P.W.M. Van Dijck

The preferential interactions of cholesterol with different classes of phospholipids

1. By differential scanning calorimetry a preferential affinity of cholesterol for sphingomyelin was established in mixtures of sphingomelin and phosphatidylcholine where sphingomyelin was either the higher or the lower melting phospholipid. 2. A preferential affinity of cholesterol for sphingomyelin was also found in mixtures of sphingomyelin and phosphatidylethanolamine where sphingomyelin was either the higher or the lower melting phospholipid. The sphingomyelin used was isolated from beef erythrocytes or synthetic palmitoyl sphingomyelin. 3. In mixtures of phosphatidylserine with phosphatidylethanolamine, or phosphatidylserine with phosphatidylcholine, cholesterol showed the highest affinity for the lower melting phospholipid. 4. In a previous paper (van Dijck et al. (1976) Biochim. Biophys. Acta 455, 576-588) it was established that cholesterol has a higher affinity for phosphatidylcholine than for phosphatidylethanolamine. The affinity order of cholesterol for the neutral phospholipids which can be deduced form these experiments is sphingomyelin greater than phosphatidylcholine greater than phosphatidylethanolamine.

The preference of cholesterol for phosphatidylcholine in mixed phosphatidylcholine-phosphatidylethanolamine bilayers.

The following phosphatidylethanolamines were studied by differential scanning calorimetry: 1,2-dipalmitoleoyl-, 1,2-dioleoyl-, 1,2-dilauroyl-, 1,2-dielaidyl-, 1,2-dimyristoyl- and 1,2-dipalmitoyl-sn-glycero-3-phosphoryl-ethanolamine. The saturated and trans-unsaturated species underwent thermotropic phase transitions at temperatures about 20-30 degrees C higher than the corresponding phosphatidylcholines but the enthalpy changes were nearly identical. The transition temperatures for the cis-unsaturated species were about the same as those of the corresponding phosphatidylcholines but here the enthalpy change was markedly decreased as compared with the phosphatidylcholines. Freeze-fracture electron microscopy revealed phase changes from a lamellar to a hexagonal phase for 1,2-dipalmitoleoyl- and 1,2-dioleoyl-sn-glycero-phosphorylethanolamine at 20 and 0 degrees C respectively. At these temperatures no transitions were apparent in the calorimeter scan. Incorporation of increasing amounts of cholesterol into phosphatidylethanol-amine bilayers gradually decreased the enthalpy changes of the phase transition in the same manner as was demonstrated before for phosphatidylcholine/cholesterol mixtures. This was studied both for 1,2-dipalmitoleoyl- and 1,2-dimyristoyl-sn-glycerophosphorylethanolamine. In an equimolar mixture of 1,2-dioleoyl- and 1,2-dipalmitoylphosphoryl-ethanolamine, which showed phase separation, cholesterol preferentially decreased the transition of the lowest melting component. In equimolar mixtures of phosphatidylethanolamines and phosphatidylcholines, which showed phase separation, cholesterol preferentially abolished the transition of the phosphatidylcholine component present. This occurred both in experiments where the phosphatidylcholine was the lowest melting and where it was the highest melting component present in the mixture. These experiments strongly suggest that in phosphatidylcholine-phosphatidylethanolamine mixtures at temperatures where both components are in the liquid-crystalline state cholesterol is preferently associated with the phosphatidylcholine component in the mixture.

Comparative studies on the effects of pH and Ca2+ on bilayers of various negatively charged phospholipids and their mixtures with phosphatidylcholine

(1) The thermotropic behaviour of dimyristoyl phosphatidylglycerol, phosphatidylserine, phosphatidic acid and phosphatidylcholine was investigated by differential scanning calorimetry and freeze-fracture electron microscopy as a function of pH and of Ca2+ concentration. (2) From the thermotropic behaviour as a function of pH, profiles could be constructed from which apparent pK values of the charged groups of the lipids could be determined. (3) Excess Ca2+ induced a shift of the total phase transition in 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-glycerophosphoglycerol mixtures. In 14 : 0/14 : 0-glycerophosphocholine bilayers containing 16 : 0/16 : 0-glycerophosphoglycerol lateral phase separation was induced by Ca2+. (4) Up to molar ratios of 1 : 2 of 14 : 0/14 : 0-glycerophosphoserine to 14 : 0/14: 0-glycerophosphocholine, excess Ca2+ induced lateral phase separation. Addition to mixtures of higher molar ratios caused segregation into different structures: the liposome organization and the stacked lamellae/cylindrical organization. (5) Addition of excess Ca2+ to mixtures of 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-phosphatidic acid caused, independent of the molar ratio, separation into two structural different organizations. (6) The nature of Ca2+-induced changes in bilayers containing negatively charged phospholipids is strongly dependent on the character of the polar headgroup of the negatively charged phospholipid involved.

Influence of fatty acid and sterol composition on the lipid phase transition and activity of membrane-bound enzymes in Acholeplasma laidlawii

1. 1. The temperature dependency of NADH-oxidase, p-nitrophenylphosphatase and Mg2+-dependent ATPas was studied in Acholeplasma laidlawii cell membranes with varying fatty acid and sterol composition. In these membranes the gel → liquid crystalline phase transition of the membrane lipids was measured by differential scanning calorimetry. 2. 2. The Arrhenius plots of the NADH oxidase and p-nitrophenylphosphatase activities showed no discontinuities although the membrane lipids underwent a phase transition in the temperature range studied. 3. 3. Distinct breaks in the Arrhenius plot of the ATPase activity were observed and found to be dependent upon the fatty acid composition of the membrane lipids; these breaks occurred at the lower ends of the lipid phase transitions. We conclude from these results that at these temperatures the ATPase is associated with molecular lipid species with the lowest transition temperature. 4. 4. Incorporation of cholesterol into the membrane decreased the temperature of the break in the ATPase activity and the temperature of the lower end of the lipid phase transition. This effect is due to the lipid-cholesterol interaction since (a) the effect is reversed by the polyene antibiotic filipin which complexes the cholesterol and (b) incorporation of epichoolesterol which does not interact with other lipids did not influence the temperature of the break in the Arrhenius plot.

Non-random distribution of cholesterol in phosphatidyl-choline bilayers

Abstract 1. 1. The effect of cholesterol upon the phase transition occurring in various mixtures of synthetic disaturated phosphatidylcholines with fatty acid constituents of 12, 14, 16, and 18 carbon atoms was measured by differential scanning calorimetry. 2. 2. Mixtures which differ 2 carbon atoms show cocrystallisation of the paraffin chains. In these mixtures cholesterol interacts randomly with the various phosphatidylcholine species. 3. 3. Mixtures which differ 4 or more carbon atoms show monotectic behaviour (phase separation). In these mixtures cholesterol interacts preferentially with the phosphatidylcholine species with the lowest transition temperature. This results in a non-random distribution of cholesterol at temperatures at which phase separation occurs. The implications of these findings for cholesterol-containing biological membranes are discussed.

Miscibility properties of binary phosphatidylcholine mixtures. A calorimetric study.

From data obtained by differential scanning calorimetry phase diagrams were constructed, using a thermodynamically based fitting method. The following binary mixtures of phosphatidylcholines in water were studied: 14:0/14:0-glycerophosphocholine/16:0/16:0-glucerophosphocholine, 14:0/14:0-glycerophosphocholine/18:0/18:0-glycerophosphocholine, 12:0/12:0-glycerophosphocholine/16:0/16:0-glycerophosphocholine, 18:1t/18:1t-glycerophosphocholine/14:0/14:0-glycerophosphocholine and 18:1t/18:1t-glycerophosphocholine/16:0/16:0-glycerophosphocholine. A comparison is made of the present results with those obtained using probe techniques and the differences are discussed.

Effects of cholesterol on the properties of equimolar mixtures of synthetic phosphatidylethanolamine and phosphatidylcholine. A 31P NMR and differential scanning calorimetry study

1. 1. The interaction of cholesterol with equimolar mixtures of 18 : 1c18 : 1c phosphatidylethanolamine and 16 : 016 : 0 phosphatidylcholine has been investigated employing differential scanning calorimetry and 31P NMR. At temperatures where lateral phase separation occurs cholesterol interacts preferentially with the (gel state) 16 : 016 : 0 phosphatidylcholine component, in agreement with previous studies (van Dijck et al. (1976) Biochim. Biophys. Acta 455, 576–587). Bilayer structure is maintained in such situations, and the effects do not therefore arise due to a preference of cholesterol for phospholipids in bilayer or non-bilayer phases. Further, the 31P NMR results indicate that when both species are in the liquid crystalline state the preferential interaction of cholesterol with the phosphatidylcholine component is maintained. 2. 2. The addition of cholesterol to (liquid crystalline) equimolar mixtures of 18 : 1c18 : 1c phosphatidylethanolamine and 16 : 016 : 0 phosphatidylcholine stabilizes the bilayer phase. Alternatively, cholesterol destabilizes the bilayer phase in equimolar mixtures of 18 : 1c18 : 1c phosphatidylethanolamine and 18 : 1c18 : 1c phosphatidylcholine, reducing the temperature at which non-bilayer phases are formed. Intermediate cholesterol concentrations encourage formation of a phase which possibly has cubic structure, whereas higher cholesterol contents (and higher temperatures) encourage formation of the hexagonal (H11) phase. 3. 3. The temperature-dependent polymorphic phase behaviour of equimolar mixtures of 18 : 1c18 : 1c phosphatidylethanolamine and 18 : 1c18 : 1c phosphatidylcholine exhibits a very pronounced hysteresis which is progressively reduced by addition of cholesterol.

Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. II. Temperature dependence of the polyene antibiotic-sterol complex formation

1. 1. The effect of filipin, amphotericin B, nystatin, etruscomycin and pimacirin upon the energy content of the gel → liquid-crystalline phase transition and upon the temperature dependence of the ATPase activity was studied in cholesterol-containing membranes of Acholeplasma laidlawii cells grown on elaidic acid. These experiments prove that the polyene antibiotics form complexes with cholesterol in the A. laidlawii cell membrane. 2. 2. The effect of temperature and different fatty acid composition upon the polyene antibiotic-cholesterol interaction in A. laidlawii cells, lecithin liposomes and in an aqueous dispersion of microcystalline dispersed cholesterol was investigated by (a) ultraviolet spectroscopy, (b) binding experiments, (c) freeze-etch electron microscopy, (d) K+ permeability. From the results of these studies it is suggested that filipin interacts first with cholesterol throughout the membrane forming primary filipin-cholesterol complexes. These complexes subsequently rearrange in the membrane to 150–250 A diameter aggregates which ultimately fragment the cell membrane. The results obtained for amphotericin B, nystatin and etruscomycin indicte that these antibiotics form much smaller complexes with cholesterol. 3. 3. The primaricin-cholesterol complexes formed in the A. laidlawii and liposomal membranes do not affect membrane permeability. A possible explanation of the haemolytic, fungicidic and fungistatic properties of this polyene antibiotic is suggested.

Effect of glycophorin incorporation on the physico-chemical properties of phospholipid bilayers.

1. The thermotropic behaviour of phospholipid molecules in reconstituted glycophorin-containing vesicles has been investigated by means of differential scanning calorimetry. Each glycophorin molecule is able to perturb the properties of 80--100 phospholipid molecules in such a way that these lipid molecules no longer participate in the cooperative gel to liquid-crystalline phase transition. This number of perturbed phospholipid molecules was discovered to be independent of the lipid charge. 2. By means of freeze-facture electron microscopy it could be demonstrated that glycophorin is not excluded from the solid lipid phase upon cooling the lipids below their gel to liquid-crystalline phase transition temperature. In mixtures of phosphatidylcholines which show solid-solid immiscibility, glycophorin is preferentially associated with the lower-melting lipid component upon phase separation, as could be demonstrated by both differential scanning calorimetry and freeze-fracture electron microscopy. 3. The effect of glycophorin on the mobility of phospholipids has been investigated by means of 31 P NMR. Glycophorin, incorporated into sonicated vesicles of dioleoylphosphatidic acid, is able to immobilize nine lipid molecules very strongly in their phosphate region. Evidence for an electrostatic inter-action between the protein and this negatively charged phospholipid has been presented. 4. The presence of glycophorin causes discontinuities in the lipid bilayer. This results in higher susceptibility of the bilayer towards attack by lipolytic enzymes and in enhanced membrane permeability.

Phase transitions in phospholipid model membranes of different curvature

1. Nuclear magnetic resonance, light scattering and freeze fracturing electron microscopic techniques were used to characterize the size of unilamellar phospholipid vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. 2. Differential scanning calorimetric and light scattering analyses showed that very small unilamellar vesicles obtained by the sonication method exhibit a downward shifted, largely broadened phase transition with a slightly decreased enthalpy change when compared with multilayered liposomes. 3. The phase transition of vesicles with variable diameter as obtained by injection methods resembled the pattern of multilayered liposomes the more the diameter was increased. 4. Repeated cycling through the lipid phase transition was shown to have a progressive effect on a fusion process. This effect was strongly increased when the osmolarity of the medium was enhanced (e.g. by the addition of cryoprotectors). Furthermore it was shown that ice-water of the systems caused abrupt fusion of the lipid structures. 5. Controversial results in the literature on the thermotropic behavior of vesicles could be explained in terms of these fusion processes.