Johannes de Gier

Protection by sugars against phase transition-induced leak in hydrated dimyristoylphosphatidylcholine liposomes

The disaccharides trehalose and sucrose have small effects on temperature and enthalpy of the pre- and main phase transition in hydrated DMPC bilayers. In contrast, these sugars cause a considerable retention of carboxyfluorescein when large unilamellar vesicles of DMPC are heated through the main transition. This effect is sugar specific, as the monosaccharides glucose and fructose are less effective and ethyleneglycol has no effect at all.

2H-NMR, 31P-NMR and DSC characterization of a novel lipid organization in calcium-dioleoylphosphatidate membranes. Implications for the mechanism of the phosphatidate calcium transmembrane shuttle

2H-NMR, 31P-NMR and DSC investigations are presented on the structure and dynamics of the Ca2+-dioleoylphosphatidate complex which is formed upon addition of calcium to dispersions of pure dioleoylphosphatidate or of dioleoylphosphatidate in mixtures with dioleoylphosphatidylcholine (DOPC). It is concluded that the phosphate region in the polar headgroup of dioleoylphosphatidate is immobilized, while the oleate chains remain liquid and have increased disorder. In mixtures of dioleoylphosphatidate and DOPC in the presence of calcium a dioleoylphosphatidate-rich phase is segregated, in which the molecular behaviour of phosphatidate is rather similar to that of the pure Ca2+-dioleoylphosphatidate complex. A hypothetical model is proposed for the structure of this complex and this is correlated with the dioleoylphosphatidate-mediated transmembrane transport of calcium (Smaal, E.B., Mandersloot, J.G., De Kruijff, B. and De Gier, J. (1986) Biochim. Biophys. Acta 860, 99-108). Data indicate that this transmembrane shuttle is an inverted organization of phosphatidate molecules enclosing calcium ions in an anhydrous core.

Determination of the size of the packing defects in dimyristoylphosphatidylcholine bilayers, present at the phase transition temperature

Abstract Multilamellar liposomes of dimyristoylphosphatidylcholine, containing 4 mol% egg phosphatidic acid show at the phase transition temperature an increased permeability for non-electrolytes of M r values up to 900. This indicates that the packing defects occurring at the liquid crystalline/gel state phase boundary have a similar pore diameter (15–18 A) as the packing defects present in glycophorin—dioleoylphos-phatidylcholine vesicles. This suggests that packing defects at the protein—lipid interphase are the major permeation pathway of the glycophorin—dioleoylphosphatidylcholine vesicles.Multilamellar liposomes of dimyristoylphosphatidylcholine, containing 4 mol% egg phosphatidic acid show at the phase transition temperature an increased permeability for non‐electrolytes of M r values up to 900. This indicates that the packing defects occurring at the liquid crystalline/gel state phase boundary have a similar pore diameter (15–18 A) as the packing defects present in glycophorin—dioleoylphos‐phatidylcholine vesicles. This suggests that packing defects at the protein—lipid interphase are the major permeation pathway of the glycophorin—dioleoylphosphatidylcholine vesicles.

Consequences of the interaction of calcium with dioleoylphosphatidate-containing model membranes: calcium-membrane and membrane-membrane interactions

Calcium binds to dioleoylphosphatidate/dioleoylphosphatidylcholine (DOPA/DOPC) (20:80, mol%) multilamellar vesicles in the presence of a calcium ionophore with stoichiometry of about 0.6 nmol calcium per nmol phosphatidate and an apparent dissociation constant of about 1.7 mM. Experiments on the behaviour of monomolecular films at an air/water interface show that calcium-phosphatidate binding results in a decrease in the area of the polar region of the phosphatidate molecule, probably caused by headgroup dehydration and partial charge neutralization. At calcium concentration higher than about 3 mM calcium neutralizes the negatively charged membrane surface of DOPA/DOPC (20:80, mol%) large unilamellar vesicles, and vesicle aggregation is observed. At 10 mM of calcium this results in a low level of vesicle fusion. These observed processes are not attended with calcium-induced phosphatidylcholine transbilayer movement in the membranes of DOPA/DOPC (20:80, mol%) large unilamellar vesicles. When these findings are compared with the results of a previous study on the permeability behaviour of large unilamellar vesicles of the same phospholipid composition under comparable conditions (Smaal, E.B., Mandersloot, J.G., De Kruijff, B. and De Gier, J. (1986) Biochim. Biophys. Acta 860, 99-108) the following conclusions can be drawn. At low millimolar calcium concentrations (less than 2.5 mM) calcium does not occupy all the binding sites of the membrane, no membrane-membrane interactions are observed and a selective translocation of calcium and calcium-chelating anions is appearing. The mechanism of this translocation may be explained by the formation of uncharged dehydrated complexes of calcium, phosphatidate and calcium chelator, which can pass the membrane via transient occurring non-bilayer structures. Between 3 and 10 mM of calcium an a selective permeability increase of the vesicular membrane is found, which is not a consequence of vesicle fusion but apparently of vesicle aggregation, possibly causing packing defects in the membrane.

A novel property of a mitochondrial presequence : its ability to induce cardiolipin-specific interbilayer contacts which are dissociated by a transmembrane potential

A new property of the presequence of the mitochondrial precursor protein cytochrome oxidase subunit IV is presented. This mitochondrial presequence induces interbilayer contacts between large unilamellar vesicles consisting of phosphatidylcholine and cardiolipin. The presequence‐vesicle aggregates can be dissociated by applying a membrane potential across the bilayers (negative inside). These effects require the presence of cardiolipin and are not observed for other negatively charged phospholipids. We propose a role for the presequence in the formation and dissociation of mitochondrial contact sites.

Essential adaptation of the calcium influx assay into liposomes with entrapped arsenazo III for studies on the possible calcium translocating properties of acidic phospholipids

An adapted version of the Ca2+-influx assay of Weissmann et al. (Weissmann, G., Anderson, P., Serhan, C., Samuelson, E. and Goodman, E. (1980) Proc. Natl. Acad. Sci. USA 77, 1506-1510) is presented for studies on the possible ionophoretic properties of acidic phospholipids. This method is based on the use of the metallochromic dye arsenazo III enclosed in liposomal vesicles, to indicate the Ca2+ influx. An essential control is introduced to discriminate between Ca2+-arsenazo III complex formation inside the vesicles, as a consequence of Ca2+ influx, and outside the vesicles, as a consequence of arsenazo III leakage from the vesicles. Furthermore, some minor improvements are added, like the use of large unilamellar vesicles instead of multilamellar vesicles, and the use of dual wavelength spectrophotometry. Using this method, it was found that dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidylglycerol, were impermeable to Ca2+. In this system a selective Ca2+ permeability could be induced by the addition of the fungal Ca2+ ionophore A23187. In contrast, dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidic acid, incubated in the presence of Ca2+ were permeable to both Ca2+ and arsenazo III.

Calcium-induced changes in permeability of dioleoylphosphatidylcholine model membranes containing bovine heart cardiolipin

At calcium concentrations up to about 4 mM a selective permeability increase of cardiolipin/dioleoylphosphatidylcholine (50:50, mol%) membranes for calcium and its chelator arsenazo III is observed. Under these conditions calcium does not occupy all the binding sites of cardiolipin at the membrane interface and no vesicle-vesicle interactions are found. Lowering of the cardiolipin content of the vesicles to 20 mol% extends the calcium concentration range in which a selective permeability for calcium and arsenazo III is appearing up to about 12 mM. We suggest that the observed selective permeability increase is caused by transient formation of inverted micellar structures in the membrane with cardiolipin as translocating membrane component for calcium and arsenazo III. At calcium concentrations of 4 mM and higher for 50 mol% cardiolipin-containing vesicles a general permeability increase is found together with calcium-cardiolipin binding in a 1:1 stoichiometry, vesicles aggregation and, above 8 mM of calcium, vesicle fusion. The loss of barrier function of the membrane under these conditions is correlated with vesicle aggregation and may be explained by a transition from a bilayer into a hexagonal HII organization of the phospholipids.

Consequences of the interaction of calcium with dioleoylphosphatidate-containing model membranes: changes in membrane permeability

The permeability behaviour of dioleoylphosphatidate/dioleoylphosphatidylcholine (20:80, mol%) large unilamellar vesicles at low millimolar calcium concentrations is different for various solutes. Between 0.5 mM and 2.5 mM of calcium a selective influx of calcium and efflux of enclosed calcium chelating anions is observed. At higher calcium concentrations the membrane loses its barrier function for a large variety of solutes. These permeability increases are a specific consequence of calcium phosphatidate interactions, because control experiments in which calcium was replaced by magnesium or in which dioleoylphosphatidate was replaced by dioleoylphosphatidylglycerol showed under the same conditions no permeability changes. These results are discussed on the basis of various putative mechanistic models for phosphatidate-mediated calcium translocation across membranes. Furthermore a kinetical model is presented by which the observed selective calcium and calcium-chelator translocation can be explained.

The full length of a mitochondrial presequence is required for efficient monolayer insertion and interbilayer contact formation

The peptide specificity of both presequence-monolayer interactions and the ability of presequences to induce interbilayer contacts between large unilamellar vesicles was investigated. A range of different synthetic peptides that are documented for their mitochondrial protein import abilities were used for this purpose. Both monolayer insertion and vesicle aggregation were found to be strongly dependent on the primary structure of the studied presequence peptides. The combination of monolayer data and results of vesicle aggregation experiments leads to the overall suggestion that monolayer insertion and interbilayer contact formation are mechanistically related. For maximal effects the full length of a presequence peptide is required. The cardiolipin specificity of presequence-induced interbilayer contact formation previously reported was found to be a more general property among presequence peptides. The peptides ability to induce vesicle-vesicle contacts seems to parallel the efficiency of its import ability into mitochondria. These results lead to an extended hypothesis on the role of presequence-induced contact site formation during the mitochondrial protein import process.

The cryoprotectant trehalose destabilises the bilayer organisation of Escherichia coli-derived membrane systems at elevated temperatures as determined by 2H and 31P-NMR

In this study, 2H and 31P-NMR techniques were used to study the effects of trehalose and glycerol on phase transitions and lipid acyl chain order of membrane systems derived from cells of E. coli unsaturated fatty acid auxotroph strain K1059, which was grown in the presence of [11,11-2H2]-oleic acid or [11,11-2H2]-elaidic acid. From an analysis of the temperature dependence of the quadrupolar splitting it could be concluded that neither 1 M trehalose or glycerol generally had any significant effect on the temperature of the lamellar gel to liquid-crystalline phase transition. In the case of the oleate-containing hydrated total lipid extract, glycerol but not trehalose caused a 5 degrees C increase of this transition temperature. In general, both cryoprotectants induced an ordering of the acyl chains in the liquid-crystalline state. Trehalose and glycerol both decrease the bilayer to non-bilayer transition temperature of the hydrated lipid extract of oleate-grown cells by about 5 degrees C, but only trehalose in addition induces an isotropic to hexagonal (HII) phase transition. In the biological membranes, trehalose and not glycerol destabilised the lipid bilayer, and in the case of the E. coli spheroplasts, part of the induced non-bilayer structures is ascribed to a hexagonal (HII) phase in analogy with the total lipids. Interestingly, 1 mM Mg2+ was a prerequisite for the destabilisation of the lipid bilayer. In the hydrated total lipid extract of E. coli grown on the more ordered elaidic acid, both transition temperatures were shifted about 20 degrees C upwards compared with the oleate-containing lipid, but the effect of trehalose on the lipid phase behaviour was similar. The bilayer destabilising ability of trehalose might have implications for the possible protection of biological systems by (cryo-)protectants during dehydration, in that protection is unlikely to be caused by preventing the occurrence of polymorphic phase transitions.