J.A.F. Op den Kamp

Action of pancreatic phospholipase A2 on phosphatidylcholine bilayers in different physical states

1. Saturated and unsaturated phosphatidylcholines, dispersed as liposomes in water, can be hydrolysed by phospholipase A2 from pig pancreas. A pure saturated phosphatidylcholine is hydrolysed only near its transition temperature. An unsaturated phosphatidylcholine is hydrolysed preferentially near its transition temperature, but hydrolysis can occur also above the transition temperature, albeit at a much lower rate. 2. An equimolar mixture of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, which shows cocrystallization of the paraffin chains, is hydrolyzed between 25 and 40 degrees C with a maximum at 32 degrees C, in agreement with the calorimetric scan of the phase transition. 3. An equimolar mixture of dilauroyl phosphatidylcholine and distearoyl phosphatidylcholine, which shows a monotectic behaviour, is hydrolysed at all temperatures. Hydrolysis is maximal at 0 and 40 degrees C, at which temperatures dilauroyl phosphatidylcholine and distearoyl phosphatidylcholine undergo their phase transition, respectively. 4. Both in the mixture showing cocrystallization and in the mixture in which phase separation occurs, the phosphatidylcholine species with the shorter fatty acid chains is hydrolysed at a higher rate than the longer chain fatty acid species. 5. Hydrolysis is inhibited by the presence of cholesterol in liposomes prepared of saturated phosphatidylcholine. Inhibition is complete at a cholesterol concentration of 35 mol %. Subsequent addition of filipin and amphotericin B to the mixed cholesterol-phosphatidylcholine liposomes overcomes the inhibitory effect of cholesterol.

Hydrolysis of phosphatidylcholine liposomes by pancreatic phospholipase A2 at the transition temperature.

Abstract Dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine dispersed as liposomes in water are hydrolysed by phospholipase A 2 from pig pancreas only near the transition temperature of these lipids. Both above and below the transition temperature of the lipids the rate of hydrolysis in these model membranes is negligible. By contrast, hydrolysis of these liposomes with bee venom phospholipase A 2 occurs below, at the above the transition temperature. The concept of lateral compressibility of lipids at the transition temperature is firmly substantiated by these observations.

RATIO-FLUORESCENCE MICROSCOPY OF LIPID OXIDATION IN LIVING CELLS USING C11-BODIPY581/591

A ratio‐fluorescence assay was developed for on‐line localization and quantification of lipid oxidation in living cells. The assay explores the oxidative sensitivity of C11‐BODIPY581/591. Upon oxidation, the fluorescence of this fluorophore shifts from red to green. The probe incorporates readily into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid. Using confocal microscopy, the cumene hydroperoxide‐induced oxidation of C11‐BODIPY581/591 was visualized at the sub‐cellular level in rat‐1 fibroblasts. Pre‐loading of the cells with tocopherol retarded this oxidation. The data demonstrate that C11‐BODIPY581/591 is a valuable tool to quantify lipid oxidation and anti‐oxidant efficacy in single cells.

Parinaric acid as a sensitive fluorescent probe for the determination of lipid peroxidation

The decrease in fluorescence of conjugated polyenic acyl chains is used as a sensitive assay for lipid peroxidation. The fatty acid cis-trans-trans-cis-9,11,13, 15-octadecatetraenoic acid (cis-parinaric acid) is introduced into liposomal membranes as free fatty acid or, by using the PC specific transfer protein from bovine liver, as 1-palmitoyl-2-cis-parinaroyl-sn-glycero-3-phosphocholine. The peroxidation process as monitored by the decrease in fluorescence intensity is compared with other peroxidation assay systems. Applications of the new assay system are discussed.

Changes in permeability of Staphylococcus aureus and derived liposomes with varying lipid composition

The physical and barrier properties of the phospholipids phosphatidylglycerol and lysylphosphatidylglycerol were studied in membrane model systems. Packing in monolayers at the air-water interface showed a larger area per molecule for lysylphosphatidylglycerol than for phosphatidylglycerol. The non-electrolyte permeability of liposomes prepared with lysylphosphatidylglycerol was higher than of those prepared with phosphatidylglycerol. On the other hand, the permeability of 86Rb+ was higher for liposomes of phosphatidylglycerol than for those of lysylphosphatidylglycerol. Valinomycin was able to increase the permeability of this cation in the phosphatidylglycerol liposome only. Studies on the effect of the environmental pH on the lysylphosphatidylglycerol to phosphatidylglycerol ratio in intact cells of Staphylococcus aureus showed that the total amount of lysylphosphatidylglycerol, cardiolipin and neutral lipids in the membrane did not change when the pH of the medium was varied between pH 6.5 and 5.0; but the total amount of phosphatidylglycerol decreased when the pH of the medium was lowered. The permeability of the intact cells for erythritol appeared to increase with increasing lysylphosphatidylglycerol to phosphatidylglycerol ratio; whereas the valinomycin mediated exchange of 86Rb+ over the cell membrane appeared to decrease when this ratio was increased. From the correlation between the permeability properties of cells and liposomes the conclusion is drawn that in S. aureus the chemical nature of the phospholipids determines to a great extent the properties of the permeability barrier.

Studies on sickled erythrocytes provide evidence that the asymmetric distribution of phosphatidylserine in the red cell membrane is maintained by both ATP-dependent translocation and interaction with membrane skeletal proteins

In order to study factors which are involved in maintenance of phosphatidylserine (PS) asymmetry within the human red cell membrane, we measured the effect of ATP-depletion and of membrane skeleton/lipid bilayer uncoupling induced by sickling on the distribution of PS within the membrane bilayer of sickle cells. Trace amounts of radiolabeled PS were introduced into the outer membrane leaflet of both fresh and ATP-depleted reversibly sickled cells (RSCs), using a non-specific lipid transfer protein purified from bovine liver. The equilibration of the newly introduced PS over the two halves of the bilayer was monitored by treatment of the cells with phospholipase A2 which selectively hydrolyzes only those molecules present in the outer membrane leaflet. Within 1 h after insertion into fresh RSCs, only 10% of the labeled PS was accessible to the action of phospholipase A2. This fraction was markedly increased when the cells were subsequently deoxygenated. Prolonged deoxygenation of RSCs, deprived of their ATP after incorporation of radiolabeled PS, caused enhanced phospholipase A2-induced hydrolysis of radiolabeled PS. Similarly, phospholipase A2-induced hydrolysis of endogenous PS in intact RSCs was markedly enhanced when ATP-depleted, but not when fresh cells, were incubated under nitrogen for 3.5 h. Deoxygenated ATP-depleted RSCs markedly enhanced the rate of thrombin formation in the presence of purified coagulation factors Xa, Va, prothrombin and Ca2+. This enhancement appeared to be dependent on the duration of incubation under nitrogen. This phenomenon, indicating the presence of increasing amounts of endogenous PS in the outer membrane leaflet, was not observed when either fresh RSCs or ATP-depleted normal erythrocytes were incubated under nitrogen. Our present observations provide evidence that, in addition to the interaction of PS with the skeletal proteins, an ATP-dependent translocation of PS is required to maintain its absolute asymmetric distribution in the human erythrocyte membrane.

The use of cis-parinaric acid to determine lipid peroxidation in human erythrocyte membranes. Comparison of normal and sickle erythrocyte membranes

The recently developed parinaric acid assay is shown to offer possibilities for studying peroxidation processes in biological membrane systems. Taking the human erythrocyte membrane as a model, several initiating systems were investigated, as well as the effect of residual hemoglobin in ghost membrane preparations. The effectivity of a radical generating system appeared to be strongly dependent upon whether radicals are generated at the membrane level or in the water phase. Thus, cumene hydroperoxide at concentrations of 1.0-1.5 mM was found to be a very efficient initiator of peroxidation in combination with submicromolar levels of hemin-Fe3+ as membrane-bound cofactor. In combination with cumene hydroperoxide, membrane-bound hemoglobin appeared to be about 6-times more effective in promoting peroxidation than hemoglobin in the water phase. Results comparing the behaviour of normal and sickle erythrocyte ghost suspensions in the peroxidation assay suggest that the increased oxidative stress on sickle erythrocyte membranes could be due to enhanced membrane binding of sickle hemoglobin, but also partly to a characteristically higher capability of sickle hemoglobin to promote peroxidation. The order of peroxidation-promoting capabilities that could be derived from the experiments was hemin greater than sickle hemoglobin greater than normal hemoglobin.

The membrane of intact human erythrocytes tolerates only limited changes in the fatty acid composition of its phosphatidylcholine

Using the phosphatidylcholine specific transfer protein from bovine liver, native phosphatidylcholine from intact human erythrocytes was replaced by a variety of different phosphatidylcholine species without altering the original phospholipid and cholesterol content. The replacement of native phosphatidylcholine by the disaturated species, 1,2-dipalmitoyl- and 1,2-distearoylphosphatidylcholine, proceeded at a low rate and extensive replacement could only be achieved by repeatedly adding fresh donor vesicles. The replacement by disaturated molecules was accompanied by a gradual increase in osmotic fragility of the cells, finally resulting in hemolysis when 40% of the native PC had been replaced. Up to this lytic concentration, the replacement did not affect the permeability of the membrane for potassium ions. Essentially, all of the PC in the outer monolayer of the membrane could be replaced by 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine. These replacements did not alter the osmotic fragility of the cells, nor the K+ permeability of the membrane. Increasing the total degree of unsaturation of the phosphatidylcholine species modified the properties of the membrane considerably. Replacement by 1,2-dilinoleoylphosphatidylcholine resulted in a progressive increase in osmotic fragility and hemolysis started to occur after 30% of the native PC had been replaced by this species. K+ permeability was found to be slightly increased in this case. Cells became leaky for K+ upon the introduction of 1-palmitoyl-2-arachidonoylphosphatidylcholine in the membrane. The increased permeability was also reflected by an apparent increase in the resistance of the cells against osmotic shock. The conclusions to be drawn are that (i) 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine are species which fit most optimally into the erythrocyte membrane; (ii) loss of membrane stability results from an increase in the degree of saturation of phosphatidylcholine (unsaturation index greater than 0.5) and (iii) the permeability is enhanced by increasing the content of highly unsaturated species (unsaturation index greater than 1.0).

The sphingomyelin pools in the outer and inner layer of the human erythrocyte membrane are composed of different molecular species

Analyses of the fatty acid composition of the outer and inner pools of sphingomyelin in the human erythrocyte membrane revealed significant differences in molecular species composition of these two pools. The sphingomyelin in the inner monolayer, representing 15-20% of the total sphingomyelin content of this membrane, is characterized by a relatively high content (73%) of fatty acids, which have less than 20 carbon atoms, whereas these account for only 31% of the total fatty acids in the sphingomyelin in the outer leaflet. On the other hand, the ratio saturated/unsaturated fatty acids in the two pools is similar. Significant differences are also observed for the fatty acid composition of the sphingomyelin in human serum when compared to that in the outer monolayer of the corresponding red cell. These results are interpreted to indicate an (almost) complete absence of transbilayer movements of sphingomyelin molecules in the human erythrocyte membrane, whereas an exchange of this phospholipid between the red cell membrane and serum is either virtually absent, or affects only a minor fraction of the sphingomyelin in the outer membrane layer.

Studies on the phospholipids and morphology of protoplasts of Bacillus megaterium

Abstract 1. 1. The phospholipids of the membrane fraction of cells of Bacillus megaterium (MK 10D) cultured at pH 7.0 were found to consist of cardiolipin (5%), phosphatidyl ethanolamine (40%), phosphatidyl glycerol (40%) and O -lysyl phosphatidyl glycerol (15%). The content of phosphatidyl glycerol was decreased to 8% in cells harvested at pH 5.0, whereas glucosaminyl phosphatidyl glycerol represented 32% of the total phospholipids. The content of other phospholipids remained constant. 2. 2. Protoplasts derived from cells harvested from different media displayed a different behaviour during lysis experiments in hypotonic sucrose. 3. 3. Electron microscopy demonstrated that cells grown at pH 7.0 gave spherical protoplasts, whereas from cells exposed to pH 5.0, rod-shaped protoplasts were produced by lysozyme. In the latter protoplasts the original structure of the bacteria was maintained to a great extent even after exposure to hypotonic conditions. Similar protoplasts, when derived from cultures in which overnight the pH dropped from 7.0 to 5.0, also tend to preserve more of the original structural organisation. 4. 4. Environmental conditions may induce differences in chemical make-up or physical properties of lipoprotein structures resulting in significant variation in the morphology of bacterial protoplasts.