A stopped-flow kinetic study of the interaction of potential-sensitive oxonol dyes with lipid vesicles

The interaction of the dyes oxonol V and oxonol VI with unilamellar dioleoylphosphatidylcholine vesicles was investigated using a fluorescence stopped-flow technique. On mixing with the vesicles, both dyes exhibit an increase in their fluorescence, which occurs in two phases. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid phase appears to be due to a second-order binding of the dye to the lipid membrane, which is very close to being diffusion-controlled. The slow phase is almost independent of vesicle concentration, and it is suggested that this may be due to a change in dye conformation or position within the membrane, possibly diffusion across the membrane to the internal monolayer. The response times of the dyes to a rapid jump in the membrane potential has also been investigated. Oxonol VI was found to respond to the potential change in less than 1 s, whereas oxonol required several minutes. This has been attributed to lower mobility of oxonol V within the lipid membrane.     

Calcium-dependent and calcium-independent interactions of prothrombin fragment 1 with phosphatidylglycerol/phosphatidylcholine unilamellar vesicles

We have measured the phase behavior of mixed dipentadecanoylphosphatidylglycerol (DC15PG)/dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUV) in the presence of saturating (greater than 98% occupancy of binding sites) concentrations of bovine prothrombin fragment 1 and 5 mM Ca2+. Binding of fragment 1 in the presence of Ca2+ was verified by an increase in 90 degrees light scattering. Only in the cases of DC15PG/DMPC SUV below their phase transition and of pure DMPC SUV were such light scattering measurements not reversible upon addition of ethylenediaminetetraacetic acid to complex Ca2+. Phase-behavior changes of DC15PG/DMPC SUV as monitored by diphenylhexatriene fluorescence anisotropy occurred in concert with the binding of fragment 1. The major effects of peptide binding on SUV phase behavior were to raise the phase-transition temperature by 2-15 degrees C, depending on vesicle composition, and, in general, to make the phase diagram for these small vesicles closely resemble that of large vesicles. No evidence was obtained for the existence of lateral membrane domains with distinct compositions induced by the binding of prothrombin fragment 1 plus Ca2+. Surprisingly, fragment 1 without Ca2+ also altered the phase behavior of DC15PG/DMPC SUV. Most striking was the effect of fragment 1 (with or without Ca2+) on DMPC SUV phase behavior. Freeze-fracture electron microscopy demonstrated that pure DMPC vesicles were induced to fuse in the presence of fragment 1, while vesicles containing DC15PG remained intact. The rate of DMPC SUV fusion (followed by 90 degrees light scattering) increased with increasing fragment 1 concentration but was not saturable up to 40 microM fragment 1, suggesting a weak, nonspecific interaction between fragment 1 and the neutral phospholipid vesicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature dependent intermediate structures during the main phase transition of dimyristoyl phosphatidylcholine vesicles a combined iodine laser-temperature jump and time resolved cryo-electron microscopy study

The kinetics of the main phase transition of dimyristoylphosphatidyl choline (DMPC) unilamellar vesicles were investigated in the time range from microseconds to seconds. Iodine laser-temperature jump (ILTJ) experiments showed three discrete relaxation phenomena. Time resolved cryo-electron microscopy (CEM) was applied to produce images of intermediate states typical for the relaxation times of lipid vesicles in the micro- to millisecond time window. A careful measurement of the rate of temperature decrease observed during the production of vitrified lamellae of aqueous samples on a copper grid was performed. The best conditions resulted in average rates of cooling of 3 x 10(4) K/s. By comparing the images from CEM of DMPC vesicle samples vitrified above, at, and below the phase transition temperature a structural model was designed, which explains the temperature jump relaxation times in the micro- to millisecond time range by the formation and disappearance of coexisting clusters of crystalline, intermediate, and fluid lipid areas inside the DMPC bilayers.     

The binding of a neutral aromatic molecule to a negatively-charged lipid membrane. II. Kinetics and mechanism

The kinetics of the binding of the fluorescence indicator N-phenyl naphthylamine to bilayer vesicles of C12-methyl-phosphatidic acid have been investigated by means of the temperature-jump relaxation technique utilizing fluorescence light detection. Single-exponential relaxation curves were observed, with time constants in the range 0.2-3 ms. The concentration dependence of the relaxation time yielded an apparent association rate constant (expressed in terms of monomeric phospholipid) of k(on) = 5 x 10(6) M(-1) s(-1) in aqueous solution at 25 degrees . The activation energy and viscosity dependence associated with the binding rate show that this process is actually diffusion-controlled. The theory of diffusion-controlled reactions then allows a determination of the average size of the bilayer vesicles and of the true rate constant for the association of the indicator molecules with the vesicles. Assuming spherical geometry for the vesicles, the values are: r(ves) = 190 A, which corresponds to 20000 lipid molecules per vesicle and k'(on) = 1 x 10(11) M(-1) s(-1) (25 degrees). The correctness of this size-determination was confirmed semi-quantitatively by electron microscopy. Since in fact a distribution of vesicle sizes must be present, a discussion is included of the relaxation function which the system is expected to take in the general case. Biological implications of diffusion control for the transport of non-polar substances and for lipid mixing are indicated.     

Interaction of dextran sulfate with phospholipid surfaces and liposome aggregation and fusion

The binding of dextran sulfate to phospholipid liposomes was investigated by microelectrophoresis experiments. The polyanion binds to neutral phospholipid liposomes (DMPC and PE) only in the presence of Ca2+. If positively charged stearylamine is incorporated in the vesicles dextran sulfate is bound without Ca2+. Negatively charged phospholipids as PS do not bind dextran sulfate, even in the presence of millimolar concentrations of Ca2+. The adsorption of dextran sulfate results in an aggregation of vesicles due to a bridging mechanism. In all cases the aggregation is followed by a disaggregation toward higher dextran sulfate concentrations. The disaggregation process starts at polymer concentrations smaller than the concentration of the onset of saturation of the adsorption. By use of the probe dilution method a fusion of small DMPC and DMPC/PE vesicles in the presence of Ca2+ and dextran sulfate was found.     

A membrane-bound fluorescent probe to detect phospholipid vesicle-cell fusion

Summary Pyrenesulfonylphosphatidylethanolamine has been incorporated into sonicated phospholipid vesicles to provide a fluorescent signal from a membrane-bound probe whose spectrum is sensitive to the local concentration of dye molecules. When vesicle material was taken up by viable mouse splenocytes, the disappearance of the pyrene excimer fluorescence emission peak that accompanied dilution of the vesicle membrane lipid could be quantitated. One can thus measure, by a simple and rapid procedure, a new parameter which is related to the extent of vesicle-cell fusion and which is independent of the transfer of aqueous vesicle contents to the cell cytoplasm.Abbreviations used 6-CF 6-carboxyfluorescein - HBSS Hanks Balanced Salt Solution - DMPC dimyristoylphosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - EYL egg yolk lecithin - PE phosphatidylethanolamine - PEG polyethylene glycol - PLV phospholipid vesicle - PSPE pyrenesulfonylphosphatidylethanolamine     

Use of the fluorescent weak acid dansylglycine to measure transmembrane proton concentration gradients

The amphiphilic fluorescent dye N-[(5-dimethylamino)naphth-1-ylsulfonyl]glycine (dansylglycine) can be used to monitor the magnitude and stability of transmembrane proton gradients. Although freely soluble in aqueous media, the dye readily adsorbs to the surfaces of lipid vesicles. Because membrane-bound dye fluoresces at a higher frequency, and with greater efficiency, than dye in aqueous solution, it is easy to isolate the fluorescence emission from those dye molecules adsorbed to the lipid surface. When dansylglycine is mixed with phospholipid vesicles, the dye molecules attain a partition equilibrium between buffer and the outer, proximal surface of the vesicles. This is a rapid, diffusion-limited process that is indicated by a fast phase of fluorescence intensity increase monitored at 510 nm. In a second step, the inner, distal surface of each vesicle becomes populated with dye, a process that involves permeation through the lipid bilayer and that is generally much slower than the original adsorption step. Dansylglycine is a weak acid that permeates as an electrically neutral species; the flux of dye across the bilayer is thus strongly dependent on the degree of protonation of the dye's carboxylate moiety. When the external pH is lower than that of the vesicle lumen, the inward flux of dye is greater than that in the opposite direction, and dye accumulates in the lumen. This leads to a local elevation of dansylglycine concentration in the inner membrane monolayer, which in turn results in an elevated fluorescence intensity proportional to the membrane pH gradient.     

Alpha-lactalbumin binding to membranes: evidence for a partially buried protein

The self-incorporation of apo-alpha-lactalbumin (alpha-LA) into single unilamellar vesicles (SUV) of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine was demonstrated by column chromatographic analyses on Sephadex G-200 (10 mM Tris-HCl, pH 7.4, 26-28 degrees C) and by intrinsic fluorescence emission of SUV-bound alpha-LA. It was shown that apo-alpha-LA slowly incorporated into the DMPC vesicle bilayer after equilibrating different mixtures of protein and SUV for several hours. The intrinsic fluorescence properties of the bound apo-alpha-LA were altered only slightly (lambda maxem = 333 nm vs. 337 nm in aqueous solution). The large blue shift in apo-alpha-LA fluorescence in solution induced by monovalent cations, such as Na(I), was almost completely prevented when apo-alpha-LA was membrane bound. Furthermore, the addition of calcium caused a slow conversion from apo-alpha-LA to Ca(II)-alpha-LA by a mechanism consistent with passive diffusion of Ca(II) into the bilayer interior to the (buried) calcium binding site. The release of Ca(II)-alpha-LA from the membrane is discussed with reference to alpha-LA release from the smooth endoplasmic reticulum in vivo.     

Effect of proteins on fluorophore lifetime heterogeneity in lipid bilayers.

The effect of three different membrane proteins on the fluorescence lifetime heterogeneity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in phospholipid vesicle systems was investigated. For large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) at 37 degrees C, the fluorescence decay was essentially monoexponential (8.6 and 8.2 ns, respectively) except for a minor component typical of DPH. For gramicidin D reconstituted into DMPC vesicles at a protein/lipid molar ratio of 1/7, the most appropriate analysis of the data was found to be in the form of a bimodal Lorentzian distribution. Centers of the major lifetime components were almost identical with those recovered for vesicles without proteins, while broad distributional widths of some 4.0 ns were recovered. Variation of the protein/lipid molar ratio in sonicated POPC vesicles revealed an abrupt increase in distributional width at ratios approximating 1/15-1/20, which leveled off at about 2.5 ns. For bacteriorhodopsin in DMPC vesicles and cytochrome b5 in POPC, the most appropriate analysis of the data was again found to be in the form of a bimodal Lorentzian also with broad distributional widths in the major component. Lifetime centers were decreased for these proteins due to fluorescence energy transfer to the retinal of the bacteriorhodopsin and heme of the cytochrome b5. Fluorescence energy transfer is distance dependent, and since a range of donor-acceptor distances would be expected in a membrane, lifetime distributions should therefore be recovered independently of other effects for proteins possessing acceptor chromophores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of biocidal guanidine hydrochloride polymer analogs with model membranes: a comparative biophysical study

Four synthesized biocidal guanidine hydrochloride polymers with different alkyl chain length, including polyhexamethylene guanidine hydrochloride and its three new analogs, were used to investigate their interactions with phospholipids vesicles mimicking bacterial membrane. Characterization was conducted by using fluorescence dye leakage, isothermal titration calorimetry, and differential scanning calorimetry. The results showed that the gradually lengthened alkyl chain of the polymer increased the biocidal activity, accompanied with the increased dye leakage rate and the increased binding constant and energy change value of polymer-membrane interaction. The polymer-membrane interaction induced the change of pretransition and main phase transition (decreased temperature and increased width) of phospholipids vesicles, suggesting the conformational change in the phospholipids headgroups and disordering in the hydrophobic regions of lipid membranes. The above information revealed that the membrane disruption actions of guanidine hydrochloride polymers are the results of the polymer's strong binding to the phospholipids membrane and the subsequent perturbations of the polar headgroups and hydrophobic core region of the phospholipids membrane. The alkyl chain structure significantly affects the binding constant and energy change value of the polymer-membrane interactions and the perturbation extent of the phospholipids membrane, which lead to the different biocidal activity of the polymer analogs. This work provides important information about the membrane disruption action mechanism of biocidal guanidine hydrochloride polymers.     

Interaction of sphingomyelins and phosphatidylcholines with fluorescent dehydroergosterol

The fluorescent sterol dehydroergosterol was used as a cholesterol analogue in conjunction with multifrequency phase and modulation (1-250 MHz) fluorometry to examine whether sterols (1) interact preferentially with fluid- or solid-phase phospholipids and (2) interact preferentially with sphingomyelin in phase-separated or phase-miscible cosonicated phospholipid membranes. Cosonicated small unilamellar vesicles (SUV) were produced by mixing lipids in organic solvents, drying the mixture, adding buffer, sonicating, and separating SUV. Phospholipids of synthetic as well as biological origin were utilized. In phase-separated, cosonicated SUV of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC, 1:1 molar ratio), the fluorescent sterol (0.5 mol %) interacted preferentially with the fluid-phase lipid (partition coefficient, Kf/s = 2.6-3.4) according to four criteria. First, dehydroergosterol detected only the phase transition of DMPC, the phospholipid with the lower phase transition temperature. Second, the dehydroergosterol fluorescence polarization, limiting anisotropy, order parameter, and rotational relaxation time in the cosonicated vesicle were similar to those of dehydroergosterol in SUV composed only of DMPC. Third, the number of dehydroergosterol fluorescence lifetime components as well as the distribution in the cosonicated SUV was similar to that of dehydroergosterol in SUV composed of DMPC. Fourth, dehydroergosterol concentration-dependent self-quenching was detected in DSPC SUV at much lower dehydroergosterol concentration than in DMPC SUV. Preference of dehydroergosterol for fluid-phase lipids was also observed by monitoring dehydroergosterol exchange between individually sonicated DMPC SUV and DSPC SUV after the two types of vesicles were mixed in equal proportions. In these SUV mixtures, the dehydroergosterol also partitioned into the more fluid SUV, 99:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Video fluorescence microscopy studies of phospholipid vesicle fusion with a planar phospholipid membrane. Nature of membrane-membrane interactions and detection of release of contents

Video fluorescence microscopy was used to study adsorption and fusion of unilamellar phospholipid vesicles to solvent-free planar bilayer membranes. Large unilamellar vesicles (2-10 microns diam) were loaded with 200 mM of the membrane-impermeant fluorescent dye calcein. Vesicles were ejected from a pipette brought to within 10 microns of the planar membrane, thereby minimizing background fluorescence and diffusion times through the unstirred layer. Vesicle binding to the planar membrane reached a maximum at 20 mM calcium. The vesicles fused when they were osmotically swollen by dissipating a KCl gradient across the vesicular membrane with the channel-forming antibiotic nystatin or, alternatively, by making the cis compartment hyperosmotic. Osmotically induced ruptures appeared as bright flashes of light that lasted several video fields (each 1/60 s). Flashes of light, and therefore swelling, occurred only when channels were present in the vesicular membrane. The flashes were observed when nystatin was added to the cis compartment but not when added to the trans. This demonstrates that the vesicular and planar membranes remain individual bilayers in the region of contact, rather than melding into a single bilayer. Measurements of flash duration in the presence of cobalt (a quencher of calcein fluorescence) were used to determine the side of the planar membrane to which dye was released. In the presence of 20 mM calcium, 50% of the vesicle ruptures were found to result in fusion with the planar membrane. In 100 mM calcium, nearly 70% of the vesicle ruptures resulted in fusion. The methods of this study can be used to increase significantly the efficiency of reconstitution of channels into planar membranes by fusion techniques.     

Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes.

The fusion of sea urchin egg secretory vesicles to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescent microscopy, in combination with electrical recordings of membrane conductance. A strong binding of vesicles to protein-free planar membranes was observed in the absence of calcium. Calcium-induced fusion of vesicles was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy: and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a dye. In both cases, no increase in the membrane conductance was observed unless vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles are devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion. Fusion was also detected when vesicles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane (native cortices) were applied to a phospholipid membrane, despite good binding of vesicles to the planar membrane and fusion of vesicles to plasma membrane. It is suggested that cortical vesicles (CVs) have sufficient calcium-sensitive proteins for fusion to lipid membranes, but in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites on vesicles access to new membranes.     

Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes

The fusion of sea urchin egg secretory vesicles to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescent microscopy, in combination with electrical recordings of membrane conductance. A strong binding of vesicles to protein-free planar membranes was observed in the absence of calcium. Calciuminduced fusion of vesicles was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy; and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a dye. In both cases, no increase in the membrane conductance was observed unless vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles are devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion. Fusion was also detected when vesicles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane (native cortices) were applied to a phospholipid membrane, despite good binding of vesicles to the planar membrane and fusion of vesicles to plasma membrane. It is suggested that cortical vesicles (CVs) have sufficient calcium-sensitive proteins for fusion to lipid membranes, but in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites on vesicles access to new membranes.     

Effect of membrane association on the stability of complexes between ionophore A23187 and monovalent cations

The monovalent cation complexation properties of ionophore A23187 in methanol-water (65-95% w/w) and bound to unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) are contrasted. In both solution and vesicle-containing systems, 1:1 complexes between the ionophore and Li+ or Na+ predominate. The analogous complexes with K+, Rb+, and Cs+, which exist in methanol, are not detected on DMPC vesicles by changes in the absorption or fluorescence emission spectra of the ionophore. In solution, the logarithms of stability constants (log KMA) for both the LiA and NaA complexes increase by 1.5 units over the range of solvent polarity encompassed by 65% methanol-water to methanol. Selectivity for Li+ vs. Na+ is constant at a ratio of 5 in these solutions. On DMPC vesicles, selectivity for Li+ vs. Na+ is improved 15-fold with log KbLiA (3.23 +/- 0.03, T = 25 degrees C, mu = 0.05 M) being comparable to the value obtained in 80% methanol-water. In the latter solvent, increasing ionic strength (0.005-0.085 M) has little effect on log KLiA or log KHA but increases these constants by 0.4-0.5 unit in the DMPC vesicle system. Transition from the vesicle liquid-crystalline to gel-phase state reduces log KbLiA and log KbNaA by approximately 0.6 unit but has no effect on log KbHA. Thermodynamic parameters for formation of HA, LiA, and NaA in 80% methanol-water and on DMPC vesicles are reported. Analysis of these data and related considerations suggests that differences in the membrane interaction energies of particular ionophore species dominate in establishing the observed difference in complexation properties between the solution and vesicle-containing systems.     

Anthracenyl crown ethers and cryptands as fluorescent probes for solid-phase transitions of phosphatidylcholines: syntheses and phospholipid membrane studies

Three structurally related crown compounds and cryptands have been synthesized that differ by the number and linkage of coronand units and anthracene moieties. The interaction of the fluorescent dyes with sonicated dimyristoylphosphatidylcholine (DMPC) vesicles is characterized by the relative quantum yields, uptake kinetics, binding curves, lifetimes, fluorescence titrations with water- and lipid-soluble quenching agents, fluorescence anisotropy, and equilibrium cooling curves. The most lipophilic compound II, which displays a similar quantum yield as the parent fluorophore 9,10-dimethylanthracene, shows a nearly equal distribution between solid and fluid lipid and is located at the bilayer surface. The least lipophilic compound IV is excluded from the hydrocarbon phase. The anthracenophane cryptand III preferentially partitions into solid-phase lecithins with the highest affinity for the phases L epsilon and L beta. The binding constant to DMPC amounts to (5.4 +/- 1.3) X 10(2) M-1 at 0 degrees C. From fluorescence quenching titrations it is concluded that the average position of the anthracenoyl dye III discontinuously shifts during the gel to liquid crystalline transition from the glycerol backbone to the choline head group. Electron microscopy and NMR experiments revealed that the anthracenophane induces in the liquid crystalline phase the fusion of small unilamellar DMPC vesicles to unilamellar medium-sized vesicles and macrovesicles, which subsequently fuse at the transition temperature to large multilamellar coacervates. Due to its large change of fluorescence intensity, the anthracenophane cryptand is a very sensitive probe for the detection of the pretransition of symmetrically substituted and of the subtransition of asymmetrically substituted phosphatidylcholines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between hydroxystilbamidine and DNA. II. Temperature jump relaxation study. Dynamics of nucleic acids and polynucleotides.

A temperature-jump relaxation study of the interaction of hydroxystilbamidine with DNA and synthetic polynucleotides has been performed. Two concentration dependent relaxation times tau1 and tau2 have been observed in the submillisecond range when detecting relaxation effects by means of light absorption. The longer of these two times (tau1) is also observed when using "blue" or "red" fluorescence detection. In the longer time scale the "red" fluorescence shows no other relaxation but the blue fluorescence shows two additional relaxation processes (tau3 and tau4) which correspond to an increase of fluorescence with temperature and which are independent of concentration. The experimental results clearly indicate that tau1 and tau2 are associated with the binding of the dye to strong and weak binding sites, respectively. A kinetic model is given to explain the results. It allows the determination of the four rate constants for the two binding reactions and yields equilibrium association constants in good agreement with those obtained from stoichiometric studies. The study of the effect of temperature, nature of the polymer, ionic strength and fraction of bound dye on tau3 and tau4 indicates that the dye acts only as a "blue" fluorescence probe of some processes involving the DNA or polynucleotide alone. These processes appear to be related with the dynamic structure of the polymers.     

Interaction of Salmonella typhimurium with phospholipid vesicles. Incorporation of exogenous lipids into intact cells.

Incubation of intact cells of Salmonella typhimurium with bilayer phospholipid vesicles results in significant transfer of vesicle lipids to the cells. The transfer requires Ca2+ or spermine, and is dependent on time, temperature, the concentration and composition of the vesicles, and the nature of the cellular lipopolysaccharide. The process results in bulk transfer of vesicle lipids to the cells rather than reciprocal molecular exchange between vesicles and the outer membrane. All components of mixed lipid vesicles, including cholesteryl oleate and lipopolysaccharide, are transferred to the cells in a ratio similar to that of the donor vesicles. The properties of the transfer process are consistent with direct fusion of vesicles with the outer membrane of the cell.