B. Roelofsen

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.

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.

Localization of red cell membrane constituents

Abstract 1. 1. The action of phospholipases, proteolytic enzymes and groupspecific labels towards erythrocytes and ghost has been reviewed with special regard to the localization lipids and proteins within the membrane structure . 2. 2. Direct evidence is presented that the outer part of the erythrocyte membrane consists predominantly of choline-containing phospholipids (lecithin and sphingomyelin), almost to the exclusion of other phospholipids. Apart from acetylcholinesterase it is likely that only a few (glyco)proteins are (even partially) oriented at the exterior of the membrane. 3. 3. (Na + + K + )-ATPase in the human erythrocyte ghost can be inactive by phospholipase treatment and reactivated by phosphatidylserine. Indirect evidence suggests that both the (Na + + K + )-ATpase activity and the phosphatidylserine are localized only at the interior of the membrane. 4. 4. A new concept for the haemolytic behaviour of mixtures of phospholipases is proposed, based upon a highly asymmetric distribution of the phospholipids in a bimolecular leaflet, and taking into account some aspects of the action of phospholipases towards red cell ghosts.

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 human MDR3 P-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice.

The mouse mdr2 P‐glycoprotein (P‐gp) and its human MDR3 homologue are present in high concentrations in the canalicular membrane of hepatocytes. Mice lacking this protein are unable to secrete phosphatidylcholine (PC) into bile, suggesting that this P‐gp is a PC translocator. We have tested this in fibroblasts from transgenic mice expressing the MDR3 gene under a vimentin promoter. Transgenic and control fibroblasts were incubated with [14C]choline to label PC. When the labeled cells were incubated with a PC transfer protein and acceptor liposomes, transfer of radioactive PC was enhanced in transgenic cells relative to the wild type controls. We conclude that the MDR3 P‐glycoprotein is able to promote the transfer of PC from the inner to the outer leaflet of the plasma membrane, supporting the idea that this protein functions as a PC flippase.

Abnormalities in membrane phospholipid organization in sickled erythrocytes.

In contrast to the wealth of information concerning membrane phospholipid asymmetry in normal human erythrocytes, very little is known about membrane phospholipid organization in pathologic erythrocytes. Since the spectrin-actin lattice, which has been suggested to play an important role in stabilizing membrane phospholipid asymmetry, is abnormal in sickled erythrocytes, we determined the effects of sickling on membrane phospholipid organization. We used two enzymatic probes: been venom phospholipase A2 and Staphylococcus aureus sphingomyelinase C, which do not penetrate the membrane and react only with phospholipids located in the outer leaflet of the bilayer. Our results suggest that the distribution of glycerophospholipids within the membrane of sickled cells is different from that in nonsickled cells. Compared with the normal erythrocyte, the outer membrane leaflet of the deoxygenated, reversibly sickled cells (RSC) and irreversibly sickled cells (ISC) was enriched in phosphatidyl ethanolamine in addition to containing phosphatidyl serine. These changes were compensated for by a decrease in phosphatidyl choline in that layer. The distribution of sphingomyelin over the two halves of the bilayer was unaffected by sickling. In contrast to ICS, where the organization of phospholipids was abnormal under both oxy and deoxy conditions, reoxygenation of RSC almost completely restored the organization of membrane phospholipids to normal. These results indicate that the process of sickling induces an abnormality in the organization of membrane phospholipids to normal. These results indicate that the process of sickling induces an abnormality in the organization of membrane lipids in RSC which become permanent in ISC.

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.

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.

Kinetics and site specificity of hydroperoxide-induced oxidative damage in red blood cells

To provide a detailed description of the time course and the site specificity of hydroperoxide-induced oxidative stress in red blood cells (RBCs), we have characterized the action of a membrane-soluble (cumene hydroperoxide [cumOOH]) and a water-soluble (hydrogen peroxide [H2O2]) oxidant. The fluorescent polyunsaturated fatty acid (PUFA) parinaric acid (PnA) was used to probe peroxidation processes in the membrane, and oxidation of hemoglobin (Hb) was measured spectrophotometrically as an indicator of cytosolic oxidative stress. The observed degradation patterns of PnA and Hb were clearly distinct for each oxidant. At comparable oxidant concentrations, the cumulative oxidative stress on the RBC membrane was always much higher with cumOOH, whereas much more Hb oxidation was measured with H2O2. The kinetics of Hb oxidation as well as the nature of the products formed were different for each oxidant. The main Hb oxidation product generated gradually by cumOOH was metHb, whereas H2O2 caused the rapid formation of ferrylHb. CumOOH caused more oxidation of endogenous PUFAs and of vitamin E, while the degradation pattern of vitamin E closely resembled that of PnA. At high oxidant concentrations, extensive cell lysis was observed after prolonged incubation. Butylated hydroxytoluene (BHT) completely prevented oxidation of endogenous PUFAs but did not completely prevent hemolysis, indicating that factors other than lipid peroxidation are also important in causing lysis of RBCs. The action of cumOOH is characterized by a gradual reaction with Hb, generating radicals that produce an oxidative stress primarily directed at the membrane, which increases in time to a maximum and then gradually decreases. In contrast, H2O2 crosses the RBC membrane and reacts rapidly with Hb, generating a very reactive radical species that has Hb, not the membrane, as a prime target. H2O2-induced oxidative stress is at a maximum immediately after addition of this oxidant and decreases rapidly to zero in a short time. These findings provide further insight into the mode of action of hydroperoxides and the mechanism of compartmentalization of RBC oxidative damage.