W.S.M. Geurts van Kessel

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.

The properties of polyunsaturated lecithins in monolayers and liposomes and the interactions of these lecithins with cholesterol.

Abstract 1. 1. The force-area characteristics of monolayers of synthetic lecithins with one to six double bonds in one acyl chain have been studied. 2. 2. The area per molecule increases stepwise. The most significant increase is observed after the introduction of the first double bond. The subsequent introduction of two, three or four double bonds or polyunsaturated chains at both ester positions produces some further increase. 3. 3. The interaction with cholesterol depends on the unsaturation and the distribution of the double bonds between the acyl chains. 4. 4. A condensing effect with cholesterol was evident for (1-stearoyl-2-oleoyl)-3-lecithin, (1-palmitoyl-2-lineoyl)-3-lecithin, (1-palmitoyl-2-linolenoyl)-3-lecithin, (1-palmitoyl-2-arachidonoyl)-3-lecithin at 22°. No effect is observed for (1,2-dilinoleoyl)-3-lecithin and (1,2-dilinolenoyl)-3-lecithin. (1-palmitoyl-2-docosahexaenoyl)-3-lecithin shows a limited effect at 22°, but no effect at 37°. 5. 5. No significant differences in behavior are found for the two structural isomers with a mono- or disaturated chain at the 1- or 2-position. 6. 6. The permeability of liposomes, derived from the above mentioned lecithins, to glucose, erythritol and glycerol increases in the same order as the area per molecule at the air-water interface. 7. 7. The presence of cholesterol reduced the permeability to glucose, erythritol, glycerol only for the lecithins which showed a condensation effect. 8. 8. The unsaturation and the distribution of the double bonds appear to be of critical importance for the barrier properties of lecithins and for the interaction with cholesterol.

Studies on the biological properties of polyene antibiotics: Comparison of other polyenes with filipin in their ability to interact specifically with sterol

1. 1. The interaction fo the five polyene antibiotics filipin, etruscomycin, pimaricin, nystatin and amphotericin B with sterol, primarily free, liposomal and membrane bound cholesterol has been examined. Each of these antibiotics has a characteristic ultraviolet absorption spectrum in aqueous or organic solvents with three or four ultraviolet absorption maxima. Addition of free cholesterol to aqueous solutions of these antibiotics results in a change of the ratio of the ultraviolet absorbance maxima. The order of effectiveness of interaction with cholesterol as judged by this criterion was filipin, amphotericin B, etruscomycin and pimaricin. 2. 2. The same alteration in ultraviolet absorption spectra of filipin etruscomycin and amphotericin B observed with addition of free cholesterol to aqueous solutions of these antibiotics also occurs upon addition of these antibiotics to liposomal, erythrocyte or Acholeplasma membrane bound cholesterol. No spectral change was found in membranes devoid of cholesterol. This spectra alteration of the antibiotic was accompanied by a binding of the antibiotic to the membrane. Both nystatin and pimaricin showed little change in spectrum with sterol in these systems, either the artificial or natural membranes which contained cholesterol. 3. 3. The structural requirements of the sterol for the spectral change with filipin, etruscomycin and amphotericin B include a planar sterol nucleus, and intact side chain at C-17 and 3β-hydroxyl group. The spectral change was not affected by the pH except in the case of amphotericin B. 4. 4. Measurements by differential scanning calorimetry of the effect of these polyene antibiotics on the phase transition of lecithin and lecithin-cholesterol showed that all polyenes can reduce the ecithin cholesterol interaction.

High-performance liquid chromatographic separation and photometric detection of phospholipids.

A rapid and efficient method for the separation of (phospho)lipids by high-performance liquid chromatography using n-hexane-2-propanol-water mixtures as the solvent system is described. The lipid separation occurs on silica gel columns and the individual components are monitored directly by UV absorption at 206 nm. Of a total lipid extract from erythrocytes as well as suboesophageal ganglia of the snail Helix pomatia, a complete separation is achieved of cholesterol, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, lysophosphatidylcholine and lysophosphatidylethanolamine, whereas phosphatidylcholine and sphingomyelin are partly separated under these circumstances. In addition to separation of phospholipids in different classes, separation of molecular species can also be achieved in some instances, as is shown for phosphatidylcholines and sphingomyelins.

The protection of A1 myelin basic protein against the action of proteolytic enzymes after interaction of the protein with lipids at the air-water interface

1. 1. The specific interaction of bovine myelin A1 basic protein with lipids at the air-water interface was studied. The interaction was measured by recording the changes in surface pressure and surface radioactivity using 131I-labelled A1 basic protein. The highest affinity of the A1 basic protein was found for cerebroside sulphate, a lipid which is characteristic for the myelin lipids. 2. 2. The proteolytic degradation of the A1 basic-protein-lipid complex by proteolytic enzymes such as trypsin, chymotrypsin A4, subtilopeptidase A and pronase E, showed that specific regions of the protein molecule are protected after the interactions with lipids. 3. 3. The A1 basic protein-cerebroside sulphate complex was collected from the interface after tryptic hydrolysis. Peptide maps showed that the N-terminal part of the protein molecule (positions 20–113) is preserved in the lipid phase. A schematic model of the A1 basic protein lipid interaction is presented.

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.

The interaction of the “Folch-Lees” protein with lipids at the air-water interface

Abstract 1. 1. The interaction of “Folch-Lees” apoprotein with different lipids has been studied at the air-water interface. Measurements of the change in surface pressure showed a high affinity of the Folch-Lees protein for a variety of myelin lipids such as cerebroside sulphate, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cholesterol. Lipids such as sphingomyelin, cerebroside and lecithin show markedly less affinity for the Folch-Lees protein. 2. 2. The remarkable interaction of the Folch-Lees protein with cholesterol is dependent on the sterol structure. Epicholesterol, cholest-4-en-3-one, coprostanol, lanosterol and androstanol showed a reduced binding in this order. 3. 3. The two major proteins, the Folch Lees protein and the A 1 basic protein, show a different lipid affinity. The subsequent injection of A 1 basic protein and Folch-Lees protein underneath a cholesterol monolayer shows a preferential binding of Folch-Lees protein with cholesterol and a rejection of A 1 basic protein from the interface. The subsequent injection of A 1 basic protein and Folch-Lees protein underneath a cerebroside sulphate monolayer shows that both proteins can bind to cerebroside sulphate. The A 1 basic protein shows however the highest affinity for this typical myelin lipid.

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.

Interbilayer swelling and the Lα-HII phase transition in phosphatidylethanolamine-phosphatidic acid mixtures

Abstract 31 P-NMR, small angle X-ray scattering and DSC techniques show that in aqueous dispersions of dielaidoylphosphatidylethanolamine, incorporation of dielaidoylphosphatidic acid causes bilayer stabilization, most likely due to interbilayer repulsive electrostatic forces. The results further indicate that attractive forces exist between phosphatidylethanolamine bilayers which prevent swelling of the multilamellar system at low phosphatidic acid concentrations.

PHOSPHOLIPIDS. SOME RECENT STUDIES ON LIPID-PROTEIN INTERACTION IN RELATION TO BIOMEMBRANES

1. With a view to possible specific associations between lipids and protein the topography of phospholipids within the human erythrocyte membrane was investigated. Using the action of various phospholipases in combination with freeze-etch electronmicroscopy an asymmetric distribution of phospholipids in this membrane was demonstrated. The outer layer of the membrane appeared to consist mainly of the two choline-containing phospholipids, namely phosphatidylcholine (lecithin) and sphingomyelin; in addition a small amount of phosphatidylethanolamine is present. The inner layer of the human erythrocyte membrane was found to contain predominantly phosphatidylethanolamine and phosphatidylserine.