The interaction of 1-anilino-8-naphthalenesulfonate with lipids: A nuclear magnetic resonance study

The proton magnetic resonance (PMR) and phosphorus magnetic resonance (PhMR) spectra of egg phosphatidylcholine in the presence of 1-anilino-8-naphthalenesulfonate (ANS) have been studied. At low ratios of ANS to phospholipid, the spectra indicate that ANS molecules are in the lipid interface region where they interact with the head-group protons. ANS also penetrates into the hydrocarbon region to some extent. As the ANS/phospholipid ratio approaches one, a significant splitting of the head-group signal occurs. This splitting is associated with head-group signals from inner and outer molecules of the phospholipid vesicles. As the ANS/phospholipid ratio is further increased, a gel phase often occurs. The spectra for this gel phase suggest a highly mobile head-group. Further ANS addition results in a PMR spectrum suggestive of ANS—phospholipid micelle formation. The results for a phospholipid—cholesterol complex and for the total lipid extract from a cell membrane show that the ANS effect is more complicated in these cases.     

A study of carbachol-atropine interaction on intestinal smooth muscle vesicles, using a fluorescent probe.

Plasma membrane vesicles were prepared from guinea pig ileum longitudinal muscle. The vesicles were characterized by electron microscopy and analysis of lipid and protein content. They were shown to be free of gross contamination from actomyosin, sarcoplasmic reticulum, and mitochondria. 8-Anilino-1-naphthalene sulphonic acid (ANS) binding characteristics were similar to those found in other membranes. Both carbachol and atropine increased the fluorescence of ANS bound to this membrane, the maximum increase for atropine being greater than that for carbachol. Since neither drug effected the apparent affinity constant for the ANS-membrane interaction. It may be assumed that the increased fluorescence was due to an increase in the number of ANS binding sites. The carbachol-dependent increase in ANS fluorescence was blocked noncompetitively by atropine but not by tubocurarine or diphenhydramine. These latter two antagonists also increased ANS fluorescence but at much higher concentrations than either carbachol or atropine. Neither atropine nor carbachol increased ANS fluorescence on either erythrocyte ghosts or liposomes (prepared from a lipid extract of the muscle membrane).     

Reactions of fluorescent probes with normal and chemically modified myelin basic protein and proteolipid. Comparisons with myelin.

Basic (encephalitogenic) protein and water-soluble proteolipid apoprotein isolated from bovine brain myelin bind 8-anilino-1-naphthalenesulfonate and 2-p-toluidinylnaphthalene-6-sulfonate with resulting enhancement of dye fluorescence and a blue-shift of the emission spectrum. The dyes had a higher affinity and quantum yield, when bound to the proteolipid (Kans=2.3x10--6,=0.67) than to the basic protein (Kans=3.3x10--5,=0.40). From the efficiency of radiationless energy transfer from trytophan to bound ANS the intramolecular distances were calculated to be 17 and 27 A for the proteolipid and basic protein, respectively. Unlike myelin, incubation with proteolytic enzymes (e.g., Pronase and trypsin) abolished fluorescence enhancement of ANS or TNS by the extracted proteins. In contrast to myelin, the fluorescence of solutions of fluorescent probes plus proteolipid was reduced by Ca-2+,not affected by La-3+, local anesthetics, or polymyxin B, and only slightly increased by low pH or blockade of free carboxyl groups. The reactions of the basic protein were similar under these conditions except for a two- to threefold increase in dye binding in the presence of La-3+, or after blockade of carboxyl groups. N-Bromosuccinimide oxidation of tryptophan groups nearly abolished native protein fluorescence, but did not affect dye binding. However, alkylation of tryptophan groups of both proteins by 2-hydroxy (or methoxy)-5-nitrobenzyl bromide reduced the of bound ANS (excited at 380 nm) to 0.15 normal. The same effect was observed with human serum albumin. The fluorescence emission of ANS bound to myelin was not affected by alkylation of membrane tryptophan groups with the Koshland reagents, except for abolition of energy transfer from tryptophan to bound dye molecules. This suggests that dye binding to protein is negligible in the intact membrane. Proteolipid incorporated into lipid vesicles containing phosphatidylserine did not bind ANS or TNS unless Ca-2+, La-3+, polymyxin B, or local anesthetics were added to reduce the net negative surface potential of the lipid membranes. However, binding to protein in the lipid-protein vesicles remained less than for soluble protein. Basic protein or bovine serum albumin dye binding sites remained accessible after equilibration of these proteins with the same lipid vesicles. It is proposed that in the intact myelin membrane the proteolipid is probably strongly associated with specific anionic membrane lipids (i.e., phosphatidylserine), and most likely deeply embedded within the lipid hydrocarbon matrix of the myelin membrane. Also, in the intact myelin membrane the fluorescent probes are associated primarily, if not solely with the membrane lipids as indicated by the binding data. This is particularly the case for TNS where the total number of myelin binding sites is three to four times the potential protein binding sites.     

Molecular mechanism of general anesthesia: I. Fluorescence studies in mitochondrial membranes

We have tested the working hypothesis that anesthetics, by labilizing lipid-protein interactions, induce conformational changes in membrane proteins involved in the transmission of neural impulses. In the first communication of this series we report that general anesthetics induce changes in the fluorescence of the probes ANS and NPN in model membranes, lipid vesicles and mitochondria. The changes observed concern the quantumyield but not the position of the emission maximum. Such changes may be interpreted as due to fluidization of the membrane core (NPN), accompanied by variable effects in the membrane surface(ANS).     

Cell selectivity correlates with membrane-specific interactions: a case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and 31P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. 31P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.     

Mechanisms of apical protein sorting in polarized thyroid epithelial cells.

The process leading to thyroid hormone synthesis is vectorial and depends upon the polarized organization of the thyrocytes into the follicular unit. Thyrocyte membrane proteins are delivered to two distinct domains of the plasma membrane using apical (AP) and basolateral (BL) sorting signals. A recent hypothesis for AP sorting proposes that apically destined proteins cluster with glycosphingolipids (GSLs) and cholesterol, into microdomains (or rafts) of the Golgi membrane from which AP vesicles originate. In MDCK cells the human neurotrophin receptor, p75hNTR, is delivered to the AP surface through a sorting signal, rich in O-glycosylated sugars, identified in its ectodomain. We have investigated whether this signal is functional in the thyroid-derived FRT cell line and whether p75hNTR clusters into lipid rafts to be sorted to the AP membrane. We found that p75hNTR is apically delivered via a direct pathway and does not associate with rafts during its transport to the surface of FRT cells. Therefore, although the same signal could be recognized by different cell types thyroid cells may possess a tissue-specific sorting machinery.     

S100A13-lipid interactions-role in the non-classical release of the acidic fibroblast growth factor

S100A13 is a 98-amino acid, calcium binding protein. It is known to participate in the non-classical secretion of signal peptide-less proteins, such as the acidic fibroblast growth factor. In this study, we investigate the lipid binding properties of S10013 using a number of biophysical techniques, including multidimensional NMR spectroscopy. Isothermal titration calorimetry and steady state fluorescence experiments show that apoS100A13 exhibits preferential binding to small unilamelar vesicles of l-phosphatidyl serine (pS). In comparison, Ca2+-bound S100A13 is observed to bind weakly to unilamelar vesicles (SUVs) of pS. Equilibrium thermal unfolding and limited trypsin digestion analysis reveal that apoS100A13 is significantly destabilized upon binding to SUVs of pS. Results of the far UV circular dichroism and ANS (8-anilino-1-napthalene sufonate) binding experiments indicate a subtle conformational change resulting in the increase in the solvent-accessible hydrophobic surface in the protein. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 facilitates its interaction with the lipid vesicles. Our data suggest that Ca2+ binding dictates the membrane binding affinity of S100A13. Based on the results of this study, a model describing the sequence of molecular events that possibly can occur during the non-classical secretion of FGF-1 is presented.     

Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and ³¹P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. ³¹P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.     

Lipid protein interactions in mitochondria. Spin and fluorescence probe studies on the effect of n-alkanols on phospholipid vesicles and mitochondrial membranes.

The effect of n-butanol on the mobility of phospholipids in phospholipid vesicles and beef heart mitochondrial membranes has been studied using three stearic acid spin labels having a paramagnetic doxyl group in positions 5,12, and 16, respectively, and the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS). The mobility of the spin labels in the phospholipid aliphatic chains increases from the polar heads toward the methyl groups both in vesicles and in mitochondrial membranes; however, in the latter there is a higher constriction of rotational mobility observed at all levels in the lipid bilayer. Butanol determines a moderate increase in mobility of phospholipids in lipid vesicles, but the effect is more striking in the mitochondrial membranes, where the protein-induced constraint of mobility of the fatty acyl chains is removed at low concentrations of the alcohol. Butanol also enhances the mobility of tightly bound phospholipids residual in lipid-depleted mitochondrial preparations, although higher concentrations of butanol are required for this effect. The effect of the series of aliphatic n-alcohols is related to their hydrophobicity.Alcohols induce a decrease of the fluorescence of ANS bound to both lipid vesicles and mitochondrial membranes. The fluorescence decrease is not the result of a decreased partition of ANS from the aqueous medium to the bilayer, but depends upon a change in the chromophore environment. Since no shift of the emission maximum is observed after alcohol addition, such a change must be ascribed to increased mobility of the probe, in accord with the spin label data.As for the spin label data, the effect of the series of aliphatic n-alcohols is related to their hydrophobicity; at difference with the electron spin resonance results, however, the effects are maximal for pure phospholipid vesicles. It is calculated that alcohols affect both the long-range interactions between phospholipids and proteins in mitochondrial membranes (as detected by spin labels) and the order of phospholipid bilayers near the glycerol region (as detected by ANS). The differences between the two kinds of probes may be related to their differing localization in the lipid bilayer.     

Change in membrane fluidity induced by lectin-mediated phase separation of the membrane and agglutination of phospholipid vesicles containing glycopeptides

Changes in membrane fluidity induced by lectin addition to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles containing synthetic glycopeptides were measured by depolarization of the fluorescent probes 8-anilino-1-naphthalenesulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH). In the present synthesized glycopeptides, N-acetylglucosamine (GlcNAc) and a tripeptide were connected by aliphatic chains of different lengths. A pyrenyl group, which is introduced to the peptide moiety, acted as a probe to characterize the distribution of glycopeptides in the membrane on the basis of its excimer formation. The glycopeptide was shown to be distributed to DPPC vesicles with the peptide moiety buried in the hydrophobic core of the lipid bilayer and the glyco moiety exposed to the outside of the membrane. By the addition of wheat germ agglutinin (WGA) to the vesicles containing the glycopeptides, intravesicular cross-linking of glycopeptides in the membrane and aggregation of vesicles were observed. The intravesicular cross-linking was antagonized by GlcNAc above the phase transition temperature. However, the dissociation of aggregation required the addition of a stronger antagonist, N,N'-diacetylchitobiose. The addition of the glycopeptide to DPPC vesicles above the phase transition temperature decreased the membrane fluidity. However, a succeeding addition of WGA caused a large increase of membrane fluidity at either the surface or the hydrophobic core of the lipid bilayer membrane. This increase of membrane fluidity was attributed to two factors by use of two kinds of antagonists having different potencies: one is a WGA-mediated cross-linking of glycopeptides in the membrane, and the other is a close contact of vesicles on aggregation.     

S100A13-lipid interactions—role in the non-classical release of the acidic fibroblast growth factor

S100A13 is a 98-amino acid, calcium binding protein. It is known to participate in the non-classical secretion of signal peptide-less proteins, such as the acidic fibroblast growth factor. In this study, we investigate the lipid binding properties of S10013 using a number of biophysical techniques, including multidimensional NMR spectroscopy. Isothermal titration calorimetry and steady state fluorescence experiments show that apoS100A13 exhibits preferential binding to small unilamelar vesicles of l-phosphatidyl serine (pS). In comparison, Ca²⁺-bound S100A13 is observed to bind weakly to unilamelar vesicles (SUVs) of pS. Equilibrium thermal unfolding and limited trypsin digestion analysis reveal that apoS100A13 is significantly destabilized upon binding to SUVs of pS. Results of the far UV circular dichroism and ANS (8-anilino-1-napthalene sufonate) binding experiments indicate a subtle conformational change resulting in the increase in the solvent-accessible hydrophobic surface in the protein. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 facilitates its interaction with the lipid vesicles. Our data suggest that Ca²⁺ binding dictates the membrane binding affinity of S100A13. Based on the results of this study, a model describing the sequence of molecular events that possibly can occur during the non-classical secretion of FGF-1 is presented.     

Effect of ionic strength on the membrane fluidity of rabbit intestinal brush-border membranes. A fluorescence probe study

The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles (out greater than in) with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47 degrees C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density.     

Tryptophan fluorescence study on the interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with model membranes

The interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with different phospholipid vesicles was investigated by fluorescence techniques, using a synthetic mutant signal peptide in which valine at position -8 in the hydrophobic sequence was replaced by tryptophan. First it was established that this mutation in the signal sequence of prePhoE does not affect in vivo and in vitro translocation efficiency and that the biophysical properties of the synthetic mutant signal peptide are similar to those of the wild-type signal peptide. Next, fluorescence experiments were performed which showed an increase in quantum yield and a blue shift of the emission wavelength maximum upon interaction of the signal peptide with lipid vesicles, indicating that the tryptophan moiety enters a more hydrophobic environment. These changes in intrinsic fluorescence were found to be more pronounced in the presence of phosphatidylglycerol (PG) or cardiolipin (CL) than with phosphatidylcholine (PC). In addition, quenching experiments demonstrated a shielding of the tryptophan fluorescence from quenching by the aqueous quenchers iodide and acrylamide upon interaction of the signal peptide with lipid vesicles, a shielding in the case of acrylamide that was more pronounced in the presence of negatively charged lipids. Finally it was found that acyl chain brominated lipids incorporated into phospholipid bilayers were able to quench the tryptophan fluorescence of the signal peptide, with the quenching efficiency in CL vesicles being much higher than in PC vesicles. The results clearly demonstrate that the PhoE signal peptide interacts strongly with different lipid vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of chlorpromazine HCl on the structural parameters of bovine brain membranes

Fluorescence probes located in different membrane regions were used to evaluate the effects of chlorpromazine .HCl on structural parameters (transbilayer lateral mobility, annular lipid fluidity, protein distribution, and lipid bilayer thickness) of synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortex. The experimental procedure was based on the selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) by trinitrophenyl groups, radiationless energy transfer from the tryptophan of membrane proteins to Py-3-Py, and energy transfer from Py-3-Py monomers to 1-anilinonaphthalene-8-sulfonic acid (ANS). In this study, chlorpromazine .HCl decreased the lateral mobility of Py-3-Py in a concentration dependent-manner, showed a greater ordering effect on the inner monolayer than on the outer monolayer, decreased annular lipid fluidity in a dose dependent-manner, and contracted the membrane lipid bilayer. Furthermore, the drug was found to have a clustering effect on membrane proteins.     

Temperature effects on 1,8-anilinonaphthalene sulfonic acid fluorescence with sarcolemma vesicles.

Increased temperature produces a red shift and decreased fluorescence intensity of the emission peak of 1,8-anilinonaphthalene sulfonic acid (ANS) in suspensions of biomembrane vesicles. These changes have been attributed to a conjectured increase in polarity of the microenvironment of ANS. If the conjecture is correct, fluorescence lifetimes must be decreased with warming. We showed than ANS binds to both protein and lipid protein of sarcolemma, that there are two kinds of sarcolemma-lipid ANS-binding sites, and that there are three fluorescence lifetimes of excited sarcolemma-bound ANS. The three fluroescence lifetimes were unchanged on warming, or decreased too little to account for the observations. Fluorescence lifetime data were consistent with the notion that the effect of increasing temperature is to decrease the amount of ANS bound to sarcolemma. From studies of liposomes prepared from lipid extracts of sarcolemma, and of proteins from sarcolemma it was deduced that warming reducted the amount of ANS bound to both of these sarcolemma components, probably mainly by reducing binding capacity. There might also be a shift of affinities such that the ratio, KA sarcolemma lipid/KA sarcolemma protein, is larger at higher temperature. Except at very small concentration ratios of ANS/sarcolemma, more than twice as much ANS was bound to sarcolemma lipids as to proteins.     

Aggregation and hemi-fusion of anionic vesicles induced by the antimicrobial peptide cryptdin-4

We show that cryptdin-4 (Crp4), an antimicrobial peptide found in mice, induces the aggregation and hemi-fusion of charged phospholipid vesicles constructed of the anionic lipid POPG and the zwitterionic lipid POPC. Hemi-fusion is confirmed with positive total lipid-mixing assay results, negative inner monolayer lipid-mixing assay results, and negative results from contents-mixing assays. Aggregation, as quantified by absorbance and dynamic light scattering, is self-limiting, creating finite-sized vesicle assemblies. The rate limiting step in the formation process is the mixing of juxtaposed membrane leaflets, which is regulated by bound peptide concentration as well as vesicle radius (with larger vesicles less prone to hemi-fusion). Bound peptide concentration is readily controlled by total peptide concentration and the fraction of anionic lipid in the vesicles. As little as 1% PEGylated lipid significantly reduces aggregate size by providing a steric barrier for membrane apposition. Finally, as stable hemi-fusion is a rare occurrence, we compare properties of Crp4 to those of many peptides known to induce complete fusion and lend insight into conditions necessary for this unusual type of membrane merger.     

Effect of ionic strength on the membrane fluidity of rabbit intestinal brush-border membranes: A fluorescence probe study

The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles ($\text{out > in}$) with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47°C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density.     

Aggregation and hemi-fusion of anionic vesicles induced by the antimicrobial peptide cryptdin-4

We show that cryptdin-4 (Crp4), an antimicrobial peptide found in mice, induces the aggregation and hemi-fusion of charged phospholipid vesicles constructed of the anionic lipid POPG and the zwitterionic lipid POPC. Hemi-fusion is confirmed with positive total lipid-mixing assay results, negative inner monolayer lipid-mixing assay results, and negative results from contents-mixing assays. Aggregation, as quantified by absorbance and dynamic light scattering, is self-limiting, creating finite-sized vesicle assemblies. The rate limiting step in the formation process is the mixing of juxtaposed membrane leaflets, which is regulated by bound peptide concentration as well as vesicle radius (with larger vesicles less prone to hemi-fusion). Bound peptide concentration is readily controlled by total peptide concentration and the fraction of anionic lipid in the vesicles. As little as 1% PEGylated lipid significantly reduces aggregate size by providing a steric barrier for membrane apposition. Finally, as stable hemi-fusion is a rare occurrence, we compare properties of Crp4 to those of many peptides known to induce complete fusion and lend insight into conditions necessary for this unusual type of membrane merger.     

Protein translocation into Escherichia coli membrane vesicles is inhibited by functional synthetic signal peptides

A synthetic peptide corresponding to the signal sequence of wild type Escherichia coli lambda-receptor protein (LamB) inhibits in vitro translocation of precursors of both alkaline phosphatase and outer membrane protein A into E. coli membrane vesicles (half-maximal inhibition at 1-2 microM). By contrast, the inhibitory effect was nearly absent in a synthetic peptide corresponding to the signal sequence from a mutant strain that harbors a deletion mutation in the LamB signal region and displays an export-defective phenotype for this protein in vivo. Two peptides derived from pseudorevertant strains that arose from the deletion mutant and exported LamB in vivo were found to inhibit in vitro translocation with effectiveness that correlated with their in vivo export ability. Controls indicated that these synthetic signal peptides did not disrupt the E. coli membrane vesicles. These results can be interpreted to indicate that the presequences of exported proteins interact specifically with a receptor either in the E. coli inner membrane or in the cytoplasmic fraction. However, biophysical data for the family of signal peptides studied here reveal that they will spontaneously insert into a lipid membrane at concentrations comparable to those that cause inhibition. Hence, an indirect effect mediated by the lipid bilayer of the membrane must be considered.     

Molecular events associated with Macrovipera lebetina obtusa and Montivipera raddei venom intoxication and condition of biomembranes

Studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including BLMs, SUVs and GUVs. The present study was undertaken to elucidate how the plastic properties (namely, its microviscosity, thickness, permeability) of model membranes from native lipids of different tissues of rats change in the course of Macrovipera lebetina obtusa (MLO), Montivipera raddei (MR) and Naja kaouthia (NK) venoms processing. The presence of viper venom in organism leads to increasing of the electrical resistance of BLMs from liver and muscle lipids approximately on a sequence, while the BLMs from brain lipids have not shown noticeable differences of plastic properties compared to the control. Giant unilamellar vesicles (GUVs) with a mean diameter of 30μm have a minimum curvature and mimic cell membranes in this respect. Snake venom was added to the sample chamber before the vesicles were formed. The membrane fluorescence probes, ANS and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. Fluorescent spectra were acquired on a Varian fluorometer instrument. ANS and pyrene allow us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. The presence of viper venom in GUVs media reveals a noticeable decreasing of membrane fluidity compared to the control, while the binding of fluorophores with GUVs modified by venom leads to the appearance of channel activity. These studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom.