Mixed-chain phosphatidylcholine bilayers: structure and properties

Calorimetric and X-ray diffraction data are reported for two series of saturated mixed-chain phosphatidylcholines (PCs), 18:0/n:0-PC and n:0/18:0-PC, where the sn-1 and sn-2 fatty acyl chains on the glycerol backbone are systematically varied by two methylene groups from 18:0 to 10:0 (n = 18, 16, 14, 12, or 10). Fully hydrated PCs were annealed at -4 degrees C and their multilamellar dispersions characterized by differential scanning calorimetry and X-ray diffraction. All mixed-chain PCs form low-temperature "crystalline" bilayer phases following low-temperature incubation, except 18:0/10:0-PC. The subtransition temperature (Ts) shifts toward the main (chain melting) transition temperature (Tm) as the sn-1 or sn-2 fatty acyl chain is reduced in length; for the shorter chain PCs (18:0/12:0-PC, 12:0/18:0-PC, and 10:0/18:0-PC), Ts is 1-2 degrees C greater than Tm, and the subtransition enthalpy (delta Hs) is much greater than for the longer acyl chain PCs. Tm decreases with acyl chain length for both series of PCs except 18:0/10:0-PC, while for the positional isomers, n:0/18:0-PC and 18:0/n:0-PC, Tm is higher for the isomer with the longer acyl chain in the sn-2 position of the glycerol backbone. The conversion from the crystalline bilayer Lc phase to the liquid-crystalline L alpha phase with melted hydrocarbon chains occurs through a series of phase changes which are chain length dependent. For example, 18:0/18:0-PC undergoes the phase changes Lc----L beta'----P beta'----L alpha, while the shorter chain PC, 10:0/18:0-PC, is directly transformed from the Lc phase to the L alpha phase. However, normalized enthalpy and entropy data suggest that the overall thermodynamic change, Lc----L alpha, is essentially chain length independent. On cooling, the conversion to the Lc phases occurs via bilayer gel phases, L beta', for the longer chain PCs or through triple-chain interdigitated bilayer gel phases, L beta, for the shorter chain PC 18:0/12:0-PC and possibly 10:0/18:0-PC. Molecular models indicate that the bilayer gel phases for the more asymmetric PC series, 18:0/n:0-PC, must undergo progressive interdigitation with chain length reduction to maintain maximum chain-chain interaction. The L beta phase of 18:0/10:0-PC is the most stable structure for this PC below Tm. The formation and stability of the triple-chain structures can be rationalized from molecular models.     

The phosphatidylcholine transfer protein from bovine liver discriminates between phosphatidylcholine isomers. A monolayer study

The activity of the phosphatidylcholine transfer protein from bovine liver toward phosphatidylcholine isomers carrying a long and a short fatty acyl chain on either the sn-1- or sn-2-position was determined by way of the monolayer-vesicle assay. In this assay equimolar mixtures of the isomers were spread at the air/water interface and their transfer measured to the vesicles in the subphase initiated by addition of the transfer protein. The following isomers were tested: 1-decanoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine (C10:0/[3H]C18:1-PC) and 1-oleoyl-2-decanoyl-sn-glycero-3-phospho[14C]choline (C18:1/C10:0-[14C]PC); 1-lauroyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine (C12:0/[3H]C18:1-PC) and 1-oleoyl-2-[14C]lauroyl-sn-glycero-3-phosphocholine (C18:1/[14C]C12:0-PC); 1-myristoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine (C14:0/[3H]C18:1-PC) and 1-oleoyl,2-myristoyl-sn-glycero-3-phospho[14C]choline (C18:1/C14:0-[14C]PC). It was found that the protein transferred C10:0/[3H]C18:1-PC twice as fast as C18:1/C10:0-[14C]PC. Similar differences in rate were observed for C12:0/[3H]C18:1-Pc and C18:1/[14C]C12:0-PC but not for the isomers carrying myristic acid. We propose that the transfer protein can discriminate between PC isomers due to the presence of distinct binding sites for the sn-1- and sn-2-acyl chain (Berkhout et al. (1984) Biochemistry, 23, 1505-1513).     

Bilayers of arachidonic acid containing phospholipids studied by 2H and 31P NMR spectroscopy

The configurational properties and dynamics of the arachidonic acyl chains of phospholipid bilayers have been investigated for the first time by solid-state 2H NMR techniques, with the goal of achieving a better understanding of the biological roles of polyunsaturated phospholipids. Vinyl perdeuterated arachidonic acid (20:4 delta 5,8,11,14-d8) was prepared from eicosatetraynoic acid (ETYA) and was esterified with 1-palmitoyl-sn-glycero-3-phosphocholine to yield 1-palmitoyl-2-vinylperdeuterioarachidonoyl-sn-glycero-3-phosphocho line [(16:0)(20:4-d8)PC]. 31P NMR spectra of aqueous dispersions of (16:0)(20:4-d8)PC as well as 1-perdeuteriopalmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine [(per-2H-16:0)(20:4)PC] were characteristic of the lamellar liquid-crystalline state. The dispersions had similar 31P chemical shift anisotropies, with little apparent motional averaging of the lineshapes due to macroscopic reorientation of liposomes or lateral diffusion of phospholipids about their curved surfaces. Comparison to other phosphatidylcholines indicated that both samples comprised the fully hydrated L alpha phase plus excess water. However, the dispersion of (16:0)(20:4-d8)PC yielded relatively narrow powder-type 2H NMR spectra, compared to (per-2H-16:0)(20:4)PC in the liquid-crystalline state. The differences in the 2H NMR powder patterns thus reflect differences in the configurational properties of the polyunsaturated sn-2 arachidonic acyl chain compared to the saturated sn-1 palmitic chain. When the powder-type 2H NMR spectra of the (16:0)(20:4-d8)PC bilayer were dePaked (theta = 0 degrees), they showed three kinds of deuterons upon integration: one with a large splitting (approximately 25-35 kHz), two with intermediate splittings (approximately 10-15 kHz), and the remainder with smaller splittings (approximately 0.3-5 kHz).(ABSTRACT TRUNCATED AT 250 WORDS)     

Raman spectroscopic study of polycrystalline mono- and polyunsaturated 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines: bilayer lipid clustering and acyl chain order and disorder characteristics

Polycrystalline lipid samples of a series of mono- and polyunsaturated, double bond positional isomers of 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines [C(20-d(39)):C(20:1 Delta(j))PC, with j = 5, 8, 11, or 13; C(20-d(39)):C(20:2 Delta(11,14))PC; and C(20-d(39)):C(20:3 Delta(11, 14,17))PC] were investigated using vibrational Raman spectroscopy to assess the acyl chain packing order-disorder characteristics and putative bilayer cluster formation of the isotopically differentiated acyl chains. Perdeuteration of specifically the saturated sn-1 acyl chains for these bilayer systems enables each chain's intra- and intermolecular conformational and organizational properties to be evaluated separately. Various saturated chain methylene CD(2) and carbon-carbon (C&bond;C) stretching mode peak height intensity ratios and line width parameters for the polycrystalline samples demonstrate a high degree of sn-1 chain order that is unaffected by either the double bond placement or number of unsaturated bonds within the sn-2 chain. In contrast, the unsaturated sn-2 chain spectral signatures reflect increasing acyl chain conformational disorder as either the cis double bond is generally repositioned toward the chain terminus or the number of double bonds increases from one to three. The lipid bilayer chain packing differences observed between the sn-1 and sn-2 chains of this series of monounsaturated and polyunsaturated 20 carbon chain lipids suggest the existence of laterally distributed microdomains predicated on the formation of highly ordered, saturated sn-1 chain clusters.     

Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of ¹³C-labeled diC8PC ((methyl-¹³C)₃-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-¹³C)₃-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.     

Influence of N-dodecyl-N,N-dimethylamine N-oxide on the activity of sarcoplasmic reticulum Ca(2+)-transporting ATPase reconstituted into diacylphosphatidylcholine vesicles: efects of bilayer physical parameters

Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 degrees C, the specific activity of ATPase displays a maximum (31.5+/-0.8 IU/mg) for dioleoylphosphatidylcholine (diC18:1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22:1PC), the zwitterionic surfactant N-dodecyl-N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2+/-0.6 to 32.5+/-0.8 IU/mg in the range of molar ratios C12NO:diC22:1PC=0/1.2. In dilauroylphosphatidylcholines (diC12:0PC) and diC18:1PC, the effect of C12NO is twofold-the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18:1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO:diC18:1PC< or =1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4+/-0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N(+)-P- accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO:diC18:1PC=1.3/2.5, where mixed tubular micelles are detected by SANS in C12NO+diC18:1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca(2+)-ATPase activity.     

Properties of the binding sites for the sn-1 and sn-2 acyl chains on the phosphatidylinositol transfer protein from bovine brain

We have studied the properties of the fatty acyl binding sites of the phosphatidylinositol transfer protein (PI-TP) from bovine brain, by measuring the binding and transfer of pyrenylacyl-containing phosphatidylinositol (PyrPI) species and pyrenylacyl-containing phosphatidylcholine (PyrPC) species as a function of the acyl chain length. The PyrPI species carried a pyrene-labeled acyl chain of variable length in the sn-2 position and either palmitic acid [C(16)], palmitoleic acid [C(16:1)], or stearic acid [C(18:1)] in the sn-1 position. Binding and transfer of the PI species increased in the order C(18) less than C(16) less than C(16:1), with a distinct preference for those species that carry a pyrenyloctanoyl [Pyr(8)] or a pyrenyldecanoyl [Pyr(10)] chain. The PyrPC species studied consisted of two sets of positional isomers: one set contained a pyrenylacyl chain of variable length and a C(16) chain, and the other set contained an unlabeled chain of variable length and a Pyr(10) chain. The binding and transfer experiments showed that PI-TP discriminates between positional isomers with a preference for the species with a pyrenylacyl chain in the sn-1 position. This discrimination is interpreted to indicate that separate binding sites exist for the sn-1 and sn-2 acyl chains. From the binding and transfer profiles it is apparent that the binding sites differ in their preference for a particular acyl chain length. The binding and transfer vs chain length profiles were quite similar for C(16)Pyr(x)PC and C(16)Pyr(x)PI species, suggesting that the sn-2 acyl chains of PI and PC share a common binding site in PI-TP.     

Binary mixtures of saturated and unsaturated mixed-chain phosphatidylcholine. A differential scanning calorimetry study

High-resolution differential scanning calorimetry (DSC) has been used to study the aqueous dispersions of mixed-chain phosphatidylcholines prepared from colyophilized mixtures of C(18):C(11:1 delta 10) PC/C(18):C(10)PC and C(18):C(11:1 delta 10) PC/C(18):C(11)PC of various molar ratios. These mixed-chain phospholipids are characterized by a marked disparity in their acyl-chain lengths; however, the sn-1 acyl chain in the fully extended conformation is about twice as long as the sn-2 acyl chain. Their thermotropic behavior was determined, and the phase diagrams of these two mixtures were constructed from the calorimetric data. Results indicate that C(18):C(11:1 delta 10)PC/C(18):C(10)PC and C(18):C-(11:1 delta 10)PC/C(18):C(11)PC are miscible in all proportions with a near-ideal behavior of mixing in the gel and liquid-crystalline phases. Equimolar mixtures of diC(14)PC/C(18):C(11:1 delta 10)PC, diC(14)PC/C(18):C(10)PC, and diC(14)PC/C(18):C(11)PC have also been studied by DSC. These phosphatidylcholines in the 1:1 mixture differ in Tm by less than 11 degrees C; however, they exhibit gel-phase immiscibility in the plane of the bilayer. Taken together, these studies suggest that C(18):C(11)PC and C(18):C(11:1 delta 10)PC are packed similarly to C(18):C(10)PC in excess water as mixed interdigitated bilayers, at T less than Tm, which transform into partially interdigitated bilayers when heated above Tm.     

Yeast cells accumulate excess endogenous palmitate in phosphatidylcholine by acyl chain remodeling involving the phospholipase B Plb1p

In the yeast Saccharomyces cerevisiae, the molecular species profile of the major membrane glycerophospholipid phosphatidylcholine (PC) is determined by the molecular species-selectivity of the biosynthesis routes and by acyl chain remodeling. Overexpression of the glycerol-3-phosphate acyltransferase Sct1p was recently shown to induce a strong increase in the cellular content of palmitate (C16:0). Using stable isotope labeling and mass spectrometry, the present study shows that wild type yeast overexpressing Sct1p incorporates excess C16:0 into PC via the methylation of PE, the CDP-choline route, and post-synthetic acyl chain remodeling. Overexpression of Sct1p increased the extent of remodeling of PE-derived PC, providing a novel tool to perform mechanistic studies on PC acyl chain exchange. The exchange of acyl chains occurred at both the sn-1 and sn-2 positions of the glycerol backbone of PC, and required the phospholipase B Plb1p for optimal efficiency. Sct1p-catalyzed acyl chain exchange, the acyl-CoA binding protein Acb1p, the Plb1p homologue Plb2p, and the glycerophospholipid:triacylglycerol transacylase Lro1p were not required for PC remodeling. The results indicate that PC serves as a buffer for excess cellular C16:0.     

Lipoprotein lipase-catalyzed hydrolysis of phospholipid monolayers: effect of fatty acyl composition on enzyme activity

The phospholipase A1 activity of lipoprotein lipase (LpL) was determined with monomolecular phospholipid films. Rates of phospholipid hydrolysis were dependent on apolipoprotein C-II (the activator protein for LpL) phospholipid fatty acyl composition, and lipid-packing density. In sphingomyelin: cholesterol (2:1, molar) monolayers containing 5 mol % disaturated phosphatidylcholines (PC) and at a surface pressure of 22 mNm-1, rates of LpL hydrolysis of diC14:0PC, diC16:0PC, and diC18:0PC were 74, 207, and 65 nmol h-1 mg LpL-1, respectively. At 22 mNm-1, phospholipids containing unsaturated fatty acyl chains were hydrolyzed at rates 5-10 times greater than saturated lipids. At higher lipid packing densities, the difference in hydrolysis rates between saturated and unsaturated lipids was less apparent. Comparison of molecular areas indicate no simple dependency between the rate of LpL catalysis and phospholipid fatty acyl chain length and saturation/unsaturation.     

Thermal behavior of liposomes containing PCs with long and very long chain PUFAs isolated from retinal rod outer segment membranes

About one-fourth the phosphatidylcholines (PC) from retina photoreceptor rod outer segment (ROS) membranes contain docosahexaenoic acid (22:6n-3) at sn-2 and a very long chain polyunsaturated fatty acid (VLCPUFA) (C24 to C36) at the sn-1 position of the glycerol backbone. In order to study the thermotropic behavior of these PCs, subfractions and molecular species of PC (16:0/22:6, 18:0/22:6, 22:6/22:6, 32:5/22:6, 32:6/22:6, 34:5/22:6), were isolated from bovine ROS, and liposomes containing different proportions of these PCs and dimyristoyl-PC (DMPC) or dipalmitoyl PC (DPPC) were compared using the fluorescence probes Laurdan and 1,6-diphenyl-1,3,5-hexatriene (DPH). With both probes, the 22:6n-3 containing PCs from ROS, in all proportions tested, decreased the transition temperature (Tt) of both DMPC and DPPC. Below the transition temperature, coexistence of phases was evidenced in all cases. Liposomes formed with 100% of any of these PCs did not show phase transitions in the temperature range studied (8 degrees C to 50 degrees C). At physiological temperatures, as it is likely to be the case in ROS membranes, all of these PC species were in the liquid-crystalline state. With Laurdan, all dipolyunsaturated PCs seemed to behave similarly: despite the large number of double bonds per molecule, all of them decreased the Tt of DPPC less than did the hexaenoic PCs. With DPH, an ample difference was detected between the dipolyunsaturates, 22:6/22:6-PC and VLCPUFA/22:6-PCs, and between the latter and hexaenoic PCs throughout the temperature range studied. This difference is consistent with the interpretation that the largest "disorder" produced by PCs containing a VLCPUFA like 32:6n-3 at the sn-1 position occurs toward the center of the membrane.     

Distribution of phosphatidylcholine molecular species between mixed micelles and phospholipid-cholesterol vesicles in human gallbladder bile: dependence on acyl chain length and unsaturation.

The partitioning of phosphatidylcholine (PC) molecular species between mixed micelles and vesicles was studied in each of seven human gallbladder biles. Biles were fractionated by Sephacryl S-300 SF gel filtration chromatography, and PC species in the micellar and vesicular fractions were quantitated by high performance liquid chromatography. Micelles were enriched in species containing unsaturated acyl groups (e.g., 16:1-18:2, 18:1-18:2, and 18:1-18:3); vesicles were enriched in more highly saturated species (e.g., 16:0-16:1, 16:0-18:1, and 18:0-18:1). Separate multivariate analyses for each bile demonstrated that the distribution of PC species between vesicles and micelles was related to the degree of sn-1 and sn-2 unsaturation, and sn-1, but not sn-2, chain length. In addition, the tendency to partition into the micellar phase was particularly marked when unsaturation was present at both the sn-1 and sn-2 positions. When this interaction was included in the multivariate analyses, the regression models accounted for virtually all of the variation in PC partitioning (for each of the seven patients r2 = 0.92-0.98, P less than 0.03). These results suggest that the partitioning of PC species between micelles and vesicles is strictly determined by sn-1 chain length and the degree of unsaturation at both the sn-1 and sn-2 positions. In light of recent reports that fatty acyl composition influences the cholesterol content of vesicles and micelles in model biles, these results raise the possibility that diet-induced alterations in the phospholipid species and the relative proportions of biliary lipid particles may influence the cholesterol-carrying capacity of bile.     

Mimicry of phorbol ester responses by diacylglycerols. Differential effects on phosphatidylcholine biosynthesis, cell-cell communication and epidermal growth factor binding

The biosynthesis of phosphatidylcholine (PC) in HEL-37 cells was followed by measuring the incorporation of [32P]Pi into PC. Incorporation was stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and by the synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (diC8), but not by sn-1-oleoyl-2-acetylglycerol or sn-1,2-dihexanoylglycerol (diC6). DiC8 was rapidly metabolised by HEL-37 cells to the corresponding PC and phosphatidic acid derivatives. diC8, diC6 and oleoylacetylglycerol effectively displaced [3H]phorbol-12,13-dibutyrate bound to a soluble cell extract from HEL-37 cells, but only diC8 was able to displace the labelled phorbol ester from prelabelled cells. TPA, diC8, diC6 and oleoylacetylglycerol were all effective inhibitors of 125I-labelled epidermal growth factor binding to, and gap junctional communication between, HEL-37 cells. It is concluded that only cell-permeable diacylglycerols stimulate PC biosynthesis which may therefore require interaction with membranes other than the plasma membrane.     

Acyl chain-length asymmetry alters the interfacial elastic interactions of phosphatidylcholines.

Phosphatidylcholines (PCs) with stearoyl (18:0) sn-1 chains and variable-length, saturated sn-2 acyl chains were synthesized and investigated using a Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at various constant temperatures between 10 degrees C and 30 degrees C. Over this temperature range, 18:0-10:0 PC displayed only liquid-expanded behavior. In contrast, di-14:0 PC displayed liquid-expanded behavior at 24 degrees C and 30 degrees C, but two-dimensional phase transitions were evident at 20 degrees C, 15 degrees C, and 10 degrees C. The average molecular area of 18:0-10:0 PC was larger than that of liquid-expanded di-14:0 PC at equivalent surface pressures, and the shapes of their liquid expanded isotherms were somewhat dissimilar. Analysis of the elastic moduli of area compressibility (Cs(-1)) as a function of molecular area revealed shallower slopes in the semilog plots of 18:0-10:0 PC compared to di-14:0 PC. At membrane-like surface pressures (e.g., 30 mN/m), 18:0-10:0 PC was 20-25% more elastic (in an in-plane sense) than di-14:0 PC. Other PCs with varying degrees of chain-length asymmetry (18:0-8:0 PC, 18:0-12:0 PC, 18:0-14:0 PC, 18:0-16:0 PC) were also investigated to determine whether the higher in-plane elasticity of fluid-phase 18:0-10:0 PC is a common feature of PCs with asymmetrical chain lengths. Two-dimensional phase transitions in 18:0-14:0 PC and 18:0-16:0 PC prevented meaningful comparison with other fluid-phase PCs at 30 mN/m. However, the Cs(-1) values for fluid-phase 18:0-8:0 PC and 18:0-12:0 PC were similar to that of 18:0-10:0 PC (85-90 mN/m). These values showed chain-length asymmetrical PCs to have 20-25% greater in-plane elasticity than fluid-phase PCs with mono- or diunsaturated acyl chains.     

Partitioning of a fluorescent phospholipid between fluid bilayers: dependence on host lipid acyl chains

The partition coefficient Kp was measured for a headgroup-labeled phospholipid (12:0,12:0)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PE (12-NBD-PE), equilibrated between LUV of a series of phosphatidylcholines (PC). Fluorescence resonance energy transfer between the 12-NBD-PE and a headgroup-rhodamine-labeled PE was used to find the equilibrium concentration of the 12-NBD-PE in the different LUV. Reliable equilibrium concentrations were obtained by monitoring the approach to equilibrium starting from a concentration below and from a concentration above the ultimate values. Using (16:0,18:1delta9)-PC as the reference lipid, Kp ranged from a high value of 1.65 favoring (16:0,18:1delta9)-PC over (16:1delta9,16:1delta9)-PC, to a low value of 0.90, favoring (22:1delta13,22:1delta13)-PC over (16:0,18:1delta9)-PC. The Kp values enabled calculation of the acyl chain contribution to the excess free energy of mixing for (12:0,12:0) acyl chains at infinite dilution in the L alpha phase of PC having acyl chains of (16:0,18:1delta9), (16:1delta9,16:1delta9), (18:1delta9,18:1delta9), (18:1delta6,18:1delta6), (20:1delta11,20:1delta11), and (22:1delta13,22:1delta13). (14:1delta9,14:1delta9)-PC was found to transfer so rapidly between LUV as to preclude reliable Kp measurement.     

Consequences of hydrophobic mismatch between lipids and melibiose permease on melibiose transport

The structural and functional consequences of a mismatch between the hydrophobic thickness d(P) of a transmembrane protein and that d(L) of the supporting lipid bilayer were investigated using melibiose permease (MelB) from Escherichia coli reconstituted in a set of bis saturated and monounsaturated phosphatidylcholine species differing in acyl-chain length. Influence of MelB on the midpoint gel-to-liquid-phase transition temperature, T(m), of the saturated lipids was investigated through fluorescence polarization experiments, with 1,6-diphenyl-1,3,5-hexatriene as the probe, for varying protein/lipid molar ratio. Diagrams in temperature versus MelB concentration showed positive or negative shifts in T(m) with the short-chain lipids DiC12:0-PC and DiC14:0-PC or the long-chain lipids DiC16:0-PC and DiC18:0-PC, respectively. Theoretical analysis of the data yielded a d(L) value of 3.0 +/- 0.1 nm for the protein, similar to the 3.02 nm estimated from hydropathy profiles. Influence of the acyl chain length on the carrier activity of MelB was investigated in the liquid phase, using the monounsaturated PCs. Binding of the sugar to the transporter showed no dependence on the acyl chain length. In contrast, counterflow and Deltapsi-driven experiments revealed strong dependence of melibiose transport on the lipid acyl chain length. Similar bell-shaped transport versus acyl chain length profiles were obtained, optimal activity being supported by diC16:1-PC. On account of a d(P) value of 2.65 nm for the lipid and of various local constraints which would all tend to elongate the acyl chains in contact with the protein, one can conclude that maximal activity was obtained when the hydrophobic thickness of the bilayer matched that of the protein.     

Incorporation of newly synthesized fatty acids into cytosolic glycerolipids in pea leaves occurs via acyl editing

In expanding pea leaves, over 95% of fatty acids (FA) synthesized in the plastid are exported for assembly of eukaryotic glycerolipids. It is often assumed that the major products of plastid FA synthesis (18:1 and 16:0) are first incorporated into 16:0/18:1 and 18:1/18:1 molecular species of phosphatidic acid (PA), which are then converted to phosphatidylcholine (PC), the major eukaryotic phospholipid and site of acyl desaturation. However, by labeling lipids of pea leaves with [(14)C]acetate, [(14)C]glycerol, and [(14)C]carbon dioxide, we demonstrate that acyl editing is an integral component of eukaryotic glycerolipid synthesis. First, no precursor-product relationship between PA and PC [(14)C]acyl chains was observed at very early time points. Second, analysis of PC molecular species at these early time points showed that >90% of newly synthesized [(14)C]18:1 and [(14)C]16:0 acyl groups were incorporated into PC alongside a previously synthesized unlabeled acyl group (18:2, 18:3, or 16:0). And third, [(14)C]glycerol labeling produced PC molecular species highly enriched with 18:2, 18:3, and 16:0 FA, and not 18:1, the major product of plastid fatty acid synthesis. In conclusion, we propose that most newly synthesized acyl groups are not immediately utilized for PA synthesis, but instead are incorporated directly into PC through an acyl editing mechanism that operates at both sn-1 and sn-2 positions. Additionally, the acyl groups removed by acyl editing are largely used for the net synthesis of PC through glycerol 3-phosphate acylation.     

Phosphatidylcholine fluidity and structure affect lecithin:cholesterol acyltransferase activity

The purpose of this study was to test the hypothesis that lipid fluidity regulates lecithin:cholesterol acyltransferase (LCAT) activity. Phosphatidylcholine (PC) species were synthesized that varied in fluidity by changing the number, type (cis vs. trans), or position of the double bonds in 18 or 20 carbon sn-2 fatty acyl chains and recombined with [(3)H]cholesterol and apolipoprotein A-I to form recombinant high density lipoprotein (rHDL) substrate particles. The activity of purified human plasma LCAT decreased with PC sn-2 fatty acyl chains containing trans versus cis double bonds and as double bonds were moved towards the methyl terminus of the sn-2 fatty acyl chain. The decrease in LCAT activity was significantly correlated with a decrease in rHDL fluidity (measured by diphenylhexatriene fluorescence polarization) for PC species containing 18 carbon (r(2) = 0.61, n = 18) and 20 carbon (r(2) = 0.93, n = 5) sn-2 fatty acyl chains. rHDL were also made containing 10% of the 18 carbon sn-2 fatty acyl chain PC species and 90% of an inert PC ether matrix (sn-1 18:1, sn-2 16:0 PC ether) to normalize rHDL fluidity. Even though fluidity was similar among the PC ether-containing rHDL, the order of PC reactivity with LCAT was significantly correlated (r(2) = 0.71) with that of 100% PC rHDL containing the same 18 carbon sn-2 fatty acyl chain species, suggesting that PC structure in the active site of LCAT determines reactivity in the absence of measurable differences in bilayer fluidity. We conclude that PC fluidity and structure are major regulators of LCAT activity when fatty acyl chain length is constant.     

X-ray diffraction evidence for fully interdigitated bilayers of 1-stearoyllysophosphatidylcholine

X-ray diffraction experiments have been performed on 1-stearoyllysophosphatidylcholine or C(18):C(0)PC as a function of hydration at temperatures below the order/disorder transition (Tm = 26.2 degrees C). At these temperatures, hydrated C(18):C(0)PC forms lamellae. The bilayer thickness, as determined by the saturation hydration method and electron-density profile, is 35-36 A, and the average area per C(18):C(0)PC molecule at the lipid/water interface is 45.5 A2. The packing geometry of C(18):C(0)PC in the lamella is proposed to adopt a fully interdigitated model in which the long C(18) acyl chain extends across the entire hydrocarbon width of the bilayer. Thus far, three different types of interdigitated bilayers are known for phosphatidylcholines. These various types of chain interdigitation are discussed in terms of the chain length difference between the sn-1 and sn-2 acyl chains.     

Differential scanning calorimetric study of a homologous series of fully hydrated saturated mixed-chain C(X):C(X + 6) phosphatidylcholines

The successive high-resolution differential scanning calorimetric (DSC) thermograms for aqueous dispersions of a homologous series of mixed-chain phosphatidylcholines, C(X):C(X + 6)PC, have been recorded and analyzed. In this series of saturated mixed-chain phosphatidylcholines, the total number of carbon atoms in the sn-1 acyl chain increases from 11 to 20, and the sn-2 acyl chain is always 6 methylene units longer than the sn-1 acyl chain. In the initial heating DSC thermograms, two prominent endothermic transitions are detected for all the samples prepared from the various C(X):C(X + 6)PCs except C(12):C(18)PC. In contrast, a single exothermic transition is observed on cooling for all the samples except C(13):C(19)PC. The temperature difference between the two endothermic transitions increases linearly as the acyl chain length of C(X):C(X + 6)PC becomes progressively longer. Interestingly, the main phase transition occurs before the subtransition for C(11):C(17)PC dispersions. Our DSC data further demonstrate that the thermodynamic parameters (Tm, delta H, and delta S) associated with the main phase transition for fully hydrated C(13):C(19)PC and other identical MW phosphatidylcholines are inversely related to the corresponding values of the chain-length inequivalence (delta C/CL) for these lipids. This linear relationship can be employed to map the Tm values for aqueous dispersions prepared from a large number of mixed-chain phosphatidylcholines whose values of delta C/CL are within the range of 0.1-0.4.