Rates of amphotericin B and filipin association with sterols. A study of changes in sterol structure and phospholipid composition of vesicles

The influence of structural modifications in sterols and phospholipids on the rate of polyene antibiotic-sterol interaction was studied. For filipin and amphotericin B association with sterols in vesicles, a preferential interaction was found with sterols whose side chain length is close to that of cholesterol. Introduction of trans double bonds into the sterol side chain did not alter the rate of interaction in vesicles. The delta 7-bond of the sterol appears to be of critical importance in amphotericin B-sterol interaction, whereas the delta 5-bond is not essential. These observations are relevant to the well-known effects of amphotericin B on cell membranes containing ergosterol compared with those containing cholesterol. The dependence of the rates of sterol-polyene antibiotic interaction on the phospholipid composition of the vesicles indicates that phospholipid vesicles may be an inadequate model for reaching a comprehensive understanding of the effects exerted on biological membranes by these agents.     

Interactions of the cholesterol side-chain with egg lecithin. A spin label study.

The effect in egg lecithin liposomes of cholesterol and cholesterol analogues with side-chains of reduced length on the order parameters of two steroid spin labels has been studied. Analogues with side-chains shorter than cholesterol by more than three carbon cause significantly less ordering than cholesterol. Liposomes containing a cholesterol analogue in which the side-chain is absent cause very little increase in the ordering of a new sterol spin label in which the nitroxide is incorporated into the side-chain. The results suggest that the sterol side-chain exerts a great influence on membrane rigidity within its immediate environment.     

Effect of filipin on liposomes prepared with different types of steroids

The action of the polyene antibiotic filipin on the permeability of liposomes prepared with lecithin and several plant and other steroids was studied. The effect of filipin was found to be dependent upon the type of steroid incorporated into the membrane. The interaction of filipin with steroids was found to be related both to the functional group at the 3-position and the aliphatic chain of the steroid. Complex formation of the steroid with filipin in aqueous solutions, as detected by ultraviolet spectrophotometry, correlated with the ability of filipin to increase the permeability of the liposomes.     

The Effect of Polyene Antibiotics on Photosynthetic Electron Transfer

The effect of four polyene antibiotics and digitonin on photosyntheticelectron transfer by maize mesophyll chloroplasts has been investigated.All five compounds, at concentrations between 0.1 mM and 20mM, inhibited photosystem 2 activity as measured by the photo-reductionof ferricyanide and 2,6-dichlorophenolindophenol from water.Etruscomycin, amphotericin B, and digitonin were more inhibitorythan filipin and nystatin. Photosystem 1 activity was inhibitedby 1 mM concentrations of etruscomycin, amphotericin B, anddigitonin but not by filipin and nystatin. In all cases whereinhibition occurred, it was temperature dependent. The inhibition of photosystem 1 activity could be relieved byplastocyanin. Etruscomycin and digitonin, at concentrationsof 0.5 mM and above, caused disintegration of the chloroplasts,and this disintegration was accompanied by a two- to three-foldincrease in photosystem 1 activity in the presence of plastocyanin.It is concluded that the action of polyene antibiotics resultsin the release of plastocyanin from its site in the photosyntheticelectron transfer chain. The results are discussed in termsof the abilities of polyene antibiotics and digitonin to formcomplexes with sterols.     

Nitroxide spin-probe study of amphotericin B-ergosterol interaction in egg phosphatidylcholine multilayers

The interaction between the polyene macrolide antibiotic, amphotericin B, and ergosterol in egg phosphatidylcholine multilayers was investigated using head group and acyl chain nitroxide spin-labelled phosphatidylcholine as probes. At physiological concentrations of less than 15 mol% sterol in egg phosphatidylcholine multilayers amphotericin B accumulates near the head group region until an amphotericin B : ergosterol ratio of approximately 0.7 is achieved. As the proportion of amphotericin B is increased above this value, formation of an acyl chain disordering complex occurs which has an approximate antibiotic:sterol ratio of unity. Dicetyl phosphate was used to increase the solubility of ergosterol past its normal limit in pure egg phosphatidylcholine (approximately 15 mol%). At concentrations of ergosterol higher than 15 mol% a complex of two ergosterol molecules and one amphotericin B was postulated when there was insufficient antibiotic to form a 1:1 complex.     

Interaction of the polyene antibiotic etruscomycin with large unilamellar lipid vesicles: binding and proton permeability inducement

The effect of the polyene antibiotic etruscomycin on the permeability of large unilamellar lipid vesicles was investigated. Proton leakage was induced in egg-yolk phosphatidylcholine (EPC) vesicles only when sterol was present in the membrane; the extent of leakage was limited. High etruscomycin/lipid ratios (R) were necessary (R greater than 0.1). Higher percentages of sterol increased the permeability, slightly more strongly for ergosterol than for cholesterol. Dipalmitoylphosphatidylcholine (DPPC) vesicles were more sensitive to permeability inducement, even in the absence of sterol in the bilayer (inducement for R greater than 0.06). The interactions of etruscomycin with the vesicles were examined by circular dichroism, fluorescence and 31P-NMR. In the range of antibiotic concentration where permeability was induced, R greater than 0.1 for EPC vesicles, R greater than 0.06 for DPPC vesicles, etruscomycin exhibited characteristic circular dichroism spectra independent of the presence of sterol. Under the same conditions, 31P-NMR and fluorescence studies indicated a destruction or a fusion of the vesicle bilayer. At lower etruscomycin concentrations (R less than 0.03), the etruscomycin circular dichroism spectra were different, indicating that the interaction with membranes containing ergosterol differed from that with membranes containing cholesterol. From correlating the increase in fluorescence intensity with this interaction, as well as from exchange experiments, it was inferred that etruscomycin at a low antibiotic/lipid ratio is more strongly bound to ergosterol-containing vesicles than to cholesterol-containing vesicles. These results and their comparison with the results obtained with other polyene antibiotics indicate that at low R etruscomycin resembles amphotericin rather than filipin in its preferential binding to ergosterol-containing vesicles. At higher R, that is in conditions where permeability is induced, the selectivity is different. The corresponding mechanism seems not to involve the formation of an etruscomycin-sterol channel, since the hydrophobic chain of the complex would be too short to form a channel.     

A reverse-phase HPLC assay for measuring the interaction of polyene macrolide antifungal agents with sterols.

A quick and simple affinity chromatography method for gauging the interaction of polyene antifungal agents with sterols has been developed. The required affinity columns are prepared from a standard C-18 reverse-phase HPLC column by injecting a measured quantity of sterol under conditions where it is completely retained. After the assay, the sterol is eluted with a less polar solvent and the column reused. By comparing the elution volume of a polyene injected onto the sterol-free column (Ve) with that of the polyene injected onto the sterol-doped column (V), an association constant (Ka) for the polyene-sterol complex was determined. Association constants of different amphotericin B-sterol and pimaricin-sterol complexes were determined and correlated with the polyene's ability to induce membrane permeability and its antifungal properties. This procedure provides a new tool for screening polyene macrolides for antifungal therapy.     

Interactions of the cholesterol side-chain with egg lecithin a spin label study

The effect in egg lecithin liposomes of cholesterol and cholesterol analogues with side-chains of reduced length on the order parameters of two steroid spin labels has been studied. Analogoues with side-chains shorter than cholesterol by more than three carbons cause significantly less ordering than cholesterol. Liposomes containing a cholesterol analogue in which the side-chain is absent cause very little increase in the ordering of a new sterol spin label in which the nitroxide is incorporated into the side-chain. The results suggest that the sterol side-chain exerts a great influence on membrane rigidity within its immediate environment.     

Optimum interaction of sterol side chains with phosphatidylcholine.

The specificity of the interaction between the cholesterol side chain and egg phosphatidylcholine was precisely defined by examining the effect of three new analogues of cholesterol with modified side chains on the ordering of two steroid spin labels in liposomes. The complete side chain of cholesterol was shown to be required for maximum ordering. Sterols with side chains shorter or longer than cholesterol caused significantly less ordering.     

Optimum interaction of sterol side chains with phosphatidylcholine

The specificity of the interaction between the cholesterol side chain and egg phosphatidylcholine was precisely defined by examining the effect of three new analogues of cholesterol with modified side chains on the ordering of two steroid spin labels in liposomes. The complete side chain of cholesterol was shown to be required for maximum ordering. Sterols with side chains shorter or longer than cholesterol caused significantly less ordering.     

Effect of variation of retinal polyene side-chain length on formation and function of bacteriorhodopsin analogue pigments

The effect of the length of the retinal polyene side chain on bacterioopsin pigment formation and function has been investigated with two series of synthetic retinal analogues. Cyclohexyl derivatives with polyene chains one carbon longer and one or more carbons shorter than retinal and linear polyenes with no ring have been synthesized and characterized. Compounds of six carbons or less in the polyene chain form pigments very poorly or not at all with bacterioopsin. Compounds containing at least seven carbons in the chain are found to form reasonably stable bacterioopsin pigments that show a small shift in absorbance on irradiation. However, photocycling and proton photorelease are not detected. The analogue with nine carbons in the polyene chain (one less than retinal) forms a stable pigment with an M-type intermediate but demonstrates reduced amounts of photocycling and light-activated proton release. The analogue with a polyene chain identical with that of retinal, but containing no ring, forms a pigment that shows both an efficient light-activated proton photocycle and release. The pigment containing the chromophore with the polyene chain one carbon longer than retinal is likewise fully active. We thus conclude that the length of the polyene chain must be at least 9 carbons for the formation of a stable pigment that photocycles and must be 10 carbons for both the photocycle and light-activated proton release to have a high quantum efficiency.     

Interaction of the polyene antibiotics with lipid bilayer vesicles containing cholesterol

The interaction of the polyene antibiotics, amphotericin B, nystatin and filipin with cholesterol-containing single bilayer lipid vesicles has been characterized using gel permeation chromatography and proton magnetic resonance. All three antibiotics bind to vesicles at low concentrations without causing a large amount of vesicle destruction. The strength of binding as determined by gel permeation studies is greater for filipin and amphotericin than for nystatin. Nystatin and amphotericin B at these low concentrations induce a rapid loss of internal vesicle contents consistent with pore formation. Filipin induces no leakage beyond that expected from partial vesicle destruction or general detergent action.At antibiotic levels above 1 : 1 antibiotic : cholesterol ratios the NMR results show all three antibiotics to cause extensive vesicle destruction. The onset of this behavior, which appears to be independent of the total antibiotic concentration, indicates a well defined antibiotic : cholesterol interaction stoichiometry. Despite the fact that cholesterol is required for antibiotic activity, the NMR spectra prior to vesicle destruction show no changes indicative of an antibiotic-induced reversal of cholesterol restriction of phosphatidylcholine mobility. The contrast with polyene antibiotic behavior in more extended bilayers is discussed.     

Effects of amphotericin B on membrane permeability--kinetics of spin probe reduction

The effect of the polyene antibiotic amphotericin B on the permeability of both unilamellar and multilamellar model membranes is investigated. The method measures the loss of the electron paramagnetic resonance signal of a spin probe, trapped in the aqueous compartment of a lipid dispersion, upon addition of ascorbate ions to the bulk aqueous phase. Amphotericin B causes large increases in the permeability of cholesterol-containing egg phosphatidylcholine membranes, whereas the effects are small in the absence of sterol and do not depend on surface charge. The effect of amphotericin depends upon the antibiotic:sterol mole ratio. The antibiotic appears to be unable to cross the membrane, acting only on the outermost bilayer of a multibilayer dispersion. When a phospholipid in the gel phase is used, amphotericin B causes large increases in permeability, independently of the presence or absence of sterol. It is suggested that the mechanism of action of amphotericin B is different for lipids in the liquid crystalline or gel states.     

The importance of the phospholipid bilayer and the length of the cholesterol molecule in membrane structure.

The properties of mixtures of phosphatidylcholine and analogues of cholesterol bearing side chains of varying lengths were examined by a variety of methods. The incorporation of the analogues into sonicated liposomes and their effect on the rate of osmotic shrinking of multilamellar liposomes were determined. The ordering of a steroid spin label was studied in an oriented multibilayer system and the effect of the analogues on the phase transition of dipalmitoyl phosphatidylcholine monitored using the spin label TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl). Mixtures of analogues and phospholipid were also studied in monolayers. In all the bilayer systems studied cholesterol caused the greatest 'rigidifying' effect, the analogues with shorter or longer side chains being less effective. However, in the monolayer experiments the length of the sterol molecule was found to be much less critical. It is suggested that cholesterol is anchored in position in a phospholipid bilayer by virtue of the molecule being the precise length required to maximise interactions between neighbouring molecules without disturbing the bilayer structure.     

Interaction of the polyene antibiotics with lipid bilayer vesicles containing cholesterol.

The interaction of the polyene antibiotics, amphotericin B, nystatin and filipin with cholesterol-containing single bilayer lipid vesicles has been characterized using gel permeation chromatography and proton magnetic resonance. All three antibiotics bind to vesicles at low concentrations without causing a large amount of vesicle destruction. The strength of binding as determined by gel permeation studies is greater for filipin and amphotericin than for nystatin. Nystatin and amphotericin B at these low concentrations induce a rapid loss of internal vesicle contents consistents consistent with pore formation. Filipin induces no leakage beyond that expected from partial vesicle destruction or general detergent action. At antibiotic levels above 1:1 antibiotic: cholesterol ratios the NMR results show all three antibiotics to cause extensive vesicle destruction. The onset of this behavior, which appears to be independent of the total antibiotic concentraion, indicates a well defined antibiotic : cholesterol interaction stoichiometry. Despite the fact that cholesterol is required for antibiotic activity, the NMR spectra prior to vesicle destruction show no changes indicative of an antibiotic-induced reversal of cholesterol restriction of phosphatidylcholine mobility. The contrast with polyene antibiotic behavior in more extended bilayers is discussed.     

A new paramagnetic analogue of cholesterol as a tool for studying molecular interactions of genuine cholesterol

The synthesis of a new paramagnetic (nitroxide) analogue of cholesterol is described. This compound (called CNO) contains a doxyl group in the lateral chain at position 25. Our results show that CNO retains three molecular interactions which characterize authentic cholesterol: It assumes an orientation perpendicular to the phospholipid bilayer with the doxyl group buried in the membrane core, as seen by ESR spectroscopy. It widens the transition temperature of dimyristoylphosphatidylcholine, to the same extent as cholesterol, as measured by Raman and ESR spectroscopies. It interacts with polyene antibiotics, such as amphotericin B and filipin, in the same manner as its model. This was proved on the one hand by the change in fluorescence of self quenched vesicle-entrapped calcein, after dilution in the external medium, provoked by filipin, and on the other hand by fluorescence quenching provoked by cobalt ions entering the vesicles under the influence of amphotericin B. We concluded that CNO, although it has a side chain different from genuine cholesterol, can help to solve many physiologically meaningful questions related to the distribution and movement rate of cholesterol itself.     

Permeability Changes by Peroxidation of Unsaturated Liposomes with Ascorbic Acid/Fe2+

Abstract

Liposomes composed of phosphatidylcholine having a polyunsaturated fatty acid side chain were peroxidized in ascorbic acid/Fe²⁺ solution. Lipid peroxidation and the change in membrane permeability were monitored by the formation of thiobarbituric acid reactive substance (TBARS) and the release of entrapped fluorescein isothiocyanate-labeled superoxide dismutase (FITC-SOD), respectively. Peroxidation of liposomes composed of dipalmitoylphosphatidylcholine and 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) having 4 double bonds on one fatty acid side chain showed high TBARS value and caused the release of FITC-SOD. This release started when TBARS reached a definite value. But liposomes composed of phosphatidylcholine having 1 or 2 double bond(s) on one fatty acid side chain caused little increase in lipid peroxidation and FITC-SOD release. During the peroxidation of PAPC-liposomes, the breakdown of PAPC and formation of lysophosphatidylcholine (or like substance) were detected by HPLC analysis. Increase in the release of FITC-SOD thus appears to be due to the breakdown of the fatty acid side chain of phospholipids of liposomes. Liposomes composed of phosphatidylcholine having a polyunsaturated fatty acid side chain may be expected to be sensitive to peroxidation signals.     

Effect of amphotericin B on cholesterol-containing liposomes of egg phosphatidylcholine and didocosenoyl phosphatidylcholine. A refinement of the model for the formation of pores by amphotericin B in membranes

(1) Binding and K+-permeability measurements were performed on egg and 22 : 1c/22 : 1c-phosphatidylcholine liposomes with or without cholesterol. (2) Amphotericin B binds specifically to cholesterol in both types of liposome despite the difference in bilayer thickness. (3) Addition of amphotericin B to one side of the cholesterol-containing egg phosphatidylcholine bilayers induces a fast K+ efflux from the outermost compartment of the liposomes. In contrast, the total K+ content of sonicated unilamellar cholesterol-containing egg phosphatidylcholine vesicles is released by amphotericin B. (4) Amphotericin B addition to one side of the cholesterol-containing 22 : 1c/22 : 1c-phosphatidylcholine liposomes does not cause a change in K+ permeability. The presence of amphotericin B on both sides of the bilayer, however, induces an increase in K+ permeability. (5) A model is proposed which accounts for the effect of bilayer thickness on the amphotericin B-induced permeability changes in membranes.     

Absorption and fluorescence spectra of polyene antibiotics in the presence of cholesterol.

The alterations in the absorption and fluorescence spectra observed for the polyene antibiotics filipin and nystatin in the presence of cholesterol are due to an exciton interaction (polyene aggregates) and cannot be attributed to a specific sterol-antibiotic complex. Filipin and nystatin molecules partition into the sterol aggregates, these structures being very efficient to induce exciton interaction; the observed splitting profile indicates that the chromophores are in a stacked arrangement (parallel transition dipoles). For filipin incorporated in lipid bilayers, the sterol is able to induce the same type of aggregate, at variance with nystatin.