Thermal membrane potential across charged membranes in 2-1 and 1-2 electrolyte solutions

Measurements of thermal membrane potential across cation exchange membranes in MgCl2, CaCl2 and BaCl2 solutions and across anion exchange membranes in K2SO4, Na2SO4 and K2CO3 solutions were carried out. The magnitude of the thermal membrane potential for divalent counterions is lower than that for monovalent counterions. If the transport number of counterions in the membrane phase is unity, the slopes of the temperature coefficient of thermal membrane potential against logarithmic activities of counterion in the external solution are predicted to be--R/2F for 2-1 electrolytes with cation exchange membranes and R/2F for 1-2 electrolytes with anion exchange membranes, respectively.     

INFLUENCE OF A SLIGHT MODIFICATION OF THE COLLODION MEMBRANE ON THE SIGN OF THE ELECTRIFICATION OF WATER

1. It is shown that collodion membranes which have received one treatment with a 1 per cent gelatin solution show for a long time (if not permanently) afterwards a different osmotic behavior from collodion membranes not treated with gelatin. This difference shows itself only towards solutions of those electrolytes which have a tendency to induce a negative electrification of the water particles diffusing through the membrane, namely solutions of acids, acid salts, and of salts with trivalent and tetravalent cations; while the osmotic behavior of the two types of membranes towards solutions of salts and alkalies, which induce a positive electrification of the water particles diffusing through the membrane, is the same. 2. When we separate solutions of salts with trivalent cation, e.g. LaCl₃ or AlCl₃, from pure water by a collodion membrane treated with gelatin, water diffuses rapidly into the solution; while no water diffuses into the solution when the collodion membrane has received no gelatin treatment. 3. When we separate solutions of acid from pure water by a membrane previously treated with gelatin, negative osmosis occurs; i.e., practically no water can diffuse into the solution, while the molecules of solution and some water diffuse out. When we separate solutions of acid from pure water by collodion membranes not treated with gelatin, positive osmosis will occur; i.e., water will diffuse rapidly into the solution and the more rapidly the higher the valency of the anion. 4. These differences occur only in that range of concentrations of electrolytes inside of which the forces determining the rate of diffusion of water through the membrane are predominantly electrical; i.e., in concentrations from 0 to about M/16. For higher concentrations of the same electrolytes, where the forces determining the rate of diffusion are molecular, the osmotic behavior of the two types of membranes is essentially the same. 5. The differences in the osmotic behavior of the two types of membranes are not due to differences in the permeability of the membranes for solutes since it is shown that acids diffuse with the same rate through both kinds of membranes. 6. It is shown that the differences in the osmotic behavior of the two types of collodion membranes towards solutions of acids and of salts with trivalent cation are due to the fact that in the presence of these electrolytes water diffuses in the form of negatively charged particles through the membranes previously treated with gelatin, and in the form of positively charged particles through collodion membranes not treated with gelatin. 7. A treatment of the collodion membranes with casein, egg albumin, blood albumin, or edestin affects the behavior of the membrane towards salts with trivalent or tetravalent cations and towards acids in the same way as does a treatment with gelatin; while a treatment of the membranes with peptone prepared from egg albumin, with alanine, or with starch has no such effect.     

Relation Between the Dielectric Constant of Hydrophobic Cation Exchange Membrane and Membrane Permeability to Counterions

Filters made of cellulose acetate-nitrate when saturated with organic solvents and interposed between aqueous solutions form membranes which behave like cation exchangers. The diffusion coefficients of counterions in such membranes are strongly dependent upon the dielectric constant of the saturating solvent. The results obtained suggest that a linear relationship between the log of the cation's diffusion coefficient (or membrane conductance) and the reciprocal value of the dielectric constant of the saturating solvent exists. There is also a good correlation between the relative membrane permeability to organic cations and the solubility of the cations in the pure solvent phase. These studies indicate that there are two routes for cation movement through the membrane: (a) the bulk hydrophobic phase and (b) continuous narrow aqueous channels.     

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.     

(Na+,K+)-ATPase of mammalian brain: Effects of temperature on cation and ATP interactions regulating phosphatase activity

The effects of temperature on interactions between univalent cations or ATP and the p-nitrophenylphosphatase activity associated with brain (Na⁺,K⁺)-ATPase were examined. The apparent affinity for K⁺ activation under conditions favoring the moderate affinity site was temperature dependent, increasing with decreasing temperature. A comparison of univalent cations showed that the negative apparent ΔH and ΔS for cation binding increased with increasing apparent cation affinity. In contrast to the case with the moderate affinity sites, apparent affinity for the high affinity K⁺ site was independent of temperature. As temperature decreased, properties of moderate affinity site binding approached those of the high affinity site. The temperature dependence of ATP inhibition was opposite to that for K⁺ activation, with positive apparent ΔH and ΔS. The apparent ΔH and ΔS for cation binding approached those for the overall conformational change to K⁺-sensitive enzyme as cation affinity increased. These data suggest that E2, the K⁺-sensitive form of (Na⁺,K⁺)-ATPase, is stabilized by forces that require a decrease in entropy, explaining the predominant existence of E1 at physiologic temperatures. A conformational change leading to stabilization of E2 at higher temperatures can be produced by binding of univalent cations to a moderate affinity, presumably intracellular, site. This effect is counteracted by ATP. ATP also appears to alter the selectivity of this site to favor Na⁺ over K⁺ binding.     

Cold-induced increase of δ9- and δ6-desaturase activities in endoplasmic membranes of carp liver

Long-term warm-acdimated (30°C) carp were exposed to a sudden decrease in acclimation temperature. Kinetic properties and the time-course of activity of the δ⁹- and δ⁶-desaturases were measured in rough and smooth membranes of endoplasmic reticulum isolated from liver. During early cold exposure of fish, enhancements of desaturase activities are about 30-fold in rough and at least 18-fold in smooth membranes. Enhancements of activity are biphasic in rough endoplasmic reticulum but monophasic in the smooth membranes. They are assumed to be caused mainly by the synthesis of additional desaturase enzyme protein. The significantly higher activities in long-term cold-acclimated (10°C) carp are in accordance with the increased fatty acid unsaturation of their membrane lipids.     

Univalent ions in phospholipid model membranes: Thermodynamic and hydration aspects

By means of differential scanning calorimetry, effects of systematic series of Group I and VII ions on the phase state of model multibilayer dimyristoylphosphatidylcholine (di(14:0)PC) membranes have been studied at a lipid/ion molar ratio of 3/1. The sign-changing correlations between the ionic radii of cations and temperature shifts of di(14:0)PC phase transition were obtained. For cosmotropic Li⁺ and Na⁺, the observed shifts were positive (LiCl: ΔT _m = 0.6°C; ΔT _p = 1.9°C), whereas chaotropic K⁺ and Rb⁺ presence resulted in negative shifts (RbCl: ΔT _m = ?0.3°C; ΔT _p = ?2.5°C). The anions (Cl^?, Br^?, I^?) showed a similar effect increasing with the ion chaotropicity. An essentially weaker effect of Cs⁺ as compared to other alkali metal ions (CsCl: ΔT _m ≈ 0°C; ΔT _p = ?0.1°C) can be one of the reasons of its accumulation in living organisms. Generalization of all available data allowed us to specify some important factors of lipid-ion interactions that should be taken into account in further investigations in this field.     

The formation and properties of thin lipid membranes from HK and LK sheep red cell lipids

Lipids were obtained from high potassium (HK) and low potassium (LK) sheep red cells by sequential extraction of the erythrocytes with isopropanol-chloroform, chloroform-methanol-0.1 M KCl, and chloroform. The extract contained cholesterol and phospholipid in a molar ratio of 0.8:1.0, and less than 1% protein contaminant. Stable thin lipid membranes separating two aqueous compartments were formed from an erythrocyte lipid-hydrocarbon solution, and had an electrical resistance of ∼10⁸ ohm-cm² and a capacitance of 0.38–0.4 µf/cm². From the capacitance values, membrane thickness was estimated to be 46–132 A, depending on the assumed value for the dielectric constant (2.0–4.5). Membrane voltage was recorded in the presence of ionic (NaCl and/or KCl) concentration gradients in the solutions bathing the membrane. The permeability of the membrane to Na⁺, K⁺, and Cl^- (expressed as the transference number, T _ion) was computed from the steady-state membrane voltage and the activity ratio of the ions in the compartments bathing the membrane. T _Na and T _K were approximately equal (∼0.8) and considerably greater than T _Cl (∼0.2). The ionic transference numbers were independent of temperature, the hydrocarbon solvent, the osmolarity of the solutions bathing the membranes, and the cholesterol content of the membranes, over the range 21–38°C. The high degree of membrane cation selectivity was tentatively attributed to the negatively charged phospholipids (phosphatidylethanolamine and phosphatidylserine) present in the lipid extract.     

Ice nucleation and supercooling behavior of polymer aqueous solutions

We determined the homogeneous nucleation temperature depression, ΔT_f,hom, the equilibrium melting point depression, ΔT_m, and the value λ, which can be obtained from the linear relationship ΔT_f,hom = λΔT_m, for aqueous solutions of PEG (200-20,000 g mol⁻¹), PVP (10,000, 35,000, 40,000 g mol⁻¹), and dextran (10,000 g mol⁻¹) in the concentration range 0-40 wt% using the emulsion method. The molecular weight dependence of T_f,hom, T_m, and λ in PEG aqueous solutions was found to change in the vicinity of Mw 600-1540 at all concentrations. In addition, it was confirmed that for all of the polymers studied, there was a good linear relationship between λ and the logarithmic value of the self-diffusion coefficient D₀ of the solute molecule. These results indicate that the parameters that describe non-equilibrium freezing, such as T_f,hom and λ, are dependent on solution properties such as viscosity and self-diffusion of solute molecules.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : IX. WATER UPTAKE AND SWELLING OF COLLODION MEMBRANES IN AQUEOUS SOLUTIONS OF ORGANIC ELECTROLYTES AND NON-ELECTROLYTES

1. Dried collodion membranes are known to swell in water and to the same limited extent also in solutions of strong inorganic electrolytes (Carr and Sollner). The present investigation shows that in solutions of organic electrolytes and non-electrolytes, the swelling of dried collodion membranes is not as uniform, but depends on the nature of the solute. 2. The solutions of typically "hydrophilic" substances, e.g., glycerine, glucose, and citric acid, swell collodion membranes only to the same extent as water and solutions of strong electrolytes. In solutions of typically carbophilic substances (e.g., butyric acid, valeric acid, isobutyl alcohol, valeramide, phenol, and m-nitrophenol) the swelling of the membranes is much stronger than in water, according to the concentration used. For the brand of collodion used the swelling in 0.5 M solution was in some cases as high as 26 per cent of the original volume, as compared to 6 to 7 per cent in water. Therefore, in these solutions the "water-wetted dried" collodion membrane is not rigid, inert, and non-swelling, but behaves as a swelling membrane. 3. The solutes which cause an increased swelling of the membranes are accumulated in the latter, the degree of accumulation being markedly parallel with the degree of their specific swelling action. 4. The anomalously high permeabilities of certain carbophilic organic solutes reported by Michaelis, Collander, and Höber find an explanation in the specific interaction of these substances with collodion. 5. The use of the collodion membrane as a model of the ideal porous membrane is restricted to those instances in which no specific interaction occurs between the solute and the collodion.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : XI. THE PREPARATION AND PROPERTIES OF "MEGAPERMSELECTIVE" COLLODION MEMBRANES COMBINING EXTREME IONIC SELECTIVITY WITH HIGH PERMEABILITY

1. The electronegative membranes described in the literature which show a high degree of ionic selectivity (permitting cations to pass and restricting the anions) have serious shortcomings: their absolute permeability is extremely low, much too small for convenient experimentation; their ionic selectivity in most cases is not as perfect as would be desirable, and is moreover adversely affected by prolonged contact with electrolyte solutions. 2. A method has been worked out to prepare membranes substantially free from these defects. Porous collodion membranes were cast on the outside of rotating tubes and then oxidized with 1 M NaOH. By allowing the oxidized porous membranes to dry in air on the tubes membranes of desirable properties are obtained. These membranes are smooth, have a well defined shape, and allow considerable handling without breaking. 3. This new type membrane when tested for ionic selectivity by the measurement of the "characteristic concentration potential," consistently gives potentials of 54 to 55 mv., the maximum thermodynamically possible value (at 25°C.) being 55.1 mv. This high degree of ionic selectivity is not lost on prolonged contact with water, and is only very slowly affected by electrolyte solutions. 4. The absolute permeability of the new type membranes can be varied over a very wide range by changing the time of oxidation. Under optimum conditions membranes can be obtained with a resistance in 0.1 N KCl solution of only 0.5 ohms per 50 cm.² membrane area. The absolute rate of cation exchange through these membranes between solutions of different uni-univalent electrolytes is very high, in one case, e.g. 0.9 m.eq. cations per 4 hours, the anion leak being 0.02 m.eq. Thus, the absolute permeability of the new type membranes is two to four orders of magnitude greater than the permeability of the dried collodion membranes and the oxidized ("activated") dried collodion membranes used heretofore. Because of the characteristic properties of the new type membranes the term "megapermselective" (or "permselective") collodion membranes is proposed for them.     

Chloride Transport in Porous Lipid Bilayer Membranes

This paper describes dissipative Cl_- transport in "porous" lipid bilayer membranes, i.e., cholesterol-containing membranes exposed to 1–3 x 10^-7 M amphotericin B. P_DCl (cm·s^-1), the diffusional permeability coefficient for Cl^-, estimated from unidirectional ³⁶Cl^- fluxes at zero volume flow, varied linearly with the membrane conductance (Gm, Ω^-1·cm^-2) when the contributions of unstirred layers to the resistance to tracer diffusion were relatively small with respect to the membranes; in 0.05 M NaCl, P_DCl was 1.36 x 10^-4 cm·s^-1 when Gm was 0.02 Ω^-1·cm^-2. Net chloride fluxes were measured either in the presence of imposed concentration gradients or electrical potential differences. Under both sets of conditions: the values of P_DCl computed from zero volume flow experiments described net chloride fluxes; the net chloride fluxes accounted for ~90–95% of the membrane current density; and, the chloride flux ratio conformed to the Ussing independence relationship. Thus, it is likely that Cl^- traversed aqueous pores in these anion-permselective membranes via a simple diffusion process. The zero current membrane potentials measured when the aqueous phases contained asymmetrical NaCl solutions could be expressed in terms of the Goldman-Hodgkin-Katz constant field equation, assuming that the P_DNa/P_DCl ratio was 0.05. In symmetrical salt solutions, the current-voltage properties of these membranes were linear; in asymmetrical NaCl solutions, the membranes exhibited electrical rectification consistent with constant-field theory. It seems likely that the space charge density in these porous membranes is sufficiently low that the potential gradient within the membranes is approximately linear; and, that the pores are not electrically neutral, presumably because the Debye length within the membrane phase approximates the membrane thickness.     

Old and new data, new issues: The mitochondrial ΔΨ

New and old data pertinent to the electrochemical potentials across the inner mitochondrial membrane are reviewed with the intent of reconciling the various findings in the light of new perspectives provided by more recent knowledge. A careful scrutiny of old data permits ruling out the presence of a significant metabolically dependent electrical membrane potential. Recent technological advances make it possible to test the proposed alternatives. These proposals recast the original idea, and the possible mechanisms that are emerging also invoke a protonmotive force. Our conclusions that ΔΨ is not involved in oxidative-phosphorylation finds parallel observations in Halobacterium halobium [H. Michel, D. Oesterhelt, Electrochemical proton gradient across the cell membrane of Halobacterium halobium: comparison of the light-induced increase with the increase of intracellular adenosine triphosphate under steady-state illumination, Biochemistry 19 (1980) 4615-4619] and thylakoid vesicles [D.R. Ort, R.A. Dilley, N.E. Good, Photophosphorylation as a function of illumination time II. Effects of permeant buffers, Biochim. Biophys. Acta 449 (1976) 108-129] in which light-induced ATP synthesis occurs in the absence of an apparent ΔΨ or ΔpH, suggesting the presence of mechanisms similar to the one proposed for mitochondria.     

1H and 13C nuclear magnetic resonance spectra of the lipids in normal and SV 40 virus-transformed hamster embryo fibroblast membranes

Well resolved ¹H and ¹³C NMR spectra were obtained with normal and SV 40-transformed cell membranes. Estimation of the ratio of ¹³CT₂ values of the normal to transformed cell membranes showed an increased intermolecular motion in the transformed cell membranes. The temperature dependence of the (CH₂)_n line in the ¹H spectra in the temperature range 298–343 °K shows an activation energy for the lateral diffusion of the fluid phospholipid regions in the normal cell membranes while the transformed ones show practically no temperature dependence in this temperature range. The fluidity of the phospholipid region in the transformed cell membrane seems to be significantly higher than that observed in the normal cell material. These data support and extend the findings concerning the mobility of the concanavalin A binding/agglutinating sites on the surface of normal and virus-transformed cells and suggest further approaches to the study of the membrane alterations in tumor cells.     

Ionic Permeability of Thin Lipid Membranes : Effects of n-alkyl alcohols,polyvalent cations,and a secondary amine

Ultrathin (black) lipid membranes were made from sheep red cell lipids dissolved in n-decane. The presence of aliphatic alcohols in the aqueous solutions bathing these membranes produced reversible changes in the ionic permeability, but not the osomotic permeability. Heptanol (8 mM), for example, caused the membrane resistance (R_m) to decrease from >10⁸ to about 10⁵ ohm-cm² and caused a marked increase in the permeability to cations, especially potassium. In terms of ionic transference numbers, deduced from measurements of the membrane potential at zero current, T _cat/T _Cl increased from about 6 to 21 and T _K/T _Na increased from about 3 to 21. The addition of long-chain (C₈ndash;C₁₀) alcohols to the lipid solutions from which membranes were made produced similar effects on the ionic permeability. A plot of log R_m vs. log alcohol concentration was linear over the range of maximum change in R_m, and the slope was -3 to -5 for C₂ through C₇ alcohols, suggesting that a complex of several alcohol molecules is responsible for the increase in ionic permeability. Membrane permselectivity changed from cationic to anionic when thorium or ferric iron (10^-4 M) was present in the aqueous phase or when a secondary amine (Amberlite LA-2) was added to the lipid solutions from which membranes were made. When membranes containing the secondary amine were exposed to heptanol, R_m became very low (10³–10⁴ ohm-cm²) and the membranes became perfectly anion-selective, developing chloride diffusion potentials up to 150 mv.     

The antibacterial activity of phospholipase A2 type IIA is regulated by the cooperative lipid chain melting behavior in Staphylococcus aureus

As one of its primary physiological functions, sPLA₂-IIA appears to act as an antibacterial agent. In particular, sPLA₂-IIA shows high activity towards Gram-positive bacteria such as Staphylococcus aureus (S. aureus). This antibacterial activity results from the preference of the enzyme towards membranes enriched in anionic lipids, which is a common feature of bacterial membranes. An intriguing aspect observed in a variety of bacterial membranes is the presence of a broad but cooperative lipid chain melting event where the lipids in the membrane transition from a solid-ordered (so) into a liquid-disordered (ld) state close to physiological temperatures. It is known that the enzyme is sensitive to the level of lipid packing, which changes sharply between the so and the ld states. Therefore, it would be expected that the enzyme activity is regulated by the bacterial membrane thermotropic behavior. We determine by FTIR the thermotropic lipid chain melting behavior of S. aureus and find that the activity of sPLA₂-IIA drops sharply in the so state. The activity of the enzyme is also evaluated in terms of its effects on cell viability, showing that cell survival increases when the bacterial membrane is in the so state during enzyme exposure. These results point to a mechanism by which bacteria can develop increased resistance towards antibacterial agents that act on the membrane through a cooperative increase in the order of the lipid chains. These results show that the physical behavior of the bacterial membrane can play an important role in regulating physiological function in an in vivo system.     

Anaerobic hydrogenase activity in Anacystis nidulans H2-dependent photoreduction and related reactions

1. Anaerobic hydrogenase activity in whole cells and cell-free preparations of H₂-induced Anacystis was studied both manometrically and spectrophotometrically in presence of physiological and artificial electron acceptors.2. Up to 90% of the activity measured in crude extracts were recovered in the chlorophyll-containing membrane fraction after centrifugation (144 000 × g, 3 h).3. Reduction of methyl viologen, diquat, ferredoxin, nitrite and NADP by the membranes was light dependent while oxidants of more positive redox potential were reduced also in the dark.4. Evolution of H₂ by the membranes was obtained with dithionite and with reduced methyl viologen; the reaction was stimulated by detergents.5. Both uptake and evolution of H₂ were sensitive to O₂, CO, and thiol-blocking agents. The H₂-dependent reductions were inhibited also by the plastoquinone antagonist dibromothymoquinone, while the ferredoxin inhibitor disalicylidenepropanediamine affected the photoreduction of nitrite and NADP only. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea did not inhibit any one of the H₂-dependent reactions.6. The results present evidence for a membrane-bound ‘photoreduction’ hydrogenase in H₂-induced Anacystis. The enzyme apparently initiates a light-driven electron flow from H₂ to various low-potential acceptors including endogenous ferredoxin.     

Divalent cation effects on membrane bending in heated erythrocytes

The thermal fragmentation of human erythrocytes involves either surface wave growth and membrane externalization at the cell rim or membrane internalization at the cell dimple. In symmetrical monovalent electrolytes an increase in membrane internalization at the cell dimple correlates with the decrease in zeta potential arising from surface charge (sialic acid residue) depletion. The influence of divalent cations on thermal fragmentation is examined in this work. The erythrocyte zeta potential decreased when divalent cations replaced some Na⁺ in the cell-suspending phase. The incidence of membrane internalization increased in rank order Ca²⁺>Ba²⁺>Mg²⁺Sr²⁺. Calcium continued to influence the thermal fragmentation of cells highly depleted of sialic acid, suggesting that the ion also interacted with membrane sites other than sialic acid. The divalent cation influence on cell fragmentation was shown to be greater than that due to zeta potential decrease alone. This conclusion was supported by the observation that the divalent cation-induced changes in zeta potential showed much less cation specificity than did the changes induced in the thermal fragmentation pattern. The result implies that the specificity of the divalent cation effects was due to interactions within the erythrocyte shear layer. The possibility that the interaction is with membrane lipids is examined.     

Subcellular distribution of iodothyronine 5′-deiodinase in cerebral cortex from hypothyroid rats

We have examined iodothyronine deiodination in subcellular fractions of cerebral cortex obtained from hypothyroid rats. Enzymatic activities were measured at 37°C in the presence of 20 mM dithiothreitol with ¹²⁵I-labeled T₄ and ¹²⁵I-labeled rT₃ as substrate for 5′-deiodination and ¹³¹I-labeled T₃ as the substrate for the 5-deiodinase. Reaction products were separated by descending paper and/or ion-exchange chromatography. Cerebral cortex subcellular fractions were also characterized by marker enzyme analysis and electron microscopy. Under optimal reaction conditions more than 80% of the 5′-deiodinase was recovered after fractionation. Both 5′-deiodinase and (Na⁺ +K⁺-ATPase showed similar subcellular distributions and were enriched approx. 3-fold in the easily sedimenting membrane fraction and nerve terminal plasma membranes. Crude microsomal membranes (6·10⁶g·min pellet) also showed 2-fold enrichment of these enzymes. Nuclei and isolated mitochondria were devoid of deiodinating activity. T₄ and T₃ 5-deiodinating activity was absent in the easily sedimenting membranes and present but not enriched in particulate fractions containing microsomal membranes. These data suggest that iodothyronine 5′-deiodinase is associated with plasma membrane fractions in the cerebral cortex.