Membrane Phospholipid Organization Research Articles

Fluorescence evidence of annexin A6 translocation across membrane in model matrix vesicles during apatite formation

AbstractMatrix vesicles (MVs) are 100–300 nm spherical structures released by mineralization competent cells to initiate formation of apatite, the mineral component in bones. Among proteins present in MVs, annexin A6 (AnxA6) is thought to be ubiquitously distributed in the MVs’ lumen, on the surface of the internal and external leaflets of the membrane and also inserted in the lipid bilayer. To determine the molecular mechanism(s) that lead to the different locations of AnxA6, we hypothesized the occurrence of a pH drop during the mineralization. Such a change would induce the AnxA6 protonation, which in turn, and because of its isoelectric point of 5.41, would change the protein hydrophobicity facilitating its insertion into the MVs’ bilayer. The various distributions of AnxA6 are likely to disturb membrane phospholipid organization. To examine this possibility, we used fluorescein as pH reporter, and established that pH decreased inside MVs during apatite formation. Then, 4‐(14‐phenyldibenzo[a,c]phenazin‐9(14H)‐yl)‐phenol, a vibration‐induced emission fluorescent probe, was used as a reporter of changes in membrane organization occurring with the varying mode of AnxA6 binding. Proteoliposomes containing AnxA6 and 1,2‐Dimyristoyl‐sn‐glycero‐3phosphocholine (DMPC) or 1,2‐Dimyristoyl‐sn‐glycero‐3phosphocholine: 1,2‐Dipalmitoyl‐sn‐glycero‐3‐phosphoserine (DMPC:DPPS 9:1), to mimic the external and internal MV membrane leaflet, respectively, served as biomimetic models to investigate the nature of AnxA6 binding. Addition of Anx6 to DMPC at pH 7.4 and 5.4, or DMPC:DPPS (9:1) at pH 7.4 induced a decrease in membrane fluidity, consistent with AnxA6 interactions with the bilayer surface. In contrast, AnxA6 addition to DMPC:DPPS (9:1) at pH 5.4 increased the fluidity of the membrane. This latest result was interpreted as reflecting the insertion of AnxA6 into the bilayer. Taken together, these findings point to a possible mechanism of AnxA6 translocation in MVs from the surface of the internal leaflet into the phospholipid bilayer stimulated upon acidification of the MVs’ lumen during formation of apatite.

Effects of Polychlorinated Pesticides and Their Metabolites on Phospholipid Organization in Model Microbial Membranes.

Polychlorinated pesticides (PPs) were classified as persistent organic pollutants because of their toxicity, limited degradability in the environment, bioaugmentation, and accumulation in animal tissues. PPs accumulate in the environment mainly in the soils and water sediments where they are toxic to the decomposer organisms including soil bacteria and fungi. Therefore, there is an urgent need to search for the microorganisms capable of PP biodegradation which could be applied for soil bioremediation. The exact mechanism of PP microbial toxicity is unknown; however, there is evidence that it can be membrane related. To shed light on the interactions of PPs with microbial membranes, we applied Langmuir monolayers formed by phospholipids as model biomembranes. The model membranes were formed by phospholipids typical to microbial membranes: cardiolipins and phosphatidylglycerols the main components of Gram positive bacteria membranes, phosphatidylcholine typical to fungal membranes, as well as phosphatidylethanolamine found in the inner membranes of Gram negative bacteria. For the studies, the most ecotoxic PPs and their water-soluble metabolites were chosen. The monolayers were studied with the application of mutually complementary techniques: Langmuir technique, grazing incidence X-ray diffraction, and PM-IRRAS spectroscopy. It turned out that the cyclodiene PPs are more membrane active than monocyclic PPs and that the possibility of their incorporation is strictly related to the phospholipid structure. The membranes prepared with cardiolipin turned out to be especially resistant to PP incorporation. Regarding the metabolites, pentachlorophenol turned out to be especially structure breaking, affecting the molecular organization of all of the investigated phospholipids.

Changes in Red Blood Cell membrane lipid composition: A new perspective into the pathogenesis of PKAN.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a form of Neurodegeneration with Brain Iron Accumulation (NBIA) associated with mutations in the pantothenate kinase 2 gene (PANK2). The PANK2 catalyzes the first step of coenzyme A (CoA) biosynthesis, a pathway producing an essential cofactor that plays a key role in energy and lipid metabolism. The majority of PANK2 mutations reduces or abolishes the activity of the enzyme. In around 10% of cases with PKAN, the presence of deformed red blood cells with thorny protrusions in the circulation has been detected. Changes in membrane protein expression and assembly during erythropoiesis were previously explored in patients with PKAN. However, data on red blood cell membrane phospholipid organization are still missing in this disease. In this study, we performed lipidomic analysis on red blood cells from Italian patients affected by PKAN with a particular interest in membrane physico-chemical properties. We showed an increased number of small red blood cells together with membrane phospholipid alteration, particularly a significant increase in sphingomyelin (SM)/phosphatidylcholine (PC) and SM/phosphatidylethanolamine (PE) ratios, in subjects with PKAN. The membrane structural abnormalities were associated with membrane fluidity perturbation. These morphological and functional characteristics of red blood cells in patients with PKAN offer new possible tools in order to shed light on the pathogenesis of the disease and to possibly identify further biomarkers for clinical studies.

029 Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein organization of rooster sperm during cooling and cryopreservation using the fluorescent stains Merocyanine 540 (phospholipid order) and Rhodamine 640 (protein order). Rooster semen samples (3 roosters and breeds per pool; 4 pools total) were diluted in Lakes Low Temperature (LLT) and Sasaki diluent and cooled to 5 °C over 30 min (Step 1). The samples were then diluted with the penetrating cryoprotectants (CPA) glycerol (LLT) or methylacetamide (Sasaki), equilibrated for 30 min (Step 2), loaded into straws and cryopreserved. The cryopreservation and thawing (Step 3) was performed using the optimized procedures for each diluent. Computer automated semen analysis (CASA) was used to analyze motility characteristics and flow cytometry was used to analyze plasma membrane phospholipid, protein organization and plasma membrane integrity (PMI) at each step. Percent values from CASA and flow cytometry fluorescent medians (FM) were transformed using arcsine and Box–Cox, respectively. The GLM ANOVA was used to detect differences in motility characteristics and membrane organization and included the effects of cryopreservation step and rooster semen pool. In the total motility analyses cryopreservation diluent/CPA was a significant ( P P P P P Source of funding: USDA-ARS-NCGRP National Animal Germplasm Program. Conflict of interest: None declared. phil.purdy@ars.usda.gov

Acyl Chain Differences in Phosphatidylethanolamine Determine Domain Formation and LacY Distribution in Biomimetic Model Membranes

Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) are the two main components of the inner membrane of Escherichia coli. It is well-known that inner membrane contains phospholipids with a nearly constant polar headgroup composition. However, bacteria can regulate the degree of unsaturation of the acyl chains in order to adapt to different external stimuli. Studies on model membranes of mixtures of PE and PG, mimicking the proportions found in E. coli, can provide essential information on the phospholipid organization in biological membranes and may help in the understanding of membrane proteins activity, such as lactose permease (LacY) of E. coli. In this work we have studied how different phosphatidylethanolamines differing in acyl chain saturation influence the formation of laterally segregated domains. Three different phospholipid systems were studied: DOPE:POPG, POPE:POPG, and DPPE:POPG at molar ratios of 3:1. Lipid mixtures were analyzed at 24 and 37 °C through three different model membranes: monolayers, liposomes, and supported lipid bilayers (SLBs). Data from three different techniques, Langmuir isotherms, Laurdan generalized polarization, and atomic force microscopy (AFM), evidenced that only the DPPE:POPG system exhibited coexistence between gel (L(β)) and fluid (L(α)) phases at both 24 and 37 °C . In the POPE:POPG system the L(β)/L(α) coexistence appears at 27 °C. Therefore, in order to investigate the distribution of LacY among phospholipid phases, we have used AFM to explore the distribution of LacY in SLBs of the three phospholipid systems at 27 °C, where the DOPE:POPG is in L(α) phase and POPE:POPG and DPPE:POPG exhibit L(β)/L(α) coexistence. The results demonstrate the preferential insertion of LacY in fluid phase.

Control of Protein and Sterol Trafficking by Antagonistic Activities of a Type IV P-type ATPase and Oxysterol Binding Protein Homologue

The oxysterol binding protein homologue Kes1p has been implicated in nonvesicular sterol transport in Saccharomyces cerevisiae. Kes1p also represses formation of protein transport vesicles from the trans-Golgi network (TGN) through an unknown mechanism. Here, we show that potential phospholipid translocases in the Drs2/Dnf family (type IV P-type ATPases [P4-ATPases]) are downstream targets of Kes1p repression. Disruption of KES1 suppresses the cold-sensitive (cs) growth defect of drs2Delta, which correlates with an enhanced ability of Dnf P4-ATPases to functionally substitute for Drs2p. Loss of Kes1p also suppresses a drs2-ts allele in a strain deficient for Dnf P4-ATPases, suggesting that Kes1p antagonizes Drs2p activity in vivo. Indeed, Drs2-dependent phosphatidylserine translocase (flippase) activity is hyperactive in TGN membranes from kes1Delta cells and is potently attenuated by addition of recombinant Kes1p. Surprisingly, Drs2p also antagonizes Kes1p activity in vivo. Drs2p deficiency causes a markedly increased rate of cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER) and redistribution of endogenous ergosterol to intracellular membranes, phenotypes that are Kes1p dependent. These data suggest a homeostatic feedback mechanism in which appropriately regulated flippase activity in the Golgi complex helps establish a plasma membrane phospholipid organization that resists sterol extraction by a sterol binding protein.

Hydroxycarbamide‐induced changes in E/beta thalassemia red blood cells

In thalassemia, fetal hemoglobin (HbF) augmentation with hydroxycarbamide (also known as hydroxyurea) is not always successful. The expected parallel effects on red cell (RBC) membrane deformability, cell hydration, and membrane phospholipid organization, all important for extending RBC life span and increasing Hb, have been infrequently examined. We analyzed these characteristics in 15 nontransfused E/beta(0) thalassemia patients treated with HU (mean 10.2 months). Membrane deformability and cell hydration mildly improved in association with increased HbF levels approaching statistical significance (r = 0.51, P = 0.06). All measures improved considerably in splenctomized patients. These findings underscore the disappointing results of hydroxyurea treatment in clinical trials and the importance of examining the effect on RBC characteristics for the development and understanding of HbF-enhancing agents.

Natural Phosphatidylcholine Is Actively Translocated across the Plasma Membrane to the Surface of Mammalian Cells

The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established.

A unique phospholipid organization in bovine erythrocyte membranes.

Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies; namely, low or absent phosphatidylcholine (PC) along with high sphingomyelin levels. Here, we report another anomaly in bovine erythrocytes that affects aminophospholipids: phosphatidylethanolamine (PE) shows an extreme asymmetry, with only 2% of the total present in the outer leaflet. Furthermore, we found that phospholipase A(2), an enzyme located on the external surface of the erythrocytes, shows higher activity against PC than against PE. In addition, we observed that acylation of PE is by far the most important biosynthetic event in this system. We propose that deacylation of PE and PC by phospholipase A(2) to generate lysocompounds, followed by selective reacylation of lyso-PE in the inner leaflet, can account for the compositional and architectural peculiarities of bovine erythrocyte membranes.

Acceleration by Ceramide of Calcium-Dependent Translocation of Phospholipase A2 from Cytosol to Membranes in Platelets

The effect of ceramide on Ca2+-dependent translocation of cytosolic phospholipase A2 (cPLA2) to membranes was studied. Pretreatment of platelets with sphingomyelinase or C6-ceramide (N-hexanoylsphingosine) led to apparent enhancement of Ca2+-ionophore A23187-stimulated arachidonic acid release but did not affect the cytosolic phospholipase A2 (cPLA2) activity. Under these conditions, the cPLA2 proteins in membranes increased significantly, compared with those by A23187 alone. Sphingomyelinase and C6-ceramide, but not C6-dihydroceramide, a control analog of C6-ceramide, also facilitated the Ca2+-dependent increase in the cPLA2 protein, as well as the activity, in membranes induced by addition of Ca2+ into platelet lysate. Protein kinase Cα, which possesses a Ca2+-dependent lipid binding domain, was increased in membranes in a Ca2+-dependent manner, but the increase was not accelerated by sphingomyelinase or C6-ceramide. These findings suggest that ceramide in membranes potentiates Ca2+-dependent cPLA2 translocation from cytosol to membranes, probably through modification of membrane phospholipid organization.

RH blood group system and molecular basis of Rh-deficiency

Rhesus (Rh) antigens are defined by a complex association of membrane polypeptides that are missing or severely deficient from the red cells of rare Rhnullindividuals who suffer a clinical syndrome of varying severity characterized by abnormalities of the red cell shape, cation transport and membrane phospholipid organization. The Rhnullphenotype is an inherited condition that may arise from homozygosity either for a ‘suppressor’ gene unrelated to the RH locus (‘regulator type’) or for a silent allele at the RH locus itself (‘amorph type’).A current model suggests that the proteins of the Rh complex (Rh, RhAG, CD47, LW, GPB) are assembled by non-covalent bonds and that it is not assembled or transported to the cell surface when one subunit is missing. Rh and RhAG proteins belong to the same protein family and are quantitatively the major components that form the core of the complex, which is firmly linked to the membrane skeleton. Molecular analysis of Rhnullindividuals has revealed that abnormalities occur only at the RHAG and RH loci, without alteration of the genes encoding the accessory chains.Mutations of the RHAG gene, but not of RH, occur in all Rhnullindividuals of the regulator type (including Rhmod) investigated so far (13 cases), strongly suggesting thatRHAG mutants act as ‘suppressors’ and not as transcriptional regulators of the RH genes and that variable expression of the RHAG alleles may account for the Rhmodphenotypes (exhibiting weak expression of Rh antigens). Conversely, mutations of the RHCE gene, but not of RHAG, occur in two unrelated Rhnullindividuals of the amorph type, supporting the view that RH mutants result from a ‘silent’ allele at the RH locus. These findings strongly support the Rh complex model since when either the Rh or RhAG protein is missing, the assembly and/or transport of the Rh complex is defective. Transcriptional as well as post-transcriptional mechanisms may account for the molecular abnormalities, but experimental evidence based on expression models is required to test these hypotheses, in the hope that they may help to clarify the biological role of the Rh proteins in the red cell membrane.

Altered membrane phospholipid organization and erythrophagocytosis in Eβ-thalassemia

The underlying cause behind the accelerated destruction of erythrocytes in the bone marrow and in the peripheral circulation, accompanying the β-thalassemic syndromes is still not clearly understood. The present investigation demonstrates that increased phagocytosis of erythrocytes in Eβ-thalassemia is inhibited by the presence of phosphatidylserine (PS) vesicles, suggesting a PS- ‘PS-receptor’ type of interaction in premature recognition of these erythrocytes by macrophages. Increased exposure of both aminophospholipids phosphatidylethanolamine (PE) and PS was demonstrated by fluorescamine labeling and annexin binding, respectively. The slower rate of translocation of PS across the bilayer suggested that this contributed towards the increased exposure of PS in Eβ-thalassemic erythrocytes.

Phospholipid composition and organization in model beta-thalassemic erythrocytes.

The membrane phospholipid organization in human red blood cells (RBC) is rigidly maintained by a complex system of enzymes. However, several elements of this system are sensitive to oxidative damage. An important component in the destruction of beta-thalassemic RBC is the generation of reactive oxygen species and the release of redox-active iron by the unpaired alpha-hemoglobin chains. Consequently, we hypothesized that the presence of this oxidative stress to the RBC membrane could lead to alterations in membrane lipid organization. Model beta thalassemic RBC, prepared by the introduction of excess alpha-globin in the cell, have previously been shown to exhibit structural and functional changes almost identical to those observed in beta-thalassemic cells. After 24 hr at 37 degrees C, the model beta thalassemic cells exhibited a significant loss of deformability, as measured by ektacytometric analysis, indicative of extensive membrane damage. However, a normal steadystate distribution of endogenous phospholipids was found, as evidenced by the accessibility of membrane phospholipids to hydrolysis by phospholipases. Similarly, the kinetics of transbilayer movement of spin-labeled phosphatidylserine (PS) and phosphatidylethanolamine (PE) in all samples was in the normal range and was not affected by the presence of excess alpha-globin chains. In contrast, a faster rate of spin-labeled phosphatidylcholine (PC) transbilayer movement was observed in these cells. While control RBC exhibited a complete loss of their initial (2 mol%) lysophosphatidylcholine (LPC) levels following 24 hr of incubation at 37 degrees C, 1.5 mol% LPC was still present in model beta-thalassemic cells, suggesting an altered phospholipid molecular species turnover, possibly as a result of an increased repair of oxidatively damaged phospholipids.

Ischemia/reperfusion-induced changes in membrane proteins and lipids of gerbil cortical synaptosomes

The effects of transient bilateral carotid occlusion on the physical state of synaptosomal membrane proteins and lipids were studied in adult and aged gerbils employing electron paramagnetic resonance. Transient ischemia was produced in adult and aged gerbils by bilateral occlusion of the common carotid arteries with reperfusion times ranging from 0 to 24 h. Synaptosomes of the cerebral cortices were isolated and labeled with a protein-specific spin probe (2,2,6,6-tetramethyl-4-maleimido-piperidine-1-oxyl) and a lipid-specific spin probe (5-doxylstearic acid). Changes in the physical state of the protein peaked at 60 min reperfusion for both adult and aged gerbil models, with a more intense change in aged, but did not return to control values by 24 h. A biphasic change occurred with the lipid-specific label in both the aged and adult models. The onset of the first phase of change occurred at an earlier time (30 min reperfusion) for aged gerbil tissue than for adult tissue (between 3 and 6 h reperfusion), while the second phase of change occurred at 12 h reperfusion for both adult and aged. These results are consistent with the hypothesis that protein oxidation and lipid peroxidation are direct results of free radicals produced during the reperfusion following ischemia and that protein oxidation may be intensified by peroxidation of the surrounding lipids. Phospholipase A2 activation is implicated to cause changes in membrane phospholipid organization as seen in these studies.

Hydrogen Peroxide Oxidation Induces the Transfer of Phospholipids from the Membrane into the Cytosol of Human Erythrocytes

The effects of oxidative damage on membrane phospholipid organization were examined in human erythrocytes. Exposure to H2O2 induced shape changes in these cells; normal discocytes became echinocytic, and stomatocytes generated by foreign phosphatidylserine incorporation reverted to discoid morphology. H2O2 treatment also inhibited phosphatidylserine transport from the outer to inner membrane monolayer, consistent with earlier reports on oxidative sensitivity of the aminophospholipid translocator. The morphological changes are consistent with movement of inner monolayer lipids to the outer monolayer, as might be expected if aminophospholipid sequestration is compromised. However, lipid extraction and prothrombinase activation assays showed no increased exposure of phosphatidylserine on the cell surface. Instead, phosphatidylserine was found associated with the cytosolic fraction of H2O2-treated cells. These observations suggest that oxidative damage alters the lipid organization of erythrocyte membranes, not by randomizing the lipid classes within the bilayer, but by inducing extraction of inner monolayer components into the cytosol.

Molecular biology of the Rh antigens

The RBC Rh antigens are of large clinical importance, but until recently have been poorly understood at a molecular level. The Rh polypeptides are a family of nonglycosylated Mr 30- to 32-Kd transmembrane proteins that are core structural components of the Rh antigens and have been purified and partially characterized biochemically. Rh polypeptides are present in RBCs from normal humans and other mammalian species and are probably required for normal membrane integrity, because they appear to be missing from the RBCs of the rare Rhnull individuals that express several membrane defects. The Rh polypeptides contain an exofacial free sulfhydryl that is important for Rh antigenic reactivity and several intracellular sulfhydryls that appear to be palmitylated, but most of the molecule appears to reside between the leaflets of the phospholipid bilayer. The cDNA coding for a 416-amino acid Rh polypeptide was recently isolated but was not found to share sequence homology with any known protein, and Northern analysis indicated that Rh is erythroid specific. The Rh antigens within the native membranes are thought to exist as a complex of Rh polypeptides and multiple other membrane components, including certain Rh-related glycoproteins. While it is thought that this assembly may be important for the Rh antigenic reactivity, the structural basis of this remains to be established. While the physiologic role of Rh is yet to be defined, several clues indicate that it may play a role in the organization of membrane phospholipids or synthesis or membrane expression of various glycoproteins. While our knowledge of Rh is still very incomplete, recent research has significantly advanced the molecular understanding of these important blood group antigens.

Alterations in erythrocyte membrane phospholipid organization due to the intracellular growth of the human malaria parasite,Plasmodium falciparum

The asymmetric distribution of phospholipids in the erythrocyte membrane during the intracellular development of the human malaria parasite Plasmodium falciparum was studied. Infected cells of high parasitaemia were treated with phospholipase A2 or sphingomyelinase C, followed by isolation of the host red cell membrane using the Affigel (731) bead method. Additionally, phosphatidylserine on the surface of infected cells was probed using a phosphatidylserine-sensitive prothrombinase assay. Trophozoite-infected cells showed an increase in phosphatidylethanolamine and phosphatidylserine and a decrease in phosphatidylcholine in the outer leaflet. In addition to the changes already present in trophozoite-infected cells, schizont-infected cells showed a decrease in sphingomyelin as well as a further increase in phosphatidylserine in the outer leaflet. The results are discussed with respect to possible mechanisms and consequences of these changes.

Heat-induced alterations in monkey erythrocyte membrane phospholipid organization and skeletal protein structure and interactions

Rhesus monkey erythrocytes were subjected to heating at 50°C for 5–15 min, and the heat-induced effects on the membrane structure were ascertained by analysing the membrane phospholipid organization and membrane skeleton dynamics and interactions in the heated cells. Membrane skeleton dynamics and interactions were determined by measuring the Tris-induced dissociation of the Triton-insoluble membrane skeletons (Triton shells), the spectrin-actin extractability at low ionic strength, spectrin self-association and spectrin binding to normal monkey erythrocyte membrane inside-out vesicles (IOVs). The Tris-induced Triton shell dissociation and spectrin-actin extractability were markedly decreased by the erythrocyte heating. Also, the binding of the heated erythrocyte membrane spectrin-actin with the IOVs was much smaller than that observed with the normal erythrocyte spectrin-actin. Further, the spectrin structure was extensively modified in the heated cells, as compared to the normal erythrocytes. Transbilayer phospholipid organization was ascertained by employing bee venom and pancreatic phospholipases A 2, fluorescamine, and Merocyanine 540 as the external membrane probes. The amounts of aminophospholipids hydrolysed by phospholipases A 2 or labeled by fluorescamine in intact erythrocytes considerably increased after subjecting them to heating at 50°C for 15 min. Also, the fluorescent dye Merocyanine 540 readily stained the 15-min-heated cells but not the fresh erythrocytes. Unlike these findings, the extent of aminophospholipid hydrolysis in 5-min-heated cells by phospholipases A 2 depended on the incubation time. While no change in the membrane phospholipid organization could be detected in 10 min, prolonged incubations led to the increased aminophospholipid hydrolysis. Similarly, fluorescamine failed to detect any change in the transbilayer phospholipid distribution soon after the 5 min heating, but it labeled greater amounts of aminophospholipids in the 5-min-heated cells, as compared to normal cells, after incubating them for 4 h at 37°C. These results have been discussed to analyse the role of membrane skeleton in maintaining the erythrocyte membrane phospholipid asymmetry. It has been concluded that both the ATP-dependent aminophospholipid pump and membrane bilayer-skeleton interactions are required to maintain the transbilayer phospholipid asymmetry in native erythrocyte membrane.

Modulation of membrane surface curvature by peptide-lipid interactions.

Recent reports on the interaction of cardiotoxin and melittin with phospholipid model membranes are reviewed and analyzed. These types of peptide toxins are able to modulate lipid surface curvature and polymorphism in a highly lipid-specific way. It is demonstrated that the remarkable variety of effects of melittin on the organization of different membrane phospholipids can be understood in a relatively simple model, based on the shape-structure concept of lipid polymorphism and taking into account the position of the peptide molecule with respect to the lipids. Based on the strong preference of the peptides for negatively charged lipids and the structural consequences thereof, and on preliminary studies of signal peptide-lipid interaction, a role of inverted or concave lipid structures in the process of protein translocation across membranes is suggested.

Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase.

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.