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All classes of phospholipids present in brush border membrane are exchanged in a 1:1 ratio for egg phosphatidylcholine when brush border membrane vesicles from rabbit small intestine are incubated with small unilamellar vesicles of egg phosphatidylcholine. The exchange reaction exhibits biphasic kinetics similar to those of the hydrolysis of brush border membrane phospholipids by phospholipase A2 and sphingomyelinase C. In both reactions there is an initial fast phase followed by a markedly slower one. The phospholipid exchange appears to be catalyzed by intrinsic brush border membrane protein(s), while the digestion by phospholipases is mediated by externally added enzymes. From a comparison of the kinetics of phospholipid exchange and phospholipid hydrolysis, the following conclusions can be drawn: Both sets of experiments indicate the presence of two phospholipid pools differing in the rate of phospholipid exchange and hydrolysis. Except for sphingomyelin, the size of the two phospholipid pools derived from phospholipid exchange is in good agreement with that derived from phospholipid hydrolysis. This is the main finding of this work, and on the basis of this result the two lipid pools are tentatively assigned to phospholipid molecules located on the outer and inner layer of the brush border membrane. The slow rate of phospholipid exchange reflects the rate of transverse or flip-flop movement of phospholipids. The half-time of this motion is approximately 8 h for isoelectric (neutral) phospholipids such as phosphatidylethanolamine and approximately 80 h for negatively charged phosphatidylserine and phosphatidylinositol. Isoelectric phospholipids (phosphatidylcholine, phosphatidylethanolamine) are preferentially located on the inner (cytoplasmic) side (to about 70%) while the negatively charged phospholipids are more evenly distributed: 55-60% are located on the inner side.

The phase behaviour of lipid X at high water concentrations (> 60%) is studied using mainly differential scanning calorimetry (DSC) and X-ray diffraction. The critical micellar concentration (CMC) decreases from 8 × 10−5 M at −2 °C to 4 × 10−5 M at 20 °C. The critical micellar temperature (CMT) is 0 °C and decreases slightly with increasing lipid X concentration. Above the CMC and below the CMT, lipid X forms a lamellar gel phase (Lβ). Above 0 °C and at concentrations ranging from the CMC up to about 0.2 M (20%), lipid X forms small micelles. At even higher concentrations there is a transition to a hexagonal phase, probably hexagonal I. Addition of excess NaCl to lipid X dispersions at concentrations 0.7 M NaCl, the bilayer repeat distance decreases to about 43 Å (1 Å = 0.1 nm).Key words: lipid X, phase behaviour, effects of NaCl, differential scanning calorimetry, X-ray diffraction.

Lipid X forms small micelles of average diameter of 13 ± 2 nm in excess H2O pH 7 to 8, and under physiological conditions. The critical micellar concentration is 4 × 10–5 M, and the critical micellar temperature coincides with the ice melting temperature. Freezing of micellar dispersions of lipid X induces the conversion of the micellar phase to a lamellar gel phase with hexagonal hydrocarbon chain packing. Conversely, melting of ice in frozen lipid X dispersions induces the transition from the lamellar gel phase to a liquid crystalline phase. At room temperature different liquid crystalline phases are observed depending on the water content of lipid X dispersions. In the order of increasing water contents cubic, hexagonal and micellar phases are formed. At room temperature lipid X has therefore a preference for phases with highly curved lipid surfaces. The preference for non-lamellar phases in the fully hydrated state of lipid X at room temperature can be rationalized in terms of hydration. About 22 mol H2O/ mol lipid X are bound which is about twice as much water as is bound to phosphatidylcholine. The large amount of water which is associated with the α-D-glucosamine-l-phosphate group of lipid X gives rise to a wedge-shaped molecule. Molecules of this shape have been shown to have a preference for non-lamellar phases. The hydrocarbon chains of lipid X in the smectic (lamellar) phase pack in a typical, hexagonal lattice. Interdigitation of the hydrocarbon chains in the lamellar phase is not observed.

The effect of increasing concentrations of lipid X (2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine 1-phosphate) on the phase behaviour of EPC (egg phosphatidylcholine) and EPE (egg phosphatidylethanolamine) is studied at a pH greater than or equal to 7 where lipid X carries one to two negative charges. Small amounts of lipid X (molar ratio approximately 0.01) induce continuous swelling of EPC and EPE bilayers and consequently the formation of large unilamellar vesicles in excess water. In many respects, the effect of lipid X on EPC and EPE bilayers is similar to that of phosphatidic acid. However, lipid X/EPC mixtures form micelles in excess lipid X whereas mixtures of phosphatidic acid/EPC vesiculate at all ratios. The same is true for lipid X/EPE mixtures. Small unilamellar vesicles of an average diameter of 40 nm form spontaneously upon dispersion of a dry lipid X/EPE film (molar ratio = 10). Unsonicated dispersions of lipid X/EPC (molar ratio = 1) are subjected to pH-jump treatment which involves raising of the pH to 11-12 and subsequent lowering of the pH to between 7.5 and 8.5. Such a treatment has little effect on the vesicle size and size distribution as compared to a control dispersion at pH 8.2. The mean size is determined to be 92 +/- 60 nm. Electron micrographs of freeze-fractured samples of lipid X/EPC (molar ratio = 1) reveal the presence of mainly micelles at pH 12. Upon lowering the pH to neutrality these micelles become unstable and aggregate/fuse rapidly to unilamellar vesicles (average diameter 95 +/- 40 nm). Sonication of equimolar mixtures of lipid X and EPC at pH 7 yields small unilamellar vesicles of a diameter of 20-25 nm as well as mixed micelles of a size between 15 and 17 nm. This behaviour is again different from that of mixed EPC/phosphatidic acid dispersions which form small unilamellar vesicles. The presence of lipid X in such mixtures does not prevent the aggregation/fusion to larger vesicles during freezing of the dispersion. As with pure EPC bilayers, stabilization is, however, achieved in the presence of 10% sucrose. This indicates that the covalently bonded glucosamine group of lipid X cannot substitute water of hydration in neighbouring EPC molecules.

Two independent parameters, two characteristic temperatures, one indicating the change in the molecular organization of the core, T e and the other in the surface layer, T p were measured for a number of natural and triglyceride-enriched porcine low-density lipoprotein (LDL 1 (buoyant density 1.020–1.063 g/ml) and LDL 2 (buoyant density 1.063–1.080 g/ml) samples T c was determined by differential scanning calorimetry (DSC), whereas T 3 was measured by Mn(II) binding to the lipoprotein surface followed by electron spin resonance (ESR) spectroscopy. A significant causal relationship between T c and T s is both LDL subfractions demonstrates the surface-core interaction in LDL. The significance of that interaction is emphasized as a possible link in the chain diet → lipoprotein changes → atherosclerosis.

The B, or binding, subunit of cholera enterotoxin forms a pentameric ring structure in the intact toxin, and also when the subunit is isolated from the A subunit. The thermal denaturation of the B subunit ring was examined by differential scanning calorimetry in the presence and absence of ganglioside GM1, its natural 'receptor'. In the absence of ganglioside an irreversible endotherm was observed with maximal excess apparent heat capacity, Cmax, at 74.6 degrees C. When the ganglioside was added in increasing amounts, multiple transitions were observed at higher temperatures, the most prominent having a Cmax at 90.8 degrees C. At high ganglioside concentrations, the 74.6 degrees C transition was not observed. In addition to the thermodynamic results a model is proposed for the interaction of GM1 and B subunit pentamer. This model is derived independently of the calorimetric results (but is consistent with such data) and is based upon considerations of the geometry of the GM1 micelle B subunit pentamer.

The phase behaviour of aqueous dispersions of lipid X, a precursor of bacterial lipopolysaccharides has been investigated by a variety of physico-chemical techniques. The results are consistent with the presence of disk-shaped micelles with an average diameter of 13 +/- 1.8 nm. The critical micellar concentration in water and physiological saline is 4 x 10(-5) M. Consistent with the formation of micelles in water and physiological saline is the finding that lipid X is in the liquid-crystalline state at temperatures higher than 0 degrees C. The packing and the dynamics of lipid X are characteristic of micelles. Close to the polar group the hydrocarbon chains are significantly more mobile and disordered than in the corresponding region of lipid bilayers. From monolayer studies an estimate of the molecular area of lipid X is derived; under physiological conditions the area/molecule is about 0.50 nm2 at 30 mN/m indicating that lipid X has a wedge-like shape. The two pK values of the primary phosphate group of lipid X are pK1 approximately 1.3 and pK2 = 8.2. At pH values less than 7, the area/molecule decreases, i.e. the packing of the lipid X molecules becomes tighter, and there is also a decrease in the solubility of lipid X. As is characteristic of charged lipids, the state of aggregation (phase behaviour) of lipid X depends on pH, the ionic strength and the nature of the counterion.