R.F.A. Zwaal

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.

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.

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.

Action of highly purified phospholipases on blood platelets. Evidence for an asymmetric distribution of phospholipids in the surface membrane

1. 1. Phospholipase C (phosphatidylcholine choline phosphohydrolase, EC 3.1.4.3) from Bacillus cereus is unable to produce either lysis or phospholipid hydrolysis in intact pig platelets, whereas sphingomyelinase C (sphingomyelin choline phosphohydrolase) from Staphylococcus aureus can degrade more than 55% of platelet sphingomyelin without producing lysis. Degradation of glycerophospholipids by B. cereus phospholipase C can be achieved after or during sphingomyelin hydrolysis by sphingomyelinase C. Simultaneous breakdown of both sphingomyelin and glycerophospholipids is also brought about by the separate action of Clostridium welchii phospholipase C. In both cases, the production of ceramides and diacylglycerols in the platelet membranes is followed by aggregation and cell lysis. 2. 2. Phospholipase A2 (phosphatide acylhydrolase, EC 3.1.1.4) from Naja naja hardly degrades phospholipids in intact pig platelets suspended in albumin-containing medium. In the absence of albumin, phospholipase A2 induces extensive aggregation of platelets followed by lysis, both of which are inhibited by aspirin or o-phenanthroline. The small amount of glycerophospholipid hydrolysis observed under these conditions is increased by subsequent incubation in the presence of sphingomyelinase C without causing lysis. 3. 3. Isolation and analysis of plasma membranes from pig platelets, which were treated first with sphingomyelinase C under non-lytic conditions, allows to estimate that 63% of the pig platelet phospholipids are located in the plasma membrane. The distribution of phospholipids between the two halves of the plasma membrane has been deduced by using this value and by comparing the degradative action of N. naja phospholipase A2 plus S. aureus sphingomyelinase C on lysed and intact platelets. It is concluded that 46% of the plasma membrane phospholipids, comprising 91% of sphingomyelin, 40% of lecithin, 34% of phosphatidylethanolamine and less than 6% of phosphatidylserine, form the outer half of the plasma membrane. A similar phospholipid distribution is proposed for the human platelet plasma membrane. 4. 4. Investigating the action of seven purified phospholipases on intact platelets (both from human and pig) has revealed that membrane phospholipids are available to enzymatic breakdown only if the phospholipase can produce degradation in monolayers spread at an initial surface pressure of at least 34 dynes/cm. By comparing these results with previous data obtained on human red cells (Zwaal, R.F.A., Roelofsen, B., Comfurius, P. and van Deenen, L.L.M. (1975) Biochim. Biophys. Acta 406, 83–96), it is concluded that the lipid packing in the platelet surface membrane is comparable with a lateral surface pressure close to 34 dynes/cm.

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.

Lytic and non-lytic degradation of phospholipids in mammalian erythrocytes by pure phospholipases.

Abstract 1. 1. Sphingomyclinase (sphingomyelin cholinephosphohydrolase) from Staphylococcus aureus hydrolysed 75–80% of the sphingomyelin in human and pig erythrocytes and 50–60% of the sphingomyelin in ox and sheep erythrocytes, without producing haemolysis of the cells. 2. 2. Although phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus alone was not lytic, the combination of sphingomyelinase and phospholipase C produced haemolysis of human and pig erythrocytes and extensive degradation of all the phospholipid classes in the ghosts thus produced. In contrast, the combination of these two enzymes failed to produce lysis of ox and sheep erythrocytes; in these cells the only phospholipid degraded was sphingomyelin. 3. 3. Human erythrocytes treated with sphingomyelinase showed increased osmotic fragility relative to control cells. 4. 4. In the intact erythrocytes studied, phosphoglyceride molecules are not accessible to phospholipases A 2 and C. Removal of choline phosphate residues from sphingomyelin exposes the phosphoglycerides to phospholipases A 2 and C in human and pig erhythrocytes but not in ox and sheep red 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.