L.L.M. Van Deenen

The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy

Abstract 1. 1. Phospholipase A2 (phosphatide acylhydrolase, EC 3.1.1.4) from Naja naja hydrolyses 68% of the lecithin of the intact human erythrocyt without changing the freeze fracture faces of the membrane. Phospholipase A2 (Naja naja) treatment of ghosts produces complete breakdown of the glycerophospholipids and induces aggregation of particles on the freeze-fracture faces of the membrane. 2. 2. Phospholipase C (phosphatidylcholine choline phosphohydrolase, EC 3.1.4.3) from Bacillus cereus does not attack intact cells and no change in freeze-etch morphology is observed. The glycerophospholipids of ghosts are almost completely degraded by this enzyme, which causes a reduction in tangentially-splitted membranes and a formation of large diglyceride droplets, which are also visible by phase-contrast microscopy. 3. 3. Sphingomyelinase (sphingomyelin choline phosphohydrolase) from Staphylococcus aureus, hydrolyses 80–85% of the sphingomyelin of the intact human red cel, and produces aggregation of the particles and the formation of small spheres (75 A and 200 A in diameter) on the outer fracture face with corresponding pits on the inner fracture face. Treatment of ghosts with this enzyme causes a complete degradation of the sphingomyelin and produces, in addition to aggregation of particles, the formation of droplets (1000–3000 A in diameters) whcih are adherent to the membrane and are not visible by phase-contrast microscopy. 4. 4. When the cells are treated successively with phospholipase A2 (Naja naja) and sphingomyelinase (Staphylococcus aureus) no lysis occurs although the osmotic fragility is markedly increased. By this treatment, up to 48% of the total phospholipids are degradd. It is concluded that this phospholipid fraction (which contains the majority of the choline-containing phospholipids and some phosphatidylethanolamine) forms the outer monolayer of the membrane.

Lipid composition and permeability of liposomes.

Abstract 1. 1. Layered latticed liquid crystals (liposomes) in dilute electrolyte solutions have been prepared from a number of synthetic lecithins with variations in length and number of double bonds of the paraffin chains. Using the property of these structures to act as practical, ideal osmometers, their permeability behaviour towards glycerol and erythritol has been studied. The penetration of such non-electrolytes into the liposome appears to be strongly temperature dependent in a way similar to that demonstrated for glycerol permeation into erythrocytes. 2. 2. Introduction of double bonds into the paraffin chains causes a marked enhancement of the permeability. The diffusion rate of glycerol into the liposomes of (distearoyl)lecithin, (1-stearoyl-2-oleoyl)lecithin, (dioleoyl)lecithin and (dilineoyl)lecithin, increases in that order. 3. 3. Decreasing chain length also increases the permeability which is demonstrated by comparing the swelling rate of liposomes prepared from (distearoyl)lecithin, (dipalmitoyl)lecithin and (dimyristoyl)lecithin and that of those prepared from (1-stearoyl-2-myristoyl)lecithin and (1-stearoyl-2-decanoyl)lecithin. 4. 4. Comparison of the permeability of the liposomes from (1-stearoyl-2-decanoyl)lecithin and (1-stearoyl-2-myristoyl)lecithin with those obtained from (dimyristoyl)lecithin and (dipalmitoyl)lecithin, respectively, suggests that, certainly at lower temperatures, the model structures of phospholipids with asymmetric chains are much more permeable than those of lecithins with chains of equal length but with the same total number of paraffin carbon atoms. 5. 5. Liposomes of mixtures of phospholipid and cholesterol normally demonstrate a decrease in permeability which is proportional to the concentration of cholesterol.

Purification and properties of phospholipase a from porcine pancreas

1. 1. Freshly prepared homogenates of pig pancreatic tissue contain a small amount of phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4); during autolysis, however, a considerable rise in lipolytic activity occurs. 2. 2. By use of heat treatment, (NH4)2SO4 precipitation and chromatographic procedures, the enzyme has been purified about 200 times and characterized by chemical and enzymatic procedures. 3. 3. The protein, which has a molecular weight of about 13 800 ± 500 appears to consist of one single polypeptide chain terminating in alanine (NH2) and cystine (COOH), and cross-linked intramolecularly by 7 disulphide bridges. 4. 4. The enzyme acts stereospecifically on all common types of 3-sn-phosphoglycerides, hydrolysing exclusively fatty acid ester bonds at the glycerol-C-2 position, regardless of chain length or degree of unsaturation. In contrast to the snake venom phospholipase A, the pancreatic enzyme shows a marked preference for anionic phospholipids such as phosphatidic acid, cardiolipin and phosphatidyl glycerol.

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 preferential interactions of cholesterol with different classes of phospholipids

1. By differential scanning calorimetry a preferential affinity of cholesterol for sphingomyelin was established in mixtures of sphingomelin and phosphatidylcholine where sphingomyelin was either the higher or the lower melting phospholipid. 2. A preferential affinity of cholesterol for sphingomyelin was also found in mixtures of sphingomyelin and phosphatidylethanolamine where sphingomyelin was either the higher or the lower melting phospholipid. The sphingomyelin used was isolated from beef erythrocytes or synthetic palmitoyl sphingomyelin. 3. In mixtures of phosphatidylserine with phosphatidylethanolamine, or phosphatidylserine with phosphatidylcholine, cholesterol showed the highest affinity for the lower melting phospholipid. 4. In a previous paper (van Dijck et al. (1976) Biochim. Biophys. Acta 455, 576-588) it was established that cholesterol has a higher affinity for phosphatidylcholine than for phosphatidylethanolamine. The affinity order of cholesterol for the neutral phospholipids which can be deduced form these experiments is sphingomyelin greater than phosphatidylcholine greater than phosphatidylethanolamine.

Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases.

1. The action of eight purified phospholipases on intact human erythrocytes has been investigated. Four enzymes, e.g. phospholipases A2 from pancreas and Crotalus adamanteus, phospholipase C from Bacillus cereus, and phospholipase D from cabbage produce neither haemolysis nor hydrolysis of phospholipids in intact cells. On the other hand, both phospholipases A2 from bee venom and Naja naja cause a non-haemolytic breakdown of more than 50% of the lecithin, while sphingomyelinase C from Staphylococcus aureus is able to produce a non-lytic degradation of more than 80% of the sphingomyelin. 2. Phospholipase C from Clostridium welchii appeared to be the only lipolytic enzyme tested, which produces haemolysis of human erythrocytes. Evidence is presented that the unique properties of the enzyme itself, rather than possible contaminations in the purified preparation, are responsible for the observed haemolytic effect. 3. With non-sealed ghosts, all phospholipases produce essentially complete breakdown of those phospholipids which can be considered as proper substrates for the enzymes involved. 4. Due to its absolute requirement for Ca2+, pancreatic phospholipase A2 can be trapped inside resealed ghosts in the presence of EDTA, without producing phospholipid breakdown during the resealing procedure. Subsequent addition of Ca2+ stimulates phospholipase A2 activity at the inside of the resealed cell, eventually leading to lysis. Before lysis occurs, however, 25% of the lecithin, half of the phosphatidylethanolamine and some 65% of the phosphatidylserine can be hydrolysed. This observation is explained in relation to an asymmetric phospholipid distribution in red cell membranes.

The preference of cholesterol for phosphatidylcholine in mixed phosphatidylcholine-phosphatidylethanolamine bilayers.

The following phosphatidylethanolamines were studied by differential scanning calorimetry: 1,2-dipalmitoleoyl-, 1,2-dioleoyl-, 1,2-dilauroyl-, 1,2-dielaidyl-, 1,2-dimyristoyl- and 1,2-dipalmitoyl-sn-glycero-3-phosphoryl-ethanolamine. The saturated and trans-unsaturated species underwent thermotropic phase transitions at temperatures about 20-30 degrees C higher than the corresponding phosphatidylcholines but the enthalpy changes were nearly identical. The transition temperatures for the cis-unsaturated species were about the same as those of the corresponding phosphatidylcholines but here the enthalpy change was markedly decreased as compared with the phosphatidylcholines. Freeze-fracture electron microscopy revealed phase changes from a lamellar to a hexagonal phase for 1,2-dipalmitoleoyl- and 1,2-dioleoyl-sn-glycero-phosphorylethanolamine at 20 and 0 degrees C respectively. At these temperatures no transitions were apparent in the calorimeter scan. Incorporation of increasing amounts of cholesterol into phosphatidylethanol-amine bilayers gradually decreased the enthalpy changes of the phase transition in the same manner as was demonstrated before for phosphatidylcholine/cholesterol mixtures. This was studied both for 1,2-dipalmitoleoyl- and 1,2-dimyristoyl-sn-glycerophosphorylethanolamine. In an equimolar mixture of 1,2-dioleoyl- and 1,2-dipalmitoylphosphoryl-ethanolamine, which showed phase separation, cholesterol preferentially decreased the transition of the lowest melting component. In equimolar mixtures of phosphatidylethanolamines and phosphatidylcholines, which showed phase separation, cholesterol preferentially abolished the transition of the phosphatidylcholine component present. This occurred both in experiments where the phosphatidylcholine was the lowest melting and where it was the highest melting component present in the mixture. These experiments strongly suggest that in phosphatidylcholine-phosphatidylethanolamine mixtures at temperatures where both components are in the liquid-crystalline state cholesterol is preferently associated with the phosphatidylcholine component in the mixture.

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.

The effect of sterol structure on the permeability of lipomes to glucose, glycerol and Rb+

1. 1. The effect of 3β-, 3α-hydroxysterol and ketosteroids on the permeability properties of (egg lecithin) liposomes towards glucose, glycerol and Rb+ has been studied. 2. 2. The 3β-hydroxysterols, cholesterol, cholestanol, lathosterol, 7-dehydrocholesterol and B-norcholesterol affect the most pronounced reduction in permeability of glucose, glycerol and Rb+. 3. 3. The plant sterols, ergosterol and stigmasterol are less effective whereas compounds lacking the side chain (androstan-3β-ol) or with a non-planar sterol nucleus (coprostanol) show no effect. 4. 4. The 3α-hydroxysterols, epicholesterol and androstan-3α-ol reveal no significant effect on the permeability. 5. 5. The ketosteroids either do not affect the permeability or increase the permeability of the lipid barrier. 6. 6. The reduction in permeability, as found for cholesterol, is dependent on: (a) planar sterol nucleus, (b) 3β-hydroxy group, (c) intact side chain. 7. 7. The effect of sterols on the permeability properties of liposomes are in good agreement with the effect on the mean molecular area measured in monolayers, and the effect of these sterols on biological membranes such as erythrocytes and Mycoplasma.

The substrate specificity of phospholipase A

Investigations on variously modified analogues of phospholipids elucidated the following substrate characteristics for phospholipase A (Crotalus adamanteus). 1. 1. Within the class of α-phosphoglycerides l-isomers are readily hydrolysed, while d-α-phospholipids appeared not to be attacked. 2. 2. l-α-Lecithins containing fatty acids with greatly varying chain length are susceptible to phospholipase A action; however, certain water-soluble short-chain compounds are hydrolysed at a very slow rate only. 3. 3. Aside from effects on the surface charge of the phosphoglyceride micelles the nature of the polar headgroup esterified to the phosphoryl moiety turned out not to form any prerequisite, and its presence even appeared to be dispensible. 4. 4. Contrary to a blocking of the amino function, protection of the hydroxyl function of phosphatidylethanolamine caused an inactivation of the substrate properties. 5. 5. Both, a γ-benzyl-β-acyl-α-phosphoglyceride and a β-acyl-lyso derivative, were hydrolysed, whereas the corresponding structural isomers carrying the fatty acid in γ-position appeared not to be susceptible to phospholipase A action. 6. 6. Glycol analogues were demonstrated to exhibit substrate activity. 7. 7. Phospholipase A catalyses the hydrolysis of a symmetric β-lecithin into an optical-active lysolecithin. 8. 8. An isolated specimen of cardiolipin was hydrolysed, whereas sphingomyelin resisted phospholipase A action.