P. Comfurius

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.

The enzymatic synthesis of phosphatidylserine and purification by CM-cellulose column chromatography

Phosphatidylserine has been prepared from phosphatidylcholine by a one-step transphosphatidylation catalyzed by phospholipase D in the presence of L-serine. The resulting mixture of phosphatidylserine and phosphatidic acid is easily and rapidly separated by CM-cellulose column chromatography using step=wise elution with solvents containing increasing percentages of methanol in chloroform. The over-all yield of the procedure is 40-50% depending on the scale of the preparation. CM-Cellulose column chromatography proved to be extremely useful in separating phospholipid mixtures obtained by phosphatidyltransferase reactions of phospholipase D and is also suitable for fractionation of other lipid extracts.

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.

Uncoupling of the membrane skeleton from the lipid bilayer. The cause of accelerated phospholipid flip-flop leading to an enhanced procoagulant activity of sickled cells.

We have previously reported that the normal membrane phospholipid organization is altered in sickled erythrocytes. More recently, we presented evidence of enhanced transbilayer movement of phosphatidylcholine (PC) in deoxygenated reversibly sickled cells (RSC) and put forward the hypothesis that these abnormalities in phospholipid organization are confined to the characteristic protrusions of these cells. To test this hypothesis, we studied the free spicules released from RSC by repeated sickling and unsickling as well as the remnant despiculated cells. The rate of transbilayer movement of PC in the membrane of deoxygenated remnant despiculated cells was determined by following the fate of 14C-labelled PC, previously introduced into the outer monolayer under fully oxygenated conditions using a PC-specific phospholipid exchange protein from beef liver. The rate of transbilayer movement of PC in the remnant despiculated cells was significantly slower than in deoxygenated native RSC and was not very much different from that in oxygenated native RSC or irreversibly sickled cells. The free spicules had the same lipid composition as the native cells, but were deficient in spectrin. These spicules markedly enhanced the rate of thrombin formation in the presence of purified prothrombinase (Factor Xa, Factor Va, and Ca2+) and prothrombin, indicating the exposure of a significant fraction of phosphatidylserine (PS) in the outer monolayer. This effect was not observed when the spicules in this assay were replaced by normal erythrocytes, deoxygenated native RSC, or a deoxygenated sample of RSC after repetitive sickling/unsickling. The results are interpreted to indicate that the destabilization of the lipid bilayer in sickled cells, expressed by the enhanced flip-flop of PC and the exposure of PS in the outer monolayer, occurs predominantly in those parts of the membrane that are in spicular form.

Complete purification and some properties of phospholipase C from Bacillus cereus.

Abstract 1. 1. Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) has been purified 450-fold over the growth medium of Bacillus cereus , with overall recovery of 23%. 2. 2. Purification was performed using both (NH 4 ) 2 SO 4 and ethanol precipitation, protamine sulfate fractionation, Sephadex G-100 gel filtration followed by ion exchange chromatography on DEAE- and CM-sephadex. 3. 3. The preparation appeared to be pure on disc electrophoresis at pH 2.3 in 6 M urea, whereas a molecular weight between 21 000 and 25 000 could be derived from gel filtration on Sephadex G-100. 4. 4. No hemolytic activity towards intact human erythrocytes could be established. Treatment of human red cell ghosts with pure phospholipase C resulted in a nearly complete degradation of the main phospholipid classes (except for sphingomyelin) up to 70% of total phosphorus. Similar results were obtained with liposomes derived from human erythrocyte total lipids.

Action of pure phospholipase A2 and phospholipase C on human erythrocytes and ghosts

Abstract 1. 1.|Pancreatic phospholipase A2 (phosphatide acyl-hydrolase, EC 3.1.1.4) and phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus appeared not to be lytic for human erythrocytes, either before or after treatment of the cells with trypsin, pronase or neuraminidase. No significant breakdown of phospholipids could be observed. 2. 2.|Both phospholipases were found to evoke hemolysis in the presence of sublytic concentrations of sodium deoxycholate, whereas sublytic concentrations of Triton X-100 were effective only in combination with phospholipase C. 3. 3.|Treatment of human red cell ghosts with either phospholipase A2 or phospholipase C resulted in a complete hydrolysis of lecithin, phosphatidylethanolamine and phosphatidylserine, whereas sphingomyelin was not attacked. Similar results were obtained with liposomes derived from human erythrocytes, indicating that the degree of hydrolysis depends only upon the chemical nature of the phospholipids involved. 4. 4.|In the native human erythrocyte membrane the fatty acid-ester linkage at C2 and the phosphoryl-glycerol linkage at C3 of the phosphoglyceride molecules apparently are not accessible to phospholipase A and C attack. Removal of the sialic acid residues from the membrane surface does not promote the action of these lipolytic enzymes. Changes in membrane architecture occurring during membrane isolation or as induced by nonlytic concentrations of detergents lead to exposure of membrane phosphoglycerides to phospholipases A and C.

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.

Involvement of ATP-dependent aminophospholipid translocation in maintaining phospholipid asymmetry in diamide-treated human erythrocytes

Crosslinking of membrane skeletal proteins such as spectrin by oxidation of their SH-groups can be provoked by treatment of intact erythrocytes with diamide. Shortly after exposure of human erythrocytes to diamide and despite the transverse destabilization of the lipid bilayer that was observed in these cells (Franck, P.F.H., Op den Kamp, J.A.F., Roelofsen, B. and Van Deenen, L.L.M. (1986) Biochim. Biophys. Acta 857, 127-130), no abnormalities could be detected regarding the asymmetric distribution of the phospholipids when probed by either the prothrombinase assay or brief exposure of the cells to a modified phospholipase A2 with enhanced membrane penetrating capacity. This asymmetry appeared to undergo dramatic changes however, when the ATP content of the cytosol had decreased to less than 10% of its original level during prolonged incubation of the treated cells. These observations indicate that the initial maintenance of phospholipid asymmetry in diamide-treated erythrocytes can be solely ascribed to the action of the ATP-dependent aminophospholipid translocase. This view is supported by experiments involving radiolabeled phospholipids of which trace amounts had been inserted into the outer membrane leaflet of diamide-treated red cells and which still showed a preferential translocation of both aminophospholipids in favour of the inner monolayer, be it that the efficiency of the translocase was found to be impaired when compared to control cells.

ON MEMBRANE LIPIDS AND THEIR INTERACTION WITH PROTEINS

A non-random distribution of phospholipid classes between the inner and outer monolayers has been detected in a number of biological membranes (for a recent review see Ref. 1). In particular the red blood cell has been extensively studied by various investigators using either chemical labelling or degradation with phospholipases. These studies have demonstrated that the choline containing phospholipids are located predominantly at the outside and the aminophosphoglycerides, in particular phosphatidyl serine, are concentrated at the cytoplasmic side of the red blood cell membrane. In the present contribution we discuss the asymmetric distribution of phospholipids in platelet membranes as well as an experimental attempt to link phospholipid-asymmetry with the transmembrane orientation of proteins.