R.A. Demel

Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. III. Molecular structure of the polyene antibiotic-cholesterol complexes

Abstract 1. 1. Based on the analysis of the complexes of cholesterol and the polyene antibiotics filipin, amphotericin B, nystatin, etruscomycin and pimaricin, which can be built with space-filling models, and from data on the polyene antibiotic-cholesterol interaction, mechanisms of the polyene antibiotic induced permeability changes in membranes are proposed. 2. 2. The amphotericin B-cholesterol complex is visualised as a circular arrangement of 8 amphotericin B molecules interdigitated by 8 cholesterol molecules. The outside of this complex is hydrophobic, the inside is hydrophilic due to the presence of the hydroxyl groups of the amphotericin B molecules. Two such complexes (half pores) will generate a pore which traverses the membrane. The hydrophilic channel of such a pore has a diameter of about 8 A. 3. 3. The nystatin-cholesterol and etruscomycin-cholesterol complexes also are visualised as pores. However, the pimaricin-cholesterol complex cannot form a conducting pore because the length of the half pores is considerably less than the length of half the thickness of the lipid core. 4. 4. The filipin-cholesterol complex is visualised as an aggregate of 150–250 A in diameter oriented in the hydrophobic core of the membrane. This aggregate might be composed of two regular arrays of stacked filipin molecules such that the exterior of this aggregate is hydrophobic due to the presence of the double bonds of filipin to which equal amounts of cholesterol are complexed. The presence of this aggregate causes membrane fragmentation.

Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers

The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes. The phospholipases which cannot hyorolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A2 from pig pancreas and Crotalus adamanteus and phospholipase D from cabbage, can hydrolyse phospholipid monolayers at pressure below 31 dynes/cm only. The phospholipases which can hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Clostridium welchii phospholipase A2 from Naja naja and bee venom and sphingomyelinase from Staphylococcus aureus, can hydrolyse phospholipid monolayers at pressure above 31 dynes/cm. It is concluded that the lipid packing in the outer monolayer of the erythrocyte membrane is comparable with a lateral surface pressure between 31 and 34.8 dynes/cm.

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.

The properties of polyunsaturated lecithins in monolayers and liposomes and the interactions of these lecithins with cholesterol.

Abstract 1. 1. The force-area characteristics of monolayers of synthetic lecithins with one to six double bonds in one acyl chain have been studied. 2. 2. The area per molecule increases stepwise. The most significant increase is observed after the introduction of the first double bond. The subsequent introduction of two, three or four double bonds or polyunsaturated chains at both ester positions produces some further increase. 3. 3. The interaction with cholesterol depends on the unsaturation and the distribution of the double bonds between the acyl chains. 4. 4. A condensing effect with cholesterol was evident for (1-stearoyl-2-oleoyl)-3-lecithin, (1-palmitoyl-2-lineoyl)-3-lecithin, (1-palmitoyl-2-linolenoyl)-3-lecithin, (1-palmitoyl-2-arachidonoyl)-3-lecithin at 22°. No effect is observed for (1,2-dilinoleoyl)-3-lecithin and (1,2-dilinolenoyl)-3-lecithin. (1-palmitoyl-2-docosahexaenoyl)-3-lecithin shows a limited effect at 22°, but no effect at 37°. 5. 5. No significant differences in behavior are found for the two structural isomers with a mono- or disaturated chain at the 1- or 2-position. 6. 6. The permeability of liposomes, derived from the above mentioned lecithins, to glucose, erythritol and glycerol increases in the same order as the area per molecule at the air-water interface. 7. 7. The presence of cholesterol reduced the permeability to glucose, erythritol, glycerol only for the lecithins which showed a condensation effect. 8. 8. The unsaturation and the distribution of the double bonds appear to be of critical importance for the barrier properties of lecithins and for the interaction with cholesterol.

The effect of sterol structure on the permeability of lipomes to glucose, glycerol and Rb+

1. 1. The effect of 3β-, 3α-hydroxysterol and ketosteroids on the permeability properties of (egg lecithin) liposomes towards glucose, glycerol and Rb+ has been studied. 2. 2. The 3β-hydroxysterols, cholesterol, cholestanol, lathosterol, 7-dehydrocholesterol and B-norcholesterol affect the most pronounced reduction in permeability of glucose, glycerol and Rb+. 3. 3. The plant sterols, ergosterol and stigmasterol are less effective whereas compounds lacking the side chain (androstan-3β-ol) or with a non-planar sterol nucleus (coprostanol) show no effect. 4. 4. The 3α-hydroxysterols, epicholesterol and androstan-3α-ol reveal no significant effect on the permeability. 5. 5. The ketosteroids either do not affect the permeability or increase the permeability of the lipid barrier. 6. 6. The reduction in permeability, as found for cholesterol, is dependent on: (a) planar sterol nucleus, (b) 3β-hydroxy group, (c) intact side chain. 7. 7. The effect of sterols on the permeability properties of liposomes are in good agreement with the effect on the mean molecular area measured in monolayers, and the effect of these sterols on biological membranes such as erythrocytes and Mycoplasma.

The effect of partial replacements of membrane cholesterol by other steroids on the osmotic fragility and glycerol permeability of erythrocytes

Abstract 1. 1. (A) Sonicated dispersions of lecithins were incubated with aliquots of washed human erythrocytes, leading to the removal of part of the cholesterol complement, eventually followed by lysis. (B) Sonicated dispersions of lecithin and cholesterol with one of a range of other steroids were prepared and incubated with aliquots of washed human erythrocytes. By a simple exchange process, the steroid composition of the erythrocytes were modified. 2. 2. (A) The osmotic fragility of erythrocytes was considerably increased after removal of part of the cholesterol complement with pure lecithin dispersions. (B) The osmotic fragilities of the modified erythrocytes were measured, and it was found that relatively small differences were detectable in view of the substantial replacement of part of the cholesterol with one of the other steroids. 3. 3. Significant differences in the glycerol permeabilities of the modified human and pig erythrocytes were demonstrated. In the presence of 3-ketosteroids, the rates of penetration of glycerol were significantly higher than those containing 3β-hydroxy steroids. Cholesterol-depleted erythrocytes also exhibited considerably increased glycerol permeability.

Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. I. Specificity of the membrane permeability changes induced by the polyene antibiotics

1. 1. The effect of filipin, amphotericin B, nsystatin, etruscomycin and pimaricin upon the permeability properties of Acholeplasma laidlawii cells and egg lecithin liposomes was investigated. When cholesterol was present in the membrane the different polyene antibiotics produced permeability changes which were different for the various antibiotics. 2. 2. Filipin disrupted the membrane structure, after the interaction with cholesterol, so that both small ions such as K+ and large protein molecules like glucose-6-phosphate dehydrogenase are released. 3. 3. Amphotericin B, nystatin and etruscomycin produced specific permeability changes which indicate that these antibiotics create aqueous pores of specific size (about 8 A in diameter) in the membrane after the interaction with cholesterol. 4. 4. Pimaricin was not able to produce permeability changes in A. laidlawii cells and egg lecithin liposomes. 5. 5. Various sterols were incorporated in A. laidlawii and liposomal membranes after which the interaction of filipin and amphotericin B with these membranes was investigated by ultraviolet spectroscopy and K+ permeability. Only those sterols which had 3β-OH group, a planar molecule and hydrophobic side chain at C17 were able to interact with these polyene antibiotics and thereby enhance the membrane permeability.

The effect of cholesterol and epicholesterol incorporation on the permeability and on the phase transition of intact Acholeplasma laidlawii cell membranes and derived liposomes

1. 1. The effect of incorporated cholesterol and epicholesterol upon the glycerol and erythritol permeability through the membrane of Acholeplasma laidlawii (previously denoted as Mycoplasma laidlawii) is studied. Both sterols, when present in the growth medium, are incorporated to the same extent in the A. laidlawii membrane. Only the cholesterol-containing A. laidlawii membrane shows a reduced permeability towards glycerol and erythritol as compared to the sterol-free cells. The 3α-hydroxy isomer, epicholesterol, does not affect the membrane permeability. 2. 2. Liposomes prepared from lipids isolated from cells grown on cholesterol-rich media also show a reduced glycerol and erythritol permeability as compared to liposomes prepared from lipids isolated from sterol-free control cells. This permeability-lowering effect can be correlated with a condensing effect of cholesterol upon a monolayer of total A. laidlawii lipids. 3. 3. The phase transitions occurring in membranes and extracted lipids of A. laidlawii have been studied by differential scanning calorimetry. Incorporated cholesterol causes a considerable reduction of the energy content of this phase transition. This reduction in energy is the same for the intact A. laidlawii cell membrane as for the liposomal bilayer system of the extracted lipids dispersed in water. 4. 4. Using the synthetic lecithin 1-oleoyl-2-stearoyl-sn-glycero-3-phosphorylcholine the same phenomenon is observed. 32 mole % cholesterol completely eliminated the phase transition of the lecithin. Epicholesterol 5α-androstan-3β-ol and cholest-4,6-dien-3-one are unable to show such an effect, also suggesting the importance of the 3β-OH group of the sterol molecule for the specific sterol-lecithin interaction.

STRUCTURAL REQUIREMENTS OF STEROLS FOR THE INTERACTION WITH LECITHIN AT THE AIR-WATER INTERFACE

SUMMARY I. The force area characteristics of monolayers of 3fl-, 3~-hydroxysterols and ketosteroids have been studied. 2. The 3fl-hydroxysterols cholesterol, cholestanol, lathosterol, 7-dehydrocholesterol, and B-norcholesterol are perpendicularly oriented at the air-water interface and show high collapse pressures. Ergosterol, stigmasterol, and androstan-3fl-ol demonstrate a similar behavior at high pressures, but the collapse pressure is reduced. The cis-structured coprostanol shows an increased area per molecule. 3~-Hydroxysterols (epicholesterol and androstan-3~-ol) reveal strongly reduced collapse pressures when compared with the respective 3fl-hydroxysterols. All ketosteroids show high areas per molecule. 3- The force-area curves show little temperature effect. 4. The interaction of i8:I/I8:O lecithin with sterols is dependent on: (a) planar sterol nucleus, (b) 3fl-hydroxy group, (c) intact side chain. 5. Besides 3~-hydroxysterols, 3~-hydroxysterols and ketosteroids can also show some condensation effect. However, minimal area per molecule can be achieved only with 3fl-hydroxysterols. 6. The interaction of lecithin and sterol in monolayers is governed by a number of factors including van der Waals interactions, but also hydrogen bonding between the 3fl-hydroxy group and environment is considered to be important.