The oxysterol 3β-hydroxy-5-oxo-5,6-secocholestan-6-al changes the phase behavior and structure of phosphatidylethanolamine–phosphatidylcholine mixtures

An oxidized form of cholesterol, atheronal, is a form found in vivo that has been associated with human pathologies. We have studied mixtures of this oxidized sterol with the phospholipids phosphatidylethanolamine and phosphatidylcholine. We used phospholipids either with palmitoyl and oleoyl acyl chains on the C1 and C2 carbon atoms of glycerol or with both acyl chains being palmitoleoyl. We also compared the effects of atheronal on the curvature properties of these lipids with the action of cholesterol. We studied the bilayer to hexagonal phase transition temperature of mixtures of these lipids using differential scanning calorimetry as well as the dimensions of the hexagonal phase cylinders using X-ray diffraction. Disordering of the lamellar phase was also qualitatively assessed by the loss of sharp diffraction peaks. Our results demonstrate that the modulation of membrane curvature in these systems depends not only on the nature of the sterol, but also on the acyl chain composition of the phospholipids used. In addition, some of the effects of atheronal could be ascribed to reaction of the aldehyde and ketone groups of this oxidized sterol with the amino group of phosphatidylethanolamine.     

Regulation of fatty acid composition in Escherichia coli: a proposed common mechanism for changes induced by ethanol, chaotropic agents, and a reduction of growth temperature.

Growth of Escherichia coli in the presence of ethanol and chaotropic salts resulted in the synthesis of lipids containing elevated levels of unsaturated fatty acids analogous to the effect of a reduction in growth temperature. Both ethanol and chaotropic agents acted at the level of fatty acid biosynthesis and altered lipid composition by decreasing the proportion of saturated acyl chains available for the synthesis of phospholipids. A reduction in temperature causes similar effects on fatty acid biosynthesis in vivo and in vitro. Ethanol, chaotropic salts, and a decrease in temperature all weaken hydrophobic interactions. Antichaotropic salts antagonized and effects of these treatments on fatty acid synthesis in vitro. These results are consistent with a common mechanism for the effects of chaotropic agents, temperature, and ethanol on fatty acid synthesis. The biosynthesis of saturated and unsaturated acyl chains may be regulated by the strength of hydrophobic interactions. Changes in the strength of hydrophobic interactions could alter enzyme structure, substrate structure, or the equilibrium between the soluble enzymes of fatty acid synthesis and their respective acyl carrier protein substrates.     

UDP-Glucose: Sterol Glucosyltransferase and a Steryl Glucoside Acyltransferase Activity in Amyloplast Membranes from Potato Tubers

Envelope membranes were isolated from potato tuber amyloplastby a discontinuous sucrose density gradient and high speed centrifugation.These membranes catalyzed the transfer of [¹⁴C]glucose fromUDP-[¹⁴C]glucose to endogenous sterol acceptors and, in turn,catalyzed the esterification of steryl glucosides with fattyacids from an endogenous acyl donor. The synthesis of sterylglucosides was stimulated in the presence of Triton. X-100,while formation of acyl steryl glucosides was inhibited by thedetergent. However, in the presence of an added sterol acceptorand Triton X-100, the inhibition of acyl steryl glucoside synthesiswas overcome by the addition of phosphatidylethanolamine. Theenzyme involved in steryl glucoside formation was solubilizedby treatment of the envelope membranes with 0.3% Triton X-100.The solubilized enzyme had an almost absolute requirement forsterol acceptors. Key words: Solanum tuberosum, Sterol glucosylation, Steryl glucoside acylation, Amyloplast membrane     

Stimulation of ABCB4/MDR3 ATPase activity requires an intact phosphatidylcholine lipid

ABCB4/MDR3 is located in the canalicular membrane of hepatocytes and translocates PC-lipids from the cytoplasmic to the extracellular leaflet. ABCB4 is an ATP-dependent transporter that reduces the harsh detergent effect of the bile salts by counteracting self-digestion. To do so, ABCB4 provides PC lipids for extraction into bile. PC lipids account for 40% of the entire pool of lipids in the canalicular membrane with an unknown distribution over both leaflets. Extracted PC lipids end up in so-called mixed micelles. Mixed micelles are composed of phospholipids, bile salts, and cholesterol. Ninety to ninety-five percent of the phospholipids are members of the PC family, but only a subset of mainly 16.0-18:1 PC and 16:0-18:2 PC variants are present. To elucidate whether ABCB4 is the key discriminator in this enrichment of specific PC lipids, we used in vitro studies to identify crucial determinants in substrate selection. We demonstrate that PC-lipid moieties alone are insufficient for stimulating ABCB4 ATPase activity, and that at least two acyl chains and the backbone itself are required for a productive interaction. The nature of the fatty acids, like length or saturation has a quantitative impact on the ATPase activity. Our data demonstrate a two-step enrichment and protective function of ABCB4 to mitigate the harsh detergent effect of the bile salts, because ABCB4 can translocate more than just the PC-lipid variants found in bile.     

Modulation of bovine milk galactosyltransferase activity by lipids

The effect of lipids singly and in combination on the ability of galactosyltransferase to transfer galactose to N-acetyl-D-glucosamine-forming lactosamine and to glucose forming lactose has been studied. Lecithins, as egg phosphatidylcholine (PC), or saturated as dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine stimulated the activity of the enzyme to form lactosamine to different extents. Egg PC produced the greatest stimulation of all the lecithins tested. Egg phosphatidic acid (PA) inhibited the activity of the enzyme at very low concentrations of lipid. In mixed vesicles of gel phase or liquid crystalline phase lecithins and egg PA, the acidic lipid was able to overcome the stimulation produced by the lecithins. The dominant effect of the head group was demonstrated by the effects of gel phase dimyristoylphosphatidic acid (DMPA). In mixtures with PC, DMPA also was able to inhibit the enzyme for lactosamine synthesis but higher concentrations of the gel phase DMPA were required for inhibition compared to the liquid crystalline PA. Although the head group appeared to dominate the inhibition, the nature of the acyl chains of the lipid played a secondary role at least. Other acid lipids, phosphatidylserine (PS) and phosphatidylinositol (PI) were much less effective than PA. PS alone inhibited the activity of the enzyme. However, in mixed lipids (PS and egg PC), PS was unable to reverse the stimulation produced by PC while PC was able to reverse the inhibition produced by PS. PI alone had no effect on the enzyme activity. In mixtures with egg PC, the stimulating effect of PC was dominant. In the lactose synthetase reaction, the effect of lipids was similar to that of the lactosamine synthetase, i.e. PC stimulated and PA inhibited activity and in mixtures of PC and PA, the inhibitory effect of PA was dominant.     

Enzymatic synthesis of nuatigenin 3β-D-glucoside in oat (Avena sativa) leaves

Crude homogenates or acetone powder preparations from oat leaves efficiently catalyse the glucosylation of a steroidal sapogenin, nuatigenin [22,25-epoxy-(20S)(22S)(25S)-furost-5-en-3β,26-diol], using UDP-glucose as the sugar donor. The reaction product was identified as nuatigenin 3β-D-monoglucoside. In contrast to the glucosylation of phytosterols, which is also catalysed by enzyme preparations from oat leaves, the formation of nuatigenin glucoside is not stimulated by Triton X-100. This result suggests that glucosyltransferases with different specifirity patterns are involved in sterol and nuatigenin glucosylation in oat leaves. Enzymatic acylation of nuatigenin glucoside to its monoacyl derivative with the use of an endogenous acyl source was also observed with a crude homogenate or a crude membranous fraction as the enzyme preparation.     

Rapid kinetic labeling of Arabidopsis cell suspension cultures: implications for models of lipid export from plastids

Cell cultures allow rapid kinetic labeling experiments that can provide information on precursor-product relationships and intermediate pools. T-87 suspension cells are increasingly used in Arabidopsis (Arabidopsis thaliana) research, but there are no reports describing their lipid composition or biosynthesis. To facilitate application of T-87 cells for analysis of glycerolipid metabolism, including tests of gene functions, we determined composition and accumulation of lipids of light- and dark-grown cultures. Fatty acid synthesis in T-87 cells was 7- to 8-fold higher than in leaves. Similar to other plant tissues, phosphatidylcholine (PC) and phosphatidylethanolamine were major phospholipids, but galactolipid levels were 3- to 4-fold lower than Arabidopsis leaves. Triacylglycerol represented 10% of total acyl chains, a greater percentage than in most nonseed tissues. The initial steps in T-87 cell lipid assembly were evaluated by pulse labeling cultures with [(14)C]acetate and [(14)C]glycerol. [(14)C]acetate was very rapidly incorporated into PC, preferentially at sn-2 and without an apparent precursor-product relationship to diacylglycerol (DAG). By contrast, [(14)C]glycerol most rapidly labeled DAG. These results indicate that acyl editing of PC is the major pathway for initial incorporation of fatty acids into glycerolipids of cells derived from a 16:3 plant. A very short lag time (5.4 s) for [(14)C]acetate labeling of PC implied channeled incorporation of acyl chains without mixing with the bulk acyl-CoA pool. Subcellular fractionation of pea (Pisum sativum) leaf protoplasts indicated that 30% of lysophosphatidylcholine acyltransferase activity colocalized with chloroplasts. Together, these data support a model in which PC participates in trafficking of newly synthesized acyl chains from plastids to the endoplasmic reticulum.     

Asymmetric distribution of phosphatidylcholine and sphingomyelin between micellar and vesicular phases. Potential implications for canalicular bile formation.

Both phosphatidylcholine (PC) and sphingomyelin (SM) are the major phospholipids in the outer leaflet of the hepatocyte canalicular membrane. Yet, the phospholipids secreted into bile consist principally (>95%) of PC. In order to understand the physical;-chemical basis for preferential biliary PC secretion, we compared interactions with bile salts (taurocholate) and cholesterol of egg yolk (EY)SM (mainly 16:0 acyl chains, similar to trace SM in bile), buttermilk (BM)SM (mainly saturated long (>20 C-atoms) acyl chains, similar to canalicular membrane SM) and egg yolk (EY)PC (mainly unsaturated acyl chains at sn-2 position, similar to bile PC). Main gel to liquid-crystalline transition temperatures were 33. 6 degrees C for BMSM and 36.6 degrees C for EYSM. There were no significant effects of varying phospholipid species on micellar sizes or intermixed-micellar/vesicular bile salt concentrations in taurocholate-phospholipid mixtures (3 g/dL, 37 degrees C, PL/BS + PL = 0.2 or 0.4). Various phases were separated from model systems containing both EYPC and (EY or BM)SM, taurocholate, and variable amounts of cholesterol, by ultracentrifugation with ultrafiltration and dialysis of the supernatant. At increasing cholesterol content, there was preferential distribution of lipids and enrichment with SM containing long saturated acyl chains in the detergent-insoluble pelletable fraction consisting of aggregated vesicles. In contrast, both micelles and small unilamellar vesicles in the supernatant were progressively enriched in PC. Although SM containing vesicles without cholesterol were very sensitive to micellar solubilization upon taurocholate addition, incorporation of the sterol rendered SM-containing vesicles highly resistant against the detergent effects of the bile salt. These findings may have important implications for canalicular bile formation.     

Studies on the purified, lipid-reconstituted (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes. Dependence of enzyme activity on lipid headgroup and hydrocarbon chain structure

The purified (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes was successfully reconstituted with a number of different phospho- and glycolipids, and the ability of these lipids to support the function of this enzyme was evaluated by their ability to increase the specific activity of the purified enzyme and by their ability to restore its lipid-phase state-dependent properties which were lost during purification. The incorporation of this ATPase into liposomes composed of the endogenous membrane lipids of the organism, or of zwitterionic phospholipids such as phosphatidylcholine or phosphatidylethanolamine, results in a full reconstitution of its activity and its lipid-phase state-dependent properties. In contrast, anionic phospholipids alone, or in combination with zwitterionic phospholipids at concentrations higher than 10 mol % of the anionic phospholipid, cause an irreversible inhibition of this ATPase. However, when combined with neutral glycolipids, larger amounts of anionic phospholipid can be tolerated without enzyme inhibition. Phosphatidylcholines with acyl chains of 14-24 linear carbon atoms and varying degrees of branching and unsaturation successfully reconstitute the enzyme, in marked contrast to the shorter chain homologues, which were ineffective. Our results indicate that the full expression of the activity of the A. laidlawii B ATPase requires a host lipid bilayer membrane of low to moderate negative surface charge which is predominantly liquid-crystalline and of a minimal bilayer thickness. Once such requirements are met, the enzyme exhibits considerable flexibility regarding the nature of the lipids which can effectively support its function. In particular, the activity of the A. laidlawii B ATPase is not very sensitive to lipid "fluidity" in the liquid-crystalline state.     

Alterations in wheat pollen lipidome during high day and night temperature stress

Understanding the adaptive changes in wheat pollen lipidome under high temperature (HT) stress is critical to improving seed set and developing HT tolerant wheat varieties. We measured 89 pollen lipid species under optimum and high day and/or night temperatures using electrospray ionization‐tandem mass spectrometry in wheat plants. The pollen lipidome had a distinct composition compared with that of leaves. Unlike in leaves, 34:3 and 36:6 species dominated the composition of extraplastidic phospholipids in pollen under optimum and HT conditions. The most HT‐responsive lipids were extraplastidic phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol, phosphatidic acid, and phosphatidylserine. The unsaturation levels of the extraplastidic phospholipids decreased through the decreases in the levels of 18:3 and increases in the levels of 16:0, 18:0, 18:1, and 18:2 acyl chains. PC and PE were negatively correlated. Higher PC:PE at HT indicated possible PE‐to‐PC conversion, lower PE formation, or increased PE degradation, relative to PC. Correlation analysis revealed lipids experiencing coordinated metabolism under HT and confirmed the HT responsiveness of extraplastidic phospholipids. Comparison of the present results on wheat pollen with results of our previous research on wheat leaves suggests that similar lipid changes contribute to HT adaptation in both leaves and pollen, though the lipidomes have inherently distinct compositions.     

Acyl chain-based molecular selectivity for HL60 cellular phosphatidylinositol and of phosphatidylcholine by phosphatidylinositol transfer protein alpha

Mammalian phosphatidylinositol transfer protein alpha (PITP) is an intracellular lipid transporter with a binding site that can accommodate a single molecule of phosphatidylinositol (PI) or phosphatidylcholine (PC). Phospholipids are a heterogeneous population of molecular species that can be distinguished by their characteristic headgroups as well as their acyl chains at the sn-1 and sn-2 position. In this study, we have defined the acyl chain preference for PITPalpha when presented with a total population of cellular lipids. Recombinant PITPalpha loaded with bacterial lipid, phosphatidylglycerol (PG), was incubated with permeabilised HL60 cells, followed by recovery of PITPalpha by affinity chromatography. Lipids extracted from the PITPalpha were analysed by tandem electrospray ionisation mass spectrometry (ESI-MS) and showed total exchange of acquired bacterial lipids for HL60 cellular PI and PC. Detailed comparison of the molecular species composition of bound phospholipids with those in whole cells permitted the assessment of selectivity of acyl chain binding. For both phospholipid classes, progressive fractional enrichments in bound species possessing shorter acyl chains were apparent with a preference order: 16:1>16:0>18:1>18:0>20:4. A recapitulation of this specificity order was also seen from a dramatically altered range of molecular species present in HL60 cells enriched with arachidonate over many weeks of culture. We speculate that short-chain, saturate-binding preferences under both conditions may reflect properties in vivo. This is consistent with target cell membranes actively remodelling newly delivered phospholipids after transport rather than relying on the transport of the specific molecular species conventionally found in mammalian membranes.     

The catalytic domains of Clostridium sordellii lethal toxin and related large clostridial glucosylating toxins specifically recognize the negatively charged phospholipids phosphatidylserine and phosphatidic acid

Clostridium sordellii lethal toxin (TcsL) is a potent virulence factor belonging to the large clostridial glucosylating toxin family. TcsL enters target cells via receptor‐mediated endocytosis and delivers the N‐terminal catalytic domain (TcsL‐cat) into the cytosol upon an autoproteolytic process. TcsL‐cat inactivates small GTPases including Rac and Ras by glucosylation with uridine‐diphosphate (UDP)‐glucose as cofactor leading to drastic changes in cytoskeleton and cell viability. TcsL‐cat was found to preferentially bind to phosphatidylserine (PS)‐containing membranes and to increase the glucosylation of Rac anchored to lipid membrane. We here report binding affinity measurements of TcsL‐cat for brain PS‐containing membranes by surface plasmon resonance and enzyme‐linked immunosorbent assay (ELISA). In addition, TcsL‐cat bound to phosphatidic acid (PA) and, to a lesser extent, to other anionic lipids, but not to neutral lipids, sphingolipids or sterol. We further show that the lipid unsaturation status influenced TcsL‐cat binding to phospholipids, PS with unsaturated acyl chains and PA with saturated acyl chains being the preferred bindingsubstrates. Phospholipid binding site is localized at the N‐terminal four helical bundle structure (1‐93 domain). However, TcsL‐1‐93 bound to a broad range of substrates, whereas TcsL‐cat, which is the active domain physiologically delivered into the cytosol, selectively bound to PS and PA. Similar findings were observed with the other large clostridial glucosylating toxins from C. difficile, C. novyi and C. perfringens.     

Interaction of beta-lactoglobulin with phospholipid bilayers: a molecular level elucidation as revealed by infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy has been used to study, at a molecular level, the interactions between beta-lactoglobulin (BLG), the most abundant globular protein in milk, and some lipids (sphingomyelin, SM; dimyristoylphosphatidylcholine, DMPC; dipalmytoylphosphatidylcholine, DPPC; dimyristoylphosphatidylserine-sodium salt, DMPS; dipalmitoylphosphatidylserine-sodium salt, DPPS) constituting the milk fat globule membrane (MFGM). The interactions were monitored with respect to alteration in the secondary structure of BLG, as registered by the amide I' band, and phospholipid conformation, as revealed by the acyl chain and carbonyl bands. The results show that neither the conformation nor the thermotropism of neutral bilayers containing DMPC or DPPC is affected by BLG. Reciprocally, the secondary structure and thermal behaviour of pure BLG remain the same in the presence of PC. These results suggest that no interaction occurs between PC and BLG, in agreement with previous studies. However, it is found that BLG interacts with neutral bilayers constituted by milk SM lipids, increasing gauche conformers and thus conformational disorder of the lipid acyl chains. This perturbing effect has been attributed to a partial penetration of BLG into the hydrophobic core of the bilayer, which allows hydrophobic interactions between BLG and SM. Moreover, the fact that SM possesses the same headgroup of PC implies that the head group does not prevent the occurrence of BLG-lipid interactions and other lipid regions can control the binding of BLG to lipids. Furthermore, BLG was found to interact electrostatically with charged bilayers containing PS, leading to a rigidification of the lipid hydrocarbon chains and a dehydration of the interfacial region. This last effect suggests that the protein limits the accessibility of water molecules to the interfacial region of the phospholipids by its presence at the membrane surface.     

Steryl glucoside and acyl glucoside biosynthesis in maturing pea seeds

Sterol glucosyltransferase activity was found in a particulate fraction of pea seeds. The activity was stimulated by Ca²⁺ and Mg²⁺ and inhibited by Zn²⁺, Cu²⁺, Hg²⁺, EDTA and EGTA. Iodoacetamide was without effect but p-chloromercuribenzoate completely inhibited the enzyme. N -Ethylmaleimide gave 60–70 % inhibition over a wide range of concentrations. The activity was stimulated by ATP in the presence of Mg²⁺. Under such conditions, steryl acyl glucoside was formed. The acyl derivative was barely detectable in the presence of Ca²⁺ either with or without ATP. Both oleyl CoA and palmityl CoA stimulated acyl glucoside synthesis. Of the four nucleoside triphosphates, ATP, GTP, UTP and CTP both ATP and CTP stimulated acylation in the presence of Mg²⁺. The observations suggest that acyl donors other than digalactosyl diglyceride and phospholipids may function in steryl acyl glucoside synthesis in plants.     

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.     

Head group precursors modify phospholipid synthesis in Schistosoma mansoni

The two predominant phospholipids in schistosomula of Schistosoma mansoni are phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which are found in a molar ratio of 0.52 (PE/PC). The incorporation of four fatty acids (arachidonic, myristic, oleic, and palmitic) and glycerol into phospholipids of schistosomula was measured. In two different media (one containing ethanolamine, the other without), all four fatty acids were predominantly incorporated into PC with a PE/PC ratio of approximately 0.1 in a 90-min label. After a 24-h chase, PC remained the predominant labeled phospholipid but the fatty acid-labeled PE/PC ratio increased slightly, the specific activity of labeled neutral lipids decreased, and the specific activity of labeled PE increased. Glycerol was incorporated with a ratio of 0.55 in the presence of ethanolamine but only 0.19 in its absence. Schistosomula also incorporate fatty acids into phosphatidylmonomethylethanolamine (PMME) and phosphatidyldimethylethanolamine (PDME) at rates intermediate to that into PE and PC in the presence of the respective head group precursor; this incorporation was inhibited by choline. Relative to PC, oleic acid is incorporated into PE, PMME, and PDME at rates higher than for palmitic acid. These results suggest that schistosomula possess acyltransferase(s) with head group specificity and that acyl chains are transferred from neutral lipids to phospholipids over time.     

Membrane lipids of Mycoplasma orale: lipid composition and synthesis of phospholipids.

Lipid composition of Mycoplasma orale was examined and compared with that of horse serum added to the growth medium. Ratios of cholesterol/cholesterol ester and sphingomyelin/phosphatidylcholine were much higher in M. orale than in the horse serum, indicating the organism incorporates selectively cholesterol and sphingomyelin. A distinct difference between the lipids from the two sources was that in phospholipids of M. orale almost all (greater than 95%) of the fatty acyl residues were saturated whereas nearly half of the residues were unsaturated in horse serum phospholipids. Approximately one third of M. orale phospholipids was phosphatidylglycerol, which was synthesized by the organism as was demonstrated by 32P-labeling experiment. Its acyl residues consisted mainly of C16:0 and were efficiently labeled with 14C-palmitate but not with 14C-acetate. These results clearly indicate the de novo synthesis of phosphatidylglycerol by M. orale is through acylation with exogenous saturated fatty acids. On the other hand, all the phosphatidylcholine and sphingomyelin of M. orale were derived from the medium. The 14C-labeling experiment demonstrates that no fatty acid synthesis takes place nor exogenous fatty acid can be incorporated so efficiently as phosphatidylglycerol, suggesting that extremely high proportion of saturated fatty acyl residues in these phospholipids is the consequence of saturation directed to the acyl chains of the incorporated phospholipids.     

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific d-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.     

Energy-minimized structures and packing states of a homologous series of mixed-chain phosphatidylcholines: a molecular mechanics study on the diglyceride moieties.

Phosphatidylcholines or C(X):C(Y)PC, quantitatively the most abundant lipids in animal cell membranes, are structurally composed of two parts: a headgroup and a diglyceride. The diglyceride moiety consists of the glycerol backbone and two acyl chains. It is the wide diversity of the acyl chains, or the large variations in X and Y in C(X):C(Y)PC, that makes the family of phosphatidylcholines an extremely complex mixture of different molecular species. Since most of the physical properties of phospholipids with the same headgroup depend strongly on the structures of the lipid acyl chains, the energy-minimized structure and steric energy of each diglyceride moiety of a series of 14 molecular species of phosphatidylcholines with molecular weights identical to that of dimyristoylphosphatidylcholine without the headgroup are determined in this communication by molecular mechanics (MM) calculations. Results of two types of trans-bilayer dimer for each of the 14 molecular species of phosphatidylcholines are also presented; specifically, the dimeric structures are constructed initially based on the partially interdigitated and mixed interdigitated packing motifs followed subsequently by the energy-minimized refinement with MM calculations. Finally, tetramers with various structures to model the lateral lipid-lipid interactions in a lipid bilayer are considered. Results of laborious MM calculations show that saturated diacyl C(X):C(Y)PC with delta C/CL values greater than 0.41 prefer topologically to assemble into tetramers of the mixed interdigitated motif, and those with delta C/CL values less than 0.41 prefer to assemble into tetramers with a repertoire of the partially interdigitated motif. Here, delta C/CL, a lipid asymmetry parameter, is defined as the normalized acyl chain length difference between the sn-1 and sn-2 acyl chains for a C(X):C(Y)PC molecule; an increase in delta C/CL value is an indication of increasing asymmetry between the two lipid acyl chains. These computational results are in complete accord with the calorimetric data presented previously from this laboratory (H-n. Lin, Z-q. Wang, and C. Huang. 1991. Biochim. Biophys. Acta. 1067:17-28).     

The Influence of Membrane Lipids in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Gamma-Hemolysins Pore Formation

The natural target of Staphylococcus aureus bicomponent γ-hemolysins are leucocyte cell membranes. Because a proteinaceous receptor has not been found yet, we checked for the importance of the different membrane lipid compositions by measuring the activity of the toxin on several pure lipid model membranes. We investigated the effect of membrane thickness, fluidity, and presence of nonbilayer lipids and found that the toxin pore-forming ability increased in the presence of phosphocholines with short saturated acyl chains or with unsaturated chains even though not short. An increase of activity was also evident in the presence of cone-shaped lipids like phosphatidylethanolamine or diphytanoylphosphatidylcholine, whereas cylindrical lipids, like sphingomyelin, did not favor the activity. All these results suggest that γ-hemolysins could bind to the bilayer only if the phosphatidylcholine (PC) head is freely accessible. This condition is satisfied by the concurrent presence of cholesterol and certain lipids, as highlighted by the so-called umbrella model (J. Huang and G. W. Feigenson, Biophys J 76:2142–2157, 1999). According to this model, cholesterol could help to a better exposition of PC head groups only if acyl chains are short or unsaturated. In fact, phosphatidylcholines with more than 13 carbon atoms acyl chains can cover cholesterol molecules; in this way, PC head groups pack tightly, rendering them inaccessible to the toxin, which thus shows a reduced pore-forming ability.