Influence of glycophorin incorporation on Ca2+-induced fusion of phosphatidylserine vesicles.

The effect of incorporation of glycophorin, the major integral sialoglycoprotein of the erythrocyte membrane, into bovine brain phosphatidylserine (PS) vesicles on the Ca2+-induced fusion of these vesicles has been investigated. Fusion was monitored by the terbium-dipicolinic acid fluorescence assay for the mixing of aqueous contents of the vesicles and by a resonance energy transfer assay that follows the intermixing of membrane lipids. The Ca2+-induced fusion of PS vesicles is completely prevented by incorporation of glycophorin (molar ratio of PS/glycophorin = 400-500:1) for Ca2+ concentrations up to 50 mM. The ability to fuse is partially restored after treating the glycophorin-containing vesicles with neuraminidase, which removes the negatively charged sialic acid residues of glycophorin. Fusion is further facilitated by trypsin treatment, removing the entire extravesicular glycosylated head group of glycophorin. However, Ca2+-induced fusion of enzyme-treated glycophorin-PS vesicles proceeds at a slower rate and to a smaller extent than fusion of protein-free PS vesicles. The influence of the aggregation state of the glycophorin molecules on fusion has been investigated in experiments using wheat germ agglutinin (WGA). Addition of WGA to the glycophorin-PS vesicles does not induce fusion. However, upon subsequent addition of Ca2+, distinct fusion occurs concomitantly with release of vesicle contents. The inhibition of Ca2+-induced fusion of PS vesicles by incorporation of glycophorin is explained by a combination of steric hindrance and electrostatic repulsion between the vesicles by the glycosylated head group of glycophorin and a direct bilayer stabilization by the intramembranous hydrophobic part of the glycophorin molecule.

Isolation and purification of cardiolipin from beef heart.

A simple and efficient procedure is described for the preparation of cardiolipin sodium salt from beef heart. A crude phospholipid fraction is isolated by chloroform-methanol extraction of the homogenized tissue, followed by acetone precipitation and reprecipitation in 4% aqueous CaCl2-methanol. Cardiolipin is separated from the calcium salts of the acidic phospholipids by partition column chromatography on silica gel (Polygosil 60-63100) using 2-propanol-cyclohexane-water 50:43:7 (v/v/v) as eluent. Further purification of the cardiolipin is achieved by high performance liquid chromatography of the calcium salt on silica gel (Lichrosorb Si 60-5) with a neutral eluent (2-propanol-cyclohexane-water 45:50:5 (v/v/v], followed by quantitative conversion to the sodium salt. The yield of this procedure is 1.5-2.1 g of pure 99% sodium salt of cardiolipin per kg of moist ventricular tissue.

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines.

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1c phosphatidylcholine and 16:0/18:1c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using 31P-NMR, freeze fracturing and differential scanning calorimetry (DSC). The phosphatidylcholines adopt a bilayer configuration above 0 degrees C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with 31P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal HII phase. 16:0/18:1c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75 degrees C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0 degrees C which decreases with increasing unsaturation and which is lowered by approximately 10 degrees C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.

Purification of phospholipids by preparative high pressure liquid chromatography.

A method is described for the purification of a number of phospholipids by preparative high performance liquid chromatography (HPLC). Purification of digalactosyl-diglyceride from spinach and egg phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine from its reaction mixture have been resolved. The lipid separation is performed on a polygosil column and the individual compounds are monitored directly by refractive index detection. Chloroform/methanol mixtures are used as eluent systems, providing a wide polarity range to separate the classes of lipids. The developed equipment can be used for columns between 10 and 50 cm long and 4 and 50 mm inner diameter. The flow rate could be varied between 1 and 100 ml/min and applied pressures between 10 and 450 bars.

Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers.

The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes. The phospholipases which cannot hyorolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A2 from pig pancreas and Crotalus adamanteus and phospholipase D from cabbage, can hydrolyse phospholipid monolayers at pressure below 31 dynes/cm only. The phospholipases which can hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Clostridium welchii phospholipase A2 from Naja naja and bee venom and sphingomyelinase from Staphylococcus aureus, can hydrolyse phospholipid monolayers at pressure above 31 dynes/cm. It is concluded that the lipid packing in the outer monolayer of the erythrocyte membrane is comparable with a lateral surface pressure between 31 and 34.8 dynes/cm.