Phase transitions of phospholipid single-wall vesicles and multilayers. Measurement by vibrational raman spectroscopic frequency differences

Raman spectroscopic frequency differences between selected carbon-carbon stretching modes of lipid hydrocarbon chains were determined as a function of temperature for use in monitoring lipid phase transition behavior and acyl chain disorder in both multilamellar and single-wall vesicles. Transition temperatues detected by this procedure for pure dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine multilayers were observed at $\text{39±1 °}\text{C}$ and $\text{23±1 °}\text{C}$, respectively. Although the phase transition for unilamellar vesicles of dipalmitoyl phosphatidylcholine occurred at nearly the same temperature as the multilayers, the crystal-liquid crystalline transition for the single-shell vesicles appeared to span a slightly broader temperature range, a characteristic consistent with irregularities in the packing arrangement of the hydrocarbon chains. Within the precision of the Raman spectroscopic method, however, the temperature behavior of both the multilamellar and the unilamellar dimyristoyl phosphatidylcholine assemblies appeared nearly identical. The temperature profile for the Raman frequency differences of an excess water sonicate of 25 mol percent cholesterol in dipalmitoyl phosphatidylcholine served as an example of the effect upon lipid phase transition characteristics of a bilayer component intercalated between the acyl chains. For this particular cholesterol-lipid system the phase transition was broadened over a 30 °C temperature range, in contrast to the narrow 5?4 °C range observed for pure multilayer and single-shell vesicle particles.     

Laser-Raman study of valinomycin-doped and omega-CD3-labeled phosphatidylcholine liposomes

The relative intensities of the CH stretching vibrations are used to study the interaction of lecithin liposomes with valinomycin, a mobile carrier for alkali ions. In the case of dipalmitoyl lecithin liposomes, the lipid phase transition is not significantly affected by valinomycin. However, in dimyristoylphosphatidylcholine liposomes, the phase transition is broadened by the addition of 1 mol% valinomycin even at low K⁺ concentrations. This indicates that the carrier interacts with the hydrophobic core of the bilayer. In addition, these experiments showed that the lipid phase transitions which are reflected by the methylene groups and the terminal methyl groups are nearly equivalent. Therefore a reevaluation of the assignment of the CH stretching bands seemed necessary. Our Raman spectroscopic investigation of ω-deuterated dipalmitoyl lecithin liposomes improves the assignment of CH stretch vibrations to methylene and methyl groups. The deuteration displaces the methyl group vibrations to the 2050–2250 cm^?1 region and produces gross intensity changes of the bands at 2883 and 2936 cm^?1. These changes lead to the conclusion that both bands arise from vibrations which can be attributed simultaneously to the methylene and methyl groups of the fatty acid chains. The displacement of the CH₃ group vibrations from their original positions enhances the intensity ratios (per centimeter), $\text{2883}\text{2847}$ and $\text{2936}\text{2847}$, for the CH₂- groups which are used to monitor the lipid phase transition, and implies that the contributions of the CH₃ groups to the phase transition curves are unimportant. Our finding that the -CD₃ groups reflect no phase transition supports this statement.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions.This lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (?120°C to +120°C).Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids.Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H²O and ²H₂O media yielded a value of 1.013 ± 0.026 ml/g for the partial specific volume of this lipid.We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude.Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

Cooperativity of the phase transition in single- and multibilayer lipid vesicles

The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersions of dimyristoyl phosphatidylcholine, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, $\text{2,2,6,6-}\text{tetramethylpiperdine}\text{-N-}\text{oxyl}$, phosphatidylcholine and stearic acid, and also 90° light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526–535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/?σ, is a measure of the mean number of molecules, per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.     

Effects of temperature and molecular interactions on the vibrational infrared spectra of phospholipid vesicles

Infrared spectra were obtained as a function of temperature for a variety of phospholipid/water bilayer assemblies (80% water by weight) in the 3000-950 cm^?1 region. Spectral band-maximum frequency parameters were defined for the 2900 cm^?1 hydrocarbon chain methylene symmetric and asymmetric stretching vibrations. Temperature shifts for these band-maximum frequencies provided convenient probes for monitoring the phase transition behavior of both multilamellar liposomes and small diameter single-shell vesiclesof dipalmitoyl phosphatidylcholine/water dispersions. As examples of the effects of bilayer lipid/cholesterol/water (3 : 1 mol ratio) and lipid/cholesterol/amphotericin B/water (3 : 1 : 0.1 mol ratios) vesicles were examined using the methylene stretching frequency indices. In comparison to the pure vesicle form, the transition width of the lipid/cholesterol system increased by nearly a factor of two (to 8°C) while the phase transition temperature remained approximately the same (41° C). For the lipid/cholesterol/amphotericin B system, the phase transition temperature increased by about 4.5° C (to 45.5°C) with the transition width increasing by nearly a factor of four ($\text{to}\text{≈ 15°}\text{C}$) above that of the pure vesicles. The lipid/cholesterol/amphotericin B data were interpreted as reflecting the formation below 38°C of a cholesterol/amphotericin B complex whose dissociation at higher temperature (38–60°C range) significantly broades the gel-liquid crystalline phase transition.     

Lipid-protein interactions in membrane models fluorescence polarization study of cytochrome b5-phospholipids complexes

According to previous authors, cytochrome $\text{b}{}_5$, when extracted from bovine liver by a detergent method, is called cytochrome $\text{d-b}{}_5$. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome $\text{t-b}{}_5$.Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very senstive to the binding of proteins, and so is a useful method to study lipid-protein interactions.The chromophore mobility, $\text{R}$, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome $\text{d-b}{}_5$, whereas $\text{R}$ does not change for cytochrome $\text{c}$ and cytochrome $\text{t-b}{}_5$. This can be interpreted as a strengthening of the bilayer, only due to the interaction of the hydrophobic peptide tail.Interaction of dipalmitoyl phosphatidylcholine vesicles with cytochrome $\text{d-b}{}_5$ occurs either below or above the melting temperature of the aliphatic chains (41 °C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected.Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome $\text{d-b}{}_5$, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.     

Carbon-13 nuclear magnetic resonance studies of cerebroside derivatives and their properties in lecithin bilayers (1)

The ¹³C NMR chemical shifts and spin-lattice relaxation times of D-galactosylsphingosine derivatives in CDCl₃-CD₃OD and in egg-yolk lecithin vesicles in D₂O, and of N-acetylpsychosine micelles, are reported. Results with sonicated, unilamellar vesicles containing cerebroside and EYL^a show that (1) cerebrosides decrease the fluidity of the lecithin bilayer membrane and have the greatest effect on the glycerol backbone and choline methyl carbons. (2) N-acetylpsychosine experiences a greater freedom of motion in the galactose region than does cerebroside and does not reduce the fluidity of the lecithin as much as cerebroside. (3) Ac-Psy/EYL vesicles formed are permeable to Yb³⁺ but cerebroside/lecithin vesicles are not. (4) The choline groups on the inner bilayer surface are less mobile than those on the outer surface according to preliminary T₁ measurements of the Yb³⁺-separated resonances. (5) Yb³⁺-induced chemical shifts of choline methyl and choline $\text{C}\text{H}{}_2$OP peaks in mixed cerebroside-lecithin vesicle systems indicate a small preference for cerebroside in the outside monolayer. The data show that these molecules have significant effects on bilayer conformational mobilities, particularly near the surface, and thus demonstrate one mechanism for modulation of cell surface properties by glycosphingolipids.     

Microviscosity changes during differentiation of neuroblastoma cells

Microviscosity $\text{η}$ of the plasma-membrane lipid matrix was measured in exponentially growing and differentiating C1300 mouse neuroblastoma cells, attached to a glass substratum, by fluorescence polarisation of 1,6-diphenyl-1,3,5-hexatriene. Upon differentiation $\text{η}$ decreases progressively, reaching values below those observed in the growth phase. Treatment of the cells with dipalmitoyl phosphatidylcholine vesicles reversibly inhibits morphological differentiation. The results show that a high membrane fluidity is a prerequisite for differentiation.     

The affinity of cholesterol for phosphatidylcholine and sphingomyelin

Erythrocyte ghosts were incubated with sonicated vesicles and the uptake of cholesterol by vesicles allowed to proceed to equilibrium. The experiments were carried out for a series of phospholipids at different temperatures. The equilibrium partition of cholesterol between ghosts and single shelled vesicles provided a measure of the relative affinities of cholesterol for the different phospholipids studied. It was found that the affinity of cholesterol for dipalmitoyl phosphatidylcholine was the same as that for $\text{N-}\text{palmitoyl}$ sphingomyelin both at temperatures above and below the gel to liquid crystalline transition temperature of these phospholipids.     

The activation energy of incorporation of extrinsic probes in model vesicles

Time dependence of fluorescence enhancement of probes after addition to lipid vesicles has been used to investigate the position of chromophores in the lipid bilayer. Incorporation studies of a series of $\text{n-(9-}\text{anthroyloxy}$) fatty acids ($\text{n}\text{= 2, 2, 12}\text{and}\text{16}$) and 1,6-diphenylhexatriene in dipalmitoyl phosphatidylcholine vesicles are described. The activation energies for incorporation of these several lipid-mimic type fluorescent probes have been measured. Results show that the activation energy is a function of the distance of the anthracene moiety (chromophore) from the polar end of the probe and the length of the acyl portion of the probe. An average insertion energy of 0.6 kcal/carbon is seen for these fatty acid probes. The activation energy of 1,6-diphenylhexatriene, a factor of 2 greater than that of 16-(9-anthroyloxy)palmitic acid, is consistent with locating 1,6-diphenyl-hexatriene in the middle of the bilayer.     

Effect of phospholipid methylation on calcium transport and (Ca2+ + Mg2+)-ATPase activity in kidney cortex basolateral membranes

Basolateral membranes isolated from hog kidney cortex, enriched 12- to 15-fold in (Na⁺ + K⁺)-ATPase activity, were 80% oriented inside-out as determined by assay of oubain-sensitive (Na⁺ + K⁺)-ATPase activity before and after opening of the membrane vesicle preparation with a mixture of deoxycholate and EDTA. In these membrane preparations 80% of total phosphatidylethanolamine was accessible to trinitrophenylation by trinitrobenzenesulfonic acid at 4°C, while at 37°C all of phosphatidylethanolamine fraction was chemically modified. Phospholipase C treatment resulted in hydrolysis of 80% phosphatidylethanolamine, 40% phosphatidylcholine and 35% of phosphatidylserine. Sphingomyelinase treatment resulted in 20% hydrolysis of sphingomyelin, presumably derived from right-side-out oriented vesicles. Results indicate that phosphatidylethanolamine is oriented exclusively on the outer leaflet of the lipid bilayer of inside-out oriented vesicles. Methylation of phospholipids in basolateral membranes with $\text{S-}\text{adenosyl}\text{[methyl-}{}^3\text{H}\text{]}\text{methionine}$ resulted in the three successive methylation of ethanolamine moiety of phosphatidylethanolamine to phosphatidylcholine. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for $\text{S-}\text{adenosylmethionine}$ was 1·10^?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg²⁺ was without any effect between pH 5 and 10. Basolateral membranes incubated in the presence of methyl donor, $\text{S-}\text{adenosylmethionine}$, exhibited increased (12–15%) (Ca²⁺ + Mg²⁺)-ATPase activity and increased ATP-dependent uptake of calcium. ATP-dependent calcium uptake in these vesicles was insensitive to oligomycin and ouabain but was abolished completely by 50 μM vanadate. The increase in ATP-dependent calcium uptake was due to an increase in $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ and not due to a change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Ca²⁺. Preincubation of membranes with $\text{S-}\text{adenosylhomocysteine}$, a methyltransferase inhibitor, abolished the stimulatory effect of phospholipid methylation on calcium uptake. Phospholipid methylation at both low and high pH did not result in a change in bulk membrane fluidity as determined by the fluorescence polarization of diphenylhexatriene. These results suggest that phospholipid methylation may regulate transepithelial calcium flux in vivo.     

Interactions of phospholipid vesicles with rat hepatocytes in vitro influence of vesicle-incorporated glycolipids

We examined the interaction of glycolipid-containing phospholipid vesicles with rat hepatocytes in vitro. Incorporation of either $\text{N-}\text{lignoceroyldihydrolactocerebroside}$ or the monosialoganglioside, G_M1, enhanced liposomal lipid uptake 4–5-fold as judged by the uptake of radioactive phosphatidylcholine as a vesicle marker. Cerebroside enhanced phospholipid uptake only when incorporated into dimyristoyl, but not into egg phosphatidylcholine vesicles. The lack of cerebroside effect in egg phosphatidylcholine-containing vesicles appeared to be due to a limited exposure of the carbohydrate part of the glycolipid as suggested by the reduced agglutinability of those vesicles by Ricinus communis agglutinin.In contrast to the results with radioactive phosphatidylcholine, we observed only a 20% increase in vesicle-cell association as a result of glycolipid incorporation, when a trace amount of [¹⁴C]cholesteryloleate served as a marker of the liposomal lipids or when using the fluorescent dye, carboxyfluorescein, as a marker of the aqueous space of the vesicles. By the same token, intracellular delivery of vesicle-contents was only slightly enhanced (approx. 10%).The discrepancy between the association with the cells of phosphatidylcholine on the one hand and cholesteryoleate or entrapped marker on the other suggests different mechanisms of uptake for these markers. Our results are compatible with the notion that the main effect of incorporation of glycolipids into the vesicles is the enhancement of exchange or transfer of phospholipid molecules between vesicles and cells. Incubation of the cells with galactose or lactose, prior to addition of vesicles, suggests that this enhanced phospholipid exchange or transfer involves specific recognition of the terminal galactose residues of the glycolipid vesicles by a receptor present on the plasma membranes of hepatocytes.     

Phospholipid interactions with cytochrome P-450 in reconstituted vesicles Preference for negatively-charged phosphatidic acid

Cytochrome $\text{P-450}$ LM2 was reconstituted by the cholate-dialysis method into vesicles containing a mixture of either phosphatidylcholine or phosphatidylethanolamine with up to 50 mol% of phosphatidic acid. Phase transition curves in the presence or absence of cytochrome $\text{P-450}$ were obtained from electron paramagnetic resonance experiments by measuring the partitioning of 2,2,6,6-tetramethylpiperidine-1-oxyl. Protein-free phospholipid vesicles exhibit a phase separation into domains of gel phase enriched in phosphatidic acid in a surrounding fluid matrix containing mainly phosphatidylcholine. The phase transition of the phosphatidic acid domains disappeared following incorporation of cytochrome $\text{P-450}$ into the bilayers. In contrast, in vesicles containing mixtures of egg-phosphatidic acid and dimyristoyl phosphatidylcholine, the phase transition of the domains enriched in dimyristoyl phosphatidylcholine was less sharp than in the corresponding vesicles containing cytochrome $\text{P-450}$. The results of both of these experiments could be explained by a redistribution of the mol fraction of the two phospholipids in the gel phase due to preferential binding of the egg-phosphatidic acid to the cytochrome $\text{P-450}$. For comparison, incorporation of cytochrome $\text{P-450}$ into uncharged vesicles of dimyristoyl phosphatidylcholine and egg-phosphatidylethanolamine did not alter the     

Asymmetric antagonistic effects of an inhalation anesthetic and high pressure on the phase transition temperature of dipalmitoyl phosphatidic acid bilayers

The phase transition temperature ($\text{T}{}_{\mathrm{<mtext>t</mtext>}}$) of dipalmitoyl phosphatidic acid multilamellar liposomes is depressed 10°C by the inhalation anesthetic methoxyflurane at a concentration of 100 mmol/mol lipid. Application of 100 atm of helium pressure to pure phosphatidic acid liposomes increased $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ only 1.5°C. However, application of 100 atm helium pressure to dipalmitoyl phosphatidic acid lipsomes containing 100 mmol methoxyflurane/mol lipid almost completely antagonized the effect of the anesthetic. A nonlinear pressure effect is observed. In a previous study, a concentration of 60 mmol methoxyflurane/mol dipalmitoyl phosphatidylcholine depressed $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ only 1.5°C, exhibiting a linear pressure effect. The completely different behavior in the charged membrane is best explained by extrusion of the anesthetic from the lipid phase.     

Protein rotation and chromophore orientation in reconstituted bacteriorhodopsin vesicles

Bacteriorhodopsin has been reconstituted into lipid vesicles with dipalmitoyl and dimyristoyls phosphatidylcholine. Circular dichroism (CD) measurements show that the proteins are in a monomeric state above the main lipid phase transition temperature (T_c), 41 and 23°C for dipalmitoyl and dimyristoyl phosphatidylcholine, respectively. Below T_c, the CD spectrum is the same as that found for the purple membrane. The latter result implies that the orientation of the chromophore at these temperatures is most likely the same as in the purple membrane ($\text{70° ± 5°}$ from the normal to the membrane plane).Transient dichroism measurements show that below T_c the proteins are immobile, while above this temperature protein rotation around an axis normal to the plane of the membrane is occurring. In addition, from the data the angle of the chromophore for the rotating proteins with respect to the rotational diffusion axis can be calculated. This angle is found to be $\text{30° ± 3°}$ and $\text{29° ± 4°}$ in dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, respectively. This is considerably smaller than the value of $\text{70° ± 5°}$ for the natural biomembrane. A reversible reorientation of the chromophore above and below the respective main T_c transition temperature could explain the change of angle observed provided that all the molecules rotate above T_c.     

Magnetic resonance study of the distribution of 2,2,6,6-tetramethylpiperidine-N-oxyl in phosphatidylcholine bilayers.

With the aid of paramagentic praseodymium ions the resonances at 60 MHz of the inward and outward facing choline methyl protons of sonicated egg yolk phosphatidylcholine vesicles were resolved. The subsequent addition of 2,2,6,6,-tetramethylpiperidine-N-oxyl (TEMPO) to the vesicle suspension broadened the inner and outer resonances equally. TEMPO easily penetrates the egg yolk phosphatidylcholine vesicles and has free access to the entire lipid volume above the gel to liquid crystalline transition temperature. The electron spin resonance (ESR) spectrum of TEMPO in the egg yolk phosphatidylcholine suspension exhibits features indicating that TEMPO dissolves principally in the hydrocarbon portion of the egg yolk phosphatidylcholine bilayer. The egg yolk phosphatidylcholine methylene chain proton resonances are also broadened by TEMPO notably to a greater extent than the choline methyl resonances. These data indicate that TEMPO should be more sensitive to the melting behavior of the fatty acyl chains of phospholipid suspensions than to the polar head groups.     

Lipid vesicle interaction with EMT-6 tumor cells and effect on subsequent cell growth

Artificial lipid vesicles of varying composition were incubated with EMT-6 tumor cells, after which the transfer of vesicle lipids to the cells and their growth $\text{in}\text{vitro}$ were determined. Vesicles composed of phosphatidylcholine, cholesterol, and gangliosides could transfer phosphatidylcholine and cholesterol to the cells, but this transfer had no effect on the subsequent growth of the cells. However, preincubation of the cells with vesicles containing sterylamine or phosphatidylserine did inhibit the subsequent growth of the cells in a tissue culture assay system. It might be possible to deliver to certain cells growth inhibitory compounds carried in lipid vesicles.     

Binding of insulin to the external surface of liposomes: Effect of surface curvature,temperature, and lipid composition

The binding of insulin to the external surface of phosphatidylcholine liposomes as a function of the temperature, the surface curvature, and the composition of lipids was studied. The amount of the saturated binding of insulin to liposomes was assessed by gel-filtration chromatography. The binding of insulin to small unilamellar vesicles was highly dependent upon the temperature, favoring low temperatures. As the temperature increased, there was a distinct temperature range where the binding of insulin to small unilamellar vesicles decreased. The temperature ranges for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles were found to be 10–20°C and 21–37°C, respectively. These temperature ranges were quite different from the reported ranges of the gel → liquid crystalline phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) for DMPC or DPPC small unilamellar vesicles. In contrast to other proteins, the amount of insulin bound to DMPC and DPPC small unilamellar vesicles was negligible at or above the upper limit of the above temperature ranges, and increased steadily to 6–7 μmol of insulin per mmol of phospholipid as the temperature decreased to or below the lower limit of these temperature ranges. On the other hand, the binding of insulin to the large multilamellar liposomes cannot be detected at all temperatures tested. The affinity of insulin to neutral phosphatidylcholine small unilamellar vesicles appeared to be related to the surface curvature of the liposomes, favoring the liposomes with a high surface curvature. Furthermore, the amount of insulin bound to small unilamellar vesicles decreased as the content of the cholesterol increased. The presence of 10% molar fraction of phosphatidic acid did not appear to affect the binding of insulin to small unilamellar vesicles. However, the presence of 5% molar fraction of stearylamine in DPPC small unilamellar vesicles increased the amount of bound insulin as well as the extent of aggregation of liposomes. The results of the present study suggest that the interstitial regions of the acyl chains of phospholipids between the faceted planes of small unilamellar vesicles below $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ may be responsible for the hydrophobic interaction of insulin and small unilamellar vesicles. The tight binding of insulin to certain small unilamellar liposomes could lead to an overestimation of the true amount of insulin encapsulated in liposomes, if care is not taken to eliminate the bound insulin during the procedure of encapsulating insulin in liposomes.     

The kinetics of ionophore X-537A-mediated transport of manganese through dipalmitoylphosphatidylcholine vesicles A 1H-NMR study

We have studied the kinetics of ionophore X-537A-mediated transport of manganese ions into small unilamellar vesicles formed from dipalmitoylphosphatidylcholine. To follow the transport we used the paramagnetic effect of manganese on the ¹H-NMR signal from choline trimethylammonium groups on the inner phospholipid monolayer. The transport of only one manganese ion produces an intravesicular concentration which is high enough (approx. 1 mM) to substantially broaden this signal. The observed signal thus arises predominantly from those vesicles which contain no manganese. Therefore, as manganese is transported into the vesicles the observed signal decreases in intensity, but does not broaden. The initial time-dependence of the intensity of the signal, $\text{S(t)}$, can be approximated by the simple first-order rate law: $\text{S(t) = S}\text{(O)}\text{exp}\text{(?K′t)}$, where $\text{K′}$ is the probability per unit time for the transport of a manganese ion from the external medium to the intravesicular space. From the dependence of $\text{K′}$ on the ionophore X-537A concentration we conclude that manganese is transported into the vesicles via both 1 : 1 and 2 : 1 complexes with ionophore X-537A. At low ratios of ionophore X-537A to vesicles transport via the 1 : 1 complex predominates; at high ratios transport via the 2 : 1 complex predominates. From the dependence of $\text{K′}$ on manganese concentration we determined that under our conditions the equilibration of ionophore X-537A between vesicles is much faster than the transport of manganese through the vesicles. Lastly, from the dependence of $\text{K′}$ on temperature, we conclude that the ionophore X-537A-mediated transport of manganese into the dipalmitoylphosphatidylcholine vesicles is very sensitive to the gel-liquid crystalline phase transition.