Diacylglycerol causes major structural transitions in phospholipid bilayer membranes

We demonstrate for the first time that major structural changes are imposed on various phospholipid bilayers by diacylglycerol, a product of phosphatidylinositol metabolism. By 5 mole percent in phosphatidylethanolamine a lamellar to hexagonal transition starts that is complete at 10 mole percent. At 30 mole percent it causes the same transition in phosphatidylcholine and forms a cubic phase at 80 mole percent. Diacylglycerol disorders the phosphatidylserine lamellar phase. We view the formation of the non-lamellar phases as diagnostic of the destabilizations that diacylglycerol can cause in membranes. We suggest how DAG may act both in its specific activation of membrane enzymes and in inducing membrane fusion.     

A role for diacylglycerol in annexin A7-mediated fusion of lung lamellar bodies

Lung surfactant secretion in alveolar type II cells occurs following lamellar body fusion with plasma membrane. Annexin A7 is a Ca2+-dependent membrane-binding protein that is postulated to promote membrane fusion during exocytosis in some cell types including type II cells. Since annexin A7 preferably binds to lamellar body membranes, we postulated that specific lipids could modify the mode of annexin A7 interaction with membranes and its membrane fusion activity. Initial studies with phospholipid vesicles containing phosphatidylserine and other lipids showed that certain lipids affected protein interaction with vesicle membranes as determined by change in protein tryptophan fluorescence, protein interaction with trans membranes, and by protein sensitivity to limited proteolysis. The presence of signaling lipids, diacylglycerol or phosphatidylinositol-4,5-bisphosphate, as minor components also modified the lipid vesicle effect on these characteristics and membrane fusion activity of annexin A7. In vitro incubation of lamellar bodies with diacylglycerol or phosphatidylinositol-4,5-bisphosphate caused their enrichment with either lipid, and increased the annexin A7 and Ca2+-mediated fusion of lamellar bodies. Treatment of isolated lung lamellar bodies with phosphatidylinositol- or phosphatidylcholine phospholipase C to increase diacylglycerol, without or with preincubation with phosphatidylinositol-4,5-bisphosphate, augmented the fusion activity of annexin A7. Thus, increased diacylglycerol in lamellar bodies following cell stimulation with secretagogues may enhance membrane fusion activity of annexin A7.     

The lamellar to hexagonal phase transition in phosphatidylethanolamine liposomes: a fluorescence anisotropy study

The steady-state anisotropy of trimethylammonium diphenylhexatriene fluorescence has been used to monitor the thermotropic lamellar to HII hexagonal phase transition in an unsaturated phosphatidylethanolamine. The transition is observed in lipid aggregates when they are heated above the transition temperature Th, as well as in diluted liposomes after aggregation above Th. Changes in fluorescence anisotropy are not observed with Ca(++)-induced fusion of phosphatidylserine vesicles, a process not involving hexagonal phase formation.     

Diacylglycerol stimulates acrosomal exocytosis by feeding into a PKC- and PLD1-dependent positive loop that continuously supplies phosphatidylinositol 4,5-bisphosphate

Acrosomal exocytosis involves a massive fusion between the outer acrosomal and the plasma membranes of the spermatozoon triggered by stimuli that open calcium channels at the plasma membrane. Diacylglycerol has been implicated in the activation of these calcium channels. Here we report that this lipid promotes the efflux of intraacrosomal calcium and triggers exocytosis in permeabilized human sperm, implying that diacylglycerol activates events downstream of the opening of plasma membrane channels. Furthermore, we show that calcium and diacylglycerol converge in a signaling pathway leading to the production of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Addition of diacylglycerol promotes the PKC-dependent activation of PLD1. Rescue experiments adding phosphatidic acid or PIP(2) and direct measurement of lipid production suggest that both PKC and PLD1 promote PIP(2) synthesis. Inhibition of different steps of the pathway was reverted by adenophostin, an agonist of IP(3)-sensitive calcium channels, indicating that PIP(2) is necessary to keep these channels opened. However, phosphatidic acid, PIP(2), or adenophostin could not trigger exocytosis by themselves, indicating that diacylglycerol must also activate another factor. We found that diacylglycerol and phorbol ester stimulate the accumulation of the GTP-bound form of Rab3A. Together our results indicate that diacylglycerol promotes acrosomal exocytosis by i) maintaining high levels of IP(3) - an effect that depends on a positive feedback loop leading to the production of PIP(2) - and ii) stimulating the activation of Rab3A, which in turn initiates a cascade of protein interactions leading to the assembly of SNARE complexes and membrane fusion.     

Alterations in erythrocyte membrane fluidity in children with trisomy 21: a fluorescence study.

Membrane fluidity of erythrocytes obtained from 15 children with trisomy 21 and 20 healthy controls were studied by measuring steady-state fluorescence anisotropy and fluorescence lifetime of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in hemoglobin-free erythrocyte membranes. Our results demonstrate a significant decrease in DPH fluorescence anisotropy and a significant increase in TMA-DPH fluorescence anistropy in erythrocytes from subjects with trisomy 21. No significant differences between the two groups were observed in the fluorescence lifetime of DPH and TMA-DPH. These data suggest an increase in membrane fluidity in the interior part of the membrane and a decrease in fluidity at the lipid-water interface region. This could be in part attributed to an increased oxidative damage in trisomy 21.     

Lipids favoring inverted phase enhance the ability of aerolysin to permeabilize liposome bilayers

Channel formation by the bacterial toxin aerolysin follows oligomerization of the protein to produce heptamers that are capable of inserting into lipid bilayers. How insertion occurs is not understood, not only for aerolysin but also for other proteins that can penetrate membranes. We have studied aerolysin channel formation by measuring dye leakage from large unilamellar egg phosphatidylcholine vesicles containing varying amounts of other lipids. The rate of leakage was enhanced in a dose-dependent manner by the presence of phosphatidylethanolamine, diacylglycerol, cholesterol, or hexadecane, all of which are known to favor a lamellar-to-inverted hexagonal (L-H) phase transition. Phosphatidylethanolamine molecular species with low L-H transition temperatures had the largest effects on aerolysin activity. In contrast, the presence in the egg phosphatidylcholine liposomes of lipids that are known to stabilize the lamellar phase, such as sphingomyelin and saturated phosphatidylcholines, reduced the rate of channel formation, as did the presence of lysophosphatidylcholine, which favors positive membrane curvature. When two different lipids that favor hexagonal phase were present with egg PC in the liposomes, their stimulatory effects were additive. Phosphatidylethanolamine and lysophosphatidylcholine canceled each other's effect on channel formation.     

Influence of immune complexes on macrophage membrane fluidity: a nanosecond fluorescence anisotropy study

Time-resolved fluorescence anisotropy (TRFA) and steady-state anisotropy measurements and fluorescence intensification microscopic observations were made on RAW264 macrophages labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Microscopic analysis revealed that the fluorescent probe DPH was found in association with plasma membranes and small vesicles. Macrophages treated with immune complexes could not be distinguished from untreated cells, indicating that the same membrane compartments were labeled. The probe TMA-DPH was exclusively localized to the plasma membrane. Steady-state anisotropy measurements indicated that in vitro culture conditions did not significantly affect membrane fluidity. TRFA measurements were conducted to determine the physical properties of macrophage membranes during immune recognition and endocytosis. Data were analyzed by iterative deconvolution to yield phi, the rotational correlation time, and r infinity, the limiting anisotropy. These parameters may be interpreted as the "fluidity" and order parameter of the membrane environment, respectively. Typical values for untreated macrophages were phi = 7.8 ns and r infinity = 0.12. Binding and endocytosis of immune complexes prepared in 4-fold antigen excess increase these values to phi = 22.1 ns and r infinity = 0.15. However, receptor-independent phagocytosis of latex beads decreases these values to phi = 2.2 ns and r infinity = 0.10. Addition of catalase before, but not after, immune complex incubation with cells diminishes the effect upon membrane structure, suggesting that H2O2 participates in fluidity changes. Pretreatment of macrophages with the membrane-impermeable sulfhydryl blocker p-(chloromercuri)benzenesulfonic acid also diminished these effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the effect of a peptide sequence of the E2 protein (HGV/GBV-C) on the physicochemical properties of zwitterionic and negatively charged bilayers.

The membrane-interacting properties of a potential epitope of GB virus C/hepatitis G virus located at the region (99-118) of the E2 structural protein were investigated using several fluorescence techniques. SUV of DMPC:DPPC (1:1) or DMPG:DPPC (1:1) zwitterionic and anionic mixtures, respectively, were used as model membranes. FRET with NBD-PE as energy donor and Rho-PE as energy acceptor-labelled SUV indicated that the peptide was able to fuse both zwitterionic and anionic SUVs, the latter requiring lower peptide concentrations. However, the peptide increased the steady-state anisotropy of DPH embedded in the hydrophobic centre of the membrane with zwitterionic headgroups and to a lesser extent in anionic bilayers, suggesting that charge-charge interactions are not required for membrane interactions and also confirming the FRET results. No changes in anisotropy were observed with the probe TMA-DPH located at the surface of the bilayer. Finally, analysis of the intrinsic emission fluorescence of the tryptophan residue, upon incubation with SUV, showed a blue shift in the presence of anionic bilayers, both below and above the main transition temperature (T(m)) (gel to liquid-crystalline state) and, to a lesser extent, with the zwitterionic model membrane.     

Regulation of chloride transport in parotid secretory granules by membrane fluidity.

Zymogen granule membranes contain Cl- conductance and Cl/anion exchange activities that become important for primary fluid production after fusion with the apical plasma membrane of the acinar cell. We have used steady-state fluorescence anisotropy of diphenylhexatriene derivatives and measurements of Cl- transport in isolated secretory granules to determine the contribution of membrane fluidity to the regulation of transport across the granule membrane. Secretory granules from several unstimulated glands (rat pancreas and parotid, rabbit gastric glands) were shown to have low membrane fluidity compared to plasma membranes. In addition, Cl- transport activity in different granule preparations showed a strong correlation to the membrane fluidity when measured with 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), but not with 3-[p-(6-phenyl)-1,3,5-hexatrienyl)-phenyl]propionic acid (PA-DPH). These data suggest that TMA-DPH preferentially partitions into a specific lipid environment associated with, or which exerts an influence on, the Cl- transport proteins and that increases in the fluidity of this environment are associated with higher transport rates. Data from other types of plasma membranes indicate that TMA-DPH partitions much more than PA-DPH into the cytoplasmic leaflet, suggesting that this part of the granule membrane is involved in the observed fluidity changes. Furthermore, increasing the bulk membrane fluidity with the local anesthetics benzyl alcohol and n-alkanols increased the Cl- transport rates up to 10-fold. This increase was apparently through specific transporters as anion selectivity was maintained in spite of the higher absolute rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid domain structure correlated with membrane protein function in placental microvillus vesicles

Membrane fluidity properties of placental microvillus membrane vesicles (MVV) were determined from fluorescence anisotropy (r), dynamic depolarization, and lifetime heterogeneity studies of diphenylhexatriene (DPH), trimethylamino-DPH (TMA-DPH), and cis- and trans-parinaric acids (c-PnA and t-PnA). Plots of r against temperature for DPH and TMA-DPH in MVV had slope discontinuities at 26 degrees C (Tc, transition temperature); however, analysis of r in terms of probe rotational rate (R), limiting anisotropy (r infinity), and lifetime (tau) revealed that DPH reported a phase transition because of changes in r infinity, whereas the phase transition observed by TMA-DPH occurred primarily because of changes in R. Heterogeneity analysis using phase and modulation lifetimes at three frequencies showed that DPH and TMA-DPH lifetimes were homogeneous in MVV. Both long (greater than 25 ns) and short (less than 6 ns) lifetime components were detected for c-PnA and t-PnA in MVV, corresponding to the probes in solid and fluid lipid phases. The fractional amplitude of the long lifetimes (solid phase) decreased from 0.86 to 0.12 with increasing temperature (5-55 degrees C) as the membrane passed through the phase transition, with 50% of the change occurring at 27 degrees C (c-PnA) or 33 degrees C (t-PnA). The activation energies for alkaline phosphatase, aminopeptidase M, and sodium-proton antiporter activities all showed discontinuities in the temperature range 27-31 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arachidonoyl-CoA:lysophosphatidylcholine acyltransferase activity in ras-transformed NIH 3T3 fibroblasts depends on the membrane composition.

Investigations were performed on the influence of membrane lipids on arachidonoyl-CoA:lysophosphatidylcholine acyltransferase in microsomal membranes from control and ras-transformed NIH 3T3 fibroblasts. Of all the tested phospholipids only sphingomyelin induced activation of acyltransferase in membranes from ras-transformed cells. No specific phospholipid effect on the acyltransferase was observed in microsomal membranes from control fibroblasts. Diacylglycerol was found to inhibit acyltransferase in both cell lines, whereas ceramide accumulation induced inhibition only in membranes from the transformed cells. The effects of diacylglycerol, ceramide, sphingomyelin and sphingomyelinase are discussed with respect to their putative roles in the signal transduction pathways in oncogene-expressing cells.     

The interfacial region of dipalmitoylphosphatidylcholine bilayers is perturbed by fusogenic amphipaths.

Several structural methods were used to probe the influence of three fusogenic and four nonfusogenic amphipaths on large, unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles. For four of these structural measurements there was a correlation observed between the ability of an amphipath to favor poly(ethylene glycol) (PEG)-induced fusion and the structural perturbation reported by each method. First, the fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenyhexa-1,3,5-triene (TMA-DPH), which probes the upper region of the bilayer, decreased in the range of PEG concentrations previously found to cause fusion of membranes containing fusogenic amphipaths. For nonfusogenic amphipaths, the anisotropy increased monotonically with PEG concentration. The properties of similar probes that locate in the hydrophobic core of the bilayer showed no correlation with fusogenicity, nor did the properties of probes purported to sense the aqueous surface of the membrane. Second, the frequency of the C=O stretch increased and then decreased dramatically as fusogenic but not nonfusogenic membranes were heated through their phase transition. Third, there was a dramatic increase in the frequency of the C-O-C ester stretch at the membrane order/disorder phase transition for membranes containing fusogenic amphipaths, twice the increase observed for nonfusogenic amphipaths. The spectral characteristics of phosphate, choline, and acyl chain motions showed no such correlation with fusogenicity. Finally, calorimetric measurements showed that low levels of fusogenic amphipaths eliminated the "pretransition" (L beta-->P beta) in DPPC membranes, whereas other amphipaths shifted but did not eliminate this transition. Taken together, these results indicate that fusogenic amphipaths perturb the interface or "backbone" region of the bilayer rather than the hydrophobic core, the headgroup, or the water interface regions of DPPC bilayers.     

Effects of non-electrolyte molecules with anesthetic activity on the physical properties of DMPC multilamellar liposomes

The effects of 13 non-electrolytes with moderate anesthetic potency on the order of DMPC liposomes were examined. Changes in order were monitored by steady-state fluorescence polarization techniques using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPG). At 30 degrees C, all of the compounds tested decreased the DPH steady-state anisotropy (rs), with potencies highly correlated to their oil/water partition coefficients. However, only the most hydrophobic anesthetics decreased TMA-DPH RS. Some of the most hydrophilic compounds, including ethanol and urethane, actually increased TMA-DPH rs, suggestive of an increase in membrane order. The concept of selectivity was borrowed from partitioning theory and used to explain some effects on anesthetic potency of decreasing temperature to 18 degrees C. In the gel as opposed to the liquid crystalline phase, selectivity for decreasing membrane order (as monitored by DPH) markedly increased, suggesting that anesthetic partitioning and/or the site of anesthetic action was occurring in a more hydrophobic domain. The solute-independent difference (or capacity) between two membranes for perturbation was defined as membrane sensitivity. Sensitivity appeared to also decrease with decreasing temperature, despite the decrease in membrane partitioning. This effect is thought to result from the selective delivery of the anesthetic solute to the membrane interior and away from more hydrophilic domains where anesthetics may order membrane structure.     

Lysophosphatidylcholine modulates catalytically important motions of the Ca-ATPase phosphorylation domain

Catalytically important motions of the Ca-ATPase, modulated by the physical properties of surrounding membrane phospholipids, have been suggested to be rate-limiting under physiological conditions. To identify the nature of the structural coupling between the Ca-ATPase and membrane phospholipids, we have investigated the functional and structural effects resulting from the incorporation of the lysophospholipid 1-myristoyl-2-hydroxy-sn-glycerol-3-phosphocholine (LPC) into native sarcoplasmic reticulum (SR) membranes. Nonsolubilizing concentrations of LPC abolish changes in fluorescence signals associated with either intrinsic or extrinsic chromophores that monitor normal conformational transitions accompanying calcium activation of the Ca-ATPase. There are corresponding decreases in the rates of calcium transport coupled to ATP hydrolysis, suggesting that LPC may increase conformational barriers associated with catalytic function. Fluorescence anisotropy measurements of the lipid analogue 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) partitioned into SR membranes indicate that LPC does not significantly modify lipid acyl chain rotational dynamics, suggesting differences in headgroup conformation between LPC and diacylglycerol phosphatidylcholines. Complementary measurements using phosphorescence anisotropy of erythrosin isothiocyanate at Lys464 on the Ca-ATPase provide a measure of the dynamic structure of the phosphorylation domain, and indicate that LPC restricts the amplitude of rotational motion. These results suggest a structural linkage between the cytosolic phosphorylation domain and the conformation of membrane phospholipid headgroups. Thus, changes in membrane phospholipid composition can modulate membrane surface properties and affect catalytically important motions of the Ca-ATPase in a manner that suggests a role for LPC generated during signal transduction.     

Asymmetry of membrane fluidity in the lipid bilayer of blood platelets: Fluorescence study with diphenylhexatriene and analogs

Summary Membrane fluidity of bovine platelets was examined with diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and anionic propionic acid derivative (DPH-PA). After addition of these probes to platelet suspensions at 37°C, the fluorescence intensity of DPH-PA reached equilibrium within 2 min, whereas those of DPH and TMA-DPH increased gradually. With increase in the fluorescence intensity of TMA-DPH, its fluorescence anisotropy decreased significantly, but the fluorescence anisotropies of DPH-PA and DPH did not change during incubation. The gradual increase of fluorescence intensity of TMA-DPH was due to its penetration into the cytoplasmic side of the platelet membrane, as shown quantitatively by monitoring decrease in its extractability with albumin. Transbilayer movement of TMA-DPH was markedly temperature-dependent, and was scarcely observed at 15°C. The fluorescence intensity of TMA-DPH was much higher in platelet membranes and vesicles of extracted membrane lipids than the initial intensity in intact platelets. Moreover, the fluorescence anisotropy of TMA-DPH was much lower in the former preparations than the initial value in intact platelets. These results suggest that binding sites for TMA-DPH in the cytoplasmic side of the platelet membrane are more fluid than those in the outer leaflet of the plasma membrane. Platelet activation by ionomycin induced specific change in the fluorescence properties of TMA-DPH without causing transbilayer incorporation of the probe.     

Infrared and time-resolved fluorescence spectroscopic studies of the polymorphic phase behavior of phosphatidylethanolamine/diacylglycerol lipid mixtures

Fourier transform infrared (FTIR) and time-resolved fluorescence spectroscopy have been employed to examine the structural dynamics of lipid fatty acyl chains and lipid/water interfacial region of a binary lipid mixture containing unsaturated phosphatidylethanolamine (PE) and diacylglycerol (DG). Infrared vibrational frequencies of the CH2 symmetric stretching and the C = O stretching bands of the lipids were measured at different lipid compositions and temperatures. For 0% DG, the lamellar gel to lamellar liquid crystalline (L beta-L alpha) and the L alpha to inverted hexagonal (L alpha-HII) phase transitions were observed at approximately 15 degrees and 55 degrees C, respectively. As the DG content increased gradually from 0% to 15%, the L alpha-HII phase transition temperature decreased drastically while the L beta-L alpha phase transition temperature decreased only slightly. At 10% DG, a merge of these two phase transitions was noticed at approximately 10 degrees C. For the composition study at 23 degrees C, the L alpha-HII transition occurred at approximately 6-10% DG as indicated by abrupt increases in both the CH2 and C = O stretching frequencies at those DG contents. Using time-resolved fluorescence spectroscopy, abrupt decreases in both the normalized long time residual and the initial slope of the anisotropy decay function of lipid probes, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine, in these PE/DG mixtures were observed at the L alpha-HII phase transition. These changes in the anisotropy decay parameters suggested that the rotational dynamics and orientational packing of the lipids were altered at the composition-induced L alpha-HII transition, and agreed with a previous temperature-induced L alpha-HII transition study on pure unsaturated PE (Cheng (1989) Biophys. J. 55, 1025-1031). The fluorescence lifetime of water soluble probes, 8,1-anilinonapthalenes sulfonate acid, in PE/DG mixtures increased abruptly at the L alpha-HII phase transition, suggesting that the conformation and hydration of the lipid/water interfacial region also undergo significant changes at the L alpha-HII transition.     

Influence of dietary fatty acids on membrane fluidity and activation of rat platelets

The apparent steady-state fluorescence anisotropy of DPH- or TMA-DPH-labeled washed rat platelets is strongly affected by factors that also influence the turbidity by these platelet suspensions. Sonicated preparations from platelet lipids have a low turbidity and give anisotropy values which are hardly affected by the experimental conditions. We studied the effect of four high-fat diets on membrane fluidity, lipid composition and activation tendency of washed platelets. The diets contained 50 energy% of oils with different levels of saturated and (poly)unsaturated fatty acids. Only small diet-induced differences in DPH fluorescence anisotropy were found, which were comparable for intact platelets and platelet lipids. These differences were unrelated to the degree of saturation of the dietary fatty acids. Platelets from rats fed mainly saturated fatty acids differed significantly from other diet groups in a higher unsaturation degree of phospholipids and a lower cholesterol/phospholipid ratio, but this was not detected by DPH in terms of decreased anisotropy. These platelets aggregated less than other platelets in response to thrombin or collagen. The lower response to collagen persisted in indomethacin-treated platelets activated with the thromboxane A2 mimetic U46619, indicating a different sensitivity of these platelets for thromboxane A2. We conclude that in rat platelets: (a) the overall membrane fluidity and phospholipid unsaturation degree are subject to strong homeostatic control; (b) steady-state anisotropy with DPH or TMA-DPH label is inadequate to reveal subtile changes in lipid profile; (c) changes in platelet responsiveness to thrombin and thromboxane A2, rather than (plasma) membrane fluidity, determine the effect of dietary fatty acids on platelet aggregation.     

X-ray diffraction study of feline leukemia virus fusion peptide and lipid polymorphism

The structural effects of the fusion peptide of feline leukemia virus (FeLV) on the lipid polymorphism of N-methylated dioleoylphosphatidylethanolamine were studied using a temperature ramp with sequential X-ray diffraction. This peptide, the hydrophobic amino-terminus of p15E, has been proven to be fusogenic and to promote the formation of highly curved, intermediate structures on the lamellar liquid-crystal to inverse hexagonal phase transition pathway. The FeLV peptide produces marked effects on the thermotropic mesomorphic behaviour of MeDOPE, a phospholipid with an intermediate spontaneous radius of curvature. The peptide is shown to reduce the lamellar repeat distance of the membrane prior to the onset of an inverted cubic phase. This suggests that membrane thinning may play a role in peptide-induced membrane fusion and strengthens the link between the fusion pathway and inverted cubic phase formation. The results of this study are interpreted in relation to models of the membrane fusion mechanism.     

Interaction of amphotericin B and its N-fructosyl derivative with murine thymocytes: a comparative study using fluorescent membrane probes

The polyene antibiotics amphotericin B (AmB) and N-(1-deoxy-D-fructos-1-yl)amphotericin (N-Fru-AmB) have different activity towards murine thymocytes (N-Fru-AmB is less toxic but is a potent immunomodulator). The interactions of the drugs with these cells have been studied by fluorescence methods. Fluorescence energy transfer from 1-[4-(trimethylammonio) phenyl]-6-phenylhexa-1,3,5-triene, p-toluenesulfonate (TMA-DPH) to polyenes was used to follow the binding of the two drugs to the plasma membrane. The results, confirmed by circular dichroism measurements, indicate that at saturation the ratio AmB bound/plasma membrane lipid is low (less than 1 molecule of polyene for 170 lipids). The slightly higher binding of AmB as compared to N-Fru-AmB demonstrates that affinity of the antibiotic for plasma membrane does not account for the activity of the polyenes towards lymphocytes. The effect of the two polyenes on membrane fluidity was studied by steady-state fluorescence anisotropy. The results suggest that AmB strongly perturbs the structure of the membrane whereas only a slight decrease of the anisotropy is observed with N-Fru-AmB in the range of concentration where the biological activity has been demonstrated. Polyene location was further investigated by comparing the energy transfer efficiency obtained with TMA-DPH and with the parental compound 1,6-diphenylhexa-1,3,5-triene, p-toluene sulfonate (DPH). While AmB binds to plasma membrane, as well as to intracellular structures, N-Fru-AmB seems to accumulate into the cell and bind to intracellular membrane structures.     

Effect of nedocromil sodium on polymorphonuclear leukocyte plasma membrane

The effect of nedocromil sodium on the plasma membrane fluidity of polymorphonuclear leukocytes (PMNs) was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results show that nedocromil sodium 300 muM significantly decreased membrane fluidity of PMNs. The decrease in membrane fluidity of PMNs induced by fMLP was abolished in the presence of nedocromil sodium. These data suggest that nedocromil sodium interferes with the plasma membranes of PMNs and modulates their activities.