Effects of fatty acid alpha-hydroxylation on glycosphingolipid properties in phosphatidylcholine bilayers.

The role of glycosphingolipid fatty acid alpha-hydroxylation as a modulator of glycolipid organization and dynamics was considered by 2H-NMR in bilayer membranes. For these experiments, galactosylceramides were prepared in which the natural fatty acid mixture was replaced with perdeuterated 18-carbon hydroxylated or non-hydroxylated stearic acid. The L-stereoisomer of N-(alpha-OH-stearoyl-d34)galactosylceramide and its naturally-occurring D-alpha-OH analogue, were isolated for independent study. Bilayers were formed using 10 mol% galactosylceramide in a shorter chain phospholipid, dimyristoylphosphatidylcholine, in an attempt to reproduce several features of glycolipid-phospholipid interactions typical of cell membranes. Spectra of deuterated galactosylceramide in gel phase phospholipid membranes indicated that alpha-hydroxylation led to greater motional freedom and/or conformational disorder, with no measurable difference between D- and L-alpha-OH fatty acid derivatives. In fluid phosphatidylcholine bilayers the effects were modest. Glycolipid fatty acid hydroxylation led to broadening of the range of order parameters associated with methylene groups near the membrane surface (frequently referred to as the 'plateau region') - this effect being more marked for the naturally-occurring (D) stereoisomer. The degree of overall molecular order sensed by the glycolipid fatty acid chain in a fluid host matrix was minimally affected by alpha-hydroxylation; although the plateau region of the D isomer was slightly more ordered than that of the L isomer and the non-hydroxylated species. These results suggest that a significant aspect of the alpha-hydroxy group effect on glycosphingolipid behaviour in bilayer membranes with low glycolipid content was interference with glycolipid packing amongst host phospholipids in the upper portion of the acyl chains. For the D stereoisomer, there was some evidence that the hydroxy group led to strengthening of interlipid interaction near the membrane surface.     

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

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

Orientation and conformation of lipids in crystals of transmembrane proteins

Orientational order parameters and individual dihedral torsion angles are evaluated for phospholipid and glycolipid molecules that are resolved in X-ray structures of integral transmembrane proteins in crystals. The order parameters of the lipid chains and glycerol backbones in protein crystals are characterised by a much wider distribution of orientational order than is found in fluid lipid bilayers and reconstituted lipid–protein membranes. This indicates that the lipids that are resolved in crystals of membrane proteins are mostly not representative of the entire lipid–protein interface. Much of the chain configurational disorder of the membrane-bound lipids in crystals arises from C–C bonds in energetically disallowed skew conformations. This suggests configurational heterogeneity of the lipids at a single binding site: eclipsed conformations occur also in the glycerol backbone torsion angles and the C–C torsion angles of the lipid head groups. Conformations of the lipid glycerol backbone in protein crystals are not restricted to the gauche C1–C2 rotamers found invariably in phospholipid bilayer crystals. Lipid head-group conformations in the protein crystals also do not conform solely to the bent-down conformation, with gauche–gauche configuration of the phosphodiester, that is characteristic of phospholipid bilayer membranes. Stereochemical violations in the protein-bound lipids are evidenced by ester carboxyl groups in non-planar configurations, and even in the cis configuration. Some lipids have the incorrect enantiomeric configuration of the glycerol backbone, and many of the branched methyl groups in the phytanyl chains associated with bacteriorhodopsin have the incorrect S configuration.     

2H-NMR studies on ether lipid-rich bacterial membranes: deuterium order profile of Clostridium butyricum

Palmitic acid specifically deuterated at different carbon atoms, has been incorporated biosynthetically into the membrane lipids of Clostridium butyricum. The lipids of this organism are rich in plasmalogens and their glycerol acetals and exhibit an unusual fatty acyl and alkenyl chain distribution with saturated chains mainly at the sn-2 position and unsaturated chains at the sn-1 position. The ordering of the deuterated hydrocarbon chains in whole cells was measured with deuterium nuclear magnetic resonance and was compared to the order profiles of isolated cell membranes and membranes formed from the total phospholipid extract. The shape of the order profiles was similar for all three membranes, but the absolute values of the order profiles in whole cells and isolated membranes were lower than those of the liposomal lipids. The order profiles have the same characteristic shape as those found for the lamellar liquid-crystalline phases of synthetic diacylphospholipids.     

Structural determinants of miscibility in surface films of galactosylceramide and phosphatidylcholine: effect of unsaturation in the galactosylceramide acyl chain

The Langmuir film balance technique has been used to define the surface structure and determine the mixing behavior of galactosylceramide (GalCer) and phosphatidylcholines in surface phases. To determine the effect of unsaturation on surface behavior, chain-pure GalCer species containing either oleoyl (18:1 delta 9), eicosenoyl (20:1 delta 11), or eicosadienoyl (20:2 delta 11,14) fatty acyl chains were synthesized. Using bovine brain GalCer as a reference, surface pressure versus molecular area (phi-A) isotherms of the pure lipids were measured and analyzed by determining their compressibilities and by using an equation of state for lipid monolayers. This information, when coupled with surface potential versus molecular area (delta V-A) analyses, provides insights into GalCer surface structure in terms of molecular packing and orientation. Lipid mixing behavior was determined by classical approaches which involve analyzing the average molecular area, the average surface dipole moment, and surface pressure as a function of film composition. The results indicate that, in contrast to the complex mixing behavior displayed by bovine brain GalCer and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), chain-pure GalCer species containing either oleoyl, eicosenoyl, or eicosadienoyl fatty acyl chains are miscible with POPC over the entire composition range. Moreover, increasing amounts of GalCer containing eicosenoyl acyl chains systematically elevate dipalmitoylphosphatidylcholine's (DPPC) liquid-expanded-to-liquid-condensed transition pressure. Such behavior is consistent with GalCer being miscible with the liquid-expanded phase of DPPC. Thus, fatty acyl unsaturation is a critical parameter governing the mixing behavior of GalCer with phosphatidylcholine.     

Inactivation of microsomal triglyceride transfer protein impairs the normal redistribution but not the turnover of newly synthesized glycerolipid in the cytosol,endoplasmic reticulum and Golgi of primary rat hepatocytes

The requirements for microsomal triglyceride transfer protein (MTP) during the turnover and transfer of glycerolipids from intracellular compartments into secretory very low-density lipoprotein (VLDL) were studied by pre-labelling lipids with [³H]glycerol and [¹⁴C]oleate in primary cultures of rat hepatocytes. The intracellular redistribution of pre-labelled glycerolipids was then compared at the end of subsequent chase periods during which the MTP inhibitor BMS-200150 was either present or absent in the medium. Inhibition of MTP resulted in a decreased output of VLDL triacylglycerol (TAG) and a delayed removal of labelled TAG from the cytosol and from the membranes of the smooth endoplasmic reticulum (SER), the cis- and the trans-Golgi. Inactivation of MTP did not decrease the bulk lipolytic turnover of cellular TAG as reflected by changes in its [³H]glycerol:[¹⁴C]oleate ratios. However, a larger proportion of the resultant TAG fatty acids was re-esterified and remained with the membranes of the various subcellular fractions rather than emerging as VLDL. The effects of BMS-200150 on the pattern of phospholipid (PL) mechanism and redistribution suggested that inhibition of MTP prevented the normal lipolytic transfer of PL-derived fatty acids out of the SER, cis- and trans-Golgi membrane pools. Finally, changes in the ¹⁴C specific radioactivities of the cytosolic and membrane pools of TAG suggested that inhibition of MTP prevented a normal influx of relatively unlabelled fatty acids into these pools during the chase period.     

The Influence of Hydrogen Bonding on Sphingomyelin/Colipid Interactions in Bilayer Membranes

The phospholipid acyl chain composition and order, the hydrogen bonding, and properties of the phospholipid headgroup all influence cholesterol/phospholipid interactions in hydrated bilayers. In this study, we examined the influence of hydrogen bonding on sphingomyelin (SM) colipid interactions in fluid uni- and multilamellar vesicles. We have compared the properties of oleoyl or palmitoyl SM with comparable dihydro-SMs, because the hydrogen bonding properties of SM and dihydro-SM differ. The association of cholestatrienol, a fluorescent cholesterol analog, with oleoyl sphingomyelin (OSM) was significantly stronger than its association with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in bilayers with equal acyl chain order. The association of cholestatrienol with dihydro-OSM, which lacks a trans double bond in the sphingoid base, was even stronger than the association with OSM, suggesting an important role for hydrogen bonding in stabilizing sterol/SM interactions. Furthermore, with saturated SM in the presence of 15 mol % cholesterol, cholesterol association with fluid dihydro-palmitoyl SM bilayers was stronger than seen with palmitoyl SM under similar conditions. The different hydrogen bonding properties in OSM and dihydro-OSM bilayers also influenced the segregation of palmitoyl ceramide and dipalmitoylglycerol into an ordered phase. The ordered, palmitoyl ceramide-rich phase started to form above 2 mol % in the dihydro-OSM bilayers but only above 6 mol % in the OSM bilayers. The lateral segregation of dipalmitoylglycerol was also much more pronounced in dihydro-OSM bilayers than in OSM bilayers. The results show that hydrogen bonding is important for sterol/SM and ceramide/SM interactions, as well as for the lateral segregation of a diglyceride. A possible molecular explanation for the different hydrogen bonding in SM and dihydro-SM bilayers is presented and discussed.     

Ice-Encasement Injury to Microsomal Membranes Isolated from Winter Wheat Crowns : II. Changes in Membrane Lipids during Ice Encasement

The physical properties and chemical composition of microsomal membranes were examined during a 7 day period of ice encasement in crown tissue of winter wheat (Triticum aestivum L. cv Norstar). Membrane damage, detected as an increase in microviscosity and electrolyte leakage, began between 1 and 3 days of icing, and was associated with a reduction in the recovery of microsomal membranes from stressed tissue, an increase in the microsomal free fatty acid:total fatty acid ratio, and a decrease in the phospholipid:total fatty acid ratio. These trends were amplified between 3 and 7 days of ice encasement. Examination of the free and total fatty acid fractions showed there was a slight, but not statistically significant (P = 0.05) reduction in the degree of unsaturation of the total fatty acid fraction. The composition of the free and total fatty acid fractions were very similar during ice encasement. Furthermore, analysis of phospholipid classes revealed no significant change in the relative amounts of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, or lysophospholipids in microsomal membranes during icing. Membrane injury during ice encasement apparently involves hydrolysis of the ester bond between glycerol and the acyl groups of the phospholipid resulting in loss of the phosphate-containing polar head group and a concomitant accumulation of free fatty acids in the bilayer.     

The interaction of cholesterol with bilayers of phosphatidylethanolamine

The interaction of cholesterol with the glycerol backbone segments of phospholipids was studied in bilayers of phosphatidylethanolamine containing equimolar amounts of cholesterol. Glycerol selectively deuterated at various positions was supplied to the growth medium of Escherichia coli strain 131 GP which is defective in endogeneous glycerol synthesis. The procedure enables the stereospecific labeling of the three glycerol backbone segments of the membrane phospholipids. Phosphatidylethanolamine with wild-type fatty acid composition was purified from E. coli cells and deuterium magnetic resonance spectra were obtained either from dispersions of pure phosphatidylethanolamine or from equimolar mixtures of phosphatidylethanolamine with cholesterol. For comparative purposes 1,2-di[9,10-²H₂]elaidoyl-sn-glycero-3-phosphoethanolamine and [3-α-²H]cholesterol were synthesized in order to monitor the behavior of the fatty acyl chains and of the cholesterol molecule itself. For all deuterated segments the deuterium quadrupole splittings as well as the deuterium spin-lattice (T₁) relaxation times were measured as a function of temperature. The glycerol backbone was found to be a remarkably stable structural element of the phospholipid molecule. The quadrupole splittings of the backbone segments changed only by at most 2 kHz upon incorporation of 50 mol % cholesterol. This was in contrast to the fatty acyl chains where the same amount of cholesterol increased the quadrupole splitting by more than 20 kHz. The glycerol segments exhibited the shortest T₁ relaxation times of all CH₂ segments indicating that the glycerol backbone is the slowest motional moiety of the lipid molecule. Addition of cholesterol has no effect on the backbone motion but the fast reorientation rate of the trans-double bonds in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine is increased dramatically.     

2H and 31P NMR study of pentalysine interaction with headgroup deuterated phosphatidylcholine and phosphatidylserine

The interaction of cationic pentalysine with phospholipid membranes was studied by using phosphorus and deuterium Nuclear Magnetic Resonance (NMR) of headgroup deuterated dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS). In the absence of pentalysine, some of the deuterium and phosphorus spectra of DMPC/DMPS 5:1 (m:m) membranes gave lineshapes similar to those of partially-oriented bilayers with the planes of the bilayers being parallel to the magnetic field. The deuterium NMR data show that the quadrupolar splittings of the deuterated methylenes of the DMPC headgroup are not affected by adsorption of pentalysine on the PC/PS membranes. By contrast, the pentalysine produces significant changes in the quadrupolar splittings of the negatively charged DMPS headgroup. The results are discussed in relation to previous ²H NMR investigations of phospholipid headgroup perturbations arising from bilayer interaction with cationic molecules.Abbreviations NMR nuclear magnetic resonance - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DMPS 1,2-dimyristoyl-sn-glycero-3-phosphoserine - POPC 1-palmitoyl, 2-oleyl-sn-glycero-3-phosphocholine - POPG 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol - PC phosphatidylcholine - PS phosphatidyl serine - PG phosphatidylglycerol - HEPES N-(2-hydroxy-ethyl)piperazine-N-2-ethanesulfonic acid - TRIS tris-(hydroxymethyl)aminoethane - EDTA ethylenediamine-tetra-acetic acid     

Miscibility of Sphingomyelins and Phosphatidylcholines in Unsaturated Phosphatidylcholine Bilayers

Polyunsaturated phospholipids are common in biological membranes and affect the lateral structure of bilayers. We have examined how saturated sphingomyelin (SM; palmitoyl and stearoyl SM (PSM and SSM, respectively)) and phosphatidylcholine (PC; dipalmitoyl PC and 1-palmitoyl-2-stearoyl PC (DPPC and PSPC, respectively)) segregate laterally to form ordered gel phases in increasingly unsaturated PC bilayers (sn-1: 16:0 and sn-2: 18:1...22:6; or sn-1 and sn-2: 18:1…22:6). The formation of gel phases was determined from the lifetime analysis of trans-parinaric acid. Using calorimetry, we also determined gel phase formation by PSM and DPPC in unsaturated PC mixed bilayers. Comparing PSM with DPPC, we observed that PSM formed a gel phase with less order than DPPC at comparable bilayer concentrations. The same was true when SSM was compared with PSPC. Furthermore, we observed that at equal saturated phospholipid concentration, the gel phases formed were less ordered in unsaturated PCs having 16:0 in sn-1, as compared to PCs having unsaturated acyl chains in both sn-1 and sn-2. The gel phases formed by the saturated phospholipids in unsaturated PC bilayers did not appear to achieve properties similar to pure saturated phospholipid bilayers, suggesting that complete lateral phase separation did not occur. Based on scanning calorimetry analysis, the melting of the gel phases formed by PSM and DPPC in unsaturated PC mixed bilayers (at 45 mol % saturated phospholipid) had low cooperativity and hence most likely were of mixed composition, in good agreement with trans-parinaric acid lifetime data. We conclude that both interfacial properties of the saturated phospholipids and their chain length, as well as the presence of 16:0 in sn-1 of the unsaturated PCs and the total number of cis unsaturations and acyl chain length (18 to 22) of the unsaturated PCs, all affected the formation of gel phases enriched in saturated phospholipids, under the conditions used.     

Polysialic acid can mediate membrane interactions by interacting with phospholipids

Polysialic acid (polySia) is expressed on the surface of neural cells, neuroinvasive bacterial cells and several tumor cells. PolySia chains attached to NCAM can influence both trans interactions between membranes of two cells and cis interactions. Here, we report on the involvement of phospholipids in regulation of membrane interactions by polySia. The pH at the surface of liposomes, specific molecular area of phosphatidylcholine molecules, phase transition of DPPC bilayers, cyclic voltammograms of BLMs, and electron micrographs of phosphatidylcholine vesicles were studied after addition of polysialic acid free in solution. The results indicate that polySia chains can associate with phosphatidylcholine bilayers, incorporate into the polar part of a phospholipid monolayer, modulate cis interactions between phosphatidylcholine molecules, and facilitate trans interactions between apposing phospholipid vesicles. These observations imply that polySia attached to NCAM or to lipids can behave similarly.     

Characterization of polar membrane lipids of the extremely halophilic bacterium Salinibacter ruber and possible role of cardiolipin

The lipid composition of the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) was investigated by thin layer chromatography, gas chromatography, high performance liquid chromatography and electrospray ionization-mass spectrometry. Polar lipids represent about 80% of the total lipid extract. The main polar lipids are a sulfonic acid analogue of ceramide (or capnine analogue), phosphatidylcholine, phosphatidylserine, dimethylphosphatidylethanolamine, phosphatidylglycerol, cardiolipin or bisphosphatidylglycerol, and a glycolipid. The major acyl chains in the phospholipids are C16:1 Δ9cis and C18:1 Δ11cis, while the sulfonolipid contains an amide-bound iso C15:0 fatty acid. On changing the salinity of the culture medium, no significant differences were found in the lipid profile or the unsaturation of the lipid fatty acyl chains. The structure of the cardiolipin, which represents 20% of polar lipids, has been elucidated by gas chromatography and electrospray ionization mass spectrometry analysis.     

Transmembrane asymmetry of vesicle lipids.

The role of fatty acyl chain unsaturation in promoting asymmetry in phospholipid vesicle bilayers was investigated in mixed lipid systems with differing acyl chains and a constant phosphatidylcholine headgroup. Ratios of outside to inside components were determined by nuclear magnetic resonance spectroscopy of 13C-enriched egg phosphatidylcholine. An asymmetry or disproportionation ratio is defined and used to express quantitatively how a mixture of two lipids distributes in the outer and inner vesicle surfaces. In mixed systems with 13C-enriched egg phosphatidylcholine as one component, increasing fatty acyl unsaturation in the other component results in an increasing preference of the unsaturated chains for the outer surface.     

Lipid composition of Mycoplasma neurolyticum

The total lipid content of Mycoplasma neurolyticum comprises about 14% of the dry weight of the organisms and is about equally distributed between the phospholipid and the neutral-glycolipid fractions. The neutral lipids were identified as triglycerides, diglycerides, and cholesterol. The glycolipid fraction contained 1-O-beta-glucopyranosyl-d-2,3-diglyceride and 1-[O-beta-d-glycopyranosyl-(1-->6)-O-beta-d-glucopyranosyl]-d-2,3-diglyceride. The latter lipid is structurally identical to the diglucosyl diglyceride which occurs in Staphylococcus aureus. The phospholipids of the organism consist of a fully acylated glycerophosphoryl-glycerophosphoryl glycerol, phosphatidic acid, diphosphatidyl glycerol, phosphatidyl glycerol, and amino acyl esters of phosphatidyl glycerol. Phosphatidic acid and phosphatidyl glycerol account for greater than 90% of the phospholipids of organisms in the exponential phase of growth. The predominant fatty acids found in all of the acyl lipids were palmitic, stearic, and oleic acids.     

Reorientational and Conformational Ordering Processes at Elevated Pressures in 1,2-Dioleoyl Phosphatidylcholine: A Raman and Infrared Spectroscopic Study

Raman and infrared spectra of fully hydrated bilayers of 1,2-dioleoyl phosphatidylcholine (DOPC) were measured at increasing hydrostatic pressures up to -37 kbar. Under ambient conditions aqueous dispersions of DOPC are in the liquid crystalline state. The application of an external hydrostatic pressure induces conformational and dynamic ordering processes in DOPC, which trigger a first-order structural phase transition at 5 kbar from a disordered liquid crystalline state to a highly ordered gel state. In the gel phase the methylene chains of each molecule are fully extended and the two all-trans chain segments on both sides of the rigid cis double bond form a bent structure. The bent oleoyl chains in each molecule, as well as in neighboring molecules are packed parallel to each other. To achieve this parallel interchain packing, the double bonds of the sn-1 and sn-2 chains of each molecule must be aligned at the same position with respect to the bilayer interface which is achieved by a rotation of the C—C bonds in the glycerol moiety in the head group. The extremely strong interchain interactions in the gel phase of DOPC are unique for this lipid with cis dimono-unsaturated acyl chains. Our experimental results suggest that in the pressure-induced gel phase of DOPC the olefinic CH bonds are rotated out of the phase of the bent oleoyl chains and that the oleoyl chains of opposing bilayers bend towards opposite directions.     

Production and Analysis of Perdeuterated Lipids from Pichia pastoris Cells

Probing molecules using perdeuteration (i.e deuteration in which all hydrogen atoms are replaced by deuterium) is extremely useful in a wide range of biophysical techniques. In the case of lipids, the synthesis of the biologically relevant unsaturated perdeuterated lipids is challenging and not usually pursued. In this work, perdeuterated phospholipids and sterols from the yeast Pichia pastoris grown in deuterated medium are extracted and analyzed as derivatives by gas chromatography and mass spectrometry respectively. When yeast cells are grown in a deuterated environment, the phospholipid homeostasis is maintained but the fatty acid unsaturation level is modified while the ergosterol synthesis is not affected by the deuterated culture medium. Our results confirm that the production of well defined natural unsaturated perdeuterated lipids is possible and gives also new insights about the process of desaturase enzymes.     

Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers

Aqueous dispersions of egg-phosphatidylcholine and egg-phosphatidylcholine/30 mol% cholesterol containing deuterated tripalmitin or triolein were studied at approx. 25°C by ²H-NMR. Incorporation of tripalmitin into egg-phosphatidylcholine bilayers was found to be less than 0.1 mol%, while the incorporation of trolein is approx. 2.5 mol% in the absence and approx. 0.7 mol% in the presence of cholesterol. The profile of order parameter versus chain position for deuterated triolein suggests that the oleoyl chains of the triacylglycerol have an average orientation such that the C2 chain segments and the segments in the vicinity of the C9–C10 double bond are tilted with respect to the bilayer normal, while all other segments are parallel to the bilayer normal. Longitudinal relaxation times were also determined and indicate that the acyl chains of triolein have a motional behaviour similar to that of phospholipid acyl chains in the bilayer.     

Interactions of chlorinated hydrocarbon insecticides with membranes

Chlorinated hydrocarbon insecticides quench the fluorescence of N-alkyl derivatives of carbazole. We used phospholipids with covalently attached carbazole as probes for the interactions of chlorinated hydrocarbon insecticides with lipid bilayers, the object being to understand better the toxicities of chlorinated hydrocarbons. Fluorescence quenching measurements revealed the lipid-water partition coefficients of the chlorinated hydrocarbons, their diffusion coefficients in the membranes, and the binding capacities of the membranes for the chlorinated hydrocarbons. Active insecticides were compared with inactive analogues to test whether activities correlated with chlorinated hydrocarbon-membrane interactions. Thus DDT and methoxychlor were compared with inactive DDE, and insecticidal γ-lindane was compared with three less active stereoisomers. The partition coefficients, diffusion coefficients and membrane saturation capacities did not correlate with insecticidal potency. The partition coefficients of these chlorinated hydrocarbons were larger in bilayers containing unsaturated fatty acyl chains as compared to bilayers containing saturated fatty acyl chains. Interestingly, neural membranes are known to contain a large percentage of unsaturated lipids. Our results indicate that the activities of chlorinated hydrocarbons are not a result of specific interactions of these compounds with the lipids of membranes. However, the neurotoxicity of chlorinated hydrocarbons may be amplified by selective partitioning in the unsaturated neural membranes.     

Ether glycerolipids: novel substrates for studying specificity of enzymes involved in glycerolipid biosynthesis in higher plants

Ether glycerolipids, predominantly alkylacylglycerols and alkylacylglycerophosphocholines, are synthesized in photomixotrophic rape (Brassica napus) suspension cells from various exogenous monoalkylglycerols. The stereospecific distribution of acyl moieties was studied in these ether glycerolipids with regard to chain-length and degree of unsaturation of alkyl moieties and compared with the distribution of acyl moieties in the corresponding endogenous acyl glycerolipids. The results show the following: (1) Alkylacylglycerophosphocholines replaced up to one-half of the corresponding physiological membrane lipids, i.e. diacylglycerophosphocholines, without changing the total amount of cholineglycerophospholipids as compared to untreated cells. (2) The composition of acyl moieties in total lipids of rape cells was practically unaltered by fatty acids derived via oxidative cleavage from the various alkyl moieties of either glycerolipids. (3) In 1-O-alkyl-2-acylglycerols derived from exogenous alkylglycerols and in endogenous 1,2-diacylglycerols compositions of acyl moieties were found to be different indicating that different pathways were operative in the biosynthesis of these two neutral glycerolipids. (4) Enzymes involved in synthesizing molecular species of 1-O-alkyl-2-acylglycerophosphocholines or 2-O-alkyl-1-acylglycerophosphocholines as well as 1,2-diacylglycerophosphocholines showed similar specificities with regard to chain-length and degree of unsaturation of both alkyl and corresponding acyl moieties. Thus, ether glycerolipids formed by plant cells from exogenous alkylglycerols are suitable metabolites for studying the specificity of enzymes involved in the biosynthesis of glyerolipids.