the latter, an additional pH-dependent lipid salt formation between anionic and cationic lipids must occur, as indicated by the depletion of membrane-bound ions in a zone around pH 5. Amphoteric liposomes fuse under these conditions but form lamellar structures at both lower and higher pH values. The integration of these observations into the classic lipid shape theory yielded a quantitative link between lipid and solvent composition and the physical state of the lipid assembly. The key parameter of the new model, κ(pH), describes the membrane phase behavior of charged membranes in response to their ion loading in a quantitative way.     

Transport properties of charged membranes undergoing conformational transitions

Summary The permeability and reflection behavior of cross-linked collagen films in dilute salt solutions have been investigated by measurements of net volume flow, isotopic exchange of THO and of Ca⁴⁵, and osmotic pressure. Complementary measurements of swelling, membrane resistance, membrane potential, and streaming potential are presented. Measurements were performed in the pH range of 5 to 1.5, at temperatures between 25 and 52 °C, and in the presence of KCl, 10^–2 m or CaCl₂, 10^–3 m. Under the conditions adopted, the membrane carries a net positive charge and undergoes large changes in degree of swelling (Donnan effect) and structure (crystal amorphous transition). The results indicate that when pH is lowered the filtration coefficientL _p decreases in the crystalline state (pH 5 to 3), increases during the conformational transition (pH 3 to 2), and decreases in the amorphous state (pH<2). It appears thatL _p is affected more by such properties as structure and porosity (i.e., mechanical resistance to flow) than properties related to the charged character of the membrane. The reflection coefficient increases when pH is lowered until pH 3, and decreases upon further lowering of pH. Such behavior is described in terms of the competition between swelling (due to both the Donnan effect and the melting transition) and fixed-charge density. Values of fixed-charge density derived on the basis of a theoretical expression for were found to be in good agreement with independent titration data.     

Lateral diffusion of membrane proteins in protein-rich membranes. A simple hard particle model for concentration dependence of the two-dimensional diffusion coefficient.

A model for the effect of protein concentration on the rate of lateral diffusion of integral membrane proteins is presented, in which the proteins are represented by equivalent hard circular particles on a surface. As the density of particles increases, the probability of finding a vacancy immediately adjacent to a tracer particle into which it may diffuse decreases, resulting in a concomitant reduction of the tracer diffusion coefficient. Using scaled particle theory to calculate the concentration-dependent probabilities, a simple approximate result is obtained in closed form, that is compared with the results of previously published Monte Carlo lattice simulations and experimental observations.