In vivo turnover of phospholipids in rabbit erythrocytes.

The rate of phospholipid turnover in erythrocyte membranes in vivo has been studied using a recently developed procedure (Kuypers, F.A., Easton, E.W., van den Hoven, R., Wensing, T., Roelofsen, B., Op den Kamp, J.A.F. and van Deenen, L.L.M. (1985) Biochim. Biophys. Acta 819, 170-178). The technique is based on the application of phospholipid transfer proteins in order to introduce trace amounts of radiolabelled phospholipids in the membrane of isolated erythrocytes, followed by re-injection of the erythrocytes into the bloodstream of the animal. The most abundant species of the phosphatidylcholine (PC) class, 1-palmitoyl,2-linoleoyl PC, has, on the basis of loss of the radioactivity in its fatty acyl part, a relatively high turnover with a half-time value of 1.5 days. Other PC species studied exhibit more moderate turnover rates of about 5 days for 1-palmitoyl,2-oleoyl PC and 1-stearoyl,2-arachidonoyl PC. Dipalmitoyl PC, labelled in the polar headgroup, turns over at a slow rate with a half-time value of 9 days. From these data and the relative abundance of the various species, it can be calculated that, on a daily basis in vivo, about one third of the total PC pool in rabbit erythrocyte membranes is replaced and/or modified by de-/reacylation. The only phosphatidylethanolamine (PE) species studied so far, 1-palmitoyl,2-arachidonoyl PE, appeared to be renewed at a relatively low rate with a half-time value of 12 days. The data demonstrate that the in vivo turnover values of phospholipids in the erythrocyte membrane may depend on their polar head group structure, their localization in the membrane and, to a large extent, on their fatty acid composition.

Growth inhibition of Plasmodium falciparum in in vitro cultures by selective action of tryptophan-N-formylated gramicidin incorporated in lipid vesicles.

We studied the differential effect of tryptophan-N-formylated gramicidin on uninfected and Plasmodium falciparum-infected erythrocytes. Trp-N-formylated gramicidin induces a much faster leakage of K+ from infected cells than from uninfected cell whereas, and at an even lower concentration, gramicidin A' causes a rapid K+ leakage from both uninfected and infected cells. We also studied the effect of Trp-N-formylated gramicidin and gramicidin A' incorporated in liposomes on the growth of Plasmodium falciparum in an in vitro culture. Incorporation of Trp-N-formylated gramicidin in the membranes of so-called 'stealth' vesicles strongly decreases the concentration needed to induce 50% inhibition of parasite growth. Moreover, no decrease in the K+ content of uninfected cells was observed when cells were exposed to liposome-incorporated Trp-N-formylated gramicidin at a concentration which causes full inhibition of parasite growth. These observations strongly suggest that Trp-N-formylated gramicidin incorporated in 'stealth' vesicles ends up specifically in the infected cell, thereby inhibiting the growth of the growth of the malaria parasite.

Selective elimination of malaria infected erythrocytes by a modified phospholipase A2 in vitro.

Pig pancreatic phospholipase A2 does not act on normal erythrocytes, but the membrane penetrating capacity is enhanced by the covalent attachment of one fatty acyl chain to Lys-116 of the enzyme. Taking advantage of the impaired packing of phospholipids in the membrane of Plasmodium infected erythrocytes it was demonstrated that a lauric acid derivative of phospholipase A2 is capable of exclusively attaching the infected erythrocytes in vitro, leaving the uninfected cells undisturbed. The chemically modified phospholipase A2 appeared to cause death of the parasite in cell cultures of infected erythrocytes.

Phospholipid asymmetry in the plasma membrane of malaria infected erythrocytes.

The transbilayer distribution of glycerophospholipids in the plasma membrane of Plasmodium knowlesi infected erythrocytes was studied by using lysine-116-epsilon-N-palmitoyl amidinated pancreatic phospholipase A2. As a consequence of its superior membrane penetrating capacities, this modified enzyme rapidly degrades its substrates in the outer membrane leaflet of intact erythrocytes, a property that makes the enzyme an excellent tool to study the malaria parasitized red cell. The modified phospholipase A2 caused a nonlytic hydrolysis of up to 12-15% of the phosphatidylethanolamine and none of the phosphatidylserine in the red cell membrane, irrespective of whether the cells harboured trophozoite and schizont stages of parasites or no parasites at all. The absence of phosphatidylserine at the exterior surface of Plasmodium infected erythrocytes was confirmed by applying the prothrombinase assay on Plasmodium falciparum infected human erythrocytes. Consequently, the results from these and previous studies indicate that the plasma membrane of Plasmodium infected erythrocytes exhibit a normal transbilayer phospholipid asymmetry.

Aminophospholipid translocase in the plasma membrane of Friend erythroleukemic cells can induce an asymmetric topology for phosphatidylserine but not for phosphatidylethanolamine.

The ATP-dependent translocation of phospholipids in the plasma membrane of intact Friend erythroleukemic cells (FELCs) was studied in comparison with that in the membrane of mature murine erythrocytes. This was done by following the fate of radiolabeled phospholipid molecules, previously inserted into the outer monolayer of the plasma membranes by using a non-specific lipid transfer protein. The transbilayer equilibration of these probe molecules was monitored by treating the cells--under essentially non-lytic conditions--with phospholipases A2 of different origin. Rapid reorientations of the newly introduced aminophospholipids in favour of the inner membrane leaflet were observed in fresh mouse erythrocytes; the inward translocation of phosphatidylcholine (PC) in this membrane proceeded relatively slow. In FELCs, on the other hand, all three glycerophospholipids equilibrated over both halves of the plasma membrane very rapidly, i.e. within 1 h; nevertheless, an asymmetric distribution in favour of the inner monolayer was only observed for phosphatidylserine (PS). Lowering the ATP-level in the FELCs caused a reduction in the rate of inward translocation of both aminophospholipids, but not of that of PC, indicating that this translocation of PS and phosphatidylethanolamine (PE) is clearly ATP-dependent. Hence, the situation in the plasma membrane of the FELC is rather unique in a sense that, though an ATP-dependent translocase is present and active both for PS and PE, its activity results in an asymmetric distribution of PS, but not of PE. This remarkable situation might be the consequence of the fact that, in contrast to the mature red cell, this precursor cell still lacks a complete membrane skeletal network.

Effect of selective chemical modification and CNBr-cleavage at methionine20 in catalytic activity and substrate binding properties of porcine pancreatic phospholipase A2.

Porcine pancreatic phospholipase A2 contains 2 methionine (Met) residues located at positions 8 and 20, respectively. Reaction of the enzyme with methyliodide and iodoacetic acid resulted in the selective methylation and carboxymethylation, respectively, of Met20. It was found that porcine pancreatic iso-phospholipase A2, possessing only Met8, was not affected by either modification. Reaction of porcine phospholipase A2 with cyanogen bromide in 0.1 N hydrochloric acid gave rise to cleavage only at Met20. The enhanced reactivity of Met20 compared to that of Met8 is in agreement with the known X-ray structure of phospholipase A2 which shows that Met8 is located in the interior of the protein, while Met20 is at the surface. Both methylation and carboxymethylation of Met20 do not significantly affect catalytic and substrate binding properties of the enzyme. In contrast, the more rigorous cleavage at Met20 by CNBr resulted in the loss of catalytic activity, while substrate and Ca2+ binding was diminished only to a limited extent. Most likely CNBr cleavage at Met20 perturbs the active site despite the fact that the N-terminal fragment Ala1-Hse20 is still bound via the disulfide bridge Cys11-Cys77 to the remainder of the protein. The results obtained strongly suggest that the conformation of the sequences Ala1-Hse20 and/or Asp21-Gly26 are important for the maintenance of the special microenvironment of the active site cleft.

Phospholipid uptake by Plasmodium knowlesi infected erythrocytes.

The uptake of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in Plasmodium knowlesi infected erythrocytes has been studied. Whereas uptake of phospholipids, in the absence of phospholipid transfer proteins, is negligible in control cells, the infected cells can incorporate considerable amounts of added phospholipids. The uptake is enhanced by the presence of lipid transfer proteins. Doubly labeled [3H]oleate, [14C]choline) PC does not undergo any appreciable remodelling following uptake, which strongly suggests that plasma PC is used as such for the biogenesis of the parasite membranes. Transport of extracellularly offered PS and PE towards the intraerythrocytic parasite and utilization of these lipids by the parasite are confirmed by the observation that these lipids are converted into respectively PE and PC. The extent and rate of these conversions depend on the way the phospholipids are introduced into the infected cells.

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.

Phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection.

The phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection has been studied. Parasitized and nonparasitized erythrocytes from malaria-infected blood were separated and pure erythrocyte membranes from parasitized cells were isolated using Affi-Gel beads. In this way, the phospholipid content and composition of the membrane of nonparasitized cells, the erythrocyte membrane of parasitized cells and the parasite could be determined. The phospholipid content and composition of the erythrocyte membranes of nonparasitized and parasitized cells and erythrocytes from chloroquine-treated monkeys cured from malaria, were the same as in normal erythrocytes. The phospholipid content of the parasite increased during its development, but its composition remained unchanged. Three independent techniques, i.e., treatment of intact cells with phospholipase A2 and sphingomyelinase C, fluorescamine labeling of aminophospholipids and a phosphatidylcholine-transfer protein-mediated exchange procedure have been applied to assess the disposition of phospholipids in: erythrocytes from healthy monkeys, nonparasitized and parasitized erythrocytes from monkeys infected with Plasmodium knowlesi, and erythrocytes from monkeys that had been cured from malaria by chloroquine treatment. The results obtained by these experiments do not show any abnormality in phospholipid asymmetry in the erythrocyte from malaria-infected (splenectomized) monkeys, neither in the nonparasitized cells, nor in the parasitized cells at any stage of parasite development. Nevertheless, a considerable degree of lipid bilayer destabilization in the membrane of the parasitized cells is apparent from the enhanced exchangeability of the PC from those cells, as well as from their increased permeability towards fluorescamine.

Phospholipid asymmetry during erythropoiesis. A study on Friend erythroleukemic cells and mouse reticulocytes.

The distribution of phospholipids over the outer and inner layers of the plasma membranes of differentiated Friend erythroleukemic cells (Friend cells) and mouse reticulocytes has been determined. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol were found to be distributed symmetrically over both layers, sphingomyelin was found to be enriched in the outer layer (80-85%) and phosphatidylserine appeared to be present mainly in the inner layer (80-90%) of the plasma membranes of differentiated Friend cells. The outer layer of reticulocyte membranes contains 50-60% of the phosphatidylcholine, 20% of the phosphatidylethanolamine, 82-85% of the sphingomyelin and 40-42% of the phosphatidylinositol. All of the phosphatidylserine is present in the inner layer. The results show, that the asymmetric distribution of phospholipids, typical for erythrocyte membranes, is partially apparent already at an early stage of erythropoiesis, the proerythroblast, while the final organization of phospholipid distribution takes place at some stage during enucleation of the enormoblast and release of the reticulocyte into the blood stream.

The transbilayer distribution of phosphatidylethanolamine in erythroid plasma membranes during erythropoiesis.

Fluorescamine was used to assess the transbilayer distribution of phosphatidylethanolamine in the plasma membrane of murine erythroid progenitor cells, CFU-E (colony-forming unit erythroid), at different stages of their differentiation pathway. Intact cells were exposed to increasing concentrations of fluorescamine and the amount of labeled phosphatidylethanolamine was determined by measuring the fluorescence intensity of its fluorescamine derivative. A semilogarithmic plot of the dose-response curve revealed three different pools of phosphatidylethanolamine, representing its fractions in, respectively, the inner- and outer monolayers of the plasma membrane and subcellular membrane systems. These results show that 9-11% of the total cellular phosphatidylethanolamine is present in the outer leaflet and 9-10% of it is located in the inner leaflet of the plasma membrane in early as well as late erythroblasts. This symmetric distribution of phosphatidylethanolamine over the two halves of the bilayer in the plasma membrane of CFU-E is very similar to that observed earlier in the plasma membrane of friend erythroleukaemic cells (Rawyler, Van der Schaft, Roelofsen and Op den Kamp (1985) Biochemistry 24, 1777-1783). These observations imply that the characteristic asymmetric distribution of phosphatidylethanolamine, as is found in mature erythrocytes, is accomplished at a very late stage of erythropoiesis and possibly during enucleation of the cells or shortly thereafter.

Does diamide treatment of intact human erythrocytes cause a loss of phospholipid asymmetry?

Diamide-treated human erythrocytes have been compared with native red cells as to the accessibility of their amino phospholipids to both phospholipase A2 hydrolysis and fluorescamine labeling. In agreement with observations by others (Haest, C.W.M., Plasa, G., Kamp, D. and Deuticke, B. (1978) Biochim. Biophys. Acta 509, 21-32), treatment of intact human erythrocytes with diamide resulted in considerably enhanced degradation of amino phospholipids upon subsequent incubation of the cells with bee venom phospholipase A2. The hydrolysis of phosphatidylethanolamine (PE) in control cells reached a plateau value at 5% after 10 min. In diamide-treated cells, on the other hand, PE hydrolysis did not level off. Contrastingly, dose-response curves recorded for the labeling of PE with the very fast reacting NH2-group-specific reagent, fluorescamine, showed identical results for both native and diamide-treated erythrocytes. In each of these two cases, a plateau was reached after approx. 15% of the PE had been labeled. These results strongly suggest that the enhanced phospholipase-A2-induced hydrolysis of amino phospholipids in diamide-treated erythrocytes may reflect a destabilization of the lipid bilayer, rather than an in situ loss of phospholipid asymmetry.

The rate of uptake and efflux of phosphatidylcholine from human erythrocytes depends on the fatty acyl composition of the exchanging species.

The rate of uptake of radioactive phosphatidylcholine molecules of different fatty acid composition in intact erythrocytes as facilitated by a phosphatidylcholine-specific transfer protein has been studied. When trace amounts of radiolabeled phosphatidylcholine molecules are present in donor vesicles consisting of egg phosphatidylcholine and cholesterol, the transfer of the radiolabeled species depends strongly on their fatty acyl composition: dipalmitoylphosphatidylcholine is transferred at the lowest rate, 1-saturated-2-unsaturated species are transferred faster and the highest rate is observed for dioleoyl phosphatidylcholine. Transfer of the various phosphatidylcholine molecules was measured furthermore using donor systems in which the bulk phosphatidylcholine was varied in its fatty acyl composition. Also in this type of experiment, the transfer protein preferentially stimulated transfer of unsaturated phosphatidylcholine molecules, especially from an environment containing more saturated molecules. Finally, the efflux of labeled phosphatidylcholine from intact erythrocytes to plasma in the absence of the phosphatidylcholine-specific transfer protein was studied and it became clear that in this case the nature of the effused molecules itself, rather than the composition of the bulk lipids, determined the effuse rates. An important conclusion to be drawn from these experiments is that radiolabeled phosphatidylcholine molecules, when used as markers for phospholipid exchange or transfer, should resemble in their fatty acid composition the composition of the bulk lipid in order to provide reliable data on rates and extents of the process studied.

ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane.

Trace amounts of radiolabeled phospholipids were inserted into the outer membrane leaflet of intact human erythrocytes, using a non-specific lipid transfer protein. Phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine were transferred from the donor lipid vesicles to the membrane of the intact red cell with equal ease, whilst sphingomyelin was transferred 6-times less efficiently. The transbilayer mobility and equilibrium distribution of the labeled phospholipids were assessed by treatment of the intact cells with phospholipases. In fresh erythrocytes, the labeled amino phospholipids appeared to move rapidly towards the inner leaflet. The choline phospholipids, on the other hand, approached an equilibrium distribution which strongly favoured the outer leaflet. In ATP-depleted erythrocytes, the relocation of the amino phospholipids was markedly retarded.

Survival of rabbit and horse erythrocytes in vivo after changing the fatty acyl composition of their phosphatidylcholine.

The phospholipid composition and the distribution of phospholipids over the two leaflets of the membrane have been investigated for rabbit and horse erythrocyte membranes. Phosphatidylcholine (PC) comprises 39.4% and 41.3% of the total phospholipid complement of the rabbit and horse erythrocytes, respectively. In both membranes the distribution of this phospholipid is asymmetric: 70% of the PC is present in the outer layer of the rabbit membrane and 60% in that of the horse. The major species of this phospholipid class are the (1-palmitoyl-2-oleoyl)- and the (1-palmitoyl-2-linoleoyl)PC. The disaturated species, (1,2-dipalmitoyl)PC, is present in limited amounts only. Partial replacement of the native PC from intact erythrocytes was accomplished with a purified PC specific transfer protein from bovine liver. Replacement of the native PC species with (1-palmitoyl-2-oleoyl)PC up to 40% of the total PC complement had no effect on the osmotic fragility, the shape and the in vivo survival time of both erythrocyte species. Replacement of the native PC in both rabbit and horse erythrocytes with (1,2-dipalmitoyl)PC up to 20% gave rise to an increased osmotic fragility, a shape change from discocytic to echinocytic and a significant reduction in survival time measured after reinjection of the modified cells. At 30% replacement with (1,2-dipalmitoyl)PC the resulting spheroechinocytes appeared to be cleared from the circulation within 24 h after reinjection. The conclusion can be drawn that the repair mechanisms which may exist in vivo are insufficient to cope with the drastic changes in properties of the erythrocyte membrane which are induced by replacing more than 15% of the native PC by the dipalmitoyl species.

Lipid molecular shape affects erythrocyte morphology: a study involving replacement of native phosphatidylcholine with different species followed by treatment of cells with sphingomyelinase C or phospholipase A2.

In a previous report it was shown that the replacement of native erythrocyte phosphatidylcholine (PC) with different PC species which have defined acyl chain compositions can lead to morphological changes (Kuypers, F.A., W. Berendsen, B. Roelofsen, J. A. F. Op den Kamp, and L.L.M. van Deenen, 1984, J. Cell Biol., 99:2260-2267). It was proposed that differences in molecular shape between the introduced PC species and normal erythrocyte PC caused the membrane to bend outwards or inwards, depending on the shape of the PC exchanged. To support this proposal, two requirements would have to be fulfilled: the exchange reaction would take place only with the outer lipid monolayer of the erythrocyte, and the extent of lipid transbilayer movement would be restricted. If this theory is correct, any treatment causing unilateral changes in lipid molecular shape should lead to predictable morphological changes. Since this hypothesis is a refinement of the coupled bilayer hypothesis, but so far lacks experimental support, we have sought other means to change lipid molecular shape unilaterally. Shape changes of human erythrocytes were induced by the replacement of native PC by various PC species using a phosphatidylcholine-specific transfer protein: by hydrolysis of phospholipids in intact cells using sphingomyelinase C or phospholipase A2, and by the combination of both procedures. The morphological changes were predictable; additive when both treatments were applied, and explicable on the basis of the geometry of the lipid molecules involved. The results strongly support the notion that lipid molecular shape affects erythrocyte morphology.

Abnormal transbilayer mobility of phosphatidylcholine in hereditary pyropoikilocytosis reflects the increased heat sensitivity of the membrane skeleton.

We determined whether the membrane defect in hereditary pyropoikilocytosis (HPP) is associated with thermally induced changes in the lipid bilayer, the stability of which was probed by the rate of translocation of phosphatidylcholine (PC) over the two leaflets. [14C]PC was incorporated into the outer leaflet of the lipid bilayer of the intact erythrocytes using a PC-specific phospholipid exchange protein. The transbilayer equilibration of this PC was determined by measuring the time-dependent changes in its accessibility to exogenous phospholipase A2. The rate of transbilayer equilibration of PC was increased in HPP cells at 37 degrees C when compared to normal erythrocytes (rate constants, 0.07 +/- 0.02 and 0.03 +/- 0.01 h-1, respectively). A further dramatic increase in PC transbilayer equilibration was noted in HPP cells incubated at 44 degrees C (rate constant, 0.15 +/- 0.02 h-1). A similar marked acceleration in transbilayer movement of PC was also seen in normal erythrocytes when incubated at 46 degrees C (rate constant, 0.13 +/- 0.03 h-1). Despite the enhanced transbilayer mobility of PC in HPP cells when compared to normal erythrocytes, no major alteration in the asymmetric distribution could be observed when probed with phospholipase A2. Since changes in transbilayer mobility of PC and cell morphology occur in HPP cells at lower temperature than in normal red cells, it may be concluded that the enhanced thermal sensitivity of spectrin is the major factor responsible for these changes. Our results therefore support the view that the structural integrity of the skeletal network is essential for stabilization of the lipid bilayer of the red cell membrane.