Study of water permeability through phospholipid vesicle membranes by 17O NMR.

Vesicle suspensions of up to 5% egg lecithin and 2.5% cholesterol have been found to have no effect on the NMR relaxation times of 17O from water. Addition of 1-5 mM Mn2+ to an equimolar vesicle suspension of egg lecithin and cholesterol permitted resolution of the free induction decay into two exponential components, a fast one arising from the external water and a slow one arising from the intravesicular fluid. From the rates of relaxation the mean life time of the water molecules within the vesicles was calculated to be 1+/- 0.1 ms at 22 degrees C. The size of the vesicle was estimated from electron micrographs to be about 500 A in diameter. These data yield an equilibrium water permeability, Pw, of about 8 mus-1 for the vesicle membranes. From the temperature dependence of Pw an activation energy of 12+/-2 kcal/mol was obtained. The longitudinal relaxation time (T1) of water within vesicles remained the same as in pure water.     

A structural transition in egg lecithin-cholesterol bilayers at 12 °C

The thermal coefficient of expansion of egg lecithin bilayer thickness, αd₁, was measured as a function of its cholesterol content up to mole ratio lecithin/cholesterol of 1:1, and over the temperature range 0–40 °C. At all cholesterol contents αd₁ changes abruptly at approximately 12 °C indicating a structural transition at this temperature. Above 12 °C, αd₁ decreases monotonically from −2·10⁻³ for pure egg lecithin to −1·10^–3 at mole ratio 1:1. Below 12 °C αd₁ is walways higher than above 12 °C and shows a sharp, anomalously high value of −6·10⁻³ at the mole ratio 2:1. The results have been interpreted as the movement of cholesterol into the bilayer or the formation of lecithin-cholesterol “complexes” at temperatures below 12 °C. Similar studies with phosphatidylinositol containing cholesterol showed no structural transition and lysolecithin containing cholesterol behaved differently giving two lamellar phases in equilibrium.     

NMR studies of glycerol permeability in lipid vesicles, erythrocytes and the alga Dunaliella

Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T₁ relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (ΔH^‡) were determined for glycerol at 25°C: for phosphatidylcholine vesicles 5·10⁻⁶ cm/s and 11±2 kcal/mol; for pig erythrocytes 7·10⁻⁸ cm/s and 18±3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17°C was found to be exceptionally low and only a lower limit (P<5·10⁻¹¹cm/s) could be calculated. At temperatures above 50°C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.     

Water lecithin binding in lecithin-water lamellar phases at 20°C

¹H and ³¹P continuous wave and spin-echo NMR measurements have been made on lecithin-water mixtures as a function of water content at 20 °C. An analysis of the data demonstrates the existence of two water environments, lecithin bound and free. Conclusions are presented concerning the stoichiometry and kinetics of the binding. The results indicate that six molecules of water are bound to one lecithin molecule and the lifetime of the bound water is 6·10⁻⁵±3·10⁻⁵ s.     

Water in normal muscle and muscle with a tumor

The total water content, the amount of non-freezable water, and the Na⁺ and K⁺ contents in the gastrocnemius muscle of albino mice with and without a solid tumor were determined. The spin-lattice relaxation time (T₁) for the water protons in the two kinds of muscle were measured at six resonance frequencies ranging from 4.5 to 60 MHz over the temperature range +37 to −65°C. Quantitatively calculated T₁ values are given. The difference in T₁ for the two types of muscle at temperatures above −5°C is attributed to the difference in the distribution ratio of water between hydration and free states, and bears no direct relation to the concentration of Na⁺.     

Nonelectrolyte diffusion through lecithin-water lamellar phases and red-cell membranes

The longitudinal diffusion of a homologous series of monoamides through lecithin-water lamellar phases with aqueous channel widths of 16–27 Å has been studied. The diffusion coefficients relative to water of the hydrophilic amides, formamide and acetamide, depend logarithmically on solute molar volume, as previously demonstrated in human red cells. Aqueous diffusion of amides in red-cell membranes is similar to that in a lecithin-water phase of aqueous channel width less than 16 Å, the smallest channel width used. Partition coefficients of the lipophilic amides, valeramide and isovaleramide, between lecithin vesicles and water are 1.64 and 1.15 at 20 °C. These data enabled us to compute a valeramide diffusion coefficient of 6.5 · 10⁻⁷cm² · s⁻¹ at 20 °C in the lipid region of a lamellar phase containing 30% water about one order of magnitude greater than the diffusion coefficient of spin-labelled analogs of phosphatidylcholine. The discrimination between the permeability coefficients of valeramide and isovaleramide is more than twice as great in the human red cell as between lipid diffusion coefficients in a phase containing 8% water. This suggests that the lipid region of the human red cell is more highly organized than lipid in the lecithin-water lamellar phase.     

Measurement of the curvature-elastic modulus of egg lecithin bilayers

The first determination of the curvature-elastic modulus k of a bilayer is presented. The method is based on the microscopic study of thermally fluctuating bilayer tubes. For egg lecithin at room temperature we obtain k = (2.3 ± 0.3) · 10⁻¹²erg.     

Elastic Torques about Membrane Edges: A Study of Pierced Egg Lecithin Vesicles

The shape of mechanically pierced giant vesicles is studied to obtain the elastic modulus of Gaussian curvature of egg lecithin bilayers. It is argued that such experiments are governed by an apparent modulus, ¯κ_app, not the true modulus of Gaussian curvature, ¯κ. A theory of ¯κ_app is proposed, regarding the pierced bilayer vesicle as a closed monolayer vesicle. The quantity measured, i.e. ¯κ_app/κ, where κ is the rigidity, agrees satisfactorily with the theory. We find ¯κ_app = -(1.9 ± 0.3) · 10^-12 erg (on the basis of κ = (2.3 ± 0.3) · 10^-12 erg). The result may have implications for bilayer fusion.     

In vitro studies of the effect of Environmental Conditions on the anthacnose pathogen of bananas, Colletotrichum musae

In vitro studies were undertaken to determine the effect of pH, temperature, water availability and carbon dioxide (CO₂) concentration on germination and growth of Colletotrichum musae, the causal pathogen of anthracnose of bananas. The optimum pH for germination and growth varied between 4·0 and 5·0 depending on temperature. At low pH (< 3·0) and 15°C, both germination and growth were significantly reduced, with a marked increase in the lag time, in days, prior to growth. C. musae germinated and grew over a wide range of water activities (a_w; 0·995−0·94 and 0·995−0·92, respectively) at 20, 25 and 30°C. In all cases where germination occurred appresoria were subsequently produced. Optimum growth occurred at 30°C and 0·995 a_w, although this changed to 0·98 a_w at 35°C. Increasing CO₂ concentration to 15% or reducing oxygen concentration to 1% resulted in a significant (P < 0·05) reduction in growth, but did not inhibit growth completely.     

Characterization of galactomannans isolated from legume endosperms of Caesalpinioideae and Faboideae subfamilies by multidetection aqueous SEC

Galactomannans isolated from legume seed endosperms, including those of commercial interest, have been characterized by multidetection aqueous SEC. Galactomannans derived from seeds of the Faboideae subfamily had substantially higher M_w than those from Caesalpinioideae seeds (M_w,Fab = 2.4–3.1 × 10⁶ g/mol, M_w,Caes. = 0.86–2.1 × 10⁶ g/mol) and within the latter botanical subfamily, an apparent correlation between M_w and the degree of galactose substitution DG was found. The molar mass distributions were unimodal and differed primarily by a scale factor, with distributional widths narrower than a true Flory ‘most-probable distribution’; good fits to Schulz–Zimm model were obtained. Across subfamilies no differences were found in the exponents of [η]–M and R_v–M relationships (0.61 ± 0.02, 0.54 ± 0.01, respectively), the Flory chain stiffness ratio (C_∞ = 20 ± 1 (BSF analysis)), or the persistence length (L_p = 5.5 ± 0.2 nm) obtained from SEC fraction data. However, it was found that prefactors in the [η]–M and R_v–M relationships as well as the unperturbed parameter K_Θ decrease in proportion to DG and therefore chain density. Generalized relationships incorporating galactose-dependent prefactors were therefore developed to model SEC fraction data of native galactomannans ([η]_GM = (1800 ± 200) × M_o^−1.61 × M^0.61±0.02, R_v,GM = 0.63 ± 0.05 × M_o^−0.54 × M^0.54±0.01) as well as lower-M fractions obtained by ultrasonication ([η]_GM = (730 ± 100) × M_o^−1.71 × M_w^0.71±0.02, R_v,GM = 0.49 ± 0.05 × M_o^−0.57 × M_w^0.57±0.01, M ≈ 1 × 10⁵-native). As a consequence of this dependence and the observed patterns in molar mass variation, [η] varies within a narrow range for galactomannans as a whole despite substantial M_w differences.     

Cholesterol affects divalent cation-induced fusion and isothermal phase transitions of phospholipid membranes

The influence of cholesterol on divalent cation-induced fusion and isothermal phase transitions of large unilamellar vesicles composed of phosphatidylserine (PS) was investigated. Vesicle fusion was monitored by the terbium/dipicolinic acid assay for the intermixing of internal aqueous contents, in the temperature range 10–40°C. The fusogenic activity of the cations decreases in the sequence Ca²⁺ > Ba²⁺ > Sr²⁺ Mg²⁺ for cholesterol concentrations in the range 20–40 mol%, and at all temperatures. Increasing the cholesterol concentration decreases the initial rate of fusion in the presence of Ca²⁺ and Ba²⁺ at 25°C, reaching about 50% of the rate for pure PS at a mole fraction of 0.4. From 10 to 25°C, Mg²⁺ is ineffective in causing fusion at all cholesterol concentrations. However, at 30°C, Mg²⁺-induced fusion is observed with vesicles containing cholesterol. At 40°C, Mg²⁺ induces slow fusion of pure PS vesicles, which is enhanced by the presence of cholesterol. Increasing the temperature also causes a monotonic increase in the rate of fusion induced by Ca²⁺, Ba²⁺ and Sr²⁺. The enhancement of the effect of cholesterol at high temperatures suggests that changes in hydrogen bonding and interbilayer hydration forces may be involved in the modulation of fusion by cholesterol. The phase behavior of PS/cholesterol membranes in the presence of Na⁺ and divalent cations was studied by differential scanning calorimetry. The temperature of the gel-liquid crystalline transition (T_m) in Na⁺ is lowered as the cholesterol content is increased, and the endotherm is broadened. Addition of divalent cations shifts the T_m upward, with a sequence of effectiveness Ba²⁺ > Sr²⁺ > Mg²⁺. The T_m of these complexes decreases as the cholesterol content is increased. Although the transition is not detectable for cholesterol concentrations of 40 and 50 mol% in the presence of Na⁺, Sr²⁺ or Mg²⁺, the addition of Ba²⁺ reveals endotherms with T_m progressively lower than that observed at 30 mol%. Although the presence of cholesterol appears to induce an isothermal gel-liquid crystalline transition by decreasing the T_m, this change in membrane fluidity does not enhance the rate of fusion, but rather decreases it. The effect of cholesterol on the fusion of PS/phosphatidylethanolamine (PE) vesicles was investigated by utilizing a resonance energy transfer assay for lipid mixing. The initial rate of fusion of PS/PE and PS/PE/cholesterol vesicles is saturated at high Mg²⁺ concentrations. With Ca²⁺, saturation is not observed for cholesterol-containing vesicles. The highest rate of fusion for both Ca²⁺- and Mg²⁺-induced fusion is observed with vesicles containing 30 mol% cholesterol.     

The state of water in muscle as studied by pulsed NMR

Spin-lattice relaxation times for the water protons in rat gastronemius muscle are reported over the temperature range +37 to −70°C at six resonance frequencies ranging from 4.5 to 60.0 MHz. From −8 to −70°C, the bulk of the muscle water is frozen. The unfrozen part is termed the hydrated layer and amounts to 7–12% of the total water content. Its correlation time takes teh form of a log-Gaussaian distribution function. From +37 to −8°C, the spin-lattice relaxation time is explained by the exchange of water between the hydration layer and the rest of the water, which behaves like ordinary liquid water. The fact that the observed T₂ values are smaller than the calculated values is attributed to the inner field inhomogeneity of the heterogenous system and/or the modification of T₂ due to non-zero dipolar interaction.In the presence of perdeuterated dimethylsulfoxide, the freezing point of water decreases and the amount of non-freezable water increases. T₁ of water protons for muscle containing 10, 20, and 40% dimethylsulfoxide was calculated.     

The effect of sonication on the hydrocarbon chain conformation in model membrane systems: A Raman spectroscopic study

Raman spectra are presented for egg lecithin above and below the gel-liquid crystal phase transition, and several regions of the Raman spectrum are shown to be sensitive to conformational changes in the hydrocarbon chains. These regions are used to investigate the effect of sonication on the structure of egg lecithin and dipalmitoyl lecithin vesicles.Sonication of both egg lecithin above T_m, and dipalmitoyl lecithin above and below T_m produces no change in the relative population of trans and gauche isomers in any of the systems studied. Sonication does however appear to effect interchain interactions, a possible consequence of imperfect packing towards the center of the bilayers in vesicle systems.     

Glucose transport in thymocyte plasma-membrane vesicles

Calf-thymocyte membrane vesicles, prepared by hypotonic lysis and homogenization, were isolated by standard centrifugal techniques designed for enrichment of plasma membrane. At 20°C, these vesicles equilibrated with d-glucose and 3-O-methyl-d-glucose more rapidly than with l-glucose. About 25% of the equilibrium d-sugar space (6 μl/mg protein) was very slowly penetrated by l-glucose ( ). The time course of d-sugar accumulation in excess of l-glucose accumulation indicated that this space equilibrated with d-glucose and 3-O-methyl-d-glucose with half-times of approximately 0.2–0.4 min. The remainder of the equilibrium d-sugar space (about 75%) appeared equally accessible to both glucose isomers ( to 5 min). This was confirmed in studies of efflux from preloaded vesicles, where the d-glucose space fell with a short half-time (0.2 min) to the l-glucose space, after which the two isomers exited with the same half-time. Addition of sucrose to increase osmolarity reduced both spaces (specific and non-specific) in a manner which indicated that little if any of the vesicle sugar was bound. This was confirmed by the fact that equilibrium glucose space was independent of glucose concentration and by the fact that vesicles immediately lost their sugar when diluted with water at 0°C. These data indicate the presence of two vesicle types, discriminant and indiscriminant as regards transport of the glucose isomers. Entry of d-glucose into the discriminant (stereospecific) vesicles was temperature sensitive (Q₁₀ > 2), saturable (K_m 2 mM), and was inhibited by phloretin (K_i < 200 μM), N-ethylmaleimide (K_i < 10 mM) and cytochalasin B (K_i < 2 μM), suggesting that these vesicles contain the plasma-membrane glucose carrier. Entry of l- and d-glucose into the indiscriminant vesicles showed none of these properties. The equilibrium-exchange K_m and V were about five times the entry K_m and V, indicating the substrate loading greatly facilitates carrier translocation, at least in the outward direction.     

Differences in the osmotic fragility of recycling and reserve synaptic vesicles from the cholinergic electromotor nerve terminals of Torpedo and their possible significance for vesicle recycling

In this study we demonstrate differences in the osmotic fragility of two metabolically and physically heterogeneous synaptic vesicle populations from stimulated electromotor nerve terminals. When synaptic vesicles isolated on sucrose density gradients are submitted to solutions of decreasing osmolarity 50% of VP₂-type vesicles lysed at (mean + S.E. (number of experiments)) 332 ± 14 (4) mosM and 50% of VP₁-type vesicles lysed at 573 ± 8 (3) mosM. These results indicate that recycling vesicles are more resistant to hypo-osmotic lysis and they are consistent with our earlier conclusion that changes in water content on recycling are secondary to changes in the content of the osmotically active small-molecular-mass constituents acetylcholine and ATP.     

Molecular mass and antitumor activities of sulfated derivatives of α-glucan from Poria cocos mycelia

Two kinds of water-insoluble (1 → 3)-α-d-glucan samples, ab-PCM3-I and ac-PCM3-I, isolated from different Poria cocos mycelia were sulfated, to produce two series of water-soluble derivatives ab-PCM3-I-S1–S5 and ac-PCM3-I-S1–S5, respectively. The derivatives having different weight-average molecular mass (M_w) were produced by changing reaction temperature and time as well as molar ratios between chlorosulfonic acid and number of hydroxyl groups in the glucan. The degrees of substitution (DS) of the sulfated derivatives were analyzed by elemental analysis (EA) to be 0.39–0.67 for ab-PCM3-I-S and 0.73–0.96 for ac-PCM3-I-S, respectively. The M_w and the intrinsic viscosity ([η]) of the samples ab-PCM3-I-S and the ac-PCM3-I-S were measured by size exclusion chromatography combined with laser light scattering (SEC–LLS) and viscometry in phosphate buffer solution (PBS) at 37 °C. The results indicated that their M_w ranged from 2.0 to 11.3 × 10⁴ for the samples ab-PCM3-I-S, and 4.7 to 40.0 × 10⁴ for the samples ac-PCM3-I-S. Moreover, the antitumor activities of the sulfated derivatives ab-PCM3-I-S and ac-PCM3-I-S against Sarcoma 180 tumor cell tested both in vitro and in vivo are significantly higher than those of the native α-d-glucans. Therefore, a moderate range of molecular mass from 2.0 × 10⁴ to 40.0 × 10⁴, relatively high chain stiffness and good water solubility of the sulfated derivatives are beneficial to the enhancement of their antitumor activities.     

The diffusional water permeability in the halotolerant alga Dunaliella as measured by nuclear magnetic resonance

T₁ nuclear relaxation measurements of ¹H and ¹⁷O of water have been applied to study the kinetics of the diffusional transport of water across the cytoplasmic cell membrane of Dunaliella salina and Dunaliella bardawil. The water permeability coefficients at 25°C were found to be 1.5·10⁻³ cm/s and 1.8·10⁻³ cm/s, respectively, with an activation energy of 3.7 kcal/mol. The results indicate that the cell membrane of Dunaliella exhibits high diffusional permeability to water, similar in magnitude to that found for other cells and model membranes, and a relatively low activation energy. This regularity is in contrast to the exceptionally low glycerol permeability of the membrane (Brown, F.F., Sussman, I., Avron, M. and Degani, H. (1982) Biochim. Biophys. Acta 690, 165–173).     

Evidence of multiple operational affinities for d-glucose inside the human erythrocyte membrane

1. 1. The Michaelis-Menten parameters of labelled d-glucose exit from human erythrocytes at 2°C into external solution containing 50 mM d-galactose were obtained. The K_m is 3.4 ± 0.4 mM, V 17.3 ± 1.4 mmol · 1⁻¹ cell water · min⁻¹ for this infinite-trans exit procedure.

2. 2. The kinetic parameters of equilibrium exchange of d-glucose at 2°C are K_m = 25 ± 3.4 mM, V 30 ± 4.1 mmol · 1⁻¹ cell water · min⁻¹.

3. 3. The K_m for net exit of d-glucose into solutions containing zero sugar is 15.8 ± 1.7 mM, V 9.3 ± 3.3 mol 9.3 ± 3.3 mol · 1⁻¹ cell water · min⁻¹.

4. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126–132] that there are sites with both high and low operational affinities for d-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.

Dopamine-stimulated cyclic AMP and binding of [H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10⁻⁸ M and was half-maximal at 7.9±3.4·10⁻⁷M. The increase at 1·10⁻⁵ M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10⁻⁹ M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10⁻⁵ M dopamine was 2.3±0.9·10⁻⁶M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The K_d value for high affinity binding sites was 0.43±0.1·10⁻⁷M and 4.7±1.6·10⁻⁷M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10^/t-7M noradrenaline, 2·10^/t-7 M epinine, 4.1±1.8·10^/t-6M fluphenazine, 8.0±1.6·10^/t-6M haloperidol, 4.2±1.2·10⁻⁶Mcis-flupenthixol, 2.7±0.4·10⁻⁵Mtrans-flupenthixol, >1·10⁻⁵M apomorphine, sulpiride, naloxone and isoproterenol.     

Interactions of divalent cations or basic proteins with phosphatidylethanolamine vesicles

Small unilamellar vesicles have been prepared from phosphatidylethanolamine by sonication of the lipid in aqueous buffers of low ionic strength and high pH. These vesicles and their interactions with various di- and trivalent cations have been characterized using freeze-fracture electron microscopy. Phosphatidylethanolamine from 4 sources was examined: Hens' yolk phosphatidylethanolamine, human grey matter phosphatidylethanolamine, Escherichia coli phosphatidylethanolamine and dimyristoyl phosphatidylethanolamine. The phosphatidylethanolamine from natural sources formed spherical, uniform 20–40 nm vesicles while dimyristoyl phosphatidylethanolamine formed larger, 70 × 25 nm, disc-shaped vesicles when sonicated above the phase transition temperature. Fusion of the unilamellar egg phosphatidylethanolamine, E. coli phosphatidylethanolamine and human grey matter phosphatidylethanolamine vesicles was induced by dialysis against buffers containing 2.0 nM Ca⁺ or 3.0 mM Mg²⁺. The fusion of the vesicles resulted in the precipitation of the lipid and the formation of multilamellar and, in some cases, hexagonal II structures. Dimyristoyl phosphatidylethanolamine vesicles were precipitated at 55°C by 1.0 mM Ca⁺ or 2.0 mM Mg²⁺. Treatment of the calcium- and magnesium-precipitated vesicles of hen's egg yolk phosphatidylethanolamine, E. coli phosphatidylethanolamine, human grey matter phosphatidylethanolamine and dimyristoyl phosphatidylethanolamine with EDTA resulted in resuspension of the lipid. The specific size and shape of the vesicles formed in this manner depends on the type of phosphatidylethanolamine and ion involved. Dialysis of the Ca⁺- and Mg²⁺-precipitated egg phosphatidylethanolamine vesicles against buffer containing no Ca⁺, Mg²⁺ or EDTA also resulted in dissociation of the precipitate and formation again of a new vesicle population. This evidence indicates that the Ca⁺ and Mg²⁺ are not strongly bound to the phosphatidylethanolamine.Egg phosphatidylethanolamine vesicles would fuse in the presence of many di- and trivalent ions. Egg phosphatidylethanolamine vesicles were precipitated by beryllium, aluminum, chromium, manganese, cobalt, nickel, copper, zinc, strontium, cadmium, barium, lanthanium, mercury and lead. The amount of ion required to precipitate the vesicles and the type of structure resulting from the fusion of the vesicles was found to be unique for each ion.Small unilamellar vesicles prepared from egg phosphatidylethanolamine were reacted with several basic proteins (cytochrome c, basic protein from human myelin, protamine, poly-l-lysine and cationically-modified ferritin). The basic proteins also initiated the fusion of egg phosphatidylethanolamine vesicles but these proteins did not fuse egg phosphatidylcholine vesicles nor did normal ferritin initiate fusion. Human myelin basic protein initiated the fusion of dimyristoyl phosphatidylethanolamine vesicles above and below the phase transition of this lipid.