W.J. Gerritsen

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 occurrence of lipidic particles in lipid bilayers as seen by 31P NMR and freeze-fracture electron-microscopy

A new type of lipid organization is observed in mixtures of phosphatidyl-choline with cardiolipin (in the presence of Ca2+), monoglycosyldiglyceride and phosphatidylethanolamine (in the presence of cholesterol). This phase is characterised by an isotropic 31P NMR signal and is visualised by freeze-fracturing as particles and pits on the fracture faces of the lipid bilayer. As the most favourable model for this phase we propose the inverted micelle sandwiched in between the two monolayers of the lipid bilayer.

The lipidic particle as an intermediate structure in membrane fusion processes and bilayer to hexagonal HII transitions

Small unilamellar vesicles comprised of a mixture of phosphatidylethanolamine/phosphatidylcholine/cholesterol (3 : 1 : 2) fuse to form large multilamellar vesicles on increasing the temperature from 0 to 50 degrees C. This event is associated with the appearance of lipidic particles at the fusion sites, consistent with a role as intermediary structures during the fusion process. Further, for phosphatidylcholine/cardiolipin (1 : 1) liposomes in the presence of Mn2+ a direct relationship between lipidic particles and the hexagonal (HII) phase is demonstrated which suggests that lipidic particles can also occur as intermediaries between bilayer and hexagonal (HII) structures.

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.

Ca2+-induced isotropic motion and phosphatidylcholine flip-flop in phosphatidylcholine-cardiolipin bilayers

Ca2+ induces a structural change in phosphatidylcholine-cardiolipin bilayers, which is visualised by freeze-fracturing as lipidic particles associated with the bilayer and is detected by 31P-NMR as isotropic motion of the phospholipids. In this structure a rapid transbilayer movement of phosphatidylcholine and a highly increased permeability towards Mn2+ are observed.

A 13C NMR method for determination of the transbilayer distribution of phosphatidylcholine in large, unilamellar, protein-free and protein-containing vesicles.

(1) Large unilamellar vesicles have been prepared from N-[Ne3-13C]-18 : 1c/18 : 1c-phosphatidylcholine, both with and without the major intrinsic proteins from the human erythrocyte membrane incorporated in the bilayer. (2) It is shown that the inside-outside distribution of the lipid molecules in these large unilamellar structures can be determined using 13C NMR. (3) Large vesicles of 18 : 1c/18 : 1c-phosphatidylcholine containing glycophorin show an enhanced permeability to Dy3+. It is shown that the permeability barrier of these vesicles can be restored by addition of 10 mol% 18 : 1c/18 : 1c-phosphatidylethanolamine or 1-18 : 1c-lysophosphatidylcholine.

Ca2+-induced changes in the barrier properties of cardiolipin/phosphatidylcholine bilayers

(1) A selective increase in permeability is induced in cardiolipin/phosphatidylcholine bilayers at Ca2+ concentrations of 1--3 mM. At higher concentrations of Ca2+ the permeability barrier is completely destroyed. (2) The selective increase in permeability is correlated with the formation of lipid particles visualized by freeze-fracture electron microscopy and an isotropic signal in 31P-NMR spectra. (3) Lowering the Ca2+ concentration shows reduction in permeability but the formation of the lipid particles is a non-reversible process. (4) At higher Ca2+ concentrations, 31P-NMR spectra and freeze-fracture results indicate the formation of the hexagonal phase, explaining the disappearance of the permeability barrier.

Non-bilayer Lipids and the Inner Mitochondrial Membrane

In the last decade the fluid mosaic model (Singer and Nicholson 1972) of biological membranes has become generally accepted as it provided a rationale for many structural and functional features of membranes. More recently, however it has become increasingly clear that this model is incomplete for reasons relating to lipid composition as well as functional abilities of biological membranes. First, although the chemical variation in membrane lipids is enormous, it is surprising that most of them can be divided into only two groups on structural grounds: The lipids of the first group, including PC* and sphingomyelin, will organize themselves in bilayers when they are in the fully hydrated state (bilayer lipids). It is obvious that this property has greatly contributed to the bilayer concept of biological membranes. In contrast, the lipids in the second group do not form bilayers when they are dispersed in excess buffer (non-bilayer lipids). This group includes major lipids such as PE*, monoglucosyl and monogalactosyl diglyceride and CL4 (in the presence of Ca2+) (for review and references see Cullis and de Kruijff 1979). These lipids prefer the hexagonal HII, phase (Fig. 1). This phase consists of cylinders of lipids surrounding long aqueous channels. The unique feature of the HII phase both from a structural and functional point of view is that it allows polar lipids to be organized in a low energy configuration inside a hydrophobic environment. The reason for the abundant presence of these non-bilayer lipids in membranes is difficult to understand in terms of membrane models in which the bilayer is suggested to be the only organization available to the lipids.