Permeability of lipid membranes revised in relation to freeze-thaw processes.

Water and solute activity gradients created during freeze-thaw processes produce water and solute fluxes across the cell membrane resulting in volume changes. Under these conditions, osmotic and thermal stresses affect the curvature, the phase behavior, and the surface properties of the lipid bilayer. These structural changes are not considered by the classical formalisms describing permeability of lipid membranes to water and nonelectrolytes such as the Nernst-Planck equation, Eyring's absolute rate theory, and Kedem-Katchalsky's thermodynamic of irreversible processes approach. In this paper, the influence of such changes on the glycerol permeation kinetics are reported. The results indicate that osmotic and chemical effects of the cryoprotectant on the membrane properties affect the rate of volume swelling depending on whether the membrane is in the gel or in the liquid crystalline state.     

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane

Fusion of multilamellar phospholipid vesicles with planar phospholipid bilayer membranes was monitored by the rate of appearance in the planar membrane of an intrinsic membrane protein present in the vesicle membranes. An essential requirement for fusion is an osmotic gradient across the planar membrane, with the cis side (the side containing the vesicles) hyperosmotic to the opposite (trans) side; for substantial fusion rates, divalent cation must also be present on the cis side. Thus, the low fusion rates obtained with 100 mM excess glucose in the cis compartment are enhanced orders of magnitude by the addition of 5-10 mM CaCl2 to the cis compartment. Conversely, the rapid fusion rates induced by 40 mM CaCl2 in the cis compartment are completely suppressed when the osmotic gradient (created by the 40 mM CaCl2) is abolished by addition of an equivalent amount of either CaCl2, NaCl, urea, or glucose to the trans compartment. We propose that fusion occurs by the osmotic swelling of vesicles in contact with the planar membrane, with subsequent rupture of the vesicular and planar membranes in the region of contact. Divalent cations catalyze this process by increasing the frequency and duration of vesicle-planar membrane contact. We argue that essentially this same osmotic mechanism drives biological fusion processes, such as exocytosis. Our fusion procedure provides a general method for incorporating and reconstituting transport proteins into planar phospholipid bilayer membranes.     

Statistical-mechanical theory of passive transport through semipermeable membranes.

The first general multicomponent equations for transport through semipermeable membranes are derived from basic statistical-mechanical principles. The procedure follows that used earlier for open membranes, but semipermeability is modelled mathematically by the introduction of external forces on the impermeant species. Gases are treated first in order to clarify the problems involved, but the final results apply to general nonideal solutions of any concentration. The mixed-solvent effect is treated rigorously, and a mixed-solvent osmotic pressure is defined. A useful specific identification of so-called osmotic flow is given, along with a demonstration that such an identification cannot be unique. Results are obtained both for discontinuous membrane models, and for a continuous model.     

Biochemical characteristics of the outer membranes of plant mitochondria

Like the outer membranes of liver mitochondria, those of plant mitochondria are impermeable to cytochrome c when intact and can be ruptured by osmotic shock. Isolated plant outer mitochondrial membranes are also similar to the corresponding liver membranes in terms of phospholipid and sterol content. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis experiments indicate that a single class of proteins (apparent molecular weight 30 000) comprises the bulk of the plant outer membrane protein. There are also considerable amounts of polysaccharide associated with these membranes, which may contribute to their osmotic stability.     

Biochemical characteristics of the outer membranes of plant mitochondria.

Like the outer membranes of liver mitochondria, those of plant mitochondria are impermeable to cytochrome c when intact and can be ruptured by osmotic shock. Isolated plant outer mitochondrial membranes are also similar to the corresponding liver membranes in terms of phospholipid and sterol content. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis experiments indicate that a single class of proteins (apparent molecular weight 30 000) comprises the bulk of the plant outer membrane protein. There are also considerable amounts of polysaccharide associated with these membranes, which may contribute to their osmotic stability.     

Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes

It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment.     

Influence of triphenyllead chloride on biological and model membranes

The physiological and hemolytic toxicities of triphenyllead chloride (TPhL) as well as its modifying influence on model lipid membranes were studied. The experiments allowed the determination of TPhL concentrations causing 50% inhibition of growth of Spirodela oligorrhiza, Lemna minor and Salvinia natans (EC50), 100% hemolysis of pig erythrocytes (C100) and destabilization of planar lipid membranes (CC). Also, fluidity of erythrocyte ghosts was measured by fluorescence technique and osmotic sensitivity of erythrocytes to the presence of TPhL. All parameters studied were found to be dependent on pH, of experimental solutions and the concentration of TPhL. Acidic conditions increased EC50 C100 and CC concentrations of TPhL. Fluorescence and osmotic measurements showed that osmotic stability and fluidity decreased with increasing trimethyllead concentration. A possible mechanism of TPhL toxicity is discussed. It is assumed that TPhL is interacting with the lipid phase of the models used. It is also assumed that there may exist various, ionic and nonionic, forms of TPhL as a result of its speciation under different experimental conditions. These species, due to their differentiated lipophilicity, may exert different effects on the model membranes studied.     

Assessment of binding of L-cystine and L-lysine by rat renal brush-border membranes

Cystine and lysine bind to isolated rat renal brush-border vesicles. Three methods to determine the extent of amino acid binding to the membranes have been compared, one relying on the osmotic reactivity of the vesicle, a second by trichloroacetic acid precipitation of membrane-bound material and a third by initial rate analysis. For cystine, all methods yield comparable results at early time points, indicating the trichloroacetic acid method is a simple and valuable tool for binding estimation under initial-rate or near initial-rate conditions. For lysine, initial rate analysis and osmotic perturbation are the methods of choice since lysine co-precipitates with trichloroacetic acid.     

Effect of chilling temperatures on osmotic water permeability and aquaporin activity in the plasma membranes from pea roots

The osmotic water permeability of plasma membrane vesicles was examined after isolation from the roots of 7-day-old etiolated pea ( Pisum sativum, cv. Orlovchanin) seedlings grown at optimal temperature and those exposed to 1-day chilling at 8°C in the end of the growth period. The homogenization medium for obtaining plasma membranes was supplemented with either SH-reagents or protein phosphatase inhibitors. The plasmalemma vesicles were purified from the microsome fraction by means of two-phase polymer system. The osmotic water permeability of membrane vesicles was evaluated from the rate of their osmotically induced shrinkage. The lowering of growth temperature was accompanied by the increase in osmotic water permeability of plasmalemma. These changes occurred without the corresponding increase in aquaporin content or permeability of membrane lipid matrix. The membranes from cooled seedlings were markedly depleted in the content of SH-groups. Furthermore, the treatment of membrane samples with a thiol-reducing agent, tributylphosphine did not raise the SH-group content in membranes from chilled plants, unlike such changes in membranes from warm-grown plants. When the homogenization medium contained dithiothreitol and phenylarsine oxide (an inhibitor of tyrosine protein phosphatases), the osmotic permeability of plasmalemma in preparations from warm-grown seedlings also increased. Based on these results, it is supposed that aquaporin-mediated water permeability of membranes is regulated through different pathways under optimal and adverse conditions for plant growth. Direct action of endogenous SH redox regulators on aquaporin activity is likely under optimal growth conditions, while protein phosphatase might mediate changes in aquaporin activity under unfavorable growth conditions.     

The contributions of normal and anomalous osmosis to the osmotic effects arising across charged membranes with solutions of electrolytes

The osmotic effect arising across a porous membrane separating the solution of an electrolyte from water (or a more dilute solution) is ordinarily due to both normal osmosis, as it occurs also with non-electrolytes, and to "anomalous" osmosis. It is shown that the normal osmotic component cannot be measured quantitatively by the conventional comparison with a non-electrolytic reference solute. Anomalous osmosis does not occur with electroneutral membranes. Accordingly, with membranes which can be charged and discharged reversibly (without changes in geometrical structure), such as many proteinized membranes, the osmotic effects caused by an electrolyte can be measured both when only normal osmosis arises (with the membrane in the electroneutral state) and when normal as well as anomalous osmosis occurs (with the membrane in a charged state). The difference between these two effects is the true anomalous osmosis. Data are presented on the osmotic effects across an oxyhemoglobin membrane in the uncharged state at pH 6.75 and in two charged states, positive at pH 4.0 and negative at pH 10.0, with solutions of a variety of electrolytes using a concentration ratio of 2:1 over a wide range of concentrations. The rates of the movement of liquid across the membrane against an inconsequentially small hydrostatic head are recorded instead of, as conventional, the physiologically less significant pressure rises after a standard time.     

Permeability studies on red cell membranes of dog, cat, and beef

Water permeability coefficients of dog, cat, and beef red cell membranes have been measured under an osmotic pressure gradient. The measurements employed a rapid reaction stop flow apparatus with which cell shrinking was measured under a relative osmotic pressure gradient of 1.25 to 1.64 times the isosmolar concentration. For the dog red cell the osmotic permeability coefficient is 0.36 cm⁴/(sec, osmol). The water permeability coefficient for the dog red cell under a diffusion gradient was also measured (rate constant = 0.10/msec). The ratio between the two permeabilities was used to calculate an equivalent pore radius of 5.9 A. This value agrees welt with an equivalent pore radius of 6.2 A obtained from reflection coefficients of nonelectrolyte water-soluble molecules, and is consistent with data on the permeability of the dog red cell membrane to glucose. These data provide evidence supporting the existence of equivalent pores in single biological membranes.     

Influence of lipid components of Mycoplasma laidlawii membranes on osmotic fragility of cells

Razin, S. (University of Connecticut, Storrs), M. E. Tourtellotte, R. N. McElhaney, and J. D. Pollack. Influence of lipid components of Mycoplasma laidlawii membranes on osmotic fragility of cells. J. Bacteriol. 91:609-616. 1966.-Lipid composition of Mycoplasma laidlawii membranes could be significantly changed by variations in the growth medium. The effect of these changes on the osmotic fragility of the cells was studied. Cholesterol, incorporated into the membrane from the growth medium, had no significant effect on osmotic fragility. Carotenoids, synthesized by the cells from acetate, were likewise without effect. Unsaturated long-chain fatty acids increased markedly the resistance of M. laidlawii to osmotic lysis and promoted growth. The fatty acids of the growth medium were incorporated mainly into membrane phospholipids. The ratio between saturated and unsaturated fatty acids in membrane lipids depended on that of the growth medium.     

Osmotic water permeability of small intestinal brush-border membranes

Summary A stopped-flow nephelometric technique was used to examine osmotic water flow across small intestinal brush-border membranes. Brush-border membrane vesicles (BBMV) were prepared from rat small intestine by calcium precipitation. Scattered 500 nm light intensity at 90° to incident was a linear function of the number of vesicles in suspension, and of the reciprocal of the suspending medium osmolality. When BBMV were mixed with hyperosmotic mannitol solutions there was a rapid increase in the intensity of scattered light that could be fit to a single exponential function. The rate constant for vesicle shrinking varied with temperature and the size of the imposed osmotic gradient. At 25°C and an initial osmotic gradient of 50 mOsm, the rate constant was 1.43±0.044 sec^–1. An Arrhenius plot of the temperature dependence of vesicle shrinking showed a break at about 25°C with an activation energy of 9.75±1.04 kcal/mole from 11 to 25°C and 17.2±0.55 kcal/mole from 25 to 37°C. The pore-forming antibiotic gramicidin increased the rate of osmotically driven water efflux and decreased the activation energy of the process to 4.51±0.25 kcal/mole. Gramicidin also increased the sodium permeability of these membranes as measured by the rate of vesicle reswelling in hyperosmotic NaSCN medium. Gramicidin had no effect on mannitol permeability. Assuming spherical vesicles of 0.1 m radius, an osmotic permeability coefficient of 1.2×10^–3 cm/sec can be estimated for the native brush-border membranes at 25°C. These fesults are consistent with the solubility-diffusion model for water flow across small intestinal BBMV but are inconsistent with the existence there of large aqueous pores.     

Sigmoidal stimulation by solutes of light-induced proton translocation in thylakoid membranes

A sigmoidal curve was obtained for the relationship betweenthe stimulation of light-induced proton uptake and the concentrationof salts in a suspending medium for thylakoid membranes. Substitutionof sucrose for the salts also resulted in a sigmoidal curve.It changed into a hyperbolic curve with salts when the mediumalready contained sucrose. The results are discussed in relationto the structural arrangement of the thylakoid membranes bythe osmotic effect of the solutes. (Received February 10, 1975; )     

Interaction of ions and water in gramicidin A channels: streaming potentials across lipid bilayer membranes

For very narrow channels in which ions and water cannot overtake one another (single-file transport), electrokinetic measurements provide information about the number of water molecules within a channel. Gramicidin A is believed to form such narrow channels in lipid bilayer membranes. In 0.01 and 0.1 M solutions of CsCl, KCL, and NaCl, streaming potentials of 3.0 mV per osmolal osmotic pressure difference (created by urea, glycerol, or glucose) appear across gramicidin A-treated membranes. This implies that there are six to seven water molecules within a gramicidin channel. Electroosmotic experiments, in which the water flux assoicated with current flow across gramicidin-treated membranes is measured, corroborate this result. In 1 M salt solutions, streaming potentials are 2.35 mV per osmolal osmotic pressure difference instead of 3.0 mV. The smaller value may indicate multiple ion occupancy of the gramicidin channel at high salt concentrations. Apparent deviations from ideal cationic selectivity observed while attempting to measure single-salt dilution potentials across gramicidin-treated membranes result from streaming potential effects.     

PIP1 aquaporins,sterols, and osmotic water permeability of plasma membranes from etiolated pea seedlings

Plasma membrane isolated from microsomal membranes of pea seedling root and shoot cells by means of aqueous two-phase polymer system was separated by flotation in discontinuous OptiPrep gradient into “light” (≤1.146 g/cm³) and “heavy” (≥1.146 g/cm³) fractions. Osmotic water permeability of plasma membrane and its two fractions was investigated by inducing transmembrane osmotic gradient on the vesicle membrane and recording the kinetics of vesicle osmotic shrinkage by the stopped-flow method. Rate constants of osmotic shrinkage and coefficients of osmotic water permeability of the membranes were estimated on the basis of the kinetic curve approximation by exponential dependencies and using electron microscope data on vesicles sizes. In plasma membrane and its fractions the content of sterols and PIP1 aquaporins was determined. It was found that in “light” PM fractions from both roots and shoots the content of PIP1 aquaporins and sterols was higher and the osmotic water permeability coefficient was lower than in “heavy” fractions of plasma membrane. The results indicate that plasma membrane of roots and shoots is heterogeneous in osmotic water permeability. This heterogeneity may be related with the presence of microdomains with different content of aquaporins and sterols in the membrane.     

Action of the steroid glycoside deltonin on the bacterial membranes of Micrococcus lysodeikticus

A slight detergent-like effect of steroid glycoside deltonine from Dioscorea deltoidea on the bacterial membranes of Micrococcus lysodeikticus was detected which resulted in the breaking of the osmotic barrier of protoplasts and in the loss from the membranes of small fragments containing the dehydrogenases of the respiratory chain but without cytochromes. These small fragments still retained the membrane structure.     

Permeability properties of peroxisomal membranes from yeasts

We have studied the permeability properties of intact peroxisomes and purified peroxisomal membranes from two methylotrophic yeasts. After incorporation of sucrose and dextran in proteoliposomes composed of asolectin and peroxisomal membranes isolated from the yeasts Hansenula polymorpha and Candida boidinii a selective leakage of sucrose occurred indicating that the peroxisomal membranes were permeable to small molecules. Since the permeability of yeast peroxisomal membranes in vitro may be due to the isolation procedure employed, the osmotic stability of peroxisomes was tested during incubations of intact protoplasts in hypotonic media. Mild osmotic swelling of the protoplasts also resulted in swelling of the peroxisomes present in these cells but not in a release of their matrix proteins. The latter was only observed when the integrity of the cells was disturbed due to disruption of the cell membrane during further lowering of the concentration of the osmotic stabilizer. Stability tests with purified peroxisomes indicated that this leak of matrix proteins was not associated with the permeability to sucrose. Various attempts to mimic the in vivo situation and generate a proton motive force across the peroxisomal membranes in order to influence the permeability properties failed. Two different proton pumps were used for this purpose namely bacteriorhodopsin (BR) and reaction center-light-harvesting complex I (RCLH_I complex). After introduction of BR into the membrane of intact peroxisomes generation of a pH-gradient was not or barely detectable. Since this pump readily generated a pH-gradient in pure liposomes, these results strengthened the initial observations on the leakiness of the peroxisomal membrane fragments. Generation of a membrane potential () was also not observed when RCLH_I complex was introduced into vesicles of purified peroxisomal membranes. The significance of the observed permeability of isolated yeast peroxisomal membranes to small molecules with respect to current and future in vitro import studies is discussed.Abbreviations CL cardiolinin - PE phosphatidylethanolamine - PC phosphatidylcholine - MES 2-(N-Morpholino)ethanesulfonic acid - R₁₈ octadecyl Rhodamine B Chloride - SUVs small unilamellar vesicles - RCLH_I-complex reaction center-light-harvesting complex I - BR bacteriorhodopsin - DCCD N,N-dicyclohexylcarbodiimide     

Membrane permeability of brain cell processes after death]

Experiments were conducted on rats. A study was made of persistence of semipermeability of the membranes of the cell processes of the brain (contraction) with the action of a hypertonic buffer at various periods after death. The membranes of the processes proved to retain the property of semi-permeability even 48 hours after death. Prefixation of the postmortem material in the glutaraldehyde did not influence the sensitivity of the membranes of the processes to the osmotic strength of the surrounding solution.