Heterodimeric V. nikolskii phospholipases A2 induce aggregation of the lipid bilayer.

We report that the action of the heterodimeric phospholipases A2 (PLA2s) from Vipera nikolskii, which comprises enzymatically active basic subunit and inactive acidic PLA2 homologue, on the lipid bilayer results in the aggregation and stacking of bilayers. These processes are demonstrated using two independent methods (fluorescence spectroscopy and electron microscopy). Aggregation of bilayers is possible because both subunits of the V. nikolskii heterodimer contain a membrane-binding site (also known as IBS). Thus, when the two IBSs bind to the membrane, the heterodimer acts as a connecting agent. Heterodimers induce aggregation of negatively charged bilayers composed of phosphatidylglycerol and do not induce aggregation of neutral bilayers composed of phosphatidylcholine.

An Expedient Synthesis of Fluorescent Labeled Ceramide-1-phosphate Analogues.

A synthesis for fluorescent analogs of ceramide-1-phosphate bearing 9-anthrylvinyl or 4,4-difluoro-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY) fluorophore at co-position of fatty acid residue was carried out. The key stage of the synthesis is hydrolysis of corresponding sphingomyelins catalyzed by phospholipase D from Streptomyces chromofuscus; the enzymatic yield has been raised to 50-70% by appliance of organic solvent in the incubation medium.

Anion binding to lipid bilayers: a study using fluorescent lipid probes.

Anion-induced fluorescence quenching of lipid probes incorporated into the liposomal membrane was used to study the binding of anions to the lipid membrane. Lipid derivatives bearing nonpolar fluorophore located either in the proximity of the polar headgroups (anthrylvinyl-labelled phosphatidylcholine, ApPC; methyl 4-pyrenylbutyrate, MPB) or in the polar region (rhodamine 19 oleyl ester, OR19) of the bilayer were used as probes. The binding of iodide to the bilayers of different compositions was studied. Based on the anion-induced quenching of the fluorescence, the isotherm of adsorption of the quencher (iodide) to the membrane was plotted. For anions, which are non-quenchers or weak quenchers (thiocyanate, perchlorate or trichloroacetate), the binding parameters were obtained from the data of the competitive displacement of iodide by these anions. The association constants of the anion binding to the bilayer (Ka) were determined for the stoichiometry of 1 ion/1 lipid and also for the case of independent anion binding. At the physiological concentration of the salt, which does not bind noticeably to the membrane (150 mM NaCl), anion binding could be satisfactorily described by the Langmuir isotherm. The approach applied here offers new possibilities for the studies of ion-membrane interactions using fluorescent probes.

Anion binding to lipid bilayers: determination using fluorescent membrane probe by direct quenching or by competitive displacement approaches.

An approach is described that enables anion binding to liposomal membranes to be assessed from the resulting quenching of fluorescent lipid probes included in the membranes. Lipid derivatives such as anthrylvinyl-labeled phosphatidylcholine (ApPC) and methyl 4-pyrenylbutyrate (MPB) were used because they bear nonpolar fluorophores that localize in the bilayer close to polar heads. Association constants (K(a)) of iodide binding to bilayers of different composition were determined on the basis of direct quenching experiments. For anions that are non-quenchers or weak quenchers (thiocyanate, perchlorate and trichloroacetate), K(a) values were obtained from the data of competitive displacement of iodide by these anions. This approach increases possibilities of fluorescence studies of ion-membrane interactions.

Gangliosides induce cell apoptosis in the cytotoxic line CTLL-2, but not in the promyelocyte leukemia cell line HL-60.

Gangliosides induce apoptosis in the cells of the IL-2-dependent cytotoxic mouse line CTLL-2. Upon incubation with gangliosides for 24 h, their effect resulting in appearance of apoptotic cells, falls in a series GM2 > GM3 > GM1 > GD1a > GD1b > GT1b. In the presence of rIL-2, apoptosis induced by GM1 is suppressed, whereas that induced by GM2 is enhanced (the effect of intracellular agent C2-Cer is independent of this cytokine). The GM1-induced apoptosis is cancelled by the caspase I inhibitor. The gangliosides under study are not able to induce apoptosis in the promyelocyte leukemia cell line HL-60. Physiological aspects of the phenomenon found are discussed.

Antitumour activity of cytotoxic liposomes equipped with selectin ligand SiaLe(X), in a mouse mammary adenocarcinoma model.

The overexpression of lectins by malignant cells compared with normal ones can be used for the targeting of drug-loaded liposomes to tumours with the help of specific carbohydrate ligands (vectors). Recently we have shown that liposomes bearing specific lipid-anchored glycoconjugates on a polymeric matrix bind in vitro to human malignant cells more effectively and, being loaded with a lipophilic prodrug of merphalan, reveal higher cytotoxic activity compared with unvectored liposomes. In this study, carbohydrate-equipped cytotoxic liposomes were tested in vivo in a mouse breast cancer model, BLRB-Rb (8.17)1Iem strain with a high incidence of spontaneous mammary adenocarcinoma (SMA). Firstly, a cell line of the SMA was established which was then used to determine the specificity of the tumour cell lectins. After screening of the lectin specificity of a number of fluorescent carbohydrate probes, SiaLe(X) was shown to be the ligand with the most affinity, and a lipophilic vector bearing this saccharide was synthesised. Then different liposomal formulations of the synthetic merphalan lipid derivative and SiaLe(X) vector were prepared and applied in the treatment of mice with grafted adenocarcinomas. The results of the tumorigenesis data show that the therapeutic efficacy of merphalan increases sharply after its insertion as a lipophilic prodrug into the membrane of SiaLe(X)-vectored liposomes.

Charged membrane surfaces impede the protein-mediated transfer of glycosphingolipids between phospholipid bilayers.

A lipid transfer protein that facilitates the transfer of glycolipids between donor and acceptor membranes has been investigated using a fluorescence resonance energy transfer assay. The glycolipid transfer protein (23-24 kDa, pI 9.0) catalyzes the high specificity transfer of lipids that have sugars beta-linked to either a ceramide or a diacylglycerol backbone, such as simple glycolipids and gangliosides, but not the transfer of phospholipids, cholesterol, or cholesterol esters. In this study, we examined the effect of different charged lipids on the rate of transfer of anthrylvinyl-labeled galactosylceramide (1 mol %) from a donor to acceptor vesicle population at neutral pH. Compared to neutral donor vesicle membranes, introduction of negatively charged lipid at 5 or 10 mol % into the donor vesicles significantly decreased the transfer rate. Introduction of the same amount of negative charge into the acceptor vesicle membrane did not impede the transfer rate as effectively. Also, positive charge in the donor vesicle membrane was not as effective at slowing the transfer rate as was negative charge in the donor vesicle. Increasing the ionic strength of the buffer with NaCl significantly reversed the charge effects. At neutral pH, the transfer protein (pI congruent with 9.0) is expected to be positively charged, which may promote association with the negatively charged donor membrane. Based on these and other experiments, we conclude that the transfer process follows first-order kinetics and that the off-rate of the transfer protein from the donor vesicle surface is the rate-limiting step in the transfer process.

Immunosuppressive activity of glycosphingolipids. Influence of serum factors on ganglioside inhibition of IL-4-dependent cell proliferation.

Gangliosides have been shown to inhibit proliferation of the interleukin-4 (IL-4) responsive cell line CT.4R. Kinetic analysis has revealed that ganglioside GT1b is a competitive inhibitor of proliferation, while GM and GM3 show a mixed pattern of inhibition, i.e., exhibit more than one inhibition type. Contribution of the competitive cell inhibition for GM1 and GM3 depends on serum factors added: the higher is the percentage of FCS, the larger is the contribution of competitive inhibition. The pattern of proliferation inhibition shown for GT1b does not depend on the FCS content. We have also studied the interaction of the recombinant IL-4 with fluorescent (anthrylvinyl-labelled) gangliosides GM1 and GM3 and lactosylceramide incorporated into liposomes. Dissociation constants of the IL-4-ganglioside complexes have been determined; lactosylceramide does not interact with rIL-4. The K(d) values for the lymphokine complexes with gangliosides support the conclusion based on the kinetic analysis that IL-4 has a higher affinity for GM3 (K(d) = 5 nM) than for GM1 (K(d) = 0.28 microM).

New fluorescent cholesterol analogs as membrane probes.

New fluorescent cholesterol analogs, (22E, 20R)-3beta-hydroxy-23-(9-anthryl)-24-norchola-5,22-die ne (R-AV-Ch), and the 20S-isomer (S-AV-Ch) were synthesized, their spectral and membrane properties were characterized. The probes bear a 9-anthrylvinyl (AV) group instead of C22-C27 segment of the cholesterol alkyl chain. Computer simulations show that both of the probes have bulkier tail regions than cholesterol and predict some perturbation in the packing of membranes, particularly for R-AV-Ch. In monolayer experiments, the force-area behavior of the probes was compared with that of cholesterol, pure and in mixtures with palmitoyloleoyl phosphatidylcholine (POPC) and N-stearoyl sphingomyelin (SSM). The results show that pure R-AV-Ch occupies 35-40% more cross-sectional area than cholesterol at surface pressures below film collapse (0-22 mN/m); whereas S-AV-Ch occupies nearly the same molecular area as cholesterol. Isotherms of POPC or SSM mixed with 0.1 mol fraction of either probe are similar to isotherms of the corresponding mixtures of POPC or SSM with cholesterol. The probes show typical AV absorption (lambda 386, 368, 350 and 256 nm) and fluorescence (lambda 412-435 nm) spectra. Steady-state anisotropies of R-AV-Ch and S-AV-Ch in isotropic medium or liquid-crystalline bilayers are higher than the values obtained for other AV probes reflecting hindered intramolecular mobility of the fluorophore and decreased overall rotational rate of the rigid cholesterol derivatives. This suggestion is confirmed by time-resolved fluorescence experiments which show also, in accordance with monolayer data, that S-AV-Ch is better accommodated in POPC-cholesterol bilayers than R-AV-Ch. Model and natural membranes can be labeled by either injecting the probes via a water-soluble organic solvent or by co-lyophilizing probe and phospholipid prior to vesicle production. Detergent-solubilization studies involving 'raft' lipids showed that S-AV-Ch almost identically mimicked the behavior of cholesterol and that of R-AV-Ch was only slightly inferior. Overall, the data suggest that the AV-labeled cholesterol analogs mimic cholesterol behavior in membrane systems and will be useful in related studies.

A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes.

A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.

Saccharide-assisted delivery of cytotoxic liposomes to human malignant cells.

The overexpression of lectins by malignant cells was applied for in vitro targeting of liposomes equipped with a saccharide vector and loaded in the lipid phase with a lipid derivative of anticancer agent sarcolysine. The lectin specificity of human leukemia HL-60 and human lung adenocarcinoma ACL cells was revealed by tests with fluorescein-labeled sugar probes. With the help of fluorescent lipid dye it was shown that active saccharide ligands increased the level of the vectored liposome binding to malignant cells by 50-80% as compared to liposomes without vector or with inactive one. The degree of liposome/cell membrane fusion was monitored fluorometrically and was shown to be complete and independent of the vectors. The targeted drug-loaded liposomes had the cytotoxic activity 2-4 times higher as compared to the vector-free ones.

New fluorescent lysolipids: preparation and selective labeling of inner liposome leaflet.

Two new fluorescent lysophosphatidylcholine probes have been synthesized for use as a donor-acceptor pair in fluorescence resonance energy transfer (FRET): 9-anthrylvinyl (LAPC) as donor and 3-perylenoyl (LPPC) as acceptor. The partition coefficients between membrane and aqueous phases were 8.3 x 10(5) and 10.5 x 10(5) for LAPC and LPPC, respectively. The inner leaflets of unilamellar lipid vesicles were labeled with these probes to assess conservation of membrane sidedness after membrane fusion. After medium-sized unilamellar vesicles (MUV) were prepared with a probe in both leaflets, probe in the outer leaflet was removed by repeatedly washing with an excess of unlabeled giant unilamellar vesicles (GUV). MUV and GUV were separated by centrifugation. The probes did not flip-flop across bilayers at 25 degrees C for at least 12 h. MUV containing the ganglioside GT1b were labeled with the LAPC/LPPC pair in the inner leaflet and incubated for 30 min at neutral pH with influenza virus. Fusion was triggered by acidification to pH 5.0 and was monitored by an increase in donor fluorescence in a FRET assay. When the inner leaflets of MUV were labeled by LAPC only, its fluorescence did not change after fusion. However, the fluorescence decreased by 60% when the LAPC was removed from the outer leaflets of the fused membranes by repeated washings with GUV. We conclude that the lipids of the inner and outer leaflets of the fused MUV/virus complexes intermixed.

Amphipathic peptide affects the lateral domain organization of lipid bilayers.

Using lipid-specific fluorescent probes, we studied the effects of amphipathic helical, membrane active peptides of the A- and L-type on membrane domain organization. In zwitterionic binary systems composed of mixtures of phosphatidylcholine and phosphatidylethanolamine, both types of peptides associated with the fluid phase. While binding with high affinity to fluid membranes, peptides were unable to penetrate into the lipid membrane in the gel state. If trapped kinetically by cooling from the fluid phase, peptides dissociated from the gel membrane on the time scale of several hours. While the geometrical shape of the alpha-helical peptides determines their interactions with membranes with non-bilayer phase propensity, the shape complementarity mechanism by itself is unable to induce lateral phase separation in a fluid membrane. Charge-charge interactions are capable of inducing lateral domain formation in fluid membranes. Both peptides had affinity for anionic lipids which resulted in about 30% enrichment of acidic lipids within several nanometers of the peptide's tryptophan, but there was no long-range order in peptide-induced lipid demixing. Peptide insertion in fluid acidic membranes was accompanied by only a small increase in bilayer surface and a decrease in polarity in the membrane core. Peptide-lipid charge-charge interactions were also capable of modulating existing domain composition in the course of the main phase transition in mixtures of anionic phosphatidylglycerol with zwitterionic phosphatidylcholine.

Selective labeling of the inner liposome leaflet by fluorescent lipid probes, and studies of liposome fusion with influenza virus.

The inner leaflet of unilamellar lipid vesicles was labeled with fluorescent lysophosphatidylcholines. The probes make a donor-acceptor pair in resonance energy transfer (RET), being labeled with 9-anthrylvinyl (L-APC, donor) and 3-perylenoyl (L-PPC, acceptor) fluorophores. They migrate rapidly between bilayers through the water phase: tau 1/2 of equilibration is approximately 5 min at 37 degrees C. The probe(s) can be removed from the outer leaflet of uniformly labeled medium-size unilamellar vesicles (MUV) by repeated washings with excess unlabeled large unilamellar vesicles (LUV) (separation by centrifugation). The probes flip-flop across bilayers rather slowly. MUV containing the ganglioside GT1b and labeled with the L-APC/L-PPC pair in the inner leaflet were fused with an equal amount of influenza virus; the process was monitored by an increase of the donor fluorescence in RET assay. If inner MUV leaflet was labeled with the anthrylvinyl probe only, the probe fluorescence decreased by half when the probe was removed from the outer leaflets of the fused membranes. This shows that the lipids of the inner and outer leaflets of the MUV randomize in the process of fusion.

Polarized fluorescence and absorption spectroscopy of 1,32-dihydroxy-dotriacontane-bis-rhodamine 101 ester. A new and lipid bilayer-spanning probe.

We report on the properties of 1,32-dihydroxy-dotriacontane-bis-rhodamine 101 ester (Rh101C32Rh101) in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and in liquid solvents. The results are compared with those of rhodamine 101 octadecanyl ester (Rh101C18). Both molecules are solubilized in the lipid bilayer and the Rh101 moieties are anchored in the lipid-water interface, so that the electronic transition dipole moments (S 0 ↔S 1) are oriented preferentially in the plane of the bilayer. At low concentrations of the dyes in lipid bilayers of DOPC, the fluorescence relaxation is single exponential with a lifetime of τ=4.9±0.2 ns. The relative fluorescence quantum yield of ΦC32/ΦC18 ≈ 0.95 in DOPC vesicles. These results strongly suggest that only a small fraction of the Rh101C32Rh101 molecules are quenched, by, for example, intra- or intermolecular dimers in the ground state at mole fractions of less than 0.1% in the lipid bilayers. For Rh101C32Rh101 in lipid vesicles, the steady-state and time-resolved fluorescence anisotropies are compatible with efficient intramolecular electronic energy transfer. It is concluded that nearly every Rh101C32Rh101 molecule is spanning across the lipid bilayer of DOPC.

Determination of membrane-bound fragments of cytochrome P-450 2B4.

Membrane-bound sites of cytochrome P-450 2B4 (LM2) were determined by means of two different methods, photoactivated binding of membrane phospholipids to the protein and epitope mapping by antibodies. Phospholipids bearing photoreactive labels at different distances from the their polar 'head' were used in the former case. Phosphatidylcholine labelled at the apolar end of the fatty acid chain bound only to the N-terminal region of the hemoprotein. Other phospholipids labelled nearer to the head group bound not only to the N-terminus but also to the segments 273-314 and 427-491. Epitope mapping of the domain next to the N-terminus (residues 21-119) of the isolated hemoprotein was performed with the help of a peptide-scanning method, a programmable peptide synthesis on pins followed by ELISA testing with the polyclonal antiserum against cytochrome P-450 2B4. This domain was shown to possess a considerable density of sites with high antigenic activity. No membrane-penetrating part of this domain was found except for the fragment 1-21. A model of structure of P-450 2B4 was computed by comparison with the structure of cytochrome P-450cam on the basis of an alignment of 47 cytochromes P-450 with the former hemoprotein. Major parts of the protein sequences photoreacting with the phospholipid probes, but not the antibody-reactive epitopes of the region 21-119, are located at the membrane-facing side in this model.

Anthrylvinyl-labeled phospholipids as membrane probes: the phosphatidylcholine-phosphatidylethanolamine system.

The phase behavior of mixtures of phosphatidylcholine (PC) with phosphatidylethanolamine (PE) identical or differing in their fatty acid composition has been investigated by using the steady-state fluorescence anisotropy of anthrylvinyl-labeled PC and PE (APC and APE) as well as of the non-lipid probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to detect temperature-dependent changes in multilayer liposomes. APC, but not APE, was able to detect the pretransition of dimyristoyl-PC. The phospholipid probes APC and APE showed the main phase transition of their unlabeled disaturated analogues at temperatures almost identical with those revealed by differential scanning calorimetry, whereas the onset of the PE phase transition recorded by DPH was several degrees higher. In PC-PE mixtures with high content of PE the phase transitions shown by APC and APE were broader than those recorded by DPH. Comparison of phase diagrams constructed on the basis of fluorescence anisotropy and calorimetric data led to the conclusion that in biphasic PE and PC-PE systems DPH tends to partition into solid regions, whereas the anthrylvinyl-labeled phospholipids distribute more evenly between coexisting phases or prefer fluid domains. The use of anthrylvinyl phospholipid probes made it possible to demonstrate that PEs and PCs identical in their fatty acids are not miscible completely, not only below but also well above Tm of the higher melting component. Generally, APC and APE fluorescence anisotropy measurements correctly reflect headgroup-dependent phase segregations in mixtures of PC with PE, but may lead to ambiguous conclusions if demixing is caused by differences in the hydrocarbon chains.

Anthrylvinyl-labeled phospholipids as fluorescent membrane probes. The action of melittin on multilipid systems.

The interaction of melittin with multicomponent lipid mixtures composed of phosphatidylcholine, sphingomyelin and phosphatidylserine or phosphatidylglycerol was investigated by measuring the intrinsic fluorescence of the peptide, steady state fluorescence anisotropy of, and Trp-fluorescence energy transfer to fluorescent analogs of the same phospholipids bearing the anthrylvinyl fluorophore in one of the aliphatic chains at various distances from the polar head group. Based on the finding that at high lipid/peptide ratio the peptide induces unequal changes in the fluorescence parameters of phospholipid probes differing structurally only in their polar head groups, it is concluded that melittin induces lipid demixing in its nearest environment. Comparison of the fluorescence energy transfer from Trp to different lipid probes indicates that the depth of penetration of melittin into the bilayer depends on the polar head group composition of the phospholipid matrix and that certain segments of the melittin chain display a specific affinity for a given lipid head group.

Use of lipophilic fluorescent probes for the isolation of hybrid cells in flow cytometry.

Flow cytometry was used for the isolation of hybrid cells immediately after fusion. Precursor cells were stained by two lipophilic fluorescent probes: perylenoyl-labeled triglyceride (perylenoyl-TG, green fluorescence, 520 nm) and rhomdaminyl-labeled triglyceride (rhodaminyl-TG, red fluorescence, greater than 580 nm). Since the maximum emission of perylenoyl-TG coincides with the maximum absorbance of rhodaminyl-TG, the two fluorescent dyes form an effective donor-acceptor pair. Cells stained by perylenoyl-TG (0.25-1 microgram/ml) at the excitation wavelength of 457 nm displayed high intensity of fluorescence in the green region (520 nm), and low intensity of fluorescence in the red region (greater than 580 nm). Using the same conditions, cells that were stained by rhodaminyl-TG displayed a low intensity of fluorescence in both regions. When cells were simultaneously labeled by perylenoyl-TG and rhodaminyl-TG (used in a concentration ratio of 1:10, respectively) essentially total energy transfer was observed, and the cells exhibited a high intensity of red fluorescence. After the fusion of cells which had been separated stained by perylenoyl-TG and rhodaminyl-TG, the hybrid cells containing the two fluorescent probes had a high intensity of red fluorescence. Resonance exitation energy transfer between the two fluorescent dyes permits effective sorting of hybrid cells by flow cytofluorometry.

The interaction of prostaglandins with serum low-density lipoproteins.

The interaction of human serum low-density lipoproteins (LDL) with various types of prostaglandins (PG) was studied using equilibrium dialysis, steady-state fluorescence polarization spectroscopy and photolabeling methods. Low concentrations (10(-13)-10(-9) M) of PGE1 and PGF2 alpha were shown to induce specific rearrangements of the lipids on the LDL surface, whereas the closely related PGE2 and PGF1 alpha had no effect. With fluorescent labeled LDL, the PGE1-induced changes of the steady-state fluorescence polarization (P) were shown to be time- and concentration-dependent, saturable and reversible. However, equilibrium dialysis revealed a very low binding capacity of LDL for PGE1 (approx. 1 prostaglandin molecule per 600 LDL particles). Approximately the same PGE1 concentration was sufficient to cause maximal changes of P, to enhance the binding to apolipoprotein B of a photoreactive sphingomyelin analogue inserted into the LDL surface and to alter the thermal phase behavior of the LDL surface lipids. It is proposed that the LDL surface rearrangement caused by prostaglandins is due to the interaction of prostaglandins with apolipoprotein B, resulting in formation of short-lived complexes. The mechanism of this interaction is discussed in terms of the non-equilibrium ligand-receptor interaction model proposed earlier to explain the interaction of prostaglandins with high-density lipoproteins (Bergelson, L.D. et al. (1987) Biochim. Biophys. Acta 921, 182-190). It is suggested that direct prostaglandin-lipoprotein interactions may play a role in the homeostasis of cholesterol.