Analysis of polar lipids from some representative enterobacteria, Plesiomonas and Acinetobacter by fast atom bombardment-mass spectrometry

Fast atom bombardment-mass spectrometry (FAB-MS) was used to analyse lipid extracts of bacteria to assess its usefulness for analysing anionic phospholipids of potential chemotaxonomic value. The following micro-organisms were tested: Acinetobacter calcoaceticus, Acinetobacter sp., Citrobacter freundii, Enterobacter cloacae (2 strains), Escherichia coli (3 strains), Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Plesiomonas shigelloides, Proteus mirabilis (3 strains), Serratia liquefaciens and Serratia marcescens. Negative-ion spectra provide data for twenty-seven major carboxylate anions (m/z 209–325) and for thirty-seven major phospholipid anions (m/z 645–774). Generally, the largest carboxylate peaks were due to 16: 1, 16: 0, cyc17 and 18: 1 while the largest phospholipid anion peaks were due to PE(32: 1), PE(33: 1), PE(34: 1), PE(34: 2), PG(30: 2), PG(31: 2), PG(32: 2), PG(34: 1) and PS (33: 0). However, quantitative differences were observed. For example, Acinetobacter lacked PE (33: 1) but had exceptionally high peaks at m/z 748, PS(33: 0), and m/z 281, octadecanoate. Unknown 'carboxylate' peaks were detected at m/z 254, 256, 261, 268, 282 and 301. In some cases, unknown peaks appeared to constitute possible homologous series being separated by Δ m/z of 14(≡ methylene). For chemotaxonomic purposes, the complexity of the data required numerical analysis. Using the Pearson coefficient of linear correlation, as a measure of association, it was possible to compare all strains analysed. Typical results for strain comparisons were as follows: Ent. cloacae vs Ent. cloacae, r = 0.90 ( Ent. cloacae vs Ac. calcoaceticus, r = 0.46). Thus FAB-MS represents an excellent means of obtaining large quantities of data on polar lipids of a range of bacterial isolates, which may be suitable for chemotaxonomic purposes.     

Phospholipid molecular species distribution of some medically important Candida species analysed by fast atom bombardment mass spectroscopy

The aim of this study was to obtain detailed information on phospholipids (PL) of the medically important Candida species and to determine their possible chemotaxonomic significance. Lipids were extracted from 22 strains representing 8 Candida species and their PL molecular species distributions were determined by Fast Atom Bombardment Mass Spectroscopy (FAB MS) in negative ion mode. Fifteen major lower mass peaks (m/z 221 to 289) were attributable to the expected presence of carboxylate anions and 24 major higher mass peaks (m/z 557 to 837) were attributable to phospholipid anions. Major carboxylate peaks were of the following m/z and identities : 253, C16:1; 255, C16:0; 277, C18:3; 279, C18:2; 281, C18:1; and 283, C18:0. The most abundant peaks consistent with the presence of phospholipid molecular species anions include those of m/z 673, 743, 833, 834 and 836 tentatively identified as phosphatidic acid (PA) (34:1), phosphatidylglycerol (PG) (34:3), phosphtidylinositol (PI) (34:2) and two unknown molecular species. This profile is diagnostic for the genus Candida. Quantitative differences were observed between different Candida species. Thus, polar lipid molecular species distribution in Candida spp. has chemotaxonomic significance, especially so in the case of carboxylate anions.     

Phospholipid analogues of Porphyromonas gingivalis

Porphyromonas has lipids containing hydroxy acids and C16:0 and iso-C15:0 major monocarboxylic acids among others. Nothing is known of its individual phospholipid molecular species. The aim of this study was to determine molecular weights and putative identities of individual phospholipid molecular species extracted from Porphyromonas gingivalis (seven strains), P. asaccharolytica (one strain) and P. endodontalis (two strains). Cultures on Blood-Fastidious Anaerobe Agar were harvested, washed and freeze-dried. Phospholipids were extracted and separated by fast atom bombardment mass spectrometry (FAB MS) in negative-ion mode. Phospholipid classes were also separated by thin layer chromatography (TLC). The major anions in the range m/z 209-299 were consistent with the presence of the C13: 0, C15: 0, C16: 0 and C18: 3 mono-carboxylate anions. Major polar lipid anion peaks in the range m/z 618-961 were consistent with the presence of molecular species of phosphatidylethanolamine, phosphatidylglycerol and with unidentified lipid analogues. Porphyromonas gingivalis differed from comparison strains of other species by having major anions with m/z 932, 946 and 960. Unusually, a feline strain of P. gingivalis had a major peak of m/z 736. Selected anions were studied by tandem FAB MS which revealed that peaks with m/z 653 and 946 did not correspond to commonly occurring classes of polar lipids. They were however, glycerophosphates. It is concluded that the polar lipid analogue profiles obtained with Porphyromonas are quite different from those of the genera Prevotella and Bacteroides but reveal heterogeneity within P. gingivalis.     

Polar lipids of Staphylococcus strains analysed by fast atom bombardment mass spectrometry

This study used fast atom bombardment mass spectrometry (FAB MS) to obtain detailed information on polar lipids of Staphylococcus by examining 23 isolates. Eighteen major anions were found in the range m/z 199–297, consistent with the presence of carboxylate anions. A further 21 major anions were found in the higher mass regions of m/z 609–805, consistent with the presence of phospholipid anions. In Staph. aureus, Staph. epidermidis, Staph. haemolyticus and Staph. hominis , the most intense peaks putatively assigned as carboxylate ions were consistent with presence of C_15:0, followed by C_17:0 except in the case of Staph. epidermidis. The major phospholipid anions were consistent with the presence of PG(30 : 0), PG(32 : 0) and PG(33 : 0). It is concluded that Staphylococcus has a characteristic polar lipid profile and that qualitative and quantitative differences may be seen between Staph. aureus and Staph. epidermidis.     

Analysis of polar lipids from some representative enterobacteria, Plesiomonas and Acinetobacter by fast atom bombardment-mass spectrometry.

Fast atom bombardment-mass spectrometry (FAB-MS) was used to analyse lipid extracts of bacteria to assess its usefulness for analysing anionic phospholipids of potential chemotaxonomic value. The following micro-organisms were tested: Acinetobacter calcoaceticus, Acinetobacter sp., Citrobacter freundii, Enterobacter cloacae (2 strains), Escherichia coli (3 strains), Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Plesiomonas shigelloides, Proteus mirabilis (3 strains), Serratia liquefaciens and Serratia marcescens. Negative-ion spectra provide data for twenty-seven major carboxylate anions (m/z 209-325) and for thirty-seven major phospholipid anions (m/z 645-774). Generally, the largest carboxylate peaks were due to 16:1, 16:0, cyc17 and 18:1 while the largest phospholipid anion peaks were due to PE(32:1), PE(33:1), PE(34:1), PE(34:2), PG(30:2), PG(31:2), PG(32:2), PG(34:1) and PS(33:0). However, quantitative differences were observed. For example, Acinetobacter lacked PE (33:1) but had exceptionally high peaks at m/z 748, PS(33:0), and m/z 281, octadecanoate. Unknown 'carboxylate' peaks were detected at m/z 254, 256, 261, 268, 282 and 301. In some cases, unknown peaks appeared to constitute possible homologous series being separated by delta m/z of 14(identical to methylene). For chemotaxonomic purposes, the complexity of the data required numerical analysis. Using the Pearson coefficient of linear correlation, as a measure of association, it was possible to compare all strains analysed. Typical results for strain comparisons were as follows: Ent. cloacae vs Ent. cloacae, r = 0.90 (Ent. cloacae vs Ac. calcoaceticus, r = 0.46). Thus FAB-MS represents an excellent means of obtaining large quantities of data on polar lipids of a range of bacterial isolates, which may be suitable for chemotaxonomic purposes.     

Phospholipids of Fusobacterium spp.

The aim of this study was to analyse individual polar lipid analogues, within each lipid family present, of fusobacteria using fast atom bombardment mass spectrometry (FAB-MS). Polar lipid extracts were prepared, washed and dried. Samples, dispersed in a matrix of m -nitrobenzyl alcohol, were analysed by negative ion FAB-MS using xenon as the reagent gas. Major anion peaks observed in the low mass region of mass/charge (m/z), 211, 221, 225, 227, 239, 241, 249, 251, 253, 255, 273, 277, 279, 281, 289 and 291, were consistent with the presence of C_13:1, C_14:3, C_14:1, C_14:0, C_15:1, C_15:0, C_16:3, C_16:2, C_16:1, C_16:0, unknown, C_18:3, C_18:2, C_18:1, unknown and C_19:3 carboxylate anions. In the high mass region, major anion peaks observed with m/z 644, 646, 648, 660, 662, 672, 673, 674, 686, 688, 689, 690, 698, 700, 701, 703, 714, 716, 717 and 719 were consistent with the presence of phosphatidylethanolamine (PE) (29:2), PE (29:1), PE (29:0), PE (30:1), PE (30:0), PE (31:2), first isotope of PE (31:2), PE (31:1), PE (32:2), PE (32:1), first isotope peak of PE (32:1), PE (30:0), PE (33:3), PE (33:2), phosphatidylglycerol (PG) (31:3), PG (31:2), PE (34:2), PE (34:1), PG (32:2) and PG (32:1). We conclude that FAB-MS can provide data on individual analogues of PE and PG from Fusobacterium spp. not readily obtained by other means. Furthermore, the phospholipid profile is diagnostic for the genus.     

Phospholipids of Lactobacillus spp.

Lactobacillus phospholipid molecular species were analyzed by mass spectrometry. Prominent anions were consistent with presence of the phosphatidylglycerols PG(37:2), PG(36:2), PG(35:1), PG(34:1), and PG(33:1). Diglycosyldiacylglycerol molecular species were also observed, although nitrogen-containing phospholipids were absent. An anion of m/z 759 was derived from an apparently novel type of lipid.     

Characterization of polar lipids of oral isolates of Candida, Pichia and Saccharomyces by Fast Atom Bombardment Mass Spectrometry (FAB MS)

AIMS: To characterize fatty acid and phospholipid analogue profiles of oral yeasts. METHODS AND RESULTS: Twenty-seven strains of oral yeasts were cultured on SDA and lipids of freeze-dried cells were extracted and analysed by FAB MS. The most abundant carboxylate anion was m/z 281 (C18 : 1). The most intense phospholipid analogue ions were of PE, PG, PA and PI. Pichia etchellsii contained molecular species of PG and PE, whereas Saccharomyces cerevisiae had PA, PG and PE analogues. Mass spectra revealed that S. cerevisiae and Candida glabrata were distinct from one another and from the other species tested. CONCLUSION: Oral yeasts largely differ with respect to their polar lipids. It is concluded that oral yeast species have distinctive fatty acid and phospholipid analogue anion profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: FAB MS provided novel chemotaxonomic information.     

Phospholipid profiles of Clostridium difficile.

Phospholipid molecular species present in 32 isolates of Clostridium difficile were examined by fast atom bombardment-mass spectrometry in negative-ion mode. This revealed major anions consistent with the expected presence of the following phosphatidylglycerol (PG) analogs: PG(31:2), PG(32:1), PG(33:2), PG(33:1), PG(34:2), and PG(34:1). The major phospholipid molecular species are distinct from those of other bacterial groups examined.     

Phospholipid molecular species distributions of Candida isolates from the UK and Iran

AIMS: Some species of Candida have been shown to differ with respect to their polar lipid fingerprints when analysed by fast atom bombardment mass spectrometry (FABMS). The aims of this study were to contribute to the existing body of information by (i) examining representatives of species not previously examined and (ii) seeking strains differences associated with country of origin (UK or Iran). METHODS AND RESULTS: FABMS analysis was performed on extracted lipids of 22 strains representing eight species of Candida. The most abundant anion (19 isolates) in spectra was with mass to charge (m/z) 281, corresponding to C18:1 carboxylate. The major phospholipid analogue anions were m/z 515 and 501 (13 strains). These anions were putatively identified as the phosphatidyl molecular species PA(23 : 2) and PA(22 : 2) respectively. Data for strain pairs were compared using the Pearson's coefficient of linear correlation. The values generated were used to cluster strains by nearest-neighbour linkage, using both carboxylate and phospholipid analogue anion data. Isolates of C. parapsilosis were clearly distinct from other isolates. Iranian isolates tended to cluster together when phospholipid anion data were used. However, if carboxylate anion data were used, four Iranian isolates of C. albicans were tightly clustered with three UK isolates, of which two were C. albicans and one was C. dubliniensis. CONCLUSION: It is concluded that both lower, and higher, mass peaks in FABMS spectra can be of potential value in comparing Candida isolates from different countries and from different species. SIGNIFICANCE AND IMPACT OF THE STUDY: When polar lipids of different Candida species are compared, it is important to bear in mind that geographical differences affect results as has been observed with bacteria in similar studies.     

Comparison of Candida dubliniensis and C. albicans based on polar lipid composition

AIMS: To test the hypothesis that strains of Candida dubliniensis and C. albicans can be differentiated on the basis of polar lipid profiles. METHODS AND RESULTS: Five isolates of C. dubliniensis and six isolates of C. albicans were tested by growth at 45 degrees C, production of chlamydospores on cornmeal agar, colonial colour on CHROMagar Candida medium and assimilation of DL-lactate, alpha-methyl-D-glucoside and xylose. Polar lipids were then extracted from freeze-dried cultures and analysed using fast atom bombardment mass spectrometry. Isolates were grouped by single linkage clustering based on correlation coefficients for strain pairs calculated with carboxylate and phospholipid molecular species distributions. The most intense carboxylate and phospholipid molecular species anions were of m/z 281 (C(18 : 1)) and m/z 515 (PA 23 : 2). Phosphatidylethanolamine and phosphatidylglycerol were the predominant phospholipid families in C. dubliniensis, compared with phosphatidic acid in C. albicans isolates. All of the C. dubliniensis isolates grouped together in one cluster, whereas all of the C. albicans isolates grouped in a separate cluster. CONCLUSIONS: Fast atom bombardment mass spectrometry can differentiate the two species based on analysis of polar lipid distributions. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings demonstrate that C. dubliniensis and C. albicans have distinct polar lipid profiles.     

Apparent relative retention of the phosphatidylethanolamine molecular species 18:0-20:5(n-3), 16:0-22:6(n-3) and the sum 16:0-20:4(n-6) plus 16:0-20:3(n-9) in the liver microsomes of pig on an essential fatty acid deficient diet.

Attempts at a better understanding of the cell membrane organization and functioning need to assess the physical properties which partly depend (i) on the positional distribution of the fatty acids in the membrane phospholipids (PLs) and (ii) on the way by which the PL molecular species are affected by exogenous fatty acids. To do that, the effects of essential (polyunsaturated) fatty acid (EFA) deficiency and enrichment were studied in the liver microsomes of piglets feeding on either an EFA-deficient diet or an EFA-enriched diet containing hydrogenated coconut oil or a mixture of soya + corn oils, respectively. After derivatization, the diacylated forms of choline and ethanolamine PLs were analyzed using a combination of chromatographic techniques and fast-atom bombardment-mass spectrometry. The dinitrobenzoyl-diacylglycerol derivatives corresponding to the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were identified. It appears that three factors brought about a marked apparent relative retention: the nature of (i) the base of the polar head, (ii) fatty acids at the sn-1 position and (iii) fatty acids at the sn-2 position. The highest apparent relative retentions were displayed by the 18:0-20:5(n-3)-PE and 16:0-22:6(n-3)-PE. It is noteworthy that the behavior of 20:3 n-9--which is synthesized during the EFA-deficient diet by the same bioconversion system as 20:4 n-6--was very similar to that of 20:4 n-6 during the formation of PC and PE molecular species and that the molecular species of PE containing 20:4(n-6) and 20:3(n-9), gathered together as metabolical homologues, were also apparently retained, particularly in association with 16:0. Present observations are consistent with some others showing retention or preferential distribution of EFA in PE and suggest that specific acyltransferase(s), ethanolamine phosphotransferase and methyltransferase would be mainly involved for PE and PC formation in liver endoplasmic reticulum. Fast-atom bombardment-mass spectrometry of intact phospholipids enables us to show that there is no very long chain dipolyunsaturated phospholipid in liver endoplasmic reticulum.     

Effects of Cadmium on Polar Lipid Composition and Unsaturation in Maize （Zea mays） in Hydroponic Culture

This study aimed to evaluate the effect of Cd exposure （100 μmol/L） on polar lipid composition, and to examine the level of fatty acid unsaturation in maize （Zea mays L.）. In roots, the level of 16：0 and monounsaturated fatty acids （16：1 ＋ 18：1） decreased in phosphatidylcholine （PC） and phosphatidylethanolamine （PE）. In contrast, the proportion of unsaturated 18-C fatty acid species showed an opposite response to Cd. The content, on the other hand, of PC, PE, digalactosyldiacylglycerol （DGDG）, and steryl lipids increased in roots （2.9-, 1.6-, 5.3-, and 1.7-fold increase, respectively）. These results suggest that a more unsaturated fatty acid composition than found in control plants with a concomitant increase in polar lipids may favor seedling growth during Cd exposure. However, the observed increase in the steryl lipid （SL） ： phospholipid （PL） ratio （twofold）, the decrease in monogalactosyldiacylglycerol （MGDG） ： DGDG ratio, as well as the induction of lipid peroxidation in roots may represent symptoms of membrane injury. In shoots, the unsaturation level was markedly decreased in PC and phosphatidylglycerol （PG） after Cd exposure, but showed a significant increase in sulfoquinovosyldiacylglycerol （SQDG）, MGDG and DGDG. The content of PG and MGDG was decreased by about 65%, while PC accumulated to higher levels （4.4-fold increase）. Taken together, these changes in the polar lipid unsaturation and composition are likely to be due to alterations in the glycerolipid pathway. These results also support the idea that the increase in overall unsaturation plays some role in enabling the plant to withstand the metal exposure.     

Phospholipid analogue profiles of Peptostreptococcus, Micromonas, and Finegoldia species analysed by fast atom bombardment mass spectrometry

Species of Peptostreptococcus cause a variety of infections, primarily abscesses of soft tissues, joints, and mucous membranes. The aim of this study was to compare the phospholipid analogue profiles of Peptostreptococcus species, represented by P. anaerobius, P. asaccharolyticus, P. indolicus, P. lacrimalis, and P. prevotii; Micromonas micros (P. micros) and Finegoldia magna (P. magnus). After anaerobic growth on blood-FAA, lipids extracted by chloroform-methanol (2:1 v/v) were purified, then analysed by fast atom bombardment mass spectrometry (FAB-MS) in negative ion mode. The major peaks with mass to charge (m/z) 719, 721, and 749, corresponded to phosphatidylglycerol analogues, namely PG (32:1), PG (32:0), and PG (34:0), which have been found previously in Lactobacillus spp., Clostridium difficile, and Staphylococcus spp. Other major peaks observed, with m/z 619, 647, 665, 675, 677, 687, 691, 693, 701, 703, 707, 733, and 746 have also been reported in one or more of these three species. However, other major peaks found here in Peptostreptococcus, Micromonas, and Finegoldia have not been described elsewhere; these are 501, 514, 515, 618, 659, 673, 676, 688, 690, 692, 694, 700, 706, 715, 718, 722, and 750. We conclude that Peptostreptococcus, Micromonas, and Finegoldia isolates are chemically unique.     

Structural characterization of the polar lipids of Clostridium novyi NT. Further evidence for a novel anaerobic biosynthetic pathway to plasmalogens

A study of the polar lipids of Clostridium novyi NT has revealed the presence of phosphatidylethanolamine (PE) and cardiolipin as major phospholipids with smaller amounts of phosphatidylglycerol (PG), lysyl-PG and alanyl-PG. Other minor phospholipids included phosphatidic acid, CDP-diacylglycerol, phosphatidylserine (PS) and phosphatidylthreonine (PT). PE, PG and amino acyl PG were present in both the diacyl and alk-1'-enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk-1'-enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP-diacylglycerol and PS were present almost exclusively as diacyl phospholipids. These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in Clostridium species. This novel pathway contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis and the alk-1-enyl bond is formed by an aerobic mechanism.     

Individual molecular species of phosphatidylcholine and phosphatidylethanolamine in myelin turn over at different rates.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of the myelin membrane exhibit heterogeneity with respect to metabolic turnover rate (Miller, S. L., Benjamins, J. A., and Morell, P. (1977) J. Biol. Chem. 252, 4025-4037). To test the hypothesis that this is due to differential turnover of individual molecular species (which differ in acyl chain composition), we have examined the relative turnover of individual molecular species of myelin PC and PE. Phospholipids were labeled by injection of [2-3H]glycerol into the brains of young rats. Myelin was isolated at 1, 15, and 30 days post-injection, lipids were extracted, and phospholipid classes were separated by thin-layer chromatography. The PC and PE fractions were hydrolyzed with phospholipase C, and the resulting diacylglycerols were dinitrobenzoylated and fractionated by reverse-phase high performance liquid chromatography. The distribution of radioactivity among individual molecular species was determined. The labeled molecular species of myelin PC were 16:0-16:0, 16:0-18:0, 16:0-18:1, and 18:0-18:1, with most of the label present in 16:0-18:1 and 18:0-18:1. Changes in distribution of label with time after injection indicated that 16:0-18:1 turned over more rapidly than 18:0-18:1. The labeled molecular species of myelin PE were 18:0-20:4, 18:1-18:1, 16:0-18:1, 18:0-18:2, and 18:0-18:1. As with myelin PC, 16:0-18:1 (and 18:1-18:1) turned over more rapidly than 18:0-18:1. The relative turnover of individual molecular species of PC in the microsomal fraction from forebrain was also examined. The molecular species profile was different from myelin PC, but again, 16:0-18:1 turned over more rapidly than the other molecular species. Thus, within the same membrane, individual molecular species of a phospholipid class are metabolized at different rates. Comparison of our results with previous studies of turnover of molecular classes of phospholipids indicates that in addition to polar head group composition (Miller et al., 1977), fatty acid composition is very important in determining the metabolic fate of a phospholipid.     

Apparent relative retention of the phosphatidylethanolamine molecular species 18:0–20:5(n−3), 16:0–22:6(n−3) and the sum 16:0–20:4(n−6) plus 16:0–20:3(n−9) in the liver microsomes of pig on an essential fatty acid deficient diet

Attempts at a better understanding of the cell membrane organization and functioning need to assess the physical properties which partly depend (i) on the positional distribution of the fatty acids in the membrane phospholipids (PLs) and (ii) on the way by which the PL molecular species are affected by exogenous fatty acids. To do that, the effects of essential (polyunsaturated) fatty acid (EFA) deficiency and enrichment were studied in the liver microsomes of piglets feeding on either an EFA-deficient diet or an EFA-enriched diet containing hydrogenated coconut oil or a mixture of soya + corn oils, respectively. After derivatization, the diacylated forms of choline and ethanolamine PLs were analyzed using a combination of Chromatographic techniques and fast-atom bombardmentmass spectrometry. The dinitrobenzoyl-diacylglycerol derivatives corresponding to the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were identified. It appears that three factors brought about a marked apparent relative retention: the nature of (i) the base of the polar head, (ii) fatty acids at the sn−1 position and (iii) fatty acids at the sn−2 position. The highest apparent relative retentions were displayed by the 18:0–20:5(n−3)-PE and 16:0–22:6(n−3)-PE. It is noteworthy that the behavior of 20:3 n−9 — which is synthesized during the EFA-deficient diet by the same bioconversion system as 20:4 n−6 — was very similar to that of 20:4 n−6 during the formation of PC and PE molecular species and that the molecular species of PE containing 20:4(n−6) and 20:3(n−9), gathered together as metabolical homologues, were also apparently retained, particularly in association with 16:0. Present observations are consistent with some others showing retention or preferential distribution of EFA in PE and suggest that specific acyltransferase(s), ethanolamine phosphotransferase and methyltransferase would be mainly involved for PE and PC formation in liver endoplasmic reticulum. Fast-atom bombardment-mass spectrometry of intact phospholipids enables us to show that there is no very long chain dipolyunsaturated phospholipid in liver endoplasmic reticulum.     

Study of phospholipase A2 specificity in substrate-containing structures having different supramolecular organization

The specificity of snake venom phospholipase A2(PLA2) towards a number of phospholipid (PL) substrates, e. g., phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) organized in Triton X-100 mixed micelles, liposomes and proteoliposomes was studied. PC was shown to be more rapidly hydrolyzed in micelles. For other PLs, the rate of hydrolysis decreased in the following sequence: PC greater than PI greater than PE greater than PG. The incorporation into micelles of a non-hydrolyzable by PLA2 sphinogomyelin which, similar to PC, has a choline group, resulted in an increase of PLA2 specificity towards PL that are known to be devoid of this group: PE greater than PI greater than PG greater than PC. Quite a different picture was observed in bilayer liposomal structures: PI congruent to PE greater than PC greater than PG. The incorporation of cytochrome P-450 into liposomes caused the acceleration of PE and PG hydrolysis. The course of the PLA2-catalyzed hydrolysis in model membrane structures seems to be governed primarily by the supramolecular organization and localization of the substrate in the bilayer, but not by its chemical structure.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A(2). We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A₂. We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.