C. J. A. van Echteld

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.

Effects of lysophosphatidylcholines on phosphatidylcholine and phosphatidylcholine/cholesterol liposome systems as revealed by 31P-NMR, electron microscopy and permeability studies

(1) The effect of incorporation of different lysophosphatidylcholine species on the structure, barrier properties and dynamics of bilayers made of various phosphatidylcholines both the presence and absence of cholesterol have been investigated by 31P-NMR, freeze-fracture electron microscopy and K+-permeability measurements. (2) In a dispersion of lysophosphatidylcholine : cholesterol (1 : 1) the lipids are organized in extended bilayers. Upon cooling a micellar solution of 1-palmitoyllysophosphatidylcholine below the chain-melting temperature a transition to a lamellar, most likely interdigitating organization is observed. 31P-NMR shows in both situations a marked decrease in effective chemical shift anisotropy. (3) 1-Palmitoyllysophosphatidylcholine can be incorporated up to 30 mol% into liquid crystalline bilayers of dipalmitoylphosphatidylcholine and up to 35 mol% into dioleoylphosphatidylcholine bilayers. Above this concentration micellization of the bilayers occurs. In the gel state, bilayer structure is maintained up to 60 mol% of the lysocompound. (4) 1-Oleoyllysophosphatidylcholine can be incorporated to higher concentrations into liquid crystalline phosphatidylcholine bilayers than the palmitoyl analogue, which can be explained by the more cylindrical shape of the 1-oleoyllysophosphatidylcholine. (5) In marked contrast, incorporation of only 1 mol% of 1-oleoyllysophosphatidylcholine into gel state dipalmitoylphosphatidylcholine already destabilizes bilayer structure and makes the membranes completely permeable for K+. These results are discussed with respect to the mixing properties of the various lysophosphatidylcholines. (6) In general these effects are accompanied by a loss of K+-permeability barrier, which however occurs at lower lysophosphatidylcholine concentrations than needed for the start of micellization. (7) Cholesterol incorporation counteracts the bilayer destabilizing role of lysophosphatidylcholines. (8) 31P-NMR demonstrates with increasing lysophosphatidylcholine concentrations in the bilayers of phosphatidylcholines a decrease in the effective chemical shift anisotropy. As the rigid lattice spectra of lysophosphatidylcholine and phosphatidylcholine are identical, this reflects a change in the conformational and/or motional properties of the phospholipid head groups. This phenomenon might play a role in the observed permeability changes.

Gramicidin promotes formation of the hexagonal HII phase in aqueous dispersions of phosphatidylethanolamine and phosphatidylcholine

Abstract It is shown by 31P-NMR and electron microscopy that gramicidin promotes the formation of the hexogonal HII phase in aqueous dispersions of dielaidoylphosphatidylethanolamine and dioleoylphosphatidylethanolamine, when present in molar ratios of 1 : 200 and higher. In addition gramicidin also induces the hexogonal HII phase in aqueous dispersions of dioleoylphosphatidylcholine, when present in molar ratios of 1 : 25 and higher.

Gramicidin induces the formation of non-bilayer structures in phosphatidylcholine dispersions in a fatty acid chain length dependent way

The hydrophobic peptide gramicidin is shown by 31P-NMR, freeze-fracture electron microscopy and small-angle X-ray diffraction, to induce a hexogonal HII-phase lipid organization when incorporated in liquid crystalline saturated and unsaturated synthetic and natural phosphatidylcholines if the length of the fatty acids exceeds a 16 carbon atoms chain. The amount of hexagonally organized lipid increases with increasing fatty acid chain length. With phosphatidylcholines possessing shorter fatty acid chains, as well as with the longer phosphatidylcholines in the gel state, a lamellar organization results. Of the various possible models to explain the induction of the hexagonal HII phase by gramicidin, one in which gramicidin dimers span adjacent cylinders of the hexagonal HII phase seems most plausible. In phosphatidylcholines with intermediary chain lengths gramicidin induces intermediary structures, such as lipidic particles and possibly cubic phases. These experiments suggest that the balance between the length of the hydrophobic domain of a peptide and the membrane thickness is of critical importance for the structure of the membrane. In relation to this observation, the possible involvement of non-bilayer lipid structures in insertion and anchoring of membrane proteins is discussed.

A POSSIBLE ROLE OF RHODOPSIN IN MAINTAINING BILAYER STRUCTURE IN THE PHOTORECEPTOR MEMBRANE

31P-NMR measurements demonstrate that at 37 degrees C, independent of the photolytic state of the photopigment rhodopsin, the lipids in the photo-receptormembrane are almost exclusively organised in a bilayer. In strong contrast, the 31P-NMR spectra of the extracted lipids are characteristic for the hexagonal HII phase and an isotropic phase. The isotropic phase is characterised by freeze-fracture electron microscopy as particles and pits on smooth surfaces, possibly indicating inverted micelles. These results suggest a structural role for rhodopsin in maintaining the photoreceptor membrane lipids in a bilayer configuration.

Effect of glycophorin on lipid polymorphism. A 31P-NMR study.

(1) The effect of glycophorin, a major intrinsic glycoprotein of the human erythrocyte membrane, on lipid polymorphism has been investigated by 31P-NRM (at 36.4 MHz) and be freeze-fracture electron microscopy. (2) Incorporation of glycophorin into vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) results in the formation of unilamellar vesicles ((1 000-5 000 A diameter) which exhibit 31P-NRM bilayer spectra over a wide range of temperature. A reduction in the chemical shift anisotropy ( delta sigma eff csa) and an increase in spectral linewidth in comparison to dioleoylphosphatidylcholine liposomes may suggest a decrease in phospholipid headgroup order. (3) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), in the presence of excess water, undergoes a bilayer to hexagonal (HII) phospholipid arrangement as the temperature is increased above 0 degrees C. Incorporation of glycophorin into this system stabilizes the bilayer configuration, prohibiting the formation of the HII phase. (4) Cosonication of glycophorin with DOPE in aqueous solution (pH 7.4) produced small, stable unilamellar vesicles (300-1 000 A diameter), unlike DOPE alone which is unstable and precipitates from solution. (5) The current study demonstrates the bilayer stabilizing capacity of an intrinsic membrane protein, glycophorin, most likely by means of a strong hydrophobic interaction between the membrane spanning portion of glycophorin and the hydrophobic region of the phospholipid.

Mixtures of gramicidin and lysophosphatidylcholine from lamellar structures

Abstract It is shown by 31P-NMR and freeze-fracture electron microscopy that in aqueous dispersions of mixtures of gramicidin and palmitoyllysophosphatidylcholine lamellar structures are formed which contain four lysophosphatidylcholine molecules per gramicidin monomer.

Changes in phosphorus compounds and water content in skeletal muscle due to eccentric exercise

The interrelationship of the time courses of soreness and oedema, and of force and phosphorus metabolites after eccentric exercise was studied. Eight male subjects performed 120 maximal eccentric contractions with their left forearm flexors. Soreness, maximal force, flexion and extension elbow angle, and creatine kinase and myoglobin efflux were followed for 96 h after exercise. For equal periods T1 and T2 relaxation times and muscle cross-sectional area were calculated from magnetic resonance images as indications of oedema, and inorganic phosphate (Pi) and phosphocreatine (PCr) were measured with magnetic resonance spectroscopy. Soreness on extension increased at 1 h (P = 0.043), T1 and T2 (both P = 0.01) and soreness when the arm was pressed (P = 0.028) at 24 h, and muscle cross-sectional area increased at 48 h (P = 0.01) after exercise. Soreness on extension reached a maximum at 48 h, the other four parameters at 72 h. All parameters related to oedema, and soreness, showed an increasing pattern for the period after exercise as a whole, but the largest increase between two points of measurement occurred earlier for soreness than for oedema. Creatine kinase increased significantly from baseline from 24 h onwards (P = 0.017) and myoglobin from 1 h onwards (P = 0.012). The Pi: PCr ratio differed from baseline for the first time 24 h after exercise (P = 0.018), increased to 225%, and then remained on a plateau until 72 h. Maximal isotonic force decreased to 53% at 1 h (P = 0.012), and recovered from then on. It was concluded firstly that the largest increase in soreness precedes that of oedema, and secondly that the decrease in force after eccentric exercise is not related to an altered metabolic state. The combined imaging and spectroscopy results gave the impression that changes in phosphorus metabolites were homogeneously distributed over the flexor muscles whereas oedema was not.

Differential miscibility properties of various phosphatidylcholine/lysophosphatidylcholine mixtures

Using enthalphy data from differential scanning calorimetry experiments and 13C-NMR linewidths of specifically (N-Me-13C)-labelled lipids, the miscibility properties of phosphatidylcholines and lysophosphatidylcholines in liposomal dispersions have been investigated. It was found that 16 : 0 lysophosphatidylcholine mixes homogeneously in 16 : 0/16 : 0 phosphatidylcholine bilayers. Mixtures of 16 : 0 lysophosphatidylcholine with 18 : 1c/18 : 1c phosphatidylcholine, of 18 : 1c lysophosphatidylcholine with 16 : 0/16 : 0 phosphatidylcholine and of 18 : 1c lysophosphatidylcholine with 18 : 1c/18 : 1c phosphatidylcholine exhibited immiscibility in the phosphatidylcholine gel state.