Dioleoylphosphatidylcholine Vesicles Research Articles

Fusion of dioleoylphosphatidylcholine vesicles induced by an amphiphilic cationic peptide and oligophosphates at neutral pH

Peptide E5 is an analogue of the fusion peptide of influenza virus hemagglutinin and K5 is a cationic peptide which has an arrangement of electric charges complementary to that of E5. We reported that a stoichiometric mixture of E5 and K5 caused fusion of large unilamellar vesicles (LUV) of neutral phospholipids (Murata, M., Kagiwada, S., Takahashi, S. and Ohnishi, S. (1991) J. Biol. Chem. 266, 14353–14358). K5 caused fusion of LUV composed of dioleoylphosphatidylcholine (DOPC) at pH > 10, but not at neutral pH. In the presence of oligophosphates, such as 1 mM ATP, GTP, or polyphosphate, K5 caused rapid and efficient fusion of DOPC LUV at neutral pH without hydrolysis of oligophosphate groups, but another anions such as citrate, acetate, AMP, phosphate, or EDTA were ineffective. The peptide/oligophosphate-induced fusion behaviors have been investigated by a fluorescence resonance energy transfer assay for lipid mixing of LUV and negative staining electron microscopy. At higher ionic strengths (> 0.3 M KCl) or in the presence of 5.0 mM MgCl 2, the fusion was inhibited. Even at the inhibitory conditions, the association of K5 with lipid vesicles at neutral pH was directly confirmed by the Ficoll gradient assay method and by blue shifts of the tryptophan fluorescence of the peptide. A nonhydrolyzable GTP analogue, GTPγS, also induced fusion. These observations suggested that the electrostatic interactions between the positive and negative charges of K5 and oligophosphate, respectively, induced complex formation, triggering membrane fusion.

Phase equilibria and formation of vesicles of dioleoylphosphatidylcholine in glycerol / water mixtures

The lipid 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) forms a lamellar liquid crystalline phase (L α) in arbitrary mixtures of glycerol and water. The phase has been characterized by means of X-ray diffraction, 31P-NMR spectroscopy and differential scanning calorimetry (DSC). In the L α state, and for DOPC concentrations greater than 50% (w/w), the thickness of the lipid bilayer decreases, while the area of the polar head group increases with increasing glycerol concentration. The phase transition from gel to L α state occurs in the range of 240 to 260 K. Contrary to a previous (McDaniel, R.V., McIntosh, T.J. and Simon, S.A. (1983) Biochim. Biophys. Acta 731, 97) study of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) we find that in the gel state, the thickness of the DOPC lipid bilayer is greater than that in the L α state. This suggests that in the gel state, the lipid acyl chains of DOPC are in extended configuration. The lamellar phase reaches its maximum swelling at about 50% (w/w) of DOPC. At lower DOPC concentrations a two-phase system is formed where the lamellar phase exists in equilibrium with excess of solvent. Unilamellar vesicles can be prepared from a diluted suspension of the lamellar phase either by using the sonicator or extruder technique. We show this by means of 31P-NMR, EPR and flourescence spectroscopy. The mean radius of the vesicles, prepared by a sonicator, has been determined at different glycerol/water mixtures. It is found to decrease continously from 100 Å at 100% water to a minimum of 75 Å at about 50% water in the solvent mixture. By further decreasing the water content in the solution, the radius rapidly increases, and a mean radius of 450 Å is estimated at a water content of 10%. The rotational relaxation times of a fluorescent probe and two EPR spin probes, solubilized in DOPC vesicles, have been measured at different glycerol/water mixtures. It is found that the rotational rates are always much slower in the systems containing glycerol.

Cholesterol and phosphoinositides increase affinity of the epidermal growth factor receptor

The epidermal growth factor receptor (EGF-R) has been purified from human epidermoid carcinoma A431 cells by affinity chromatography in a single step using a monoclonal antibody (528) which competes with EGF for receptor binding. The purified EGF-R exhibits EGF inducible tyrosine kinase and autophosphorylation activity. Steady-state binding of EGF to the purified receptor revealed the presence of one class of binding sites exhibiting an apparent dissociation constant ( K d ) of approx. 2 nM. When Angiotensin II was used as a receptor tyrosine kinase substrate the specific activity of the EGF induced kinase was 87 nmol/min per mg and the K m of the reaction was about 2 mM. Reconstitution of the EGF receptors into lipid vesicles was achieved by octylglucoside dialysis. Reconstitution of the receptor into pure dioleoylphosphatidylcholine (DOPC) vesicles had no effect on the EGF-binding properties in comparison to receptors in Triton X-100 micelles. Binding of EGF to the reconstituted receptor with ATP and Angiotensin II incorporated into the vesicles resulted in a five fold stimulation of the receptor kinase activity. The introduction of cholesterol, ranging from 10% to 50% (w/w), into DOPC vesicles resulted in an increase of the affinity of the receptor for its ligand. The K d for EGF decreased from 1.8 nM in pure DOPC vesicles to 0.3 nM in DOPC/cholesterol (1:1 (w/w)) vesicles. With the introduction of small amounts (2% (w/w)) of phosphatidylinositol lipids into DOPC vesicles the K d changed from 1.8 nM to 0.2 nM with phosphatidylinositol (PtdIns) and phosphatidylinositol 4,5-biphosphate ( PtdIns4,5-P 2 ) and to 0.1 nM in the case of phosphatidylinositol 4 phosphate ( PtdIns4-P ). No change in affinity was found when equal amounts of phosphatidylserine (PS) or phosphatidic acid (PA) were used.

Catalytic properties of cytochrome P-450 scc purified from the human placenta: comparison to bovine cytochrome P-450 scc

Cytochrome P-450 scc was purified from the human placenta by extraction of mitochondria with cholate and Emulgen 911, chromatography on phenyl-Sepharose and DEAE-Sephacel, and ammonium sulphate fractionation. The catalytic properties of the purified human cytochrome P-450 scc were analysed in Tween-20 micelles and compared to those of bovine adrenal cytochrome P-450 scc analysed in the same system. Both enzymes had the same K m for cholesterol and were stimulated by cardiolipin when the cholesterol concentration was subsaturating. Examination of the rates of pregnenolone synthesis from 20α-hydroxycholesterol, 22R-hydroxycholesterol and 20α,22R-dihydroxycholesterol by human and bovine cytochromes P-450 scc revealed that the first hydroxylation (22R position) was rate-limiting for both in Tween-20 micelles. The rate of the 22R-hydroxylation was further decreased when a 20α-hydroxyl group was already present on the cholesterol side-chain. The second hydroxylation occurred at about the same rate as the third hydroxylation for both enzymes. The rate of side-chain cleavage of 25-hydroxycholesterol by human cytochrome P-450 scc in Tween-20 micelles was low, the highest rate being about 1% of the Vm max for cholesterol. Substrate inhibition was seen with high concentrations of 25-hydroxycholesterol. Conversion of 25-hydroxycholesterol to pregnenolone was accompanied by a build-up of products with intact side-chains, which were probably intermediates of the reaction. Side-chain cleavage of 25-hydroxycholesterol by bovine cytochrome P-450 scc showed similar characteristics to the human enzyme, except that the highest velocity observed was approx. 25% of the V max for cholesterol. Rates of cleavage of 25-hydroxycholesterol by both enzymes were higher in dioleoylphosphatidylcholine vesicles than in Tween-20, but were still well below the V max for cholesterol and showed substrate inhibition. This study shows that there is close similarity in catalytic properties between human and bovine cytochromes P-450 scc which suggests that the active site of the cytochrome is highly conserved.

Specificity of amphiphilic anionic peptides for fusion of phospholipid vesicles

We have synthesized five amphiphilic anionic peptides derived from E5 peptide [Murata, M., Takahashi, S., Kagiwada, S., Suzuki, A., Ohnishi, S. 1992. Biochemistry 31:1986-1992. E5NN and E5CC are duplications of the N-terminal and the C-terminal halves of E5, respectively, and E5CN is an inversion of the N- and the C-terminal halves. E5P contains a Pro residue in the center of E5 and E8 has 8 Glu residues and 9 Leu residues. We studied fusion of dioleoylphosphatidylcholine (DOPC) large unilamellar vesicles assayed by fluorescent probes. The peptides formed alpha-helical structure with different degrees; E5NN, E5CN, and E8 with high helical content and E5CC and E5P with low helical content. These peptides bound to DOPC vesicles at acidic pH in proportion to the helical content of peptide. The peptides caused leakage of DOPC vesicles which increased with decreasing pH. The leakage was also proportional to the helicity of peptide. Highly helical peptides E5NN, E5CN, and E8 caused hemolysis at acidic pH but not at neutral pH. The fusion activity was also dependent on the helicity of peptides. In fusion induced by an equimolar mixture of E5 analogues and K5 at neutral pH, E8, E5NN, and E5CN were most active but E5CC did not cause fusion. In fusion induced by E5-analogue peptides alone, E5CN was active at acidic pH but not at neutral pH. Other peptides did not cause fusion. Amphiphilic peptides also appear to require other factors to cause fusion.

In vitro fusion of rabbit liver Golgi membranes with liposomes.

Fusion of Golgi membranes isolated from rabbit liver with liposomes was studied by lipid mixing of fluorescent lipid analogues and internal content mixing and by electron microscopic observation of transfer of horseradish peroxidase from liposomes into Golgi membranes. A monoclonal antibody was used to confirm fusion of Golgi membranes but not other contaminating vesicles. Fusion was rapid and efficient, reaching about 20% of the maximum after a 5-min incubation using small or large unilamellar dioleoylphosphatidylcholine vesicles. The fusion was dependent on temperature, decreasing at lower temperatures, and becoming nearly zero below 10 degrees C. The addition of ATP, GTP, cytosolic factors, or N-ethylmaleimide did not affect fusion. Treatments of Golgi membranes with 0.1 M Na2CO3 or 1 M KCl did not cause any changes in fusion. However, treatment with proteases inhibited fusion. These results suggest that Golgi integral membrane protein(s) are involved in fusion. Changing the medium to an isoosmotic substance, sucrose, in place of KCl or NaCl inhibited fusion. The binding assay of fluorescent liposomes to Golgi membranes showed that lowering the temperature or replacing salts with sucrose did not affect binding. However, treatment of Golgi membranes with proteases inhibited binding. Addition of phosphatidylserine or phosphatidylethanolamine to dioleoylphosphatidylcholine liposomes caused a 2-fold increase in binding and fusion. Fusion between Golgi membranes by themselves did not occur. These results provide some information on the mechanism of intracellular vesicular transport.

Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer.

We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 A in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 A for NBD and 31-55 A for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.

Anionic phospholipids are essential for alpha-helix formation of the signal peptide of prePhoE upon interaction with phospholipid vesicles.

The conformational consequences of the interaction of the PhoE signal peptide with bilayers of different types of phospholipids was investigated using circular dichroism. It was found that interaction of the signal peptide with anionic phospholipid vesicles of dioleoylphosphatidylglycerol and dioleoylphosphatidylserine results in induction of high amounts of alpha-helical structure of 70% and 57%, respectively. Upon addition of the signal peptide to cardiolipin vesicles, less but still significant alpha-helical structure was induced (29%). In contrast, no alpha-helix formation was observed upon the interaction of the signal peptide with zwitterionic dioleoylphosphatidylcholine vesicles. In bilayers of dioleoylphosphatidylcholine with dioleoylphosphatidylglycerol, it was shown that in the presence of 100 mM NaCl a minimum amount of 50% of negatively charged lipid was required for induction of the maximal percentage of alpha-helix, whereas in the absence of salt a minimum amount of 35% of negatively charged lipid was necessary. Induction of alpha-helix structure appeared to be correlated with functionality, since, in a less functional analogue of the PhoE signal peptide, the PhoE-[Asp-19,20] signal peptide, less alpha-helix was induced than in the wild-type PhoE signal peptide. It is proposed that the interaction with anionic phospholipids is essential for a functional conformation of the PhoE signal sequence during protein translocation.

Effects of Temperature on the Coupled Activities of the Vanadate-Sensitive Proton Pump from Maize Root Microsomes

The mechanism by which proton transport is coupled to ATP hydrolysis by vanadate-sensitive pumps is poorly understood. The effects of temperature on the activities of the vanadate-sensitive ATPase from maize (Zea mays) roots were assessed to provide insight into the coupling mechanism. The initial rate of proton transport had a bell-shaped dependence on temperature with an optimal range between 20 and 30 degrees C. However, the rate of vanadate-sensitive ATP hydrolysis increased as the temperature was raised from 4 to 43 degrees C. The differential sensitivity of proton transport to temperatures above 30 degrees C was also observed when the ATPase was reconstituted into dioleoylphosphatidylcholine vesicles. Inhibition of proton transport with temperatures above 30 degrees C was associated with higher rates of proton leakage from the membranes. In addition, proton transport was more inhibited than ATP hydrolysis at temperatures below 10 degrees C. Reduced rates of proton transport at lower temperatures were not associated with higher rate of proton conductivity across the membranes. Therefore, the preferential inhibition of proton transport at temperatures below 10 degrees C may reflect an effect of temperature on the coupling between proton transport and ATP hydrolysis within the vanadate-sensitive ATPase.

Cytochrome P-450scc induces vesicle aggregation through a secondary interaction at the adrenodoxin binding sites (in competition with protein exchange)

Addition of bovine adrenal cytochrome P-450scc to small unilamellar dioleoylphosphatidylcholine vesicles (DOPC-SUV) produces a complex sequence of interactions, indicating exceptional cytochrome mobility. First, cholesterol transfer from cytochrome to vesicles indicated rapid dissociation of P-450scc oligomers and integration of monomers into the membrane (delta A 390-420 nm; t1/2 = 2 s). After 10-15 s, P-450scc-induced aggregation of the vesicles starts, as indicated by increased turbidity (delta A 448 or 520 nm; complete in 6-8 min). Fluorescence quenching experiments indicate that this aggregation does not lead to measurable vesicle fusion during this period. Aggregation is prevented by mild heat denaturation of P-450scc, by addition of anti-P-450scc IgG, and also by 1:1 complex formation with the electron donor adrenodoxin (ADX). P-450scc, therefore, links two vesicles through two separate domains involved in, respectively, membrane integration (lipophilic) and ADX binding (charged). Although completely bound by DOPC-SUV, as evidenced by Sephadex elution, P-450scc has access within 1 min to cholesterol in secondary SUV. This is indicated by spectral changes (cholesterol complex formation) and by metabolism of secondary vesicle cholesterol. Since cholesterol equilibrates slowly between vesicles (t1/2 = 1-2 h), these changes arise from P-450scc transfer. This transfer was maximally slowed after a 5-min preincubation with primary vesicles, reflecting more extensive integration into the membrane than is necessary for the rapid initial cholesterol transfer to P-450scc. P-450scc transfer probably results from simultaneous interaction of P-450scc with two vesicles that may also initiate aggregation. Weaker integration into primary dimyristoylphosphatidylcholine vesicles facilitates exchange but prevents aggregation. Integration and aggregation are both enhanced by incorporation of 10% phosphatidylinositol into SUV, while exchange is slowed. This mobility of P-450scc is most probably a consequence of the absence of amino-terminal anchoring. P-450scc-induced association of inner mitochondrial membrane segments may contribute to the exceptionally vesiculated structure of adrenal and ovarian mitochondria that parallels increased P-450scc content.

Monolayers and bilayers of lipids

The spreading monolayers of ceramides alone and in mixtures with monooleoyl glycerol were studied at the air/water interface in the 15-30°C temperature range. The bidimensional phases were determined from the spreading isotherms of the pure components, whereas the interfacial miscibility of the compounds being studied was deduced from the spreading isotherms of the mixtures. The specific resistance and capacitance of black lipid membranes containing mono-oleoyl glycerol and monooleoyl glycerol/ceramides in squalene were studied. Langmuir-Blodgett monolayers and bilayers obtained by the transfer of spreading monolayers at the air/water interface onto chromated glass slides, were prepared. Vesicles of dioleoylphosphatidylcholine and of mixtures of dioleoylphosphatidylcholine/ceramides were also prepared. The results obtained for the different types of aggregates were compared in order to find the relations that connect the characteristics of the various systems. Results showed that the possibility of obtaining flat and curved bilayers of pure components and of mixtures was closely connected to the phase of the corresponding monolayers at the air/water interface.

Binding and diffusion kinetics of the interaction of a hydrophobic potential-sensitive dye with lipid vesicles

The interaction of the dye oxonol V with unilamellar dioleoylphosphatidylcholine vesicles has previously been investigated using a fluorescence stopped-flow technique. It has been found that the most suitable mathematical description of the equilibrium and kinetic data is obtained by assuming the presence of saturable dye binding sites in both monolayers of the vesicle membrane and a potential-dependent diffusion across the membrane interior between these two classes of sites. A kinetic model is presented which takes into account the degree of saturation of the binding sites, the degree of fluorescence quenching within the membrane, and the production of an electrical potential gradient across the membrane interior by the binding of the negatively charged dye. The model successfully predicts the time course of the fluorescence change due to binding and diffusion over the complete range of dye and vesicle concentrations as well as the fluorescence response of the dye to changing membrane potential.

Effect of the addition of ceramide to dioleoylphosphatidylcholine vesicles: an ESR and SEM study

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEffect of the addition of ceramide to dioleoylphosphatidylcholine vesicles: an ESR and SEM studyFrancesca Bonosi, Gabriella Gabrielli, Elena Margheri, and Giacomo MartiniCite this: Langmuir 1990, 6, 12, 1769–1773Publication Date (Print):December 1, 1990Publication History Published online1 May 2002Published inissue 1 December 1990https://doi.org/10.1021/la00102a011RIGHTS & PERMISSIONSArticle Views95Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Gramicidin A induced fusion of large unilamellar dioleoylphosphatidylcholine vesicles and its relation to the induction of type II nonbilayer structures

The fusogenic properties of gramicidin were investigated by using large unilamellar dioleoylphosphatidylcholine vesicles. It is shown that gramicidin induces aggregation and fusion of these vesicles at peptide to lipid molar ratios exceeding 1/100. Both intervesicle lipid mixing and mixing of aqueous contents were demonstrated. Furthermore, increased static and dynamic light scattering and a broadening of 31P NMR signals occurred concomitant with lipid mixing. Freeze-fracture electron microscopy revealed a moderate vesicle size increase. Lipid mixing is paralleled by changes in membrane permeability: small solutes like carboxyfluorescein and smaller dextrans, FD-4(Mr approximately 4000), rapidly (1-2 min) leak out of the vesicles. However, larger molecules like FD-10 and FD-17 (Mr approximately 9400 and 17,200) are retained in the vesicles for greater than 10 min after addition of gramicidin, thereby making detection of contents mixing during lipid mixing possible. At low lipid concentrations (5 microM), lipid mixing and leakage are time resolved: leakage of CF shows a lag phase of 1-3 min, whereas lipid mixing is immediate and almost reaches completion during this lag phase. It is therefore concluded that leakage, just as contents mixing, occurs subsequent to aggregation and lipid mixing. Although addition of gramicidin at a peptide/lipid molar ratio exceeding 1/50 eventually leads to hexagonal HII phase formation and a loss of vesicle contents, it is concluded that leakage during fusion (1-2 min) is not the result of HII phase formation but is due to local changes in lipid structure caused by precursors of this phase. By making use of gramicidin derivatives and different solvent conformations, it is shown that there is a close parallel between the ability of the peptide to induce the HII phase and its ability to induce intervesicle lipid mixing and leakage. It is suggested that gramicidin-induced fusion and HII phase formation share common intermediates.

Organization of microsomal UDP-glucuronosyltransferase. Activation by treatment at high pressure

Treatment of microsomes at pressures as high as 2.25 kbar led to an apparent irreversible activation of UDP-glucuronylsyltransferase when pressure was released. The response of the enzyme to pressure, as reflected by activity measured after release of pressure, appeared to be discontinuous in that no activation was seen for any preparation at pressures less than 1.2 kbar. In addition, activation was temperature dependent. Maximum activation at 2.25 kbar occurred at about 12 degrees C; the extent of activation in 10 min was less for either higher or lower temperatures. Activation was also time dependent. Maximum activation at 2.25 kbar and 9 degrees C required 90 min of pressure treatment. Activation appeared to occur more slowly at lower pressure. Pressure-induced activation was associated with a loss of sensitivity of the enzyme to allosteric activation by UDP-N-Ac-Glc and a conversion of the kinetic pattern from non-Michaelis-Menten to Michaelis-Menten. Pressure did not activate enzyme that had previously been activated maximally by adding detergent to microsomes. Pressure also did not activate pure UDP-glucuronosyltransferase reconstituted into unilamellar vesicles of dioleoylphosphatidylcholine. Pressure treatment did not release UDP-glucuronosyltransferase from microsomes into water. Pressure had a continuous effect on the polarization and excimer/monomer formation of fluorescent probes incorporated into microsomes, and the properties returned essentially to their values at 1 atm when pressure was released. Measurements of activity at 2.2 kbar showed that pressure-induced activation of UDP-glucuronosyltransferase in microsomes occurred via two intermediates that were inactive and that the activated state of the enzyme was generated during/after release of pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrinsic gramicidin fluorescence lifetimes in bilayer lipid membranes and in vesicles

Intrinsic Gramicidin A′ tryptophan steady-state fluorescence anisotropies and fluorescence lifetimes have been determined in bilayer lipid membranes (BLMs) prepared from glyceryl monooleate (GMO). In GMO BLMs, fluorescence anisotropy, the r value, was found to be 0.05 ± 0.02. Decays of Gramicidin A′ fluorescence intensities were fitted to the sum of three exponentials (τ1, τ2, and τ3) and appropriate pre-exponentials (A1, A2, and A3). These values allowed for the assessment of average fluorescence lifetimes, [Formula: see text]. These values related to those determined in vesicles prepared from dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and diphytanoylphosphatidylcholine (DPhPC). In BLMs, [Formula: see text], 3.6 ns, and 2.3 ns were determined for vertically, horizontally, and unpolarized average fluorescence lifetimes, respectively. Increasing the applied potential across the BLM from 0 to 80 mV increased [Formula: see text] from 2.2 ns to 4.9 ns and τ1 from 0.43 ± 0.05 to 0.73 ± 0.06 ns, as well as the contributions and lifetimes of the longer lived fluorescence (A2 and A3, τ2 and τ3). The emission maximum of Gramicidin A′ (334 nm in DPPC) and the absence of quenching by iodide ions indicated complete incorporation of the polypeptide into vesicles. The r values were of the order of 0.10 in vesicles prepared from DPPC and DSPC, both in the absence and in the presence of added 1.2 × 10−4 M CsCl. In vesicles prepared from DOPC and DPhPC, r values increased to 0.13 and 0.14 in water and to 0.15 and 0.20 in 1.2 × 10−4 M CsCl, respectively. At 25.0 °C, the temperature of the measurements, DPPC and DSPC are in their "solid" states, but DOPC and DPhPC are in their "liquid" states, [Formula: see text] values for Gramicidin A′ in vesicles prepared from DPPC, DOPC, and DSPC were all in the 3.0 ± 0.3 ns range. In DPhPC vesicles, [Formula: see text] was determined. Time-dependent anisotropics became observable in DOPC and DPhPC vesicles, particularly in the presence of 1.2 × 10−4 M CsCl. Keywords: gramicidin, fluorescence lifetimes, vesicles, bilayer lipid membranes, time-dependent anisotropics.

Are natural lipids UV-screening agents?

Isolated lipids from Deinococcus radiodurans were reconstituted at final concentrations of 1 mg/ml into dioleoyl phosphatidyl choline (DOPC) vesicles and assayed for the ability to protect cells of Escherichia coli against killing by UV light (254 nm). Values of D37 (UV dose required to reduce the number of surviving cells to 37% of the original number) were calculated from killing curves. E. coli was afforded the greatest protection with an individual lipid, identified as vitamin MK8 (D37=310 J//m2, compared to D37=67 J/m2 for E. coli irradiated in the presence of DOPC alone). Liposome-mediated protection was dependent on UV254 absorbance and not on turbidity-related light-scattering. BOth vitamin MK8 from D. radiodurans and vitamin K1, which is available commercially, showed a similar degree of UV254-protection for E. coli. The UV-protective properties of vitamin K1 were also investigated on mammalian cells in comparison with other natural lipids and known sunscreens. Survival curves were obtained for mouse fibroblast (L) cells irradiated at UV254 in the absence or presence of DOPC liposomes into which were incorporated various natural lipids or standard sunscreen ingredients, all at final concentrations of 1 mg/ml. Experimentally determined values of D37 were as follows: Vitamin K1, 73 J/m2; \-carotene, 44 J/m2; α-tocopherol, 20 J/m2; sulisobenzone, 156 J/m2; p-aminobenzoic acid (PABA), 113 J/m2; benzophenone, 80 J/m2; oxybenzone, 61 J/m2 and DOPC alone. 23 J/m2. Vitamin K1, the most protective lipid tested, was also compared with PABA and oxybenzone (all at concentrations of 20 mg/ml; applied topicall) for its ability to protec Skh-hairless mice from UV254-induced erythema, yielding a UV254 protection factor of 3.5. In addition, vitamin K1 (at 100 mg/ml) was able to provide hairless mice with a small degree of UVB protection, as indicated by an experimentally determined Solar Protection Factor of 1.5–2.0. Although it is concluded that vitamin K is not likely to account for the extraordinarily high degree of UV-resistance of D. radiodurans, vitamin K does show characteristics worthy of its consideration as a UV-screening agent.

Thermodynamic parameters of the binding of retinol to binding proteins and to membranes

Retinol (vitamin A alcohol) is a hydrophobic compound and distributes in vivo mainly between binding proteins and cellular membranes. To better clarify the nature of the interactions of retinol with these phases which have a high affinity for it, the thermodynamic parameters of these interactions were studied. The temperature-dependence profiles of the binding of retinol to bovine retinol binding protein, bovine serum albumin, unilamellar vesicles of dioleoylphosphatidylcholine, and plasma membranes from rat liver were determined. It was found that binding of retinol to retinol binding protein is characterized by a large increase in entropy (T delta S degrees = +10.32 kcal/mol) and no change in enthalpy. Binding to albumin is driven by enthalpy (delta H degrees = -8.34 kcal/mol) and is accompanied by a decrease in entropy (T delta S degrees = -2.88 kcal/mol). Partitioning of retinal into unilamellar vesicles and into plasma membranes is stabilized both by enthalpic (delta H degrees was -3.3 and -5.5 kcal/mol, respectively) and by entropic (T delta S degrees was +4.44 and +2.91 kcal/mol, respectively) components. The implications of these finding are discussed.

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.

Cholesterol metabolism by purified cytochrome P-450scc is highly stimulated by octyl glucoside and stearic acid exclusively in large unilamellar phospholipid vesicles.

Cholesterol side-chain cleavage (CSCC) catalyzed by purified bovine adrenal mitochondrial cytochrome P-450scc is highly dependent on the vesicles that supply cholesterol. Six-fold higher rates are achieved with large unilamellar dioleoylphosphatidylcholine vesicles (diameter 150 nm) prepared by octyl glucoside (OG) dialysis (DOPC-LUV) than with small sonicated vesicles (diameter 30 nm) (DOPC-SUV) (Vmax = 25 and 4 min-1, respectively. Extensive dialysis that may remove OG decreased Vmax rates for DOPC-LUV almost to rates seen with DOPC-SUV. These dialyzed DOPC-LUV were, however, very sensitive to addition of OG (EC50 = 2.5 microM, 4.3-fold stimulation) while DOPC-SUV were only weakly affected (EC50 = 100 microM, 1.6-fold stimulation). This enhancement of CSCC in LUV by OG only occurred when the cholesterol:DOPC exceeded 0.1 and was associated with a 15-fold increase in the Km for cholesterol. Structural changes in both SUV and LUV at high cholesterol:DOPC ratios (0.1-1) were indicated by decreases in internal volume that were insensitive to OG and did not affect the external diameters. Stearic acid produced a similar stimulation of CSCC in LUV (EC50 = 50 microM) and had no effect on SUV. The Vmax for CSCC, produced by OG activation of DOPC-LUV, is comparable to the highest attained for cytochrome P-450scc (Tween 20/cholesterol). In LUV, a minor proportion of OG (1-5% of cholesterol) is thus sufficient to generate a domain of reactive cholesterol that maintains a near-optimum turnover. This increased CSCC was paralleled by increased binding of cholesterol to P-450scc, suggesting that this cholesterol is more readily donated by the membrane to the cytochrome.