Phospholipid transfer between phosphatidylcholine-taurocholate mixed micelles

The transfer of fluorescent-labeled N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE) between phosphatidylcholine-taurocholate mixed micelles was measured by monitoring the increase in fluorescence as N-NBD-PE, initially contained in mixed micelles at self-quenching concentrations, was diluted into unlabeled mixed micelles. The half-times for transfer of a homologous series of N-NBD-PEs differing in saturated acyl chain length from 11 to 16 carbons increased with acyl chain length from 4 to 35 s. The half-times for transfer of the same N-NBD-PEs between phosphatidylcholine vesicles without taurocholate were 200-6000 times slower than those between the mixed micelles. A kinetic analysis of initial transfer rate data was used to determine the mechanistic model that best described the data. According to this analysis, the increased rate of intermicellar phospholipid transfer relative to that of intervesicular transfer is a result of (1) exchange between micelles during transient micelle collisions which is not observed between vesicles and (2) an increased rate of monomer diffusion due to a faster rate of phospholipid dissociation from mixed micelles into the water phase than from vesicles. The relative significance of dissociation from mixed micelles into the water phase than from vesicles. The relative significance of collision-dependent versus monomer diffusion transfer increases with acyl chain length and hydrophobicity.     

Low concentrations of bile salts increase the rate of spontaneous phospholipid transfer between vesicles

The rate of 1-palmitoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino] dodecanoyl] phosphatidylcholine (P-C12-NBD-PC) transfer between dioleoylphosphatidylcholine vesicles was measured by a technique based on resonance energy transfer between P-C12-NBD-PC and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine [Nichols, J. W., & Pagano, R. E. (1982) Biochemistry 21, 1720-1726]. Addition of bile salts at concentrations below their critical micelle concentrations increased the rate of spontaneous P-C12-NBD-PC transfer without disrupting the vesicles. The effectiveness in increasing the transfer rate was dependent on the structure of the bile salt. In general, conjugated bile salts were more effective than unconjugated, and mono- and dihydroxy bile salts were more effective than trihydroxy. The kinetics of intervesicular P-C12-NBD-PC transfer in the presence of cholate were found to be consistent with a mass action kinetic model based on the premise that bile salts bind to the vesicles, alter the dissociation and/or association rate constants for phospholipid monomer-vesicle interaction, and increase the rate of phospholipid transfer via the diffusion of soluble monomers through the aqueous phase. Temperature dependence studies indicated that cholate binding to vesicles is an entropy-driven process and that cholate binding lowers the free energy of activation for phospholipid monomer-vesicle dissociation by producing compensatory decreases in both the enthalpy and entropy of activation.     

Calcium/phosphate-induced immobilization of fluorescent phosphatidylserine in synthetic bilayer membranes: inhibition of lipid transfer between vesicles

Resonance energy transfer between 4-nitro-2,1,3-benzoxadiazole (NBD) acyl chain labeled phospholipid analogues and (lissamine) rhodamine B labeled phosphatidylethanolamine was used to monitor the rate of NBD-labeled lipid transfer between a variety of small unilamellar donor vesicles and dioleoylphosphatidylcholine (DOPC) acceptor vesicles. In the presence of appropriate concentrations of Ca2+ and phosphate, the transfer rate of NBD-phosphatidylserine (NBD-PS) from vesicles composed of lipid extracts from human red blood cells was reduced by approximately 10-fold, while the transfer rates of NBD-phosphatidylcholine, -ethanolamine, -glycerol, -N-succinylethanolamine, and -phosphatidic acid were essentially unaffected. A systematic evaluation of the lipid composition needed to facilitate the Ca2+/phosphate-induced inhibition of NBD-PS transfer revealed that the process was dependent upon the inclusion of both cholesterol and phosphatidylethanolamine (PE) in the donor vesicle population. Inhibition of NBD-PS transfer required the sequential addition of phosphate and Ca2+ to the vesicles, indicating that the combined interaction of Ca2+ and phosphate at the membrane surface was a prerequisite for inhibition to occur. Parallel experiments designed to determine the possible mechanism of this phenomenon showed that inhibition of NBD-PS transfer was not due to Ca2+-mediated phase separations or vesicle-vesicle fusion. However, the addition of Ca2+ and phosphate to vesicles composed of total red blood cell lipids or cholesterol/PE did result in their aggregation. On the other hand, aggregation per se did not seem to be responsible for the inhibition of transfer since NBD-PS-containing vesicles composed of DOPC or DOPC/DOPE also aggregated, although NBD-PS transfer was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of pyrene phospholipid/gamma-cyclodextrin complex

Recently, it was demonstrated that gamma-cyclodextrins (gamma-CDs) greatly accelerates transfer of hydrophobic pyrene-labeled and other fluorescent phospholipid derivatives from vesicles to cells in culture (). To understand better the characteristics of this process, we studied the interaction of gamma-CD with pyrene-labeled phosphatidylcholines (PyrPCs) using a variety of physical methods. Either one or both of the acyl chains of PC was labeled with a pyrene moiety (monoPyrPCs and diPyrPCs, respectively), and the length of the labeled chain(s) varied from 4 to 14 carbons. Fluorescent binding assays showed that the association constant decreases strongly with increasing acyl chain length. PyrPC/gamma-CD stoichiometry was 1:2 for the shorter chain species, but changed to 1:3 when the acyl chain length exceeded 8 (diPyrPCs) or 10 (monoPyrPCs) carbons. The activation energy for the formation of diPyr(10)PC/gamma-CD complex was high, i.e., +92 kJ/mol, indicating that the phospholipid molecule has to fully emerge from the bilayer before complex formation can take place. The free energy, enthalpy, and entropy of transfer of monoPyrPC from bilayer to gamma-CD complex were close to zero. The absorption, Fourier transform infrared, and fluorescence spectral measurements and lifetime analysis indicated that the pyrene moiety lies inside the CD cavity and is conformationally restricted, particularly when the labeled chain is short. The acyl chains of a PyrPC molecule seem to share a CD cavity rather than occupy different ones. The present data provide strong evidence that the ability of gamma-CD to enhance intermembrane transfer of pyrene-labeled phospholipids is based on the formation of stoichiometric complexes in the aqueous phase. This information should help in designing CD derivatives that are more efficient lipid carriers then those available at present.     

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC相似文献   

Transbilayer diffusion of phospholipids: dependence on headgroup structure and acyl chain length

The kinetics and thermodynamics of the transmembrane movement (flip-flop) of fluorescent analogs of phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were investigated to determine the contributions of headgroup composition and acyl chain length to phospholipid flip-flop. The phospholipid derivatives containing n-octanoic, n-decanoic or n-dodecanoic acid in the sn-1 position and 9-(1-pyrenyl)nonanoic acid in the sn-2 position were incorporated at 3 mol% into sonicated single-bilayer vesicles of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC). The kinetics of diffusion of the pyrene-labeled phospholipids from the outer and inner monolayers of the host vesicles to a large pool of POPC acceptor vesicles were monitored by the time-dependent decrease of pyrene excimer fluorescence. The observed kinetics of transfer were biexponential, with a fast component due to the spontaneous transfer of pyrenyl phospholipids in the outer monolayer of labeled vesicles and a slower component due to diffusion of pyrenyl phospholipid from the inner monolayer of the same vesicles. Intervesicular transfer rates decreased approx. 8-fold for every two carbons added to the first acyl chain. Correspondingly, the free energy of activation for transfer increased approx. 1.3 kcal/mol. With the exception of PE, the intervesicular transfer rates for the different headgroups within a homologous series were nearly the same, with the PC derivative being the fastest. Transfer rates for the PE derivatives were 5-to 7-fold slower than the rates observed for PC. Phospholipid flip-flop, in contrast, was strongly dependent on headgroup composition with a smaller dependence on acyl chain length. At pH 7.4, flip-flop rates increased in the order PC less than PG less than PA less than PE, where the rates for PE were at least 10-times greater than those of the homologous PC derivative. Activation energies for flip-flop were large, and ranged from 38 kcal/mol for the longest acyl chain derivative of PC to 25 kcal/mol for the PE derivatives. Titration of the PA headgroup at pH 4.0 produced an approx. 500-fold increase in the flip-flop rate of PA, while the activation energy decreased 10 kcal/mol. Increasing acyl chain length reduced phospholipid flip-flop rates, with the greatest change observed for the PC analogs, which exhibited an approx. 2-fold decrease in flip-flop rate for every two methylene carbons added to the acyl chain at the sn-1 position.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spontaneous, intervesicular transfer rates of fluorescent, acyl chain-labeled phosphatidylcholine analogs

It was recently shown that the structure of the fluorophore attached to the acyl chain of phosphatidylcholine analogs determines their mechanism of transport across the plasma membrane of yeast cells (Elvington et al., J. Biol Chem. 280:40957, 2005). In order to gain further insight into the physical properties of these fluorescent phosphatidylcholine (PC) analogs, the rate and mechanism of their intervesicular transport was determined. The rate of spontaneous exchange was measured for PC analogs containing either NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene), Bodipy 530 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene), or Bodipy 581 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene) attached to a five or six carbon acyl chain in the sn-2 position. The rate of transfer between phospholipid vesicles was measured by monitoring the increase in fluorescence as the analogs transferred from donor vesicles containing self-quenching concentrations to unlabeled acceptor vesicles. Kinetic analysis indicated that the transfer of each analog occurred by diffusion through the water phase as opposed to transfer during vesicle collisions. The vesicle-to-monomer dissociation rate constants differed by over four orders of magnitude: NBD-PC (k(dis)=0.115 s(-1); t(1/2)=6.03 s); Bodipy FL-PC (k(dis)=5.2x10(-4); t(1/2)=22.2 min); Bodipy 530-PC (k(dis)=1.52x10(-5); t(1/2)=12.6 h); and Bodipy 581-PC (k(dis)=5.9x10(-6); t(1/2)=32.6 h). The large differences in spontaneous rates of transfer through the water measured for these four fluorescent PC analogs reflect their hydrophobicity and may account for their recognition by different mechanisms of transport across the plasma membrane of yeast.     

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC < POPC < DOPC, which have 0, 1, and 2 cis double bonds, respectively.     

Novel fluorescent phospholipids for assays of lipid mixing between membranes

A series of fluorescent phospholipids has been synthesized, by a general and versatile procedure, with various fluorescent groups attached to the methyl-terminal half of one acyl chain in an otherwise normal phospholipid structure. Phospholipids labeled with (dialkylamino)coumarin moieties, and to a slightly lesser extent those labeled with a bimane group, exhibit a strong and stable blue fluorescence in phospholipid dispersions that is relatively insensitive to the physical state of the lipid phase. The fluorescence of these labeled phospholipids is efficiently quenched by resonance energy transfer to lipids labeled with a [[(dimethylamino)phenyl]azo]phenyl or a methyl(nitrobenzoxadiazolyl)amino group when these acceptors are incorporated into the same bilayer as the donor species. Acyl chain labeled phospholipid probes, both of whose chains are at least sixteen carbons in length, exchange extremely slowly between lipid vesicles (less than 1% exchange/h). These properties allow various donor-acceptor combinations of probes to be employed in sensitive and reliable assays of lipid mixing accompanying membrane fusion. We demonstrate that, in two particularly demanding applications (assays of the calcium-mediated coalescence of phosphatidylserine vesicles and of the proton-triggered coalescence of phosphatidylethanolamine vesicles), some combinations of acyl chain labeled probes offer substantial advantages over the commonly used N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine/N-(l issamine rhodamine B sulfonyl)phosphatidylethanolamine pair to monitor accurately the progress of lipid mixing between vesicles.     

A computer simulation of functional group contributions to free energy in water and a DPPC lipid bilayer

A series of all-atom molecular dynamics simulations has been performed to evaluate the contributions of various functional groups to the free energy of solvation in water and a dipalmitoylphospatidylcholine lipid bilayer membrane and to the free energies of solute transfer (Delta(DeltaG(o))X) from water into the ordered-chain interior of the bilayer. Free energies for mutations of the alpha-H atom in p-toluic acid to six different substituents (-CH3, -Cl, -OCH3, -CN, -OH, -COOH) were calculated by a combined thermodynamic integration and perturbation method and compared to literature results from vapor pressure measurements, partition coefficients, and membrane transport experiments. Convergence of the calculated free energies was indicated by substantial declines in standard deviations for the calculated free energies with increased simulation length, by the independence of the ensemble-averaged Boltzmann factors to simulation length, and the weak dependence of hysteresis effects on simulation length over two different simulation lengths and starting from different initial configurations. Calculated values of Delta(DeltaG(o))X correlate linearly with corresponding values obtained from lipid bilayer transport experiments with a slope of 1.1 and from measurements of partition coefficients between water and hexadecane or decadiene, with slopes of 1.1 and 0.9, respectively. Van der Waals interactions between the functional group of interest and the acyl chains in the ordered chain region account for more than 95% of the overall potential energy of interaction. These results support the view that the ordered chain region within the bilayer interior is the barrier domain for transport and that solvation interactions within this region resemble those occurring in a nonpolar hydrocarbon.     

Interaction of lysophospholipid/taurodeoxycholate submicellar aggregates with phospholipid bilayers.

The equilibrium partitioning and the rate of transfer of monoacylphosphatidylethanolamines (lysoPEs) between phospholipid bilayers and lysoPE/taurodeoxycholate submicellar aggregates (SMAs) were examined with a series of environment-sensitive fluorescent-labeled N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-1-monoacylphosphatidyletha nolamine (N-NBD-lysoPE) probes of differing acyl chain length. Our previous work has demonstrated the formation of SMAs between bile salts and lysophospholipids [Shoemaker & Nichols (1990) Biochemistry 29, 5837-5842]. The experiments in the current work demonstrate that SMAs can coexist with phospholipid vesicles and can function as shuttle carriers for the transfer of lysophospholipids between membranes. The formation of submicellar aggregates of N-NBD-lysoPE and taurodeoxycholate (TDC) in equilibrium with 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles was determined from the increase in fluorescence generated upon addition of TDC to POPC vesicles containing 3 mol% N-NBD-lysoPE and 3 mol% N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE) as a nonextractable fluorescence energy-transfer quencher. The fraction of lysolipid extracted increased as a function of decreasing acyl chain length of the N-NBD-lysoPE molecule. The half-time for equilibration was independent of acyl chain length and averaged 44 ms at 10 degrees C. The delivery of N-NBD-lysoPE from preformed N-NBD-lysoPE/TDC SMAs into POPC vesicles containing the energy-transfer quencher N-Rh-PE was measured by the rate of fluorescence decline. The initial rate of insertion increased with decreasing acyl chain length of the N-NBD-lysoPE molecule and as a function of vesicle concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free fatty acid transfer from rat liver fatty acid-binding protein to phospholipid vesicles. Effect of ligand and solution properties.

Fatty acid binding proteins (FABP) are a family of 14-15-kDa proteins found in many mammalian cell types in high abundance. Although their precise physiological role remains hypothetical, the transfer of free fatty acids (ffa) to intracellular membrane sites is believed to be an important function of FABP. To better understand the role of FABP in this process, we have examined how the rate of ffa transfer from liver FABP (L-FABP) to model membranes is influenced by variations in ffa structure and properties of the aqueous phase. The rate of transfer of fluorescent anthroyloxy ffa to model acceptor membranes was monitored using a resonance energy transfer assay. The results show that a monounsaturated ffa transfers 2-fold more rapidly than a saturated ffa of equivalent chain length, and a two-carbon increase in acyl chain length results in a 3-fold decrease in transfer rate. The transfer rate decreases logarithmically with increasing ionic strength, suggesting that the aqueous solubility of the ffa is an important determinant of its dissociation rate from L-FABP. Fatty acid binding and the relative partition of n-(9-anthroloxy) ffa to L-FABP as compared with phospholipid membranes both decrease as pH decreases, indicating that ionized but not protonated ffa bind to L-FABP. The rate of ffa transfer from L-FABP to membranes increases approximately 4-fold with increasing pH, suggesting that ionization of the ffa carboxyl group is also an important determinant of the transfer process. Analysis of the dependence of the transfer rate on temperature demonstrates that the delta G++ of the activated state for ffa transfer arises from both enthalpic and entropic processes. These studies demonstrate that the rate of transfer of long chain ffa from L-FABP to membranes is substantially affected by aqueous phase variables as well as properties of the ffa ligand itself.     

Polyphosphoinositide activation of cholesterol side chain cleavage with purified cytochrome P-450scc

Highly purified beef adrenal cytochrome P-450 specific for cholesterol side chain cleavage (P-450-scc) has been reconstituted with sonicated vesicles containing cholesterol and either dimyristoyl phosphatidylcholine (DMPC) or dioleoyl phosphatidylcholine (DOPC). When cholesterol was present in DMPC vesicles at 1:15 molar ratio, cardiolipin and L-alpha-phosphatidylinositol 4-monophosphate (DPI) increased side chain cleavage by at least 5-fold (0.7 min-1-3.5 min-1). In DOPC vesicles, a smaller increase was observed (2.8 min-1-5.0 min-1). Activator phospholipids increased the rate of transference of cholesterol both to and from the cytochrome when, respectively, cholesterol-free P-450scc and cholesterol-P-450scc complex are combined with either DMPC or DOPC vesicles. Transfer of cholesterol to and from cytochrome P-450 occurred with similar first order rate constants and was also independent of the concentrations of cholesterol vesicles and P-450. It is suggested that transfer in both directions is limited by the rate of insertion of P-450scc into the membrane. Phospholipid stimulatory effects for both cholesterol transfer and for activation of side chain cleavage occurred with the same ranking, even though cholesterol transfer, following reconstitution, was 5-10 times slower than the turnover of side chain cleavage. DPI increased Vmax for side chain cleavage in both DMPC and DOPC vesicles to the same rate (12 min-1) without effect on the Km for cholesterol, while cardiolipin both produced a similar increase in Vmax and decreased Km (cholesterol). This activation by DPI is attributed to more favorable incorporation of P-450scc in these membranes and is consistent with previously reported effects of acidic phospholipids on other mitochondrial proteins.     

Nod factors integrate spontaneously in biomembranes and transfer rapidly between membranes and to root hairs, but transbilayer flip-flop does not occur.

Three novel nodulation (Nod) factors were synthesized from chitotetraose and three structurally different fluorescent BODIPY-tagged fatty acids. With fluorescence spectroscopic and microscopic techniques, the following aspects were studied: whether these amphiphilic molecules insert in membranes, whether they transfer between different membranes, and whether they are able to transfer from a membrane to a legume root hair. Fluorescence correlation spectroscopy showed that fluorescent Nod factors are present as monomers in PBS buffer at a concentration of 10 nM, but that when either Triton X-100 micelles or dioleoylphosphatidylcholine (DOPC) vesicles are present, the Nod factors are associated with these particles. With time-correlated single-photon counting fluorescence spectroscopy, it was shown that upon Nod factor insertion in the membrane, the rotation of the fluorescent acyl chain was markedly reduced. A fluorescence resonance energy transfer assay was used to study the transfer of Nod factors from one membrane to the other, or from vesicles to root hairs. Nod factors transfer rapidly between membranes or from vesicles to root hair cell walls. However, they do not flip-flop between membrane leaflets. The results provide novel insights for the mode of secretion and transfer of Nod factors during the early steps of the Rhizobium-legume interaction.     

Thermodynamics of interaction of octyl glucoside with phosphatidylcholine vesicles: partitioning and solubilization as studied by high sensitivity titration calorimetry

The interaction of the surfactant octyl glucoside (OG) with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and soy bean phosphatidylcholine (soy bean PC) was studied using high-sensitivity titration calorimetry. We determined the partition coefficient of OG between water and lipid bilayers and the transfer enthalpy of the surfactant by addition of lipid vesicles to OG monomers or vice versa. Comparison with the micellization enthalpy of the surfactant gives information on differences in the hydrophobic environment of OG in a liquid-crystalline bilayer or a micelle. The average partition coefficient P in mole fraction units for x_e≈0.12–0.2 decreases slightly from 4152 at 27°C to 3479 at 70°C for DMPC and from 4260 to 3879 for soy bean PC, respectively. The transfer enthalpy ΔH^T of OG into lipid vesicles is positive at 27°C and negative at 70°C. Its temperature dependence is larger for the incorporation of OG into DMPC than into soy bean PC vesicles. It is concluded that OG in DMPC vesicles is better shielded from water than in soy bean PC vesicles or in micelles. Titration calorimetry was also used to determine the phase boundaries of the coexistence region of mixed vesicles and mixed micelles in the systems OG/DMPC, OG/DPPC, OG/DSPC, and OG/soy bean PC vesicles at 70°C in the liquid-crystalline phase. DMPC and soy bean PC solubilization was also studied at 27°C to investigate the effect of temperature. The effective surfactant to lipid ratios at saturation, R_e^sat, for all PCs studied are in the range between 1.33–1.72 and the ratios at complete solubilization, R_e^sol, are between 1.79–3.06. At 70°C, the R_e^sat values decrease with increasing chain length of the saturated PC. The ratios depend also slightly on temperature and the degree of unsaturation of the fatty acyl chains. For the OG/soy bean PC system, the coexistence range for mixed vesicles and mixed micelles is larger than for the corresponding PCs with saturated chains.     

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.     

Lipid transfer between phosphatidylcholine vesicles and human erythrocytes: exponential decrease in rate with increasing acyl chain length

The rate of phospholipid transfer from sonicated phospholipid vesicles to human erythrocytes has been studied as a function of membrane concentration and lipid acyl chain composition. Phospholipid transfer exhibits saturable first-order kinetics with respect to both cell and vesicle membrane concentrations. This kinetic behavior is consistent either with transfer during transient contact between cell and vesicle surfaces (but only if the fraction of the cell surface susceptible to such interaction is small) or with transfer of monomers through the aqueous phase. The acyl chain composition of the transferred phospholipid affects the transfer kinetics profoundly; for homologous saturated phosphatidylcholines, the rate of transfer decreases exponentially with increasing acyl chain length. This behavior is consistent with passage of phospholipid monomers through a polar phase, which might be the bulk aqueous phase( as in the monomer transfer model) or the hydrated head-group regions of a cell-vesicle complex (transient collision model). Collisional transfer also predicts that intercell transfer of phospholipids should be slow compared to cell-vesicle transfer, as surface charge and steric effects should prevent close apposition of donor and acceptor membranes. This is not found; dilauroylphosphatidylcholine transfers rapidly between red cells. Thus, the observed relationship between acyl chain length and intermembrane phospholipid transfer rates likely reflects the energetics of monomer transfer through the aqueous phase.     

Effect of apolipoprotein C-II on the temperature dependence of lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholines. A hydrophobic model for the mechanism

The lipoprotein lipase-catalyzed hydrolysis of diacylphosphatidylcholines (PC) in mixed micelles of Triton X-100/PC was studied as a function of temperature in the presence and absence of apolipoprotein C-II (apo-C-II), the activator protein for lipoprotein lipase. Dilauroyl-, dimyristoyl-, dipalmitoyl-, and distearoyl-phosphatidylcholine (di-C12-PC, di-C14-PC, di-C16-PC, and di-C18-PC, respectively) were used as substrates. No systematic relationship between substrate fatty acyl chain length and either the rates of the activation energies for hydrolysis in the presence or absence of apo-C-II was observed. However, there was a linear relationship between fatty acyl chain length and both the logarithm of the activation factor (the ratio of enzyme activity with apo-C-II to that without apo-C-II) and the difference in activation energy in the presence and absence of apo-C-II. These relationships were not the result of an alteration in the physical form of the substrate, since a mixture of di-C14-PC and di-C16-PC gave activation factors for each PC which were the same as those obtained for each individual lipid. From the temperature dependence of the activation factor, thermodynamic functions of the apo-C-II-induced change in the reaction pathway were calculated. The free energy of activation decreased linearly with increasing chain length as the result of a linear increase in activation entropy which more than offset the unfavorable increase in activation enthalpy. We propose that the apo-C-II-mediated increase in the rate of the lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholine is associated with transfer of a fatty acyl chain of the substrate or product to a more hydrophobic environment within the transition state complex.     

Permeability of alkylamines across phosphatidylcholine vesicles as studied by 1H-NMR

The exchange rate and enthalpy and entropy of activation of the diffusion of the first five n-alkylamines across egg phosphatidylcholine vesicles has been measured by 1H-NMR spectroscopy employing the 1:2 Gd3+-EDTA complex as a relaxation reagent. The permeability determined from the exchange rate of the ethyl through the pentyl derivatives increased sequentially with increasing chain length from 7.10(-7) to 4.10(-4) cm/s, respectively, at 25 degrees C. The permeability of methylamine was similar to that of ethylamine (1.10(-6) cm/s at 25 degrees C) and exhibited a relatively smaller entropy increase. The enthalpy of activation for the transfer reaction was high for all amine derivatives (20 kcal/mol). The entropy of activation increased with increasing chain length. The results indicate that the rate of diffusion is dominated by the partition into the membrane. Methylamine, being the smallest molecule in this series, can probably diffuse also through vacancies formed by the internal motions of the lipid chains.     

Potentiometric titration of poly-L-lysine: the coil-to-beta transition

We have performed potentiometric titrations of poly-L -lysine. From these data we have calculated the free energy and enthalpy changes for the folding of the random coil to the α-helix in 10% ethanol (?120 and ?120 cal/mole) and from the random coil to the β-structure in water (?140 and 870 cal/mole) and in 10% ethanol (?180 and 980 cal mole). Comparison of these values with each other and with values for the coil → α- helix transition in water (?78 and ?880 cal/mole) led to the following conclusions. The stabilization by ethanol of ethanol of the α-helix with respect to the coil is that predicted from the known free energy of transfer of the peptide group from water to 10% ethanol. Similar data to explain the enthalpy difference are not available. The thermodynamic functions for the transition from α-helix to β-structure, obtained by subtracting those for the coil → α-helix and coil → β-structure transitions, are explained from a consideration of the structural differences: non bonded interactions of the polypeptide backbone are less favorable in the β-structure than in the α-helix, causing an increase in the energy, while hydrophobic contacts between side chains raise the entropy of the β-structure as compared with the α-helix, so that the free energy difference between the two structures is small, but enthalpy and entropy differences are large. The observation of only small differences in the free energy and enthalpy changes for the transition from coil β-structure upon going from water to 10% ethanol is expected by considering both the free energy of transfer of the peptide group (as for the α-helix) and the free energy and enthalpy of transfer of the apolar part of the side chain involved in hydrophobic bond formation.