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A new and unnatural type of lipid analogs with the phosphocholine and phosphoglycerol head groups linked to the C-2 position of the glycerol moiety have been synthesized and the thermodynamic lipid membrane behavior has been investigated using differential scanning calorimetry. From the heat capacity measurements, it was observed that the pre-transition was abolished most likely due to the central position of the head groups providing better packing properties in the low temperature ordered gel phase. Activity measurements of secretory phospholipase A2 (PLA2) on unilamellar liposomal membranes revealed that the unnatural phospholipids are excellent substrates for PLA2 catalyzed hydrolysis. This was manifested as a minimum in the PLA2 lag time in the main phase transition temperature regime and a high degree of lipid hydrolysis over a broad temperature range. The obtained results provide new information about the interplay between the molecular structure of phospholipids and the lipid membrane packing constrains that govern the pre-transition. In addition, the PLA2 activity measurements are useful for obtaining deeper insight into the molecular details of the catalytic site of PLA2. The combined results also suggest new approaches to rationally design liposomal drug carries that can undergo a triggered activation in diseased tissue by overexpressed PLA2.

Differential scanning calorimetry (DSC) has been used to study the effects of repeated freezing and thawing on dipalmitoylphosphatidylcholine (DPPC) vesicles. Aqueous suspensions of both multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) were cycled between −37 and 8 °C, and for each thawing event, the enthalpy of ice-melting was measured. In the case of MLVs, the enthalpy increased each time the vesicles were thawed until a steady state was attained. In contrast, the enthalpies measured for LUV suspensions were independent of the number of previous thawing events. It was concluded that MLVs in terms of freezing characteristics contain two pools of water, namely bulk water and interlamellar water. Interlamellar water does not freeze under the conditions employed in the present study, and the MLVs therefore experience freeze-induced dehydration, which is the reason for the observed increase in ice-melting enthalpy. Furthermore, the thermodynamic results suggest that the osmotic stress resulting from the freeze-induced dehydration changes the lamellarity of the MLVs.

The influence of ionic strength and equilibration time on the appearance of the sub-main transition in fully hydrated multilamellar vesicles composed of phosphatidylcholines has been investigated by means of calorimetry and densitometry. The heat capacity measurements show that the transition enthalpy of the sub-main transition is affected by both salt concentration (KCl) and equilibration time. The small heat capacity peak appearing in vesicles made in pure water is significantly increased upon addition of salt. Furthermore, equilibration of the multilamellar vesicles at low temperatures for several weeks results in a pronounced enhancement of the transition enthalpy of the sub-main transition. Neither salt concentration nor equilibration time affected the transition temperature of the sub-main transition. In the densitometry measurements a small volume change is detectable for high salt concentrations. In order to gain further insight into the physical mechanisms involved in the sub-main transition, a Monte Carlo computer simulation study has been carried out using a microscopic model. The combined experimental and simulation results suggest that the sub-main transition involves an acyl chain disordering of phospholipids in lipid bilayer regions that are characterized by a locally decreased lateral pressure most likely caused by a curvature stress.

The association of ethanol at physiologically relevant concentrations with lipid bilayers of different lipid composition has been investigated by use of isothermal titration calorimetry (ITC). The liposomes examined were composed of combinations of lipids commonly found in neural cell membranes: dimyristoyl phosphatidylcholine (DMPC), ganglioside (GM 1 ), sphingomyelin and cholesterol. The calorimetric results show that the interaction of ethanol with fluid lipid bilayers is endothermic and strongly dependent on the lipid composition of the liposomes. The data have been used to estimate partitioning coefficients for ethanol into the fluid lipid bilayer phase and the results are discussed in terms of the thermodynamics of partitioning. The presence of 10 mol% sphingomyelin or ganglioside in DMPC liposomes enhances the partitioning coefficient by a factor of 3. Correspondingly, cholesterol (30 mol%) reduces the partitioning coefficient by a factor of 3. This connection between lipid composition and partitioning coefficient correlates with in vivo observations. Comparison of the data with the molecular structure of the lipid molecules suggests that ethanol partitioning is highly sensitive to changes in the lipid backbone (glycerol or ceramide) while it appears much less sensitive to the nature of the head group.

Simultaneous small- and wide-angle X-ray diffraction was used to investigate the structural conversions in the so-called sub-main-transition of fully hydrated multilamellar vesicles of distearoyl phosphatidylcholine (DSPC). The small-angle diffraction patterns show a modification in the supramolecular structure of the corrugated lipid bilayer and reveal that the sub-main-transition does not abolish the general features of the ripple phase ( P β ′ ). Concomitantly in the wide-angle region the diffraction patterns exhibit a rearrangement of the hydrocarbon chain packing and an onset of chain melting. The presence of KCl show an enhancement of the effects of the sub-main-transition. Moreover, in the ripple phase ( P β ′ ) below the sub-main-transition the hydrocarbon chains are positioned on an orthorhombic lattice in the presence of KCl. © 1997 Elsevier Science B.V. All rights reserved.

A recently discovered submain phase transition in multi-lamellar bilayers of long-chain saturated diacyl phosphatidylcholines (Jørgensen, K. (1995) Biochim. Biophys. Acta 1240, 111–114) is discussed in terms of a theoretical molecular interaction model using computer simulation techniques. The model interprets the transition to be due to a decoupling of the acyl-chain melting from the melting of the pseudo-two-dimensional crystalline lattice of the Pβ′ phase. A two-stage melting process is predicted by the calculations suggesting that the sub-main transition involves a lattice melting whereas the acyl-chain melting takes place at a higher temperature at the main transition. The calculated heat contents of the two transitions as well as the chain-length dependence compare favorably with experimental data for multi-lamellar phosphatidylcholine lipid bilayers.

The effects of a small molecule, the insecticide lindane, on unilamellar DMPC bilayers in the phase transition region, have been studied by means of differential scanning calorimetry and fluorescence spectroscopy. The calorimetric data show that increasing concentrations of lindane broaden the transition and lower the transition temperature, without changing the transition enthalpy significantly. Lindane therefore enhances the thermal fluctuations of the bilayer. The calorimetric data furthermore suggest that the bilayer structure is intact and not disrupted by even high concentrations (32 mol%) of lindane. Fluorescence spectroscopy was used to measure the passive permeability of unilamellar DMPC bilayers to Co2+ ions. The data show that lindane seals the bilayer for Co2+ penetration and that this effect increases with increasing lindane concentration. The results are discussed in relation to the effects on the permeability of other small molecules, e.g., anesthetics.

A molecular interaction model is used to describe the phase diagram of two-component phospholipid bilayer membranes of saturated phospholipids, DCnPC, with different acyl-chain lengths, n = 12,14,18,20. The interaction between acyl chains of different length is formulated in terms of a hydrophobic mismatch which permits the series of binary phase diagrams to be calculated in terms of a single 'universal' interaction parameter. The properties of the model are calculated by computer-simulation techniques which not only permit determination of the specific-heat function and the phase diagram but also reveal the local structure of the mixture in the different parts of the phase diagram. The local structure is described pictorially and characterized quantitatively in terms of a correlation function. It is shown that the non-ideal mixing of lipid species due to mismatch in the hydrophobic lengths leads to a progressively increasing local ordering as the chain-length difference is increased. A pronounced local structure is found to persist deep inside the fluid phase of the mixture. The local structure is discussed in relation to the features observed in the specific-heat function, for which theoretical data, as well as experimental data obtained from differential-scanning calorimetry are presented.

An extensive computer-simulation study is performed on a simple but general molecular model recently proposed (Jorgensen et al. (1991) Biochim. Biophys. Acta 1062, 227–238) to describe foreign molecules interacting with lipid bilayers. The model is a multi-state lattice model of the main bilayer transition in which the foreign molecules are assumed to intercalate at interstitial lattice positions. Specific as well as non-specific interactions between the foreign molecules and the lipid acyl chains are considered. Particular attention is paid to the fluctuating properties of the membrane and how the presence of the foreign molecules modulates these fluctuations in the transition region. By means of computer-simulation techniques, a detailed account is given of the macroscopic as well as microscopic consequences of the fluctuations. The macroscopic consequences of the fluctuations are seen in the thermal anomalies of the specific heat and the passive trans-membrane permeability. Microscopically, the fluctuations manifest themselves in lipid-domain formation in the transition region which implies an effective dynamic membrane heterogeneity. Within the model it is found that certain anaesthetics and insecticides which are characterised by specific interactions with the lipids have a strong effect on the heterogeneity of the membrane inducing regions of locally very high concentration of the foreign molecules. This leads to a broadening of the specific heat peak and a maximum in the membrane/water partition coefficient. These results are in accordance with available experimental data for volatile general anaesthetics like halothane, local anaesthetics like cocain derivatives, and insecticides like lindane.

Phospholipase A(2) (PLA(2)) is an interfacially active enzyme whose hydrolytic activity is known to be enhanced in one-component phospholipid bilayer substrates exhibiting dynamic micro-heterogeneity. In this study the activity of PLA(2) towards large unilamellar vesicles composed of DPPC:SMPC and DMPC:DSPC:SMPC is investigated using fluorescence and HPLC techniques. Phase diagrams of the mixtures are established by differential scanning calorimetry and the PLA(2) activity, monitored by the lag time, is correlated with the phase behavior of the mixtures. In addition, the degree of lipid hydrolysis in the DMPC:DSPC:SMPC lipid mixtures is detected by HPLC. The PLA(2) activity is found to be significantly increased in the temperature range of the coexistence region where the lipid mixtures exhibit lateral gel-fluid phase separation. Furthermore, in the entire temperature range it is demonstrated that PLA(2) preferentially hydrolyzes the short chain DMPC lipid. This discriminative effect becomes less pronounced when the asymmetric lipid SMPC is present in the lipid substrate. Inclusion of SMPC into either DPPC or DMPC:DSPC vesicles prolongs the lag time. The results clearly show that the PLA(2) activity is significantly enhanced by lipid bilayer micro-heterogeneity in both one-component and multi-component lipid bilayer substrates. The PLA(2) activity measurements are discussed in terms of dynamic gel-fluid lipid domain formation due to density fluctuations and static lipid domain formation due to gel-fluid phase separation.

The existence of a sub-main transition in multilamellar bilayers composed of long-chain saturated diacyl phosphatidylcholine (DC17PC, DC18PC, DC19PC, and DC20PC) is reported for the first time using high-sensitivity differential scanning calorimetry. The highly cooperative sub-main transition which takes place over a narrow temperature range positioned between the well-known pre-transition and main-transition is characterized by a heat capacity curve with a half-height width of delta T1/2 congruent to 0.15 C degrees and an enthalpy change, delta H, which is a few percent of the transition enthalpy for the main-transition of the lipid bilayer.

The permeability-enhancing effects of the two surfactants, 1-palmitoyl-2-lyso-sn-gycero-3-phosphocholine (lysoPPC) and palmitic acid (PA), on lipid membranes that at physiological temperatures are in the gel, fluid, and liquid-ordered phases were determined using the concentration-dependent self-quenching properties of the hydrophilic marker, calcein. Adding lysoPPC to lipid membranes in the gel-phase induced a time-dependent calcein release curve that can be described by the sum of two exponentials, whereas PA induces a considerably more complex release curve. However, when lysoPPC and PA were added simultaneously in equimolar concentrations, a dramatic synergistic permeability-enhancing effect was observed. In contrast, when both lysoPPC and PA are added to liposomal membranes that are in the fluid or liquid-ordered phases, no effect on the transmembrane permeation of calcein was observed.