The oxysterol 3β-hydroxy-5-oxo-5,6-secocholestan-6-al changes the phase behavior and structure of phosphatidylethanolamine–phosphatidylcholine mixtures

An oxidized form of cholesterol, atheronal, is a form found in vivo that has been associated with human pathologies. We have studied mixtures of this oxidized sterol with the phospholipids phosphatidylethanolamine and phosphatidylcholine. We used phospholipids either with palmitoyl and oleoyl acyl chains on the C1 and C2 carbon atoms of glycerol or with both acyl chains being palmitoleoyl. We also compared the effects of atheronal on the curvature properties of these lipids with the action of cholesterol. We studied the bilayer to hexagonal phase transition temperature of mixtures of these lipids using differential scanning calorimetry as well as the dimensions of the hexagonal phase cylinders using X-ray diffraction. Disordering of the lamellar phase was also qualitatively assessed by the loss of sharp diffraction peaks. Our results demonstrate that the modulation of membrane curvature in these systems depends not only on the nature of the sterol, but also on the acyl chain composition of the phospholipids used. In addition, some of the effects of atheronal could be ascribed to reaction of the aldehyde and ketone groups of this oxidized sterol with the amino group of phosphatidylethanolamine.     

Calorimetric study on the induction of interdigitated phase in hydrated DPPC bilayers by bioactive labdanes and correlation to their liposome stability: The role of chemical structure

Labd-7,13-dien-15-ol (1), labd-13-ene-8alpha,15-diol (2), and labd-14-ene-8,13-diol (sclareol) have been found to exhibit cytotoxic and cytostatic effects. Their partitioning into phospholipid bilayers may induce membrane structure modifications, crucial in the development of liposomes. DSC was used to elucidate the profile of modifications induced in DPPC bilayers by incorporating increasing concentrations of the labdanes. Labdanes 1, 2 and sclareol were incorporated into SUV liposomes composed of DPPC their physicochemical stability was monitored (4 degrees C) and was compared to liposomes incorporating cholesterol. All labdanes strongly affect the bilayer organization in a concentration dependent manner in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase, the induction of a lateral phase separation and the possible existence of interdigitated domains in the bilayer. The physicochemical stability of liposomes was strongly influenced by the chemical features of the labdanes. The liposomal preparations were found to retain their stability at low labdane concentration (10 mol%), while at higher concentrations up to 30 mol% a profound decrease in intact liposomes occurred, and a possible existence of interdigitated sheets was concluded.     

Supramolecular organization of S12363-liposomes prepared with two different remote loading processes

The S12363 anticancer drug was encapsulated into liposomes in an attempt to increase its therapeutic index. Loading of S12363 was achieved using two different processes based on the formation of either a pH gradient or an ammonium gradient between the acidic inner liposomal compartment and the basic outer phase. High encapsulation yields (> 90%) were obtained using both processes for sphingomyelin/cholesterol/cholesterol-PEG vesicles. Spectrofluorimetry measurements have shown that liposomes were characterized by an internal pH around 4 for both loading processes. This internal pH was stable over a period of at least 20 days. Differential scanning calorimetry coupled with time-resolved synchrotron X-ray diffraction was used to study the drug/carrier supramolecular organization. In ammonium sulfate, S12363 was inserted into the bilayer in the vicinity of the polar headgroup. In citrate buffer, S12363 was mainly adsorbed at the water-lipid interface. The drug partitioning into the membrane was inhomogeneous and led to the formation of drug-rich and drug-poor domains. This effect was enhanced in the presence of cholesterol, especially in ammonium sulfate. To conclude, for both processes, the encapsulated drug was found inside the liposome aqueous core but strongly interacting with the membrane.     

Oxidized phosphatidylcholines facilitate phospholipid flip-flop in liposomes

Lipid asymmetry is a ubiquitous property of the lipid bilayers in cellular membranes and its maintenance and loss play important roles in cell physiology, such as blood coagulation and apoptosis. The resulting exposure of phosphatidylserine on the outer surface of the plasma membrane has been suggested to be caused by a specific membrane enzyme, scramblase, which catalyzes phospholipid flip-flop. Despite extensive research the role of scramblase(s) in apoptosis has remained elusive. Here, we show that phospholipid flip-flop is efficiently enhanced in liposomes by oxidatively modified phosphatidylcholines. A combination of fluorescence spectroscopy and molecular dynamics simulations reveal that the mechanistic basis for this property of oxidized phosphatidylcholines is due to major changes imposed by the oxidized phospholipids on the biophysical properties of lipid bilayers, resulting in a fast cross bilayer diffusion of membrane phospholipids and loss of lipid asymmetry, requiring no scramblase protein.     

Role of leaflet asymmetry in the permeability of model biological membranes to protons, solutes, and gases.

Bilayer asymmetry in the apical membrane may be important to the barrier function exhibited by epithelia in the stomach, kidney, and bladder. Previously, we showed that reduced fluidity of a single bilayer leaflet reduced water permeability of the bilayer, and in this study we examine the effect of bilayer asymmetry on permeation of nonelectrolytes, gases, and protons. Bilayer asymmetry was induced in dipalmitoylphosphatidylcholine liposomes by rigidifying the outer leaflet with the rare earth metal, praseodymium (Pr3+). Rigidification was demonstrated by fluorescence anisotropy over a range of temperatures from 24 to 50 degrees C. Pr3+-treatment reduced membrane fluidity at temperatures above 40 degrees C (the phase-transition temperature). Increased fluidity exhibited by dipalmitoylphosphatidylcholine liposomes at 40 degrees C occurred at temperatures 1-3 degrees C higher in Pr3+-treated liposomes, and for both control and Pr3+-treated liposomes permeability coefficients were approximately two orders of magnitude higher at 48 degrees than at 24 degrees C. Reduced fluidity of one leaflet correlated with significantly reduced permeabilities to urea, glycerol, formamide, acetamide, and NH3. Proton permeability of dipalmitoylphosphatidylcholine liposomes was only fourfold higher at 48 degrees than at 24 degrees C, indicating a weak dependence on membrane fluidity, and this increase was abolished by Pr3+. CO2 permeability was unaffected by temperature. We conclude: (a) that decreasing membrane fluidity in a single leaflet is sufficient to reduce overall membrane permeability to solutes and NH3, suggesting that leaflets in a bilayer offer independent resistances to permeation, (b) bilayer asymmetry is a mechanism by which barrier epithelia can reduce permeability, and (c) CO(2) permeation through membranes occurs by a mechanism that is not dependent on fluidity.     

Redox-active tyrosine residue in the microcin J25 molecule

Microcin J25 (MccJ25) is a 21 amino acid lasso-peptide antibiotic produced by Escherichia coli and composed of an 8-residues ring and a terminal ‘tail’ passing through the ring. We have previously reported two cellular targets for this antibiotic, bacterial RNA polymerase and the membrane respiratory chain, and shown that Tyr9 is essential for the effect on the membrane respiratory chain which leads to superoxide overproduction. In the present paper we investigated the redox behavior of MccJ25 and the mutant MccJ25 (Y9F). Cyclic voltammetry measurements showed irreversible oxidation of both Tyr9 and Tyr20 in MccJ25, but infrared spectroscopy studies demonstrated that only Tyr9 could be deprotonated upon chemical oxidation in solution. Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H₂O₂ was demonstrated by Electron Paramagnetic Resonance Spectroscopy; this radical was not detected when the reaction was carried out with the MccJ25 (Y9F) mutant. These results show that the essential Tyr9, but not Tyr20, can be easily oxidized and form a tyrosyl radical.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Fine-tuning of POPC liposomal leakage by the use of β-cyclodextrin and several hydrophobic guests

The effect of entrapped β-cyclodextrin (β-CD) on the stability of multilamellar vesicles (MLVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), prepared by the dehydration-rehydration method, was studied by monitoring the release of 5(6)-carboxyfluorescein encapsulated into the liposomes. Different hydrophobic guests, such as Fullerene C₆₀, have been incorporated into the POPC bilayer in order to modify the membrane composition. The kinetic results as well as ESI-MS measurements evidenced that the destabilizing activity of β-CD is due to the formation of β-CD inclusion complexes and the consequent removal of selected bilayer constituents from the liposomal membrane. Hence, when β-CD was added to the liposomes in the form of a strong, water-soluble 2:1 β-CD/C₆₀ inclusion complex, such a destabilizing effect was not observed. However, the same β-CD/C₆₀ inclusion complex does not form as a result of C₆₀ extraction from the bilayer. This may be attributed either to the overwhelming concentration of POPC with respect to C₆₀ and/or to the fact that C₆₀ is largely aggregated in the bilayer. Turbidimetric and fluorimetric determinations of lamellarity and entrapped volume of the studied MLVs provided further evidence of the alteration of the liposomal bilayer as a consequence of the addition of β-CD and/or the presence of the studied guests.     

Membrane-permeabilizing activities of amphidinol 3, polyene-polyhydroxy antifungal from a marine dinoflagellate

Amphidinols, which are polyene-polyhydroxy metabolites produced by the marine dinoflagellate Amphidinium klebsii, possess potent antifungal and hemolytic activities. The membrane permeabilizing actions of amphidinol 3, the most potent homologue, were compared with those of polyene antibiotics, amphotericin B (AmB) and filipin, in hemolytic tests, ²³Na nuclear magnetic resonance (NMR)-based membrane permeabilizing assays, and UV spectroscopy for liposome-bound forms. In Na⁺ flux experiments using large unilamellar vesicles (LUVs), ion efflux by amphidinol 3 was inhibited by cholesterol or ergosterol, which was opposed to previous results [J. Mar. Biotechnol., 5 (1997) 124]. When the effect of the agents on the size of vesicles was examined by light scattering experiments, amphidinol 3 did not significantly alter their size while filipin and synthetic detergent Triton X-100 did. The observations implied that the activity of amphidinol 3 was mainly due to formation of large pores/lesions in liposomes rather than detergent-like disruption of membrane. The pore/lesion size was estimated to be 2.0-2.9 nm in diameter on the basis of osmotic protection experiments using blood cells. The UV spectra in liposomes, which revealed the close interaction of polyene moieties in a lipid bilayer, further implied that the membrane activity of amphidinol 3 is caused by the molecular assemblage formed in biomembrane. These results disclose that amphidinol 3 is one of few non-ionic compounds that possess potent membrane permeabilizing activity with non-detergent mechanism.     

Bilayer polarity and its thermal dependency in the ?o and ?d phases of binary phosphatidylcholine/cholesterol mixtures

Diverse variations in membrane properties are observed in binary phosphatidylcholine/cholesterol mixtures. These mixtures are nonideal, displaying single or phase coexistence, depending on chemical composition and other thermodynamic parameters. When compared with pure phospholipid bilayers, there are changes in water permeability, bilayer thickness and thermomechanical properties, molecular packing and conformational freedom of phospholipid acyl chains, in internal dipolar potential and in lipid lateral diffusion. Based on the phase diagrams for DMPC/cholesterol and DPPC/cholesterol, we compare the equivalent polarity of pure bilayers with specific compositions of these mixtures, by using the Py empirical scale of polarity. Besides the contrast between pure and mixed lipid bilayers, we find that liquid-ordered (?_o) and liquid-disordered (?_d) phases display significantly different polarities. Moreover, in the ?_o phase, the polarities of bilayers and their thermal dependences vary with the chemical composition, showing noteworthy differences for cholesterol proportions at 35, 40, and 45 mol%. At 20 °C, for DMPC/cholesterol at 35 and 45 mol%, the equivalent dielectric constants are 21.8 and 23.8, respectively. Additionally, we illustrate potential implications of polarity in various membrane-based processes and reactions, proposing that for cholesterol containing bilayers, it may also go along with the occurrence of lateral heterogeneity in biological membranes.     

Transmembrane Peptides Influence the Affinity of Sterols for Phospholipid Bilayers

Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mβCD can be measured. Comparison of how cholesterol and CTL partitioning between mβCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mβCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins can both decrease and increase the affinity of sterols for the lipid bilayers surrounding proteins. This is likely to affect the sterol distribution within the bilayer and thereby the lateral organization in biomembranes.     

Cholesterol-induced protein sorting: an analysis of energetic feasibility

The mechanism(s) underlying the sorting of integral membrane proteins between the Golgi complex and the plasma membrane remain uncertain because no specific Golgi retention signal has been found. Moreover one can alter a protein's eventual localization simply by altering the length of its transmembrane domain (TMD). M. S. Bretscher and S. Munro (SCIENCE: 261:1280-1281, 1993) therefore proposed a physical sorting mechanism based on the hydrophobic match between the proteins' TMD and the bilayer thickness, in which cholesterol would regulate protein sorting by increasing the lipid bilayer thickness. In this model, Golgi proteins with short TMDs would be excluded from cholesterol-enriched domains (lipid rafts) that are incorporated into transport vesicles destined for the plasma membrane. Although attractive, this model remains unproven. We therefore evaluated the energetic feasibility of a cholesterol-dependent sorting process using the theory of elastic liquid crystal deformations. We show that the distribution of proteins between cholesterol-enriched and cholesterol-poor bilayer domains can be regulated by cholesterol-induced changes in the bilayer physical properties. Changes in bilayer thickness per se, however, have only a modest effect on sorting; the major effect arises because cholesterol changes also the bilayer material properties, which augments the energetic penalty for incorporating short TMDs into cholesterol-enriched domains. We conclude that cholesterol-induced changes in the bilayer physical properties allow for effective and accurate sorting which will be important generally for protein partitioning between different membrane domains.     

Cholesterol, lanosterol, and ergosterol attenuate the membrane association of LL-37(W27F) and temporin L

Sterols impart significant changes to the biophysical properties of lipid bilayers. In this regard the impact of cholesterol on membrane organization and dynamics is particularly well documented and serves for comparison with other sterols. However, the factors underlying the molecular evolution of cholesterol remain enigmatic. To this end, cholesterol attenuates membrane perturbation by the so-called antimicrobial peptides (AMPs), produced ubiquitously by eukaryotic cells to combat bacterial infections by compromising the permeability barrier function of the microbial target membranes. In the present study, we addressed the effects of cholesterol, ergosterol, and lanosterol on the membrane association of two structurally and functionally diverse AMPs viz. LL-37(F27W) and temporin L (TemL) using fluorescence spectroscopy. Interestingly, sterol concentration dependent effects on the membrane association of these peptides were observed. At X_Sterol = 0.5 cholesterol was most effective in reducing the membrane intercalation of both LL-37(F27W) and TemL, the corresponding efficiencies of the three sterols decreasing as cholesterol > lanosterol ≥ ergosterol, and cholesterol > lanosterol > ergosterol. It is conceivable that part of the selection pressure for the chemical evolution of cholesterol may have derived from the ability to protect the AMP-secreting host cell from the membrane damaging action of the antimicrobial peptides.     

Liposome-Mediated transfer of bacterial RNA into carrot protoplasts

The uptake of liposome-encapsulated E. coli [³H]RNA by carrot (Daucus carota L.) protoplasts was examined. [³H]RNA extracted from protoplasts that had been incubated with [³H]RNA-containing, large, unilamellar lipid vesicles (liposomes) obtained by ether infusion, and examined by sucrose gradient centrifugation and formamide-polyacrylamide gel electrophoresis, appeared substantially degraded, with a total elimination of 23S RNA and a partial loss of 16S RNA. In contrast, no breakdown of the [³H]RNA was apparent in the liposomes after sequestration, even in the presence of externally added ribonuclease, or in the unfused liposomes remaining after incubation of protoplasts with liposomes. Thus, the degradation of the [³H]RNA extracted from the protoplasts must have occurred within the protoplasts and represents evidence for liposome-mediated RNA uptake. Naked RNA added to the protoplast culture was found to be totally degraded after incubation with the protoplasts. The uptake of liposome-sequestered RNA by protoplasts was demonstrated to be a function both of the lipid composition of the liposomal membrane and of the temperature of incubation of the liposomeprotoplast mixture. Furthermore, the mode of this uptake (fusion versus endocytosis) could be manipulated by adjusting the cholesterol content of the liposomal membrane. The implications of the ability to insert RNA into protoplasts without degradation by extracellular nucleases are discussed.     

Cholesterol depletion-induced inhibition of stretch-activated channels is mediated via actin rearrangement

Cholesterol is a critical regulator of lipid bilayer dynamics and plasma membrane organization in eukaryotes. A variety of ion channels have been shown to be modulated by cellular cholesterol and partition into cholesterol-enriched membrane rafts. However, very little is known about functional role of membrane cholesterol in regulation of mechanically gated channels that are ubiquitously present in living cells. In our previous study, the effect of methyl-beta-cyclodextrin (MbCD), cholesterol-sequestering agent, on Ca²⁺-permeable stretch-activated cation channels (SACs) has been described. Here, cell-attached patch-clamp method was employed to search for the mechanisms of cholesterol-dependent regulation of SACs and to clarify functional contribution of lipid bilayer and submembranous cytoskeleton to channel gating. Cholesterol-depleting treatment with MbCD significantly decreased open probability of SACs whereas alpha-cyclodextrin had no effect. F-actin disassembly fully restored high level of SAC activity in cholesterol-depleted cells. Particularly, treatment with cytochalasin D or latrunculin B abrogated inhibitory effect of MbCD on stretch-activated currents. Single channel analysis and fluorescent imaging methods indicate that inhibition of SACs after cholesterol depletion is mediated via actin remodeling initiated by disruption of lipid rafts. Our data reveal a novel mechanism of channel regulation by membrane cholesterol and lipid rafts.     

Plasma membrane cholesterol plays a critical role in the Salmonella-induced anti-inflammatory response in intestinal epithelial cells

Our recent study demonstrated that a phosphatidylinositol-3 kinase (PI3K)/Akt-dependent anti-inflammatory pathway was activated by Salmonella in intestinal epithelial cells. Salmonella virulence is dependent on the ability of the bacterium to invade nonphagocytic host cells and then survive and replicate within modified Salmonella-containing vacuoles where cholesterol accumulates. In addition, cholesterol in membrane lipid rafts is frequently a platform for the activation of downstream signaling pathways, including the PI3K/Akt pathway. However, the role of plasma membrane cholesterol in the Salmonella-induced anti-inflammatory response in intestinal epithelial cells has not been elucidated. Here, we show that the effect of plasma membrane cholesterol depletion on the inhibition of Akt activation allows sustained ERK activation and the subsequent upregulation of IL-8 expression. These results demonstrate that plasma membrane cholesterol plays a critical role in the PI3K-dependent anti-inflammatory pathway activated by Salmonella in intestinal epithelial cells.     

Liposome-based formulations for the antibiotic nonapeptide Leucinostatin A: Fourier transform infrared spectroscopy characterization and in vivo toxicologic study

Leucinostatin-A is a nonapeptide isolated fromPaecilomyces marquandii Paecilomyces lilacinus A257, andAcremonium sp., exerting remarkable phytotoxic, antibacterial (especially against Gram-positive) and antimycotic activities. With the aim to find alternative formulation for in vivo administration, a number of Leucinostatin-A—loaded liposomal formulations have been prepared and characterized. Both large unilamellar vesicles and multilamellar vesicles consisting of synthetic and natural lipids were evaluated. In addition, to determine the nature of peptide-membrane interactions and the stability of liposomes loaded with Leucinostatin-A, a Fourier Transform Infrared Spectroscopy study was performed. The results suggest that the mode of interaction of the peptide is dependent on its concentration, on bilayer fluidity, and on liposome type. Finally, the LD₅₀ of both free and liposome-delivered Leucinostatin-A was determined in mice. These results suggest that the incorporation of Leucinostatin-A into liposomes may result in decreased Leucinostatin-A toxicity, as the intraperitoneal administration of Leucinostatin-A—loaded liposomes reduced the LD₅₀ of Leucinostatin-A 15-fold.     

Interaction of sporidesmin,a mycotoxin fromPithomyces chartarum,with lipid bilayers

Sporidesmin, a mycotoxin fromPithomyces chartarum is a hydrophobic molecule. It can therefore be easily incorporated in the cell membrane, where it is likely to cause changes in the bilayer organization and the properties of membrane proteins. In order to understand the redox behaviour of sporidesmin in a hydrophobic environment, we have investigated the effects of oxidized and reduced sporidesmin on the phase transition properties of bilayers and on the susceptibility of bilayers to pancreatic phospholipase A₂ (PLA₂). The changes induced by sporidesmin in the thermotropic phase transition profiles of dimyristoyl-sn-3-phosphatidyl choline (DMPC) bilayers were similar to those caused by solutes known to localize in the glycerol-backbone region of the lipid bilayer, suggesting a similar localization for oxidized and reduced sporidesmin. Neither form of toxin disrupt the bilayer or membrane organization even at relatively high mole fractions. At concentrations <10 mole% both forms partitioned equally well in the gel and liquid-crystalline phases, whereas at higher concentrations (30 mole%) reduced sporidesmin is preferentially localized in the liquid-crystalline phase. These effects of sporidesmin on the phase properties of DMPC vesicles were also reported by the fluorescence behavior of 10-pyrenedecanoic acid (PDA). The effects of oxidized and reduced sporidesmins on PLA₂ kinetics are consistent with their ability to perturb bilayer organisation.     

Antioxidant Ascorbate Is Stabilized by NADH-Coenzyme Q10 Reductase in the Plasma Membrane

Plasma membranes isolated from K562 cells contain an NADH-ascorbate free radical reductase activity and intact cells show the capacity to reduce the rate of chemical oxidation of ascorbate leading to its stabilization at the extracellular space. Both activities are stimulated by CoQ₁₀ and inhibited by capsaicin and dicumarol. A 34-kDa protein (p34) isolated from pig liver plasma membrane, displaying NADH-CoQ₁₀ reductase activity and its internal sequence being identical to cytochrome b ₅ reductase, increases the NADH-ascorbate free radical reductase activity of K562 cells plasma membranes. Also, the incorporation of this protein into K562 cells by p34-reconstituted liposomes also increased the stabilization of ascorbate by these cells. TPA-induced differentiation of K562 cells increases ascorbate stabilization by whole cells and both NADH-ascorbate free radical reductase and CoQ₁₀ content in isolated plasma membranes. We show here the role of CoQ₁₀ and its NADH-dependent reductase in both plasma membrane NADH-ascorbate free radical reductase and ascorbate stabilization by K562 cells. These data support the idea that besides intracellular cytochrome b ₅-dependent ascorbate regeneration, the extracellular stabilization of ascorbate is mediated by CoQ₁₀ and its NADH-dependent reductase.     

Multiple phospholipid substrates of phospholipase C/sphingomyelinase HR2 from Pseudomonas aeruginosa

The activity of phospholipase C/sphingomyelinase HR₂ (PlcHR₂) from Pseudomonas aeruginosa was characterized on a variety of substrates. The enzyme was assayed on liposomes (large unilamellar vesicles) composed of PC:SM:Ch:X (1:1:1:1; mol ratio) where X could be PE, PS, PG, or CL. Activity was measured directly as disappearance of substrate after TLC lipid separation. Previous studies had suggested that PlcHR₂ was active only on PC or SM. However we found that, of the various phospholipids tested, only PS was not a substrate for PlcHR₂. All others were degraded, in an order of preference PC > SM > CL > PE > PG. PlcHR₂ activity was sensitive to the overall lipid composition of the bilayer, including non-substrate lipids.