The effect of potential-sensitive molecular probes on the thermal phase transition in dimyristoylphosphatidylcholine preparations

Differential scanning calorimetry (DSC) has been employed to determine the effect of five commonly employed extrinsic potential-sensitive probes on phase transitions of multilamellar suspensions of L-alpha-dimyristoylphosphatidylcholine (DMPC). At mol% values of less than five, the effect of these probes on the excess heat capacity curve in the vicinity of the gel to liquid crystal phase transition can be described by an equation based on the formation of ideal solutions in both phases. Even at up to 4 mol%, these dyes only moderately reduce the enthalpy change associated with this transition, but cause a marked decrease in the size of the cooperative unit parameter. The excess heat capacity profile for diS-C3-(5) is represented by the ideal solution equation, even at 12 mol%, whereas the suspensions with the other probes present at this level have profiles covering large temperature ranges. Multiple peaks appear at the higher levels for the negatively charged oxonols V and VI, and merocyanine 540, a result consistent with the presence of well-defined microdomains or even phase separation. The enthalpy change associated with the transition near 15 degrees C involving packing in the headgroup region is decreased significantly, indicating that the probes probably affect the lipid headgroup conformation, even at low levels. The cyanine probe diS-C3-(5) causes the heat capacity profile of small unilamellar vesicles to be transformed very rapidly into one similar to that of the vortexed lipid preparations, presumably by a dye-mediated vesicle fusion process, enhanced by the surface location of this probe. All our results are consistent with diS-C3-(5) being located on the surface of the bilayer in both phases, but a penetration of the other probes into the hydrocarbon region, at least in the liquid crystal phase.     

Effect of lipid phase transition on the binding of anions to dimyristoylphosphatidylcholine liposomes

Temperature dependence of the electrophoretic mobility of multilamellar liposomes prepared from dimyristoylphosphatidylcholine was measured in the presence of salts with different anions in aqueous solutions. It was established that specific binding of anions to liposome surface induced a pronounced zeta potential (electrostatic potential at the hydrodynamic plane of shear). A combination of Langmuir, Gouy-Chapman, and Boltzmann equations was used to describe the dependence of the zeta potential on the concentration of anions. The values of binding constants (K) and maximum numbers of binding sites per unit area (σ_max) were determined by this method. The sequence for anion affinities to liposome surface was found to be as follows: trinitrophenol >ClO₄⁻ >I⁻ >SCN⁻ >Br⁻ >NO₃⁻ >Cl⁻ SO₄²⁻. A sharp increase in the negative zeta potential was detected at the temperature of phase transition of the lipid from the gel to liquid-crystalline state. It was found that the parameter K did not change at lipid phase transition and the shifts in zeta potential might be due to alterations of σ_max. The binding sites were considered as defects in the package of lipid molecules in membranes.     

The effect of hashish compounds on phospholipid phase transition

The interaction of hashish compounds, Δ¹-tetrahydrocannabinol and cannabidiol, with dipalmitoyl phosphatidylcholine was investigated using differential scanning calorimetry. Both drugs affect the transition of dipalmitoyl phosphatidylcholine from the gel to liquid crystalline state, decreasing both the melting temperature and the enthalpy of melting. At a drug to dipalmitoyl phosphatidylcholine ratio of approx. 1:5, two peaks appear in the transition profile, suggesting a phase separation in the drug dipalmitoyl phosphatidylcholine mixture.     

A densitometric study of the effects of free fatty acids on the phase transition of dimyristoylphosphatidylcholine bilayers

The technique of scanning densitometry was applied to a study of the effect of fatty acids on the packing and phase transition properties of dimyristolyphosphaticylcholine (DMPC). It was observed that the three fatty acids studied, i.e. palmitic, palmitoleic and behenic, had the effect of broadening the phase transition and increasing the extent of the change in volume which occurs at the phase transition. Palmitic and behenic acids broaden the phase transition toward higher temperatures while palmitoleic broadens it toward lower temperatures.     

Evidence for phase boundary lipid. Permeability of Tempo-choline into dimyristoylphosphatidylcholine vesicles at the phase transition.

The existence of distinct regions of mismatch in molecular packing at the interfaces of the fluid and ordered domains during the phase transition of dimyristoylphosphatidylcholine vesicles has been demonstrated by measuring the temperature dependence of the permeability to a spin-label cation and comparing this with a statistical mechanical calculation of the fraction of interfacial lipid. The kinetics of uptake and release of the 2,2,6,6-tetramethylpiperidinyl-1-oxycholine (Tempo-choline) spin label by single-bilayer dimyristoylphosphatidylcholine vesicles were measured using electron spin resonance spectroscopy to quantitate the amount of spin label present within the vesicles after removal of the external spin-label by ascorbate at 0 degrees C. Both the uptake and release experiments show that the Tempo-choline permeability peaks to a sharp maximum at the lipid-phase transition, the vesicles being almost impermeable to Tempo-choline below the transition and having a much reduced permeability above. The temperature profile of the permeability is in reasonable quantitative agreement with calculations of the fraction of interfacial boundary lipid from the Zimm and Bragg theory of cooperative transitions, which use independent spin-label measurements of the degree of transition to determine the cooperativity parameter. The relatively high intrinsic permeability of the interfacial regions (P approximately 0.2-1.0 X 10(-8) cm/s) is attributed to the mismatch in molecular packing of the lipid molecules at the ordered-fluid boundaries, which could have important implications not only for permeability in natural membranes (e.g., in transmitter release), but also for the function of membrane-bound enzymes and transport proteins.     

Effects of cholesterol and temperature on the permeability of dimyristoylphosphatidylcholine bilayers near the chain melting phase transition

The passive leakage of glucose across bilayers of dimyristoylphosphatidylcholine (DMPC), cholesterol (variable), and dicetyl phosphate (constant 5.9 mol%) has been measured as efflux over 30 min from multilamellar vesicles. Bilayer cholesterol was varied from 20 mol% to 40 mol%. Glucose permeation rates were measured from 10 degrees C to 36 degrees C, and showed a maximum in permeability at 24 degrees C, the DMPC phase transition temperature. Increasing the bilayer cholesterol content above 20 mol% reduced that permeability peak. These results are quite consistent with a large number of similar bilayer permeability studies over the past 25 years. However, they are not consistent with a previous study of these same systems, which reported increased glucose permeability with temperature, without any maximum at or near the lipid chain melting temperature (K. Inoue, Biochim. Biophys. Acta 339 (1974) 390-402).     

Antiviral action of membranotropic compounds modified by adamantane and norbornene pharmacophores exerted on different HIV-1 strains

The anti-HIV activity of new membranotropic compounds, i.e. of the polycarboxylate matrix and of its derivatives modified by adamantane and norbonene, was studied in respect of HIV-1 strains, whose tropicity to coreceptors CCR5 and CXCR4 was different, as well as in respect of HIV-1 variants resistant to azidothymidine (AZT) in a continuous culture of human lymphoid cells (MT-4) and in mononuclear cells of peripheral blood from healthy donors. Testing of complex compounds in a culture of infected MT-4 human lymphoid cells showed an effective inhibition of viral reproduction of LAV.04 (CXCR4-tropic variant) and of HIV11(EVK) as well as AZT-resistant variants. The studied pharmacophores-modified compounds displayed, in infection of the primary culture of human mononuclear cells of the HIV-1 R5 and X4 strains, a notable antiviral activity with their HIV efficiency significantly exceeding the one of the original matrix.     

Effects of membranotropic agents on mono- and multilayer structures of dipalmitoylphosphatidylcholine

We have studied the action of some membranotropic agents (MTAs) on the parameters of mono- and multilayers of dipalmitoylphosphatidylcholine (DPPC). The MTAs used included an antimicrobial drug, decamethoxinum, the model amphiphilic agent stearoyl-L-alpha-alanine, and cholesterol as a reference substance. Using differential scanning calorimetry and the Langmuir monolayer technique, we measured the temperature and enthalpy of the main phase transition of DPPC, the mean molecular area, the collapse pressure and the free energy of the mixed monolayers of DPPC and MTA. A good correlation has been obtained between the structure of the MTA used and changes in the parameters of both mono- and multilayers. Thus, for cholesterol, its well-known condensing effect in the L alpha phase correlates with its behavior in the mixed monolayers. The disturbing action of decamethoxinum (depression of the phase transition in DPPC multilayers and relatively high free energy of mixing in monolayers) is presumably connected with interaction of its charged ammonium moieties with polar phospholipid heads. At the same time, stearoyl-L-alpha- alpha-alanine condensed the lipid layers and increased the melting point of DPPC, owing to its interaction with both polar and non-polar lipid moieties. One can conclude that the three MTAs used can really be considered as representative examples of three different types of behavior in mono- and multilayers.     

The influence of acyl chain-length asymmetry on the phase transition parameters of phosphatidylcholine dispersions

The influence of acyl chain-length asymmetry on the thermodynamic parameters (Tm, delta H, and delta S) associated with the reversible main phase transition of aqueous dispersions prepared from saturated diacyl phosphatidylcholines was studied by high-resolution differential scanning calorimetry. Two series of saturated diacyl phosphatidylcholines, grouped according to their molecular weights of 678 and 706, with a total number of 25 molecular species were examined. The normalized acyl chain-length difference between the sn-1 and sn-2 acyl chains for a given phospholipid molecule in the gel-state bilayer is expressed quantitatively by the structural parameter delta C/CL, and the values of delta C/CL for the two series of lipids under study vary considerably from 0.04 to 0.67. When the value of delta C/CL is within the range of 0.09-0.40, it was shown that the thermodynamic parameters are, to a first approximation, a linear function of delta C/CL with a negative slope. In addition, the experimental Tm values and the predicted Tm values put forward by Huang (Biochemistry (1991) 30, 26-30) are in very good agreement. Beyond the point of delta C/CL = 0.41, the influence of acyl chain-length asymmetry on the thermodynamic parameters deviates significantly from a linear function. In fact, within the range of delta C/CL values of 0.42-0.67, the thermodynamic parameters in the Tm (or delta H) vs. delta C/CL plot were shown to be bell-shaped with the maximal Tm (or delta H) at delta C/CL = 0.57. These results are discussed in terms of changes in the acyl chain packing modes of various phosphatidylcholine molecules within the gel-state bilayer in excess water.     

Determination of the size of the packing defects in dimyristoylphosphatidylcholine bilayers,present at the phase transition temperature

Multilamellar liposomes of dimyristoylphosphatidylcholine, containing 4 mol% egg phosphatidic acid show at the phase transition temperature an increased permeability for non-electrolytes of M_r values up to 900. This indicates that the packing defects occurring at the liquid crystalline/gel state phase boundary have a similar pore diameter (15–18 A) as the packing defects present in glycophorin—dioleoylphos-phatidylcholine vesicles. This suggests that packing defects at the protein—lipid interphase are the major permeation pathway of the glycophorin—dioleoylphosphatidylcholine vesicles.     

Possibility of reversing the action of the membranotropic antibiotic, gramicidin S, on bacteria

The study on the possibility of eliminating gramicidin S from the bacterial cells which had adsorbed it showed that a part of the labeled antibiotic bound by the bacteria may be washed out with buffer or salines. When the cells which had adsorbed gramicidin S were treated with lecithin emulsion, a significant part of the bound antibiotic was transferred to the lecithin liposomes. This turned the gramicidin S effect to the cells: significant but not complete reduction of the membrane barrier properties and dehydrogenase reactivation. Elimination of gramicidin S also reduced the colony forming capacity in a part of the cells.     

A fluorescence anisotropy study on the phase behavior of dimyristoylphosphatidylcholine/cholesterol mixtures

The phase behavior of L-alpha-dimyristoylphosphatidylcholine/cholesterol mixtures was studied in multilamellar vesicles by fluorescence polarization of the sterol molecule dehydroergosterol and of the polyene molecule alpha-parinaric acid. In the absence of cholesterol, dehydroergosterol exhibited an increase in polarization as DMPC vesicles were heated through the phase transition. This rise in polarization anisotropy was observed over a 0.6-1.0 degrees C increase in temperature with the midpoint of the phase transition occurring at 23.6 degrees C. Addition of 5 mol% cholesterol completely obliterated this change in polarization anisotropy through the phase transition of DMPC. alpha-Parinaric acid underwent a characteristic decrease in polarization anisotropy through the phase transition of DMPC. The change in anisotropy through the phase transition was over 4-fold greater than the values observed with dehydroergosterol. Vesicles containing 5 mol% cholesterol in the presence of alpha-parinaric acid underwent a decrease in polarization anisotropy that was over 75% of the original decrease in amplitude observed in the absence of any membrane cholesterol. The difference in sensitivity of the two fluorescent probes to the phase transition of DMPC as a function of membrane cholesterol content may be explained by a preferential partitioning of dehydroergosterol (and cholesterol) into a sterol-rich phase at low sterol concentrations. This partitioning allows dehydroergosterol to detect sterol-rich regions in the membrane bilayer.     

Effect of phase transition on the kinetics of dye transport in phospholipid bilater structures.

Binding of 8-anilino-1-naphthalenesulfonate to dimyristoyl-L-alpha-lecithin bilayers enhances the fluorescence quantum yield of the dye molecule by 100-fold. By following the generation of fluorescence after a rapid mixing in a stopped-flow apparatus (mixing time 2 msec), kinetics of the binding of the fluorescence probe to the phospholipid vesicles has been investigated in the temperature range where the crystal-liquid crystal phase transition of the bilayer structures occurs. No reactions depending on the dye or the vesicle concentrations were detected. This suggests that the initial adsorption of the dye was very rapid. Two kinetic phases which appear in the 50 msec and the second time ranges are unimolecular. The faster one has a small amplitude and is observable in the entire temperature range studied. In the phase transition region the slower reaction becomes the major kinetic phase. It also increases the apparent concentration of bound dye by a factor of 2. These observations suggest that the 50-msec reaction has detected a reorientation of the probe molecule after the initial binding, and that the slow reaction represents a transport of the dye molecule into the inner layer of the lipid vesicle. The transport reaction is extremely temperature sensitive and exhibits a maximum rate at the midpoint of the bilayer phase transition (Tm = 24.1 degrees). the Arrhenius plot of the transport reaction shows a maximum at the Tm. the same temperature dependence was also observed for the bromothymol blue transport reaction. However, no such effects were detected for less amphiphilic molecules such as tetracycline, chlortetracycline, and pyrene. In the latter systems only a slight bending of the Arrhenius plots were seen at the phase transition temperature. Since the kinetics of the transport of 8-anilino-1-naphthalenesulfonate is sensitive to the physical state of the phospholipid bilayers this reaction may be used for probing membrane structures.     

The action of various venoms on Escherichia coli

The antibacterial activity of honeybee venom ( Apis mellifera ), three snake venoms ( Naja naja sputatrix, Vipera russellii and Crotalus adamanteus ) and the polypeptide melittin was investigated against Escherichia coli . Minimum inhibitory concentration values, cell lysis and alterations in cell permeability were determined and action against E. coli was in the order: A. mellifera venom > melittin > N. naja sputatrix venom ≫ V. russellii venom > C. adamanteus venom. Cellular damage by A. mellifera and N. naja sputatrix venoms was evident in electron micrographs.     

The action of various venoms on Escherichia coli

The antibacterial activity of honeybee venom (Apis mellifera), three snake venoms (Naja naja sputatrix, Vipera russellii and Crotalus adamanteus) and the polypeptide melittin was investigated against Escherichia coli. Minimum inhibitory concentration values, cell lysis and alterations in cell permeability were determined and action against E. coli was in the order: A. mellifera venom greater than melittin greater than N. naja sputatrix venom much greater than V. russellii venom greater than C. adamanteus venom. Cellular damage by A. mellifera and N. naja sputatrix venoms was evident in electron micrographs.