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.     

Identification and characterization of kink motifs in 1-palmitoyl-2-oleoyl- phosphatidylcholines: a molecular mechanics study.

As a cis carbon-carbon double bond (delta) is introduced into the middle of an isolated all-trans hydrocarbon chain, it can be shown by molecular graphics that this delta-bond makes a bend of 130 degrees in the chain axis, thus producing a boomerang-like conformation. Such a bent structure, indeed, has been detected experimentally for oleic acid by x-ray crystallography (Abrahamson and Ryderstedt-Nahringbaur, 1962). Membrane diacyl phospholipids are largely mixed-chain lipids containing a saturated sn-1 acyl chain and an unsaturated sn-2 acyl chain. 1-Palmitoyl-2-oleoyl-phosphatidylcholine (POPC), the most abundant phospholipid in animal cell membranes, is a typical example in which the sn-2 acyl chain is the acyl chain of an oleic acid. However, this sn-2 acyl chain of POPC is unlikely to adopt a boomerang-like configuration in the gel-state lipid bilayer due to the steric hindrance imposed by neighboring chains. Instead, it has been suggested that the oleate chain in POPC is kinked in the shape of a crankshaft in the gel-state bilayer (Huang, 1977; Lagaly et al., 1977), because POPC with such a kinked sn-2 acyl chain, which is denoted here as the secondary structural element or motif, can pack efficiently against other neighboring phospholipids. In this communication, 16 different types of secondary structural elements or motifs are derived for POPC at T < Tm based on a single protocol guided by two-dimensional steric contour maps and computer-based molecular graphics. After subjecting these derived molecular species to energy minimization using the molecular mechanics method, the number of the secondary structural motifs is reduced to 13 as a result of conformational degeneracy. The structure and steric energy of each of the energy-minimized lipid rotomers are presented in this communication. Furthermore, these rotomers packed in small clusters are also simulated to mimic the lipid bilayer structure of 1-palmitoyl-2-oleoyl-phosphatidylcholines at T < Tm.     

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.     

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.     

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.     

Conformation of fatty acyl chains in alpha- and beta-phosphatidylcholine and phosphatidylethanolamine derivatives in sonicated vesicles

Mono- and dimethylated derivatives constitute important intermediates in the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in eucaryote membranes. 1H-NMR techniques were utilized to examine the conformation of the region of the fatty acyl chains that is close to the polar group in the series of alpha-phospholipids: PE, N-methyl-PE, N,N-dimethyl-PE, and PC. The same series of polar groups, but on phospholipid containing sn-1 and/or sn-3 fatty acyl chains (beta-phospholipids) were also examined. All of the phospholipids were in the form of small sonicated vesicles which are widely utilized as membrane models. The alpha-methylene group of the sn-1 and sn-2 fatty acyl chains of the alpha-phospholipids give rise to separate signals due to the non-equivalency of these chains with respect to the glycerol phosphate backbone on all alpha-phospholipids tested. Additionally, differences in the environment of the PC molecules as well as N-methyl-PE, and N,N-dimethyl-PE, but not PE itself on the inside and outside of the vesicles are reflected in the chemical shift of the alpha-methylene protons. On the other hand, all of the beta-phospholipids (including beta-PE) were found to reflect the inside/outside packing differences in their alpha-methylene groups. The bilayer packing does not induce any nonequivalence in the chemically equivalent acyl chains. In mixed micelles with detergents, beta-phospholipids showed one alpha-CH2 signal for all phospholipids. These results are consistent with a common conformational arrangement for the fatty acyl chains in all alpha-phospholipids that have been investigated no matter what aggregated form. The conformational arrangement in the beta-phospholipids is different, but again is similar for all of the compounds tested in various aggregated forms.     

Transacylase formation of bis(monoacylglycerol)phosphate

Recent work within our laboratory has focused on the enzymes we hypothesize are involved in the biosynthesis of bis(monoacylglycerol)phosphate from phosphatidylglycerol. Here we describe a transacylase, active at acidic pH values, isolated from a macrophage-like cell line, RAW 264.7. This enzyme acylates the head group glycerol of sn-3:sn-1' lysophosphatidylglycerol to form sn-3:sn-1' bis(monoacylglycerol)phosphate. Here we demonstrate that this enzyme uses two lysophosphatidylglycerol molecules, one as an acyl donor and another as an acyl acceptor, and that the acyl contributions from all other lipids tested are comparatively minor. This enzyme prefers saturated acyl chains to monounsaturates, 16 and 18 carbon fatty acids over 14 carbon fatty acids, and saturated acyl chains at the sn-1 position to monounsaturated acyl chains on the sn-2 carbon of lysophosphatidylglycerol. We present data which show the transacylase activity depends on the presence of a lipid-water interface and the lipid polymorphic state.     

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)     

Structural identification of glycerolipid molecular species isolated from cyanobacterium Synechocystis sp. PCC 6803 using fast atom bombardment tandem mass spectrometry

Our previous works have demonstrated that fast atom bombardment tandem mass spectrometry can be a valuable tool in determining the complete structure of glycoglycerolipids and glycerophospholipids. Collision-induced dissociation of sodium-adducted molecular ions ([M + Na]+ or [M - H + 2Na]+) generates diverse product ions informative on the double-bond position in fatty acyl groups as well as the polar head group and fatty acid composition. Here we report that this direct and rapid method can be applied to the structural determination of individual molecular species of each glycerolipid class purified from the total lipid extract of cyanobacterium Synechocystis sp. PCC 6803. Especially, based on the preference for the loss of the fatty acyl group positioned at the sn-2, it was proved that all of the molecular species of diacylglycerolipids contained a palmitoyl group exclusively at the sn-2 position. Additionally, lysoglycerolipids, monoacyl forms of four major membrane diacylglycerolipids, were first isolated together from a fresh extract. Using fast atom bombardment mass spectrometry and tandem mass spectrometry, it was found that each lysoglycerolipid had a molecular species with only palmitic acid as a fatty acyl group. Thus, these compounds could be synthesized by specific enzyme-catalyzed hydrolysis of the sn-1 fatty acyl group of the corresponding diacylglycerolipids.     

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.     

Membrane protein-lipid hydrogen bonding: evidence from protein kinase C, diglyceride, and tumor promotors

Membrane-bound proteins owe their retention and conformation in the lipid bilayer to hydrophobic peptide domains. Additional fixation, by protein-lipid hydrogen bonding, has been suggested, and recent reports on protein kinase C activation by diacylglycerol (DG) provide an unambiguous model for such bonding. The sn-1,2-diacylglycerol appears to donate a hydrogen bond from the sn-3 hydroxyl to the enzyme and to receive two hydrogen bonds, in the sn-1 and sn-2 ester CO groups, from the enzyme. This arrangement is confirmed in phorbol ester, a competitive inhibitor of DG for the kinase. This tumor promotor has a nearly identical spatial arrangement of hydrogen bond donor (9 alpha-OH) and acceptors (12 and 13 ester CO); so have two other tumor promotors, teleocidin and aplysiatoxin. There are reasons to believe that protein kinase C is not the only protein that is bound to membrane lipids by hydrogen bonding, and such bonding will have to be considered in membrane-associated events such as fusion, cross-membrane transport, or anesthesia.     

Lysophosphatidylcholine profiling of plasma: discrimination of isomers and discovery of lung cancer biomarkers

Lysophosphatidylcholines (lysoPCs) are a class of compounds that have a constant polar head, and fatty acyls of different chain lengths, position, degrees of saturation, and double bond location in human plasma. LysoPCs levels can be a clinical diagnostic indicator that reveals pathophysiological changes. In this work, a method was developed to discriminate between different types of lysoPCs using reversed phase ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry, using mass spectrometry MS^E. Isomeric lysoPCs were distinguished based on retention time and the peak intensity ratio of product ions, and 14 pairs of lysoPCs regioisomers were identified in human plasma. The plasma samples of 12 lung cancer patients and 12 healthy persons were collected and analyzed by principal component analysis to generate metabolic profiles of the identified lysoPCs. Both electrospray ionization ESI+ and ESI− results showed that all lung cancer patients had the same five lysoPC metabolic abnormalities, specifically in sn-1 lyso16:0, sn-2 lysoPC 16:0, sn-1 lysoPC 18:0, sn-1 lysoPC 18:1 and sn-1 lysoPC 18:2. Thus, the function of isomers with different fatty acyl positions may be related to lung cancer, and this may help elucidate the mechanism of the disease.     

Solvent-dependent conformational behaviour of lipochitoligosaccharides related to Nod factors.

The solution conformation of two lipooligosaccharides related to Nod factors or lipochitoligosaccharides have been analysed by 1D and 2D 1H and 13C NMR spectroscopy, molecular mechanics and dynamics calculations. The obtained data indicate that the glycosidic torsion angles have restricted fluctuations, but may adopt a variety of shapes. Remarkably, the relative orientation of the fatty acid chain towards the oligosaccharide backbone is solvent dependent. In water solution, the acyl residue and the oligosaccharide adopt a quasi-parallel orientation for a significant amount of time.     

Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate.

Enoyl-ACP reductases participate in fatty acid biosynthesis by utilizing NADH to reduce the trans double bond between positions C2 and C3 of a fatty acyl chain linked to the acyl carrier protein. The enoyl-ACP reductase from Mycobacterium tuberculosis, known as InhA, is a member of an unusual FAS-II system that prefers longer chain fatty acyl substrates for the purpose of synthesizing mycolic acids, a major component of mycobacterial cell walls. The crystal structure of InhA in complex with NAD+ and a C16 fatty acyl substrate, trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester, reveals that the substrate binds in a general "U-shaped" conformation, with the trans double bond positioned directly adjacent to the nicotinamide ring of NAD+. The side chain of Tyr158 directly interacts with the thioester carbonyl oxygen of the C16 fatty acyl substrate and therefore could help stabilize the enolate intermediate, proposed to form during substrate catalysis. Hydrophobic residues, primarily from the substrate binding loop (residues 196-219), engulf the fatty acyl chain portion of the substrate. The substrate binding loop of InhA is longer than that of other enoyl-ACP reductases and creates a deeper substrate binding crevice, consistent with the ability of InhA to recognize longer chain fatty acyl substrates.     

Influence of cis double bonds in the sn-2 acyl chain of phosphatidylethanolamine on the gel-to-liquid crystalline phase transition.

We have semisynthesized 19 species of mixed-chain phosphatidylethanolamines (PEs) in which the sn-1 acyl chain is derived from saturated fatty acids with varying chain lengths and the sn-2 acyl chain has different chain lengths but contains 0, 1, and 2 cis double bond(s). The gel-to-liquid crystalline phase transition temperatures (Tm) of lipid bilayers prepared from these 19 mixed-chain PEs were determined calorimetrically. When the Tm values are compared with those of saturated and monounsaturated counterparts, a common Tm profile is observed in the plot of Tm versus the number of cis double bonds. Specifically, a marked stepwise decrease in Tm is detected as the number of cis double bonds in the sn-2 acyl chain of the mixed-chain PE is successively increased from 0 to 1 and then to 2. The large Tm-lowering effect of the acyl chain unsaturation can be attributed to the increase in Gibbs free energy of the gel-state bilayer as a result of weaker lateral chain-chain interactions. In addition, we have applied molecular mechanics calculations to simulate the molecular structure of dienoic mixed-chain C(X):C(Y:2 delta n,n+3)PE in the gel-state bilayer, thus enabling the three independent structural parameters (N, delta C, and LS) to be calculated in terms of X, Y, and n, which are intrinsic quantities of C(X):C(Y:2 delta n,n+3)PE. When the Tm values and the corresponding N and delta C values of all dienoic mixed-chain PEs under study are first codified and then analyzed statistically by multiple regressions, the dependence of Tm on the structural parameters can be described quantitatively by a simple and general equation. The physical meaning and the usefulness of this simple and general equation are explained.     

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.     

A novel Delta9 acyl-lipid desaturase, DesC2, from cyanobacteria acts on fatty acids esterified to the sn-2 position of glycerolipids

Acyl-lipid desaturases are enzymes that convert a C-C single bond into a C=C double bond in fatty acids that are esterified to membrane-bound glycerolipids. Four types of acyl-lipid desaturase, namely DesA, DesB, DesC, and DesD, acting at the Delta12, Delta15, Delta9, and Delta6 positions of fatty acids respectively, have been characterized in cyanobacteria. These enzymes are specific for fatty acids bound to the sn-1 position of glycerolipids. In the present study, we have cloned two putative genes for a Delta9 desaturase, designated desC1 and desC2, from Nostoc species. The desC1 gene is highly similar to the desC gene that encodes a Delta9 desaturase that acts on C18 fatty acids at the sn-1 position. Homologues of desC2 are found in genomes of cyanobacterial species in which Delta9-desaturated fatty acids are esterified to the sn-2 position. Heterologous expression of the desC2 gene in Synechocystis sp. PCC 6803, in which a saturated fatty acid is found at the sn-2 position, revealed that DesC2 could desaturate this fatty acid at the sn-2 position. These results suggest that the desC2 gene is a novel gene for a Delta9 acyl-lipid desaturase that acts on fatty acids esterified to the sn-2 position of glycerolipids.     

Chemical characterization of cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth,Bombyx mori

Accumulation of lipid droplets within the cytoplasm is a common feature of the pheromone gland cells of many lepidopteran species. The cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth, Bombyx mori, were effectively extracted by dipping the trimmed glands in acetone for 10 min. In order to analyze the components originating from the lipid droplets, we separated the acetone extracts prepared before and after adult eclosion using HPLC, and specified the peaks showing a similar pattern of stage-dependence to that in the morphological change of the lipid droplets previously reported by Fónagy et al. (Arthropod Struct. Dev. 30 (2001) 113). Finally, we specified the peaks #1-5 and #1a-4a separated by reversed-phase HPLC as lipid droplet contents. Structure elucidation using FAB-MS and MS-MS analyses confirmed that they were triacylglycerols (TGs), and 12 species of TGs were identified as lipid droplet contents. Fatty acyl groups contained in these TGs were limited to five unsaturated C16 and C18 fatty acyl groups (delta 11-hexadecenoate, delta 10,12-hexadecadienoate, delta 9-octadecenoate, delta 9,12-ocatadecadienoate, and delta 9,12,15-ocatadecatrienoate), including the pheromone precursor delta 10,12-hexadecadienoate as a major component. Digestion with porcine pancreatic lipase confirmed that three major TGs eluted in the peaks #3-5 all contained C18 fatty acyl groups at the sn-2 position, indicating that the pheromone precursor is sequestered preferentially at the sn-1 and/or sn-3 position. Present results combined with the fact that the morphological change of the lipid droplets is under the control of PBAN indicate that the role of the cytoplasmic lipid droplets in the pheromone-producing cells is to store the pheromone precursor in the form of TGs and to provide it for pheromone production in response to the external signal of PBAN.     

Remodeling of rat hepatocyte phospholipids by selective acyl turnover

Acyl turnover of rat hepatocyte phospholipids and triacylglycerols was assessed by incubating the cells in media containing 40% H2(18)O and measuring the time-dependent incorporation of 18O into ester carbonyls by gas chromatography-mass spectrometry of hydrogenated methyl esters. Incorporation of 18O into 22-carbon acyl groups was low in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine, whereas in phosphatidylethanolamine, it was about the same as in the other acyl groups. Incorporation of 18O into individual molecular species of phosphatidylcholine and phosphatidylethanolamine was determined after phospholipase C hydrolysis, derivatization to dinitrobenzoates, and separation by high-performance liquid chromatography. In most molecular species, acyl groups at the sn-1 and sn-2 positions became 18O-labeled at drastically different rates, indicating remodeling through deacylation-reacylation. Molecular species expected to arise de novo from acylation of glycerophosphate exhibited similar rates of 18O incorporation at the sn-1 and sn-2 positions. The data suggest that hepatocyte phospholipids are continually synthesized, remodeled by deacylation-reacylation at specific turnover rates up to 10-15%/h, and degraded. This acyl turnover probably does not involve the majority of intracellular unesterified fatty acids whose 18O incorporation was found to be very low. In contrast, the oxygens of extracellular unesterified fatty acids were readily exchanged with the media. This exchange was enzyme-catalyzed, possibly by lipases released into the media from damaged cells. Incorporation of 18O into exogenously added fatty acids was also rapid and resulted in enhanced uptake of 18O-labeled fatty acids into cellular lipids, primarily triacylglycerols and phosphatidylcholine, without drastic change of the intracellular free fatty acid pool.