Focused lipidomics by tandem mass spectrometry

In this paper we performed focused analyses of phospholipids by using the data of precursor ion scanning and neutral loss scanning of their polar head groups and fatty acyl moieties for the specific search of categorical phospholipids. By using precursor ion scanning or neutral loss scanning of polar head groups in the positive ion mode, more sensitive identification were obtained than that in the negative ion mode. Precursor ion scanning of carbonic anions in the negative ion mode was also effective to identify molecular species of phospholipids having specified fatty acyl moieties. By using these analytical methods, the detection limits of individual metabolites are going up to 5-20-fold of former conventional methods. The important factor is that by focusing in some limited categories of molecules, detection limit is greatly enhanced, thus minor but important molecules can be detected. Moreover, combination of LC-MS/MS and focused scanning for head group was revealed to be useful to identify very minor molecular species in the focused class of phospholipids.     

The Chemical Nature of the Polar Functional Group of Oxidized Acyl Chain Uniquely Modifies the Physicochemical Properties of Oxidized Phospholipid-Containing Lipid Particles

Oxidative modification of phospholipids generates a variety of oxidized phospholipid (Ox-PL) species which differ considerably in their chemical compositions and molecular structures. Recent results suggest that even closely related Ox-PL species can have considerably different biological effects. However, the molecular mechanism for this is not yet clear. In truncated Ox-PLs (tOx-PLs) the fatty acyl chain is shorter in length than the parent nonoxidized phospholipid molecules and contains a polar functional group(s). In a previous study we showed that two closely related tOx-PL species having a similar polar functional group and differing only in the length of the oxidized fatty acyl chain exerts significantly different effects on the physicochemical properties of the nonoxidized phospholipid particles containing these lipids (Kar et al., Chem Phys Lipids 164:54–61, 2011). In this study we have characterized the effect of polar functional groups of oxidized fatty acyl chain on the physicochemical properties of the nonoxidized phospholipid particles containing these lipids. Our results show that Ox-PL species differing only in the chemical nature of polar functional groups in their oxidized fatty acyl chain modify the properties of nonoxidized phospholipid particles containing them in a distinctive way. These results indicate that different species of Ox-PLs induce unique changes in the physicochemical properties of lipid particles/membranes containing them and that this may lead to their different biological effects.     

A new role for apolipoprotein E: modulating transport of polyunsaturated phospholipid molecular species in synaptic plasma membranes

Phospholipids and their acyl group composition are important in providing the proper membrane environment for membrane protein structure and function. In particular, the highly unsaturated phospholipids in synaptic plasma membranes in the CNS are known to play an important role in modulating receptor function and neurotransmitter release processes. Apolipoprotein E (apoE) is a major apolipoprotein in the CNS, mediating the transport of cholesterol, phospholipids and their fatty acids, particularly in reparative mechanisms during neuronal injury. This study was performed to determine whether deficiency in the apoE gene contributes to an alteration of the phospholipids in synaptic plasma membranes. Phospholipid molecular species were identified and quantitated by HPLC/electrospray ionization-mass spectrometry. Analysis of the different phospholipid classes in membranes of apoE-deficient and C57BL/6 J mice indicated no obvious differences in the distribution of different phospholipid classes but substantial differences in composition of phospholipid molecular species. Of special interest was the prevalence of phospholipids (phosphatidylcholine, diacyl-phosphatidylethanolamine, and phosphatidylserine) with 22:6n-3 in both the sn-1 and sn-2 positions of SPM and these phospholipid species were significantly higher in apoE-deficient mice as compared to control mice. Since polyunsaturated fatty acids in neurons are mainly supplied by astrocytes, these results revealed a new role for apoE in regulating polyunsaturated phospholipid molecular species in neuronal membranes.     

Schistosoma mansoni: synthesis and release of phospholipids, lysophospholipids, and neutral lipids by schistosomula

Lipids in the two surface membranes of Schistosoma mansoni may play an important role in the parasite's defense against host immunity. In particular, lysophosphatidylcholine lyses erythrocytes attached to the parasite and alters the lateral mobilities of their membrane proteins and lipids (Golan et al. 1986). Here, we have studied the incorporation of radiolabeled precursors into the major lipid classes of schistosomula as well as into lipids released by schistosomula into the medium. Radiolabeled polar head groups (choline and ethanolamine) and fatty acid precursors (palmitate and oleate) were linearly incorporated into parasite phospholipids. Fatty acids were differentially incorporated into the various phospholipid classes, principally into phosphatidylcholine and, to a lesser extent, into phosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine. The major neutral lipid class labeled, triglycerides, had a decrease in specific activity with time after pulse labeling and the specific activity of the phospholipids increased with time. Thus, triglycerides may provide acyl chains for phospholipid synthesis. Choline was incorporated into phosphatidylcholine and lysophosphatidylcholine, and ethanolamine into phosphatidylethanolamine and lysophosphatidylethanolamine. No evidence was found for phospholipid methylation or demethylation in schistosomula. Labeled lipids were linearly and selectively released into the medium. Triglycerides were released at the highest rate with measurable quantities of phosphatidylcholine, lysophosphatidylcholine, and phosphatidylethanolamine also observed. Monopalmitoylphosphatidylcholine was the only lysophosphatidylcholine present in the medium as demonstrated by reverse-phase chromatography of released choline-labeled lysophosphatidylcholine. These studies demonstrate that schistosomula synthesize phospholipids and neutral lipids and release some of them into the culture medium. In particular, they release a single molecular species of a potent biologically active molecule, monopalmitoylphosphatidylcholine, that may play a role in the parasite's evasion of the immune response.     

Phospholipid metabolism in V79-R membranes composed of phospholipid molecular species containing trans-monoenoic fatty acids

The interrelationship between the inhibition of cell growth and changes in phospholipid molecular species was studied in the presence of elaidic, trans-11-eicosenoic, or brassidic acids in Chinese hamster V79-R cells. The addition of trans-monoenoic fatty acids to the medium inhibited cell growth and caused an increase in the total cellular content of phospholipids. However, there was no difference in the polar head group composition of these phospholipids among all the cells supplemented with trans-monoenoic fatty acids. Exogenous trans-monoenoic fatty acids were incorporated into cellular phospholipids to form novel phospholipid molecular species. Phospholipid synthesizing enzyme activities bound to the membranes composed of phospholipid molecular species of trans-monoenoic fatty acids were determined. Cholinephosphotransferase [EC 2.7.8.2] and ethanolaminephosphotransferase [EC 2.7.8.1] activities were decreased by trans-11-eicosenoic acid, but not changed by elaidic acid. Glycerophosphate acyltransferase [EC 2.3.1.15] activity was increased by elaidic acid and decreased by trans-11-eicosenoic acid. Cholinephosphate cytidylyltransferase [EC 2.7.7.15] activity was not changed by trans-monoenoic fatty acids.     

Formation of molecular species of mitochondrial cardiolipin: 2. A mathematical model of pattern formation by phospholipid transacylation

Formation of the unique molecular species of mitochondrial cardiolipin requires tafazzin, a transacylase that exchanges acyl groups between phospholipid molecular species without strict specificity for acyl groups, head groups, or carbon positions. However, it is not known whether phospholipid transacylations can cause the accumulation of specific fatty acids in cardiolipin. Here, a model is shown in linear algebra representation, in which acyl specificity emerges from the transacylation equilibrium of multiple molecular species, provided that different species have different free energies. The model defines the conditions and energy terms, under which transacylations may generate the characteristic composition of mitochondrial cardiolipin. It is concluded that acyl-specific cardiolipin patterns could arise from phospholipid transacylations in the tafazzin domain, even if tafazzin itself does not have substrate specificity.     

Effect of phospholipid composition on activity of sodium-dependent leucine transport system in Pseudomonas aeruginosa

The sodium-dependent leucine transport system of Pseudomonas aeruginosa was reconstituted into liposomes of specific polar head group composition. Na+-dependent counterflow and Na+ gradient-driven transport were measured as reconstituted transport activities. Proteoliposomes containing phosphatidylethanolamine exhibited increased transport activities. Phosphatidylglycerol, second to phosphatidylethanolamine, also enhanced the reconstituted transport activities. Proteoliposomes composed of phosphatidylcholine did not accumulate leucine. The enhanced transport activity by phosphatidylethanolamine was significantly influenced by its fatty acid composition. Dioleoylphosphatidylethanolamine was more effective in stimulating counterflow activity than dilauroylphosphatidylethanolamine. These results show that the leucine transport system of P. aeruginosa is sensitive to not only the polar head group composition but also the acyl group composition of phospholipids.     

Anthrylvinyl-labeled phospholipids as fluorescent membrane probes. The action of melittin on mulitlipid systems

The interaction of melittin with multicomponent lipid mixtures composed of phosphatidylcholine, sphingomyelin and phosphatidylserine or phosphatidylglycerol was investigated by measuring the intrinsic fluorescence of the peptide, steady state fluorescence anisotropy of, and Trp-fluorescence energy transfer to fluorescent analogs of the same phospholipids bearing the anthrylvinyl fluorophore in one of the aliphatic chains at various distances from the polar head group. Based on the finding that at high lipid/peptide ratio the peptide induces unequal changes in the fluorescence parameters of phospholipid probes differing structurally only in their polar head groups, it is concluded that melittin induces lipid demixing in its nearest environment. Comparison of the fluorescence energy transfer from Trp to different lipid probes indicates that the depth of penetration of melittin into the bilayer depends on the polar head group composition of the phospholipid matrix and that certain segments of the melittin chain display a specific affinity for a given lipid head group.     

Acyl chain and head group regulation of phospholipid catabolism in senescing carnation flowers

Microsomal membranes from the petals of senescing carnation (Dianthus caryophyllus L.) flowers contain phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol. These phospholipid classes decline essentially in parallel during natural senescence of the flower and when microsomal membranes isolated from young flowers are aged in vitro. However, measurements of changes in the endogenous molecular species composition of microsomal phospholipids during natural senescence of the flower petals and during in vitro aging of isolated membranes have indicated that the various molecular species of phospholipids have quite different susceptibilities to catabolism. Acyl chain composition and the nature of the head group are both determinants of their susceptibility to catabolism. As well, a comparison of the phospholipid catabolism data for naturally senesced membranes and for membranes aged in vitro suggests that the phospholipid composition of membranes is continuously altered during senescence by acyl chain desaturation and possibly retailoring so as to generate molecular species that are more prone to catabolism. The results collectively indicate that provision of particular molecular species of phospholipids with increased susceptibility to degradation contributes to enhanced phospholipid catabolism in the senescing carnation petal.     

Differential turnover of polyunsaturated fatty acids in plasmalogen and diacyl glycerophospholipids of isolated cardiac myocytes

To investigate the relative turnover of esterified polyunsaturated fatty acids in diacylglycerophospholipids and plasmalogens in isolated cardiac myocytes, we characterized the phospholipid composition and distribution of radiolabel in different phospholipid classes and in individual molecular species of diradyl choline (CGP) and ethanolamine (EGP) glycerophospholipids after incubation of isolated cardiac myocytes with [3H]arachidonate or [14C]linoleate. Plasmalogens in CGP (55%) and EGP (42%) quantitatively accounted for the total plasmalogen content (39%) of cardiac myocyte phospholipids. Plasmalogens comprised 86% and 51% of total arachidonylated CGP and EGP mass, respectively, and [3H]arachidonate was primarily incorporated into plasmalogens in both CGP (65%) and EGP (61%) classes. The specificity activity of [3H]arachidonylated diacyl-CGP was approximately 2- to 5-fold greater than that of [3H]arachidonylated choline plasmalogen, whereas comparable specific activities were found in the [3H]arachidonate-labeled ethanolamine plasmalogen and diacyl-EGP pools. Of the total linoleate-containing CGP and EGP mass, 54% and 57%, respectively, was esterified to plasmalogen molecular species. However, [14C]linoleate was almost exclusively incorporated into diacyl-CGP (96%) and diacyl-EGP (86%). The specific activities of [14C]linoleate-labeled diacyl-CGP and diacyl-EGP were 5- to 20-fold greater than that of the [14C]linoleate-labeled plasmalogen pools. The differential incorporation of polyunsaturated fatty acids in plasmalogens and diacylglycerophospholipids demonstrates that the metabolism of the sn-2 fatty acyl moiety in these phospholipid subclasses is differentially regulated, possibly fulfilling separate and distinct physiologic roles.     

Turnover of phospholipid fatty acyl chains in cultured neuroblastoma cells: involvement of deacylation-reacylation and de novo synthesis in plasma membranes

Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.     

Activation of D-beta-hydroxybutyrate apodehydrogenase using molecular species of mixed fatty acyl phospholipids

D-beta-Hydroxybutyrate apodehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for enzymatic function. The purified enzyme which is devoid of lipid can be reactivated with lecithin or mixtures of natural phospholipid-containing lecithin. However, it is mitochondrial phospholipid which activates the enzyme optimally and with kinetic parameters similar to that of the native membrane-bound enzyme. Mitochondrial phospholipid consists of three classes of phospholipid (lecithin:phosphatidylethanolamine:diphosphatidylglycerol in a ratio of approximately 2:2:1 by phosphorus); each class consists of a multiplicity of different molecular species due to diversity in the fatty acyl substituents. In this study, we have synthesized defined molecular species of mixed fatty acyl phospholipids to evaluate whether multiplicity of phospholipid molecular species are essential for optimal reactivation. We find that: 1) ternary mixtures of single molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylpropan-1,3-diol in the liquid crystalline state mimic the optimal reactivation of the enzyme obtained with mitochondrial phospholipids; 2) although some negatively charged phospholipid appears necessary for optimizing the efficiency of activation, diphosphatidylglycerol can be replaced by phosphatidylpropan-1,3-diol, another negatively charged phospholipid; and 3) biphasic Arrhenius plots can be correlated with the liquid crystalline and gel states of the phospholipid.     

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.     

Head group-independent interaction of phospholipids with bile salts. A fluorescence and EPR study

Bile salts are essential for phospholipid secretion into the bile. To study the relevance of the structure of phospholipids for their interaction with bile salts, we used spin-labeled or fluorescent phospholipid analogues of different head groups and acyl chain length. Those analogues form micelles in aqueous suspension. Their solubilization by bile salts resulting in the formation of mixed micelles was followed by the decrease of spin-spin interaction of spin-labeled analogues or by the relief of fluorescence self-quenching of (7-nitro-2-1,3-benzooxadiazol (NBD))-labeled analogues. Solubilization of analogue micelles occurred at and above the critical micellar concentration (CMC) of the bile salts. As revealed by stopped-flow technique, solubilization of NBD-analogues was very rapid with half times as low as 0.1 sec above the CMC of taurocholate. Both kinetics and extent of solubilization were independent of the phospholipid head group, but were significantly affected by the fatty acid chain length. Furthermore, using vesicles with varying phospholipid composition and different types of analogues in self-quenching concentrations, we could show that bile salt-mediated vesicle solubilization depended on the fatty acid chain length of phospholipids. In contrast, neither for phospholipids nor for analogues could an influence of the lipid head group on the solubilization process be observed. These findings support a head group-independent mechanism of bile salt-mediated enrichment of specific phospholipids in the bile fluid.     

Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes.

Composition and thermotropic phase behavior of sperm membrane lipids from species ranging in sensitivity to cold shock were determined. Lipids from whole sperm and sperm plasma membrane were fractionated into neutral lipid, glycolipid, and phospholipid fractions. Compositional analyses were completed for free sterols, phospholipids and phospholipid-bound fatty acids. Phase transition temperatures were determined for phospholipid and glycolipid fractions using differential scanning calorimetry. Cholesterol was the major sterol in sperm lipids of all species. Cholesterol to phospholipid molar ratios were 0.26, 0.30, 0.36, and 0.45 for sperm plasma membrane of the boar, rooster, stallion, and bull, respectively. Choline and ethanolamine phosphoglycerides and sphingomyelin were the major phospholipid classes in sperm and their proportions differed across species. Phospholipid-bound fatty acyl compositions of choline and ethanolamine phosphoglycerides were characterized by a high proportion of docosapentanoyl and docosahexanoyl groups in mammalian sperm and shorter, more saturated groups in rooster sperm. Glycolipids represented less than 10% of total polar lipids for all species. Thin-layer chromatographic analysis indicated that the major glycolipid component of rooster sperm was different from that of mammalian sperm. Peak phase transition temperatures (Tm) for sperm membrane phospholipids were 24.0, 25.4, 20.7 and 24.5, for the boar, stallion, and rooster, respectively. Corresponding Tm's for glycolipids were 36.2, 42.8, and 33.4 with no exotherm for rooster sperm glycolipids. These results demonstrate a difference in both composition and thermotropic phase behavior of glycolipids between rooster and mammalian sperm which may be related to the greater tolerance of rooster sperm to rapid cooling.     

Modulation of the Activity of Purified Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls by Various Lipids

H⁺-ATPase was solubilized from the tonoplast of mung bean (Vignaradiata L.) hypocotyls and purified by fast protein liquid chromatographyon a Mono Q ion-exchange column. The purified ATPase hardlycontained any phospholipid, but it did contain 10 to 15 moleculesof sterol and 25 to 30 molecules of glycolipid per ATPase molecule,and it had little activity without exogenously added phospholipids.Each individual polar head group, acylglyceride and fatty acidthat constituted a phospholipid was incapable by itself of activatingthe ATPase. Sterols and cerebroside had little activating effect.Maximal activation of ATPase was noted with asolectin or variousmolecular species of phosphatidylcholine (PC) at 0.005% to 0.01%(w/v). The activation by the various molecular species of PCwas dependent on the length and degree of unsaturation of fattyacyl chains. PC with two saturated and long fatty acyl chainsof more than 18 carbon atoms failed entirely to activate theATPase. PC, PS and PG with 1-palmitoyl (16:0)-2-oleoyl(18:1)fatty acyl chains all activated ATPase to nearly the same extentas asolectin, but the activation by PE and PA with the samefatty acyl composition was 52% and 15% of that by asolectin,respectively. The molecular species of PC with phase-transitiontemperatures below 50C activated ATPase, as determined at 38C.The dependence on temperature of the activation by the molecularspecies of PC indicated that the activation of the ATPase beganclose to the temperature of the phase transition of the PC added.These data indicate that phospholipids in the liquid-crystallinephase are essential for the catalytic activity of the ATPase. (Received June 4, 1992; Accepted January 18, 1993)     

The lipid whisker model of the structure of oxidized cell membranes

An essential feature of the innate immune system is maintaining cellular homeostasis by identifying and removing senescent and apoptotic cells and modified lipoproteins. Identification is achieved through the recognition of molecular patterns, including structurally distinct oxidized phospholipids, on target cells by macrophage receptors. Both the structural nature of the molecular patterns recognized and their orientation within membranes has remained elusive. We recently described the membrane conformation of an endogenous oxidized phospholipid ligand for macrophage scavenger receptor CD36, where the truncated oxidized sn-2 fatty acid moiety protrudes into the aqueous phase, rendering it accessible for recognition. Herein we examine the generality of this conformational motif for peroxidized glycerophospholipids within membranes. Our data reveal that the addition of a polar oxygen atom on numerous peroxidized fatty acids reorients the acyl chain, whereby it no longer remains buried within the membrane interior but rather protrudes into the aqueous compartment. Moreover, we show that neither a conformational change in the head group relative to the membrane surface nor the presence of a polar head group is essential for CD36 recognition of free oxidized phospholipid ligands within membranes. Rather, our results suggest the following global phenomenon. As cellular membranes undergo lipid peroxidation, such as during senescence or apoptosis, previously hydrophobic portions of fatty acids will move from the interior of the lipid bilayer to the aqueous exterior. This enables physical contact between pattern recognition receptor and molecular pattern ligand. Cell membranes thus "grow whiskers" as phospholipids undergo peroxidation, and many of their oxidized fatty acids protrude at the surface.     

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

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

Phospholipids of ethambutol-susceptible and resistant strains ofMycobacterium smegmatis

The composition, subcellular distribution and rate of synthesis of phospholipids were compared in ethambutol susceptible and resistant strains ofMycobacterium smegmatis. Significant quantitative alterations in phospholipids accompanied the acquisition of resistance, whereas fatty acyl group composition of total phospholipid remained the same in ethambutol resistant and susceptible strains. Cell wall of resistant strain exhibited an accumulation of phospholipids and a decrease in the degree of unsaturation of phospholipid fatty acyl groups. Changes in the cell wall phospholipid composition may contribute to resistance ofMycobacterium smegmatis to ethambutol.     

Metabolism of unusual membrane phospholipids in the marine sponge Microciona prolifera

Sponges are unique in regard to membrane phospholipid composition. Features virtually without parallel in other organisms are the predominance of the C26-C30 polyenoic acids (demospongic acids) in the phosphatidylethanolamines (PE) and the attachment of identical acyl groups to the glycerol moiety. The biosynthesis and disposition of these unusual phospholipids were followed in the marine sponge Microciona prolifera where PE ( delta 5,9-26:2, delta 5,9-26:2) is a major molecular species. Incorporation experiments with radiolabeled fatty acids, bases, and intact phospholipids revealed the de novo biosynthesis of the two major phosphatides, phosphatidylethanolamines (PE) and phosphatidylcholines (PC), via the cytidine pathway as in higher animals, with ethanolamine selectively incorporated into PE( delta 5,9-26:2, delta 5,9-26:2). Methylation of PE and random acyl chain migration across different phospholipid classes were marginal, but the exchange of PC for PE, apparently mediated by the action of phospholipase, was indicated after uptake of the unnatural PC( delta 9-27:1, delta 9-26:1). The present study demonstrates in the most primitive multicellular animals a phospholipid metabolic pattern similar to that in higher organisms, with unique acyl and phosphoethanolamine transferases apparently involved in the biosynthesis of the (demospongic) di-C26-acyl-PE molecular species.