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.     

Effects of various numbers and positions of cis double bonds in the sn-2 acyl chain of phosphatidylethanolamine on the chain-melting temperature

In an attempt to investigate systematically the effects of various single and multiple cis carbon-carbon double bonds in the sn-2 acyl chains of natural phospholipids on membrane properties, we have de novo synthesized unsaturated C20 fatty acids comprised of single or multiple methylene-interrupted cis double bonds. Subsequently, 15 molecular species of phosphatidylethanolamine (PE) with sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains were semi-synthesized by acylation of C20-lysophosphatidylcholine with unsaturated C20 fatty acids followed by phospholipase D-catalyzed base-exchange reaction in the presence of excess ethanolamine. The gel-to-liquid crystalline phase transitions of these 15 mixed-chain PE, in excess H2O, were investigated by high resolution differential scanning calorimetry. In addition, the energy-minimized structures of these sn-1 C20-saturated/sn-2 C20-unsaturated PE were simulated by molecular mechanics calculations. It is shown that the successive introduction of cis double bonds into the sn-2 acyl chain of C(20):C(20)PE can affect the gel-to-liquid crystalline phase transition temperature, Tm, of the lipid bilayer in some characteristic ways; moreover, the effect depends critically on the position of cis double bonds in the sn-2 acyl chain. Specifically, we have constructed a novel Tm diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds on Tm can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristic Tm profiles exhibited by different series of mixed-chain PE with increasing degree of unsaturation can be interpreted in terms of structural changes associated with acyl chain unsaturation.     

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.     

Affinity of phosphatidylcholine molecular species for the bovine phosphatidylcholine and phosphatidylinositol transfer proteins. Properties of the sn-1 and sn-2 acyl binding sites

Both the phosphatidylcholine transfer protein (PC-TP) and the phosphatidylinositol transfer protein (PI-TP) act as carriers of phosphatidylcholine (PC) molecules between membranes. To study the structure of the acyl binding sites of these proteins, the affinity of 32 distinct natural and related PC molecular species was determined by using a previously developed fluorometric competition assay. Marked differences in affinity between species were observed with both proteins. Affinity vs lipid hydrophobicity (determined by reverse-phase HPLC) plots displayed a well-defined maximum indicating that the acyl chain hydrophobicity is an important determinant of binding of a phospholipid molecule by these transfer proteins. However, besides the overall lipid hydrophobicity, steric properties of the individual acyl chains contribute considerably to the affinity, and PC-TP and PI-TP respond differently to modifications of the acyl chain structure. The affinity of PC-TP increased steadily with increasing unsaturation of the sn-2 acyl moiety, resulting in high affinity for species containing four and six double bonds in the sn-2 chain, whereas the affinity of PI-TP first increased up to two to three double bonds and then declined. These data, as well as the distinct effects of sn-2 chain double bond position and bromination, indicate that the sn-2 acyl chain binding sites of the two proteins are structurally quite different. The sn-1 acyl binding sites are dissimilar as well, since variation of the length of saturated sn-1 chain affected the affinity differently. The data are discussed in terms of the structural organization of the sn-1 and sn-2 acyl binding sites of PC-TP and PI-TP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic analysis of chain-melting transition temperatures for monounsaturated phospholipid membranes: dependence on cis-monoenoic double bond position.

Unsaturated phospholipid is the membrane component that is essential to the dynamic environment needed for biomembrane function. The dependence of the chain-melting transition temperature, T(t), of phospholipid bilayer membranes on the position, n(u), of the cis double bond in the glycerophospholipid sn-2 chain can be described by an expression of the form T(t) = T(t)(c)(1 + h'(c)|n(u) - n(c)|)/(1 + s'(c)|n(u) - n(c)|), where n(c) is the chain position of the double bond corresponding to the minimum transition temperature, T(t)(c), for constant diacyl lipid chain lengths. This implies that the incremental transition enthalpy (and entropy) contributed by the sn-2 chain is greater for whichever of the chain segments, above or below the double-bond position, is the longer. The critical position, n(c), of the double bond is offset from the center of the sn-2 chain by an approximately constant amount, deltan(c) approximately 1. 5 C-atom units. The dependence of the parameters T(t)(c), h'(c), and s'(c) on sn-1 and sn-2 chain lengths can be interpreted consistently when allowance is made for the chain packing mismatch between the sn-1 and sn-2 chains. The length of the sn-2 chain is reduced by approximately 0.8 C-atom units by the cis double bond, in addition to a shortening by approximately 1.3 C-atom units by the bent configuration at the C-2 position. Based on this analysis, a general thermodynamic expression is proposed for the dependence of the chain-melting transition temperature on the position of the cis double bond and on the sn-1 and sn-2 chain lengths. The above treatment is restricted mostly to double-bond positions close to the center of the sn-2 chain. For double bonds positioned closer to the carboxyl or terminal methyl ends of the sn-2 chain, the effects on transition enthalpy can be considerably larger. They may be interpreted by the same formalism, but with different characteristic parameters, h'(c) and s'(c), such that the shorter of the chain segments makes a considerably smaller contribution to the calorimetric properties of the chain-melting transition.     

Incorporation and remodeling of extracellular phosphatidylcholine with short acyl residues in Saccharomyces cerevisiae

The pem1/cho2 pem2/opi3 double mutant of Saccharomyces cerevisiae, which is auxotrophic for choline because of the deficiency in methylation activities of phosphatidylethanolamine, grew in the presence of 0.1 mM dioctanoyl-phosphatidylcholine (diC(8)PC). Analysis of the metabolism of methyl-(13)C-labeled diC(8)PC ((methyl-(13)C)(3)-diC(8)PC) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) revealed that it was rapidly converted to (methyl-(13)C)(3)-PCs containing C16 or C18 acyl chains. (Methyl-(13)C)(3)-8:0-lyso-PC, (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC, which are the probable intermediate molecular species of acyl chain remodeling, appeared immediately after 5 min of pulse-labeling and decreased during the subsequent chase period. These results indicate that diC(8)PC was taken up by the pem1 pem2 double mutant and that the acyl chains of diC(8)PC were exchanged with longer yeast fatty acids. The temporary appearance of (methyl-(13)C)(3)-8:0-lyso-PC suggests that the remodeling reaction may consist of deacylation and reacylation by phospholipase activities and acyltransferase activities, respectively. The detailed analyses of the structures of (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC by MS/MS and MS(3) strongly suggest that most (methyl-(13)C)(3)-8:0-16:0-PCs have a C16:0 acyl chain at sn-1 position, whereas (methyl-(13)C)(3)-8:0-16:1-PCs have a C16:1 acyl chain at either sn-1 or sn-2 position in a similar frequency, implying that the initial C16:0 acyl chain substitution prefers the sn-1 position; however, the C16:1 acyl chain substitution starts at both sn-1 and sn-2 positions. The current study provides a pivotal insight into the acyl chain remodeling of phospholipids in yeast.     

The influence of unsaturation on the phase transition temperatures of a series of heteroacid phosphatidylcholines containing twenty-carbon chains

A series of heteroacid sn-1,2 phosphatidylcholines (PC) with twenty-carbon fatty acyl chains has been synthesized. Each PC contained eicosanoate (20:0) in the sn-1 position and one of a group of eicosaenoic acids with increasing numbers of cis double bonds in the sn-2 position. The double bonds were at positions Δ11 (20:1), Δ11,14 (20:2), Δ11,14,17 (20:3), or Δ5,8,11,14 (20:4). The disaturated PC containing two eicosanoate chains was also studied. Aqueous dispersions of these PC were analyzed by differential scanning calorimetry, and data for the gel to liquid-crystalline transitions (given as PC: T_c (° C), T_max (° C), ΔH(kcal/mol)) were as follows - 20:0-20:0 PC: 66.8, 68.4, 15; 20:0-20:1 PC: 19.8, 22.2, 8; 20:0-20:2 PC: −4.3, 1.8, 5; 20:0-20:3 PC: 1.2, 4.4, 7; 20:0-20:4 PC: −10.7, −6.8, 3. Double bonds in excess of two per chain did not substantially change the transition temperatures of these heteroacid PC. There was a small effect of the location of the multiple double bonds on the transition temperature. The data is consistent with the model that the transition temperatures are determined by a balance between a decrease in the packing density in the gel and a decrease in the rotational freedom of the chains in the liquid crystal, both caused by the double bonds ((1983) Biochemistry 22, 1466–1473).     

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.     

Effect of acyl chain unsaturation on the packing of model diacylglycerols in simulated monolayers.

In a companion study of the effects of acyl chain unsaturation on a series of model sn-1,2-diacylglycerols (DGs) we showed that individual DGs could adopt one of three energy-minimized conformations depending on the number and location of cis double bonds in the sn-2 chain. Here we show that each of these conformations promotes a distinct type of packing arrangement in a simulated DG monolayer. One conformation, shown by sn-1-18:0 DGs containing an sn-2 22:6(n-3)-, 20:4(n-6)-, or 20:3(n-9)- group, determines a regular packing that resembles a known hybrid subcell, HS2, of crystalline hydrocarbon chains. The second conformation, shown by DGs containing an sn-2 18:0-, 18:2(n-6)-, or 18:3(n-3)- group, determines a regular packing that resembles a second known, distinct hydrocarbon subcell, HS1. The third conformation, that of 18:0/18:1(n-9) DG, determines a much looser, less energetically favorable packing. Stable heterogeneous packings are possible for DGs that have similar conformations, but mixed packings of DGs that have dissimilar conformations are less stable. These results raise the possibility that differences in sn-2 acyl chain unsaturation among membrane sn-1,2-diacylglycerophospholipids may promote the formation of different domains.     

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.     

Structural aspects of pressure effects on infrared spectra of mixed-chain phosphatidylcholine assemblies in D2O

The barotropic behavior of D2O dispersions of 1-stearoyl-2-caproyl-sn-glycero-3-phosphocholine, C(18):C(10)PC, a highly asymmetric phospholipid in which the length of the fully extended acyl chain at the sn-1 position of the glycerol backbone is twice as long as that at the sn-2 position, has been investigated by high-pressure Fourier transform infrared spectroscopy. This asymmetric phosphatidylcholine bilayer at room temperature displays a pressure-induced phase transition corresponding to the liquid-crystalline----gel phase transition at 1.4 kbar. A conformational ordering of the lipid acyl chains is observed to take place abruptly at the transition pressure of 1.4 kbar. However, the lamellar lipid molecules and their acyl chains remain to be orientationally disordered in the gel phase until the applied pressure reaches 5.5 kbar. In the gel phase of fully hydrated C(18):C(10)PC, the asymmetric lipid molecules assemble into mixed interdigitated bilayers with perpendicular orientation of the zigzag planes among neighboring acyl chains. The role of excess water played in the interchain structure and the behavior of excess water and bound water under high pressure are also discussed.     

Simultaneous observation of order and dynamics at several defined positions in a single acyl chain using 2H NMR of single acyl chain perdeuterated phosphatidylcholines

Deuterium nuclear magnetic resonance (2H NMR) spectra from aqueous dispersions of phosphatidylcholines in which perdeuterated palmitic acid is esterified at the sn-1 position have several very useful features. The powder spectra show six well-resolved 90 degree edges which correspond to the six positions closest to the methyl end of the acyl chain. The spectral overlap inherent in the multiple powder pattern line shape of these dispersions can be removed by using a "dePaking" procedure [Bloom, M., Davis, J.H., & Mackay, A. (1981) Chem. Phys. Lett. 80, 198-202] which calculates the spectra that would result if the lipid bilayers were oriented in the magnetic field. This procedure produces six well-resolved doublets whose NMR properties can be observed without interference from the resonances of other labeled positions. The presence of a single double bond in the sn-2 chain increases the order of the saturated 16:0 sn-1 chain at every position in the bilayer compared with a saturated sn-2 chain at the same reduced temperature. Surprisingly, addition of five more double bonds to the sn-2 chain only slightly reduces the order of the 16:0 sn-1 chain at many positions in the bilayer compared with the single double bond. Calculating oriented spectra from a spin-lattice (T1) relaxation series of powder spectra allows one to obtain the T1 relaxation times of six positions on the acyl chain simultaneously. As an example of the utility of these molecules, we demonstrate that the dependence of the spin-lattice (T1) relaxation rate as a function of orientational order for two unsaturated phospholipids differs significantly from the corresponding fully saturated analogue. Interpreting this difference using current models of acyl chain dynamics suggests that the bilayers containing either of the two unsaturated phospholipids are significantly more deformable than bilayers made from the fully saturated phospholipid.     

Acyl chain dynamics of phosphatidylethanolamines containing oleic acid and dihydrosterculic acid: 2H NMR relaxation studies

The dynamical behavior of the acyl chains of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and 1-palmitoyl-2-dihydrosterculoyl-sn-glycero-3-phosphoethanolamine has been investigated by using 2H T1 and T2 relaxation times. Lipids were labeled at the 5-,9-,10-, and 16-positions of the sn-2 acyl chain. The profile of deuterium spin-lattice relaxation rate (T1(-1) vs. chain position is characterized in all systems by a marked discontinuity at the positions of the carbon-carbon double bond and the cyclopropane ring; the deuterons at these positions have relaxation rates which are greater than at any other labeled position of the sn-2 chain. For both types of sn-2 acyl chain, assuming a single-exponential correlation time and that the motion is within the rapid regime, the phosphatidylcholine lipid systems are less mobile than their phosphatidylethanolamine analogues. Systems containing an oleoyl chain are more dynamic than their analogues containing a dihydrosterculoyl chain. The rates of motion of the sn-2 acyl chains of phosphatidylethanolamine in a bilayer structure are slower than those of the lipid in an inverted hexagonal structure. In the hexagonal phase, the motional rates of a dihydrosterculoyl chain are slower than those of the corresponding positions of an oleoyl chain.     

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.     

Lanosterol and cholesterol have different effects on phospholipid acyl chain ordering

2H nuclear magnetic resonance (2H-NMR) spectra of dioleoylphosphatidylcholine labelled at positions 9 and 10 in the acyl chains of the phospholipid were obtained in the presence of cholesterol and lanosterol. The spectra show in all cases three quadrupole splittings. One is due to the deuterium on position 10 of the sn-1 chain and another to the deuterium on position 10 of the sn-2 chain. The third deuterium quadrupole splitting arises from the deuterium at position 9 of both chains. Cholesterol, at increasing concentration, produces an increase in the quadrupole splitting from position 9, corresponding to an increase in order of that C-D bond segment arising from the inclusion of cholesterol in the membrane. Little effect is noted on the quadrupole splittings arising from position 10 of either chain. Lanosterol appears to have no effect on the quadrupole splittings from position 9. Lanosterol, likewise, has no effects on the quadrupole splittings from position 10 of both chains. These data therefore suggest little disorganization of the membrane structure due to the 14-methyl group. However, the 14-methyl group prevents lanosterol from causing the increase in motional order of the phospholipid hydrocarbon chains characteristic of cholesterol.     

2H nuclear magnetic resonance order parameter profiles suggest a change of molecular shape for phosphatidylcholines containing a polyunsaturated acyl chain.

Solid-state 2H nuclear magnetic resonance spectroscopy was used to determine the orientational order parameter profiles for a series of phosphatidylcholines with perdeuterated stearic acid, 18:0d35, in position sn-1 and 18:1 omega 9, 18:2 omega 6, 18:3 omega 3, 20:4 omega 6, 20:5 omega 3, or 22:6 omega 3 in position sn-2. The main phase transition temperatures were derived from a first moment analysis, and order parameter profiles of sn-1 chains were calculated from dePaked nuclear magnetic resonance powder patterns. Comparison of the profiles at 37 degrees C showed that unsaturation causes an inhomogenous disordering along the sn-1 chain. Increasing sn-2 chain unsaturation from one to six double bonds resulted in a 1.6-kHz decrease in quadrupolar splittings of the sn-1 chain in the upper half of the chain (or plateau region) and maximum splitting difference of 4.4 kHz at methylene carbon 14. The change in chain order corresponds to a decrease in the 18:0 chain length of 0.4 +/- 0.2 A with 18:2 omega 6 versus 18:1 omega 9 in position sn-2. Fatty acids containing three or more double bonds in sn-2 showed a decrease in sn-1 chain length of 0.7 +/- 0.2 A compared with 18:1 omega 9. The chain length of all lipids decreased with increasing temperature. Highly unsaturated phosphatidylcholines (three or more double bonds in sn-2) had shorter sn-1 chains, but the chain length was somewhat less sensitive to temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and cohesive properties of sphingomyelin/cholesterol bilayers.

Thermal, structural, and cohesive measurements have been obtained for both bovine brain sphingomyelin (BSM) and N-tetracosanoylsphingomyelin (C24-SM) in the presence and absence of cholesterol. A goal of these experiments has been to clarify the mechanisms responsible for the strong interaction between sphingomyelin and cholesterol. Differential scanning calorimetry shows that fully hydrated bilayers of BSM and C24-SM have main endothermic phase transitions at 39 and 46 degrees C, respectively, that reflect the melting of the acyl chains from a gel to a liquid-crystalline phase. For each lipid, the addition of cholesterol monotonically reduces the enthalpy of this transition, so that at equimolar cholesterol the transition enthalpy is zero. The addition of equimolar cholesterol to either BSM or C24-SM coverts the wide-angle X-ray diffraction reflection at 4.15 A to a broad band centered at 4.5 A. Electron density profiles of gel-phase C24-SM bilayers contain two terminal methyl dips in the center of the bilayer, indicating that the lipid hydrocarbon chains partially interdigitate so that the long saturated 24-carbon acyl chains in one monolayer cross the bilayer center and appose the shorter sphingosine chains from the other monolayer. The incorporation of cholesterol adds electron density to the hydrocarbon chain region near the head group and removes the double terminal methyl dip. These wide- and low-angle X-ray data indicate that cholesterol packs into the hydrocarbon chain region near the sphingomyelin head group, fluidizes the methylene chains near the center of the bilayer compared to the gel phase, and reduces the extent of methylene chain interdigitation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase

The specific volumes of seven 1,2-diacyl-sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. Experiments were conducted in the liquid crystalline lamellar phase over the temperature range from 5 to 35 degrees C. It is demonstrated that the molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chain CH2, CH, and terminal CH3 groups. A linear dependence of chain segment volumes on temperature was observed. A self-consistent set of partially temperature-dependent volumes is obtained that allows prediction of phosphatidylcholine molecular volumes within very tight error margins.     

Determination of the acyl chain specificity of the bovine liver phosphatidylcholine transfer protein. Application of pyrene-labeled phosphatidylcholine species

The phosphatidylcholine transfer protein from bovine liver has specific binding sites for the sn-1 and sn-2 acyl chains of the phosphatidylcholine molecule [Berkhout, T.A., Visser, A.J. W.G., & Wirtz, K.W.A. (1984) Biochemistry 23, 1505-1513]. In the present study, we have investigated the properties of these binding sites by determining both binding and transfer of several sets of pyrenylphosphatidylcholine species. These sets consisted of positional isomers in which the length of the pyrene-labeled acyl chain (i.e., 5-13 methylene units) or of the unlabeled saturated acyl chain (i.e., 9-19 methylene units) was varied in either the sn-1 or the sn-2 position. Binding studies showed that there was a considerable discrimination between positional isomers with the higher affinity observed for those lipids that carry the pyrenyl chain in the sn-2 position. In addition, the affinity is markedly dependent on the length of the acyl chains; pyrenyl acyl chains of 9 and 11 methylene units and the palmitoyl chain provided the most efficient binding. The affinity of the transfer protein for the strongest bound pyrene lipid was approximately 2.5 times higher than for an average egg phosphatidylcholine molecule. In general, the transfer studies were in agreement with the binding data. However, with some short-chain derivatives, transfer rates were faster than expected on the basis of the binding data. This emphasizes the importance of kinetic factors (i.e., activation energy) in the transfer process. The rates of spontaneous transfer decreased monotonically with increasing chain length and were very similar for all positional isomer pairs studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.