Position Of Glycerol Research Articles

Identification of unusual butanetriol dialkyl glycerol tetraether and pentanetriol dialkyl glycerol tetraether lipids in marine sediments

Glycerol serves as the principal backbone moiety bound to various acyl/alkyl chains for membrane lipids of Eukarya, Bacteria, and Archaea. In this study, we report a suite of unusual tetraether lipids in which one of the two conventional glycerol backbones is substituted by butanetriol or pentanetriol. Identification of these lipids was achieved via diagnostic fragments and their expected acetylation products using liquid chromatography/mass spectrometry (LC/MS), and their diagnostic ether cleavage products using gas chromatography/mass spectrometry (GC/MS). We observed structural variations in the polyol backbones and alkyl chains and term these core lipid derivatives: isoprenoidal butanetriol dialkyl glycerol tetraethers (iso-BDGTs), isoprenoidal pentanetriol dialkyl glycerol tetraethers (iso-PDGTs), and hybrid isoprenoidal/branched BDGTs and PDGTs (ib-BDGTs, ib-PDGTs). Of these, iso-BDGTs were the most abundant with a methylation at either the sn-1 or sn-3 position of glycerol and were also found as part of intact polar lipids, adjoined to mono- or diglycosidic headgroups. Iso-BDGTs and iso-PDGTs are likely produced by Archaea, as indicated by the presence of the characteristic biphytanyl moieties. Butanetriol- and pentanetriol-based tetraether lipids occur in modern estuarine and deeply buried subseafloor sediments, suggesting the presence of alternative backbones in archaeal lipids.

High resolution 13CNMR detection of short‐ and medium‐chain synthetic triacylglycerols used in butterfat adulteration

Recently in European countries, control laboratories have revealed real cases of deceptive practices in which synthetic triacylglycerols (TAGs) were used for adulterating butterfat. The complex fat blends are difficult to detect with the classical methods of analysis, especially when the adulterating TAGs contain fatty acids (FAs) in the proportions similar to that of genuine butter. Nevertheless, because butyric acid (C4) exclusively occurs at the sn‐3 position in milk fat TAGs, the determination of its distribution on the glycerol backbone provides a diagnostic indicator to differentiate genuine butter from mixtures with synthetic TAGs. Herein, we have demonstrated the straightforward application of mono‐dimensional 13C NMR spectroscopy for detecting synthetic TAGs in mixtures with authentic butterfat. The method is based on the 13C NMR determination of the regioisomeric distribution of C4 between the sn‐1,3‐ and sn‐2‐positions and boasts several analytical advantages, such as specificity, robustness and minimal sample handling. The limit of detection (LOD) and limit of quantification (LOQ) of the synthetic fats, estimated to be 1 and 2.5% w/w, respectively, easily surpass the limit of 10% at which a fraudulent practice might be economically attractive.Practical applications We have developed a robust and efficient method, based on high resolution 13C NMR spectroscopy, for detecting the adulteration of butterfat with synthetic low‐ and medium‐carbon number TAG mixtures. In milk fat TAGs, butyric acid is esterified exclusively at the sn‐3 position of the glycerol backbone. Thus, the method relies on 13C NMR monitoring of the carbonyl resonance of butyric acid at the sn‐2 glycerol position, which is diagnostic of synthetic or interesterified TAGs. The 13C NMR‐based analysis overcomes the pitfalls of traditional pattern recognition methodologies for the assessment of butter authenticity, which can be ineffective when butter is adulterated with synthetic TAGs obtained from FA in proportions similar to those of natural milk fat. The method achieves reliable quantification up to 2.5% w/w of adulterating fats (LOQ) and can detect quantities as low as 1% w/w of synthetic TAGs blended with butter (LOD).

Comparative study of post‐natal retinal vascular development in mice models of iPLA2 inhibition and plasmalogen deficiency

AbstractPurpose Plasmalogens are particular phospholipids characterized by the presence of a polyunsaturated fatty acid (PUFA) at sn‐2 position of glycerol. Plasmalogen deficiency in mouse leads to developmental abnormalities in retinal vasculature. We propose that liberation of PUFA by the specific calcium‐independent phospholipase A2 (iPLA2), is involved in the mechanism by which plasmalogens control retinal vascular development. To confirm this hypothesis, we performed a comparative study of retinal vascular development in a mouse model of retinal iPLA2 inhibition and a model of plasmalogen deficiency.Methods Vessel and astrocyte networks were visualized on flat‐mounted retinas through immunostaining methods.Results Similar abnormalities were observed in retina of both mouse models. They consisted in an increased number of vessel ramifications at PN14, and in an abnormal glial cells migration from the optic nerve, at PN14 and at PN21. An activation of microglial cells was also observed at adult age.Conclusion These results confirm the implication of plasmalogen in the control of retinal vessel development through PUFA release from their sn‐2 position.

Synthesis of CLA‐enriched TAG by Candida antarctica lipase under vacuum

AbstractConjugated linoleic acid (CLA)‐enriched triacylglycerol (TAG) of 90 wt% was successfully synthesized in 10 h by direct esterification of glycerol and CLA using an immobilized lipase from Candida antarctica under vacuum. The best operating conditions for the synthesis of TAG were investigated according to the three parameters of temperature, enzyme loading, and vacuum. The synthesis of TAG increased with increasing temperature but it did not significantly change above 60°C (p>0.05). The increase of enzyme loading lead to an enhanced conversion of TAG, but enzyme loading of more than 10% (based on the total weight of the substrates) was not effective. Moreover, when vacuum increased, the conversion of TAG increased, but the conversion rate decreased when the vacuum level was too high. The best combination of temperature, enzyme loading, and vacuum level were 60°C, 10% of the total weight of the substrates, and 0.4 kPa, respectively. During the initial 6 h of reaction, Candida antarctica lipase had more selectivity for 10t,12c‐CLA than 9c,11t‐CLA onto the glycerol backbone, and a preference for the incorporation at the sn‐1,3 positions of glycerol rather than at the sn‐2 position.

Studies of the Genetics, Function, and Kinetic Mechanism of TagE, the Wall Teichoic Acid Glycosyltransferase in Bacillus subtilis 168

The biosynthetic enzymes involved in wall teichoic acid biogenesis in gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of α-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis.

Determination of lipoprotein lipase activity using a novel fluorescent lipase assay

A novel, real-time, homogeneous fluorogenic lipoprotein lipase (LPL) assay was developed using a commercially available substrate, the EnzChek lipase substrate, which is solubilized in Zwittergent. The triglyceride analog substrate does not fluoresce, owing to apposition of fluorescent and fluorescent quenching groups at the sn-1 and sn-2 positions, respectively, fluorescence becoming unquenched upon release of the sn-1 BODIPY FA derivative following hydrolysis. Increase in fluorescence intensity at 37°C was proportional to LPL concentration. The assay was more sensitive than a similar assay using 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin ester) and was validated in biological samples, including determination of LPL-specific activity in postheparin mouse plasma. The simplicity and reproducibility of the assay make it ideal for in vitro, high-throughput screening for inhibitors and activators of LPL, thus expediting discovery of drugs of potential clinical value.

Regiospecific Analyses of Triacylglycerols of Hoki (Macruronus novaezelandiae) and Greenshell™ Mussel (Perna canaliculus)

AbstractThe lipid profiles of the two most important New Zealand marine oil sources were investigated, with particular attention to the regioisomeric compositions of triacylglycerides (TAG), using 13C‐nuclear magnetic resonance analysis. Oils from hoki (Macruronus novaezelandiae) and Greenshell™ mussel (Perna canaliculus) (GSM) were analyzed for their lipid content, lipid class and fatty acid profile. The regiospecific distribution of long chain (C ≥ 20) polyunsaturated fatty acids (LC‐PUFA) between the sn‐1,3 and sn‐2 glycerol positions was calculated from 13C responses in the carbonyl region in the triacylglycerol fraction. Rendered hoki oil (RHO) produced from the viscera and filleting discards, had a similar lipid profile to that of hoki liver oil (HLO) confirming that the liver is the major source of oil in RHO. The regioisomeric distribution of fatty acids showed differences between the two oil sources. Docosahexaenoic acid (DHA) had a regioisomeric distributional preference to the sn‐2 position in TAG from all the oils (59.2% HLO, 54.3% RHO and 63.4% GSM). Eicosapentaenoic acid (EPA) had a more even distribution along the triacylglycerol backbone in hoki TAG (29.1% HLO, 33.6% RHO) while there was a slight sn‐2 positional preference in the GSM TAG (37.6%). This regioisomeric information is vital to distinguish LC‐PUFA‐rich marine oils from other marine sources for authentication purposes.

ChemInform Abstract: Synthesis and Antineoplastic Properties of Ether-Linked Thioglycolipids.

Ether-linked glycero-alpha- and beta-D-glucopyranosides and glycero-1-thio-alpha- and beta-D-glucopyranosides have been synthesized by modifications of the Konigs-Knorr procedure, and their antitumor activities have been evaluated. The bioactivities of these compounds have been evaluated in five different cell lines (WEHI 3B, C653, X63/OMIL3, R6X-B15, and HL-60) and compared with the activities of 1-O-hexadecyl-2-O-methyl-sn-3-glycerophosphocholine (GPC) and its enantiomer, 3-O-hexadecyl-2-O-methyl-sn-1-GPC. The results indicate that a alpha-D-thioglucopyranoside [1-O-hexadecyl-2-O-methyl-3-S-(alpha-D-1'- thioglucopyranosyl-sn-glycerol)] is selective with respect to its action on target cells, with high activity for killing of WEHI 3B and C653 cells as determined by inhibition of [3H]thymidine incorporation into DNA and HL-60 cell cytotoxicity, but unable to induce aggregation of rabbit platelets at 10(-5) M. The corresponding beta-linked thioglycolipid was ineffective with respect to cytotoxicity against each cell line tested, indicating the importance of configuration at the anomeric position; the beta-thioglycoside was also ineffective with respect to inducing platelet aggregation. 1-O-Hexadecyl-2-O-methyl-sn-3-GPC and 3-O-hexadecyl-2-O-methyl-sn-1-GPC were potent inhibitors of growth of each cell line tested but also caused rabbit platelet aggregation at concentrations greater than or equal to 10(-7) M. Thus, 3-S-(alpha-thioglycopyranosyl)-sn- glycerols bearing a long-chain O-alkyl group at the sn-1 position and a methoxy group at the sn-2 position of glycerol appear to be a promising class of antineoplastic agents with lower risk of inducing thrombosis than the widely studied platelet activating factor analogue, 1-O-octadecyl-2-O-methyl-rac-3-GPC.

A DFT investigation of methanolysis and hydrolysis of triacetin

The thermodynamic and kinetic aspects of the methanolysis and hydrolysis reactions of glycerol triacetate or triacetin, a model triacylglycerol compound, were investigated by using Density Functional Theory (DFT) at the B3LYP/6-31++G(d,p) level of calculation. Twelve elementary steps of triacetin methanolysis were studied under acid-catalyzed and base-catalyzed conditions. The mechanism of acid-catalyzed methanolysis reaction which has not been reported yet for any esters was proposed. The effects of substitution, methanolysis/hydrolysis position, solvent and face of nucleophilic attack on the free energy of reaction and activation energy were examined. The prediction confirmed the facile position at the middle position of glycerol observed by NMR techniques. The calculated activation energy and the trends of those factors agree with existing experimental observations in biodiesel production.

Chemoenzymatic synthesis of symmetrically structured triacylglycerols possessing short-chain fatty acids

Synthesis of symmetrically structured triacylglycerols possessing bioactive n−3 polyunsaturated fatty acids (eicosapentaenoic acid or docosahexaenoic acid) at the 2-position and a short-chain fatty acid (C 2, C 4, C 6) located at the end-positions by a highly efficient two-step chemoenzymatic process is described. Full regiocontrol devoid of any acyl-migration side reactions was obtained in both a lipase promoted step to introduce the short-chain fatty acids exclusively into the primary alcohol positions of glycerol using activated vinyl esters at low temperature and a subsequent coupling reaction involving free EPA and DHA using EDAC as a coupling agent.

Activation of the Chloroplast Monogalactosyldiacylglycerol Synthase MGD1 by Phosphatidic Acid and Phosphatidylglycerol

One of the major characteristics of chloroplast membranes is their enrichment in galactoglycerolipids, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG), whereas phospholipids are poorly represented, mainly as phosphatidylglycerol (PG). All these lipids are synthesized in the chloroplast envelope, but galactolipid synthesis is also partially dependent on phospholipid synthesis localized in non-plastidial membranes. MGDG synthesis was previously shown essential for chloroplast development. In this report, we analyze the regulation of MGDG synthesis by phosphatidic acid (PA), which is a general precursor in the synthesis of all glycerolipids and is also a signaling molecule in plants. We demonstrate that under physiological conditions, MGDG synthesis is not active when the MGDG synthase enzyme is supplied with its substrates only, i.e. diacylglycerol and UDP-gal. In contrast, PA activates the enzyme when supplied. This is shown in leaf homogenates, in the chloroplast envelope, as well as on the recombinant MGDG synthase, MGD1. PG can also activate the enzyme, but comparison of PA and PG effects on MGD1 activity indicates that PA and PG proceed through different mechanisms, which are further differentiated by enzymatic analysis of point-mutated recombinant MGD1s. Activation of MGD1 by PA and PG is proposed as an important mechanism coupling phospholipid and galactolipid syntheses in plants.

13C NMR spectroscopy for determining the acylglycerol positional composition of lampante olive oils. Chemical shift assignments and their dependence on sample concentration

Two Lampante olive oils with free acidity expressed as oleic acid of 25.9 and 8.4 g per 100 g were used to carry out the 13C NMR study for the assignment of all resonances of free fatty acid fraction and of the acylglycerol fraction comprising 1(3)-mono- (1(3)-MAG), 1,3-di- (1,3-DAG), 1,2-di- (1,2-DAG) and triacylglycerols (TAG). The glycerol carbon resonances were found to be resolved according to the esterification degree of the glycerol molecule but no data were available on the length and the unsaturation pattern of the acyl chains. It was the spectral region where the C-1 carboxy carbons resonate that enabled the determination of free fatty acids and of different acylglycerol species. Moreover, within each acylglycerol class, the acyl chain distribution between the 1,3- and 2- glycerol positions was also calculated where the saturated, vaccenate, oleate and linoleate chains were detected as baseline resolved resonances in 1(3)-MAG, 1,3-DAG, 1,2-DAG and TAG. The variability of chemical shifts to sample concentration changes was also evidenced in all the frequency ranges being studied where the methylene C-2 and C-3 carbons underwent the most dramatic changes in their resonance patterns which prevented any resonance assignment.

Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT + Bn-fae1 transgenes in rapeseed (Brassica napus L.)

High erucic acid rapeseed (HEAR) oil is of interest for industrial purposes because erucic acid (22:1) and its derivatives are important renewable raw materials for the oleochemical industry. Currently available cultivars contain only about 50% erucic acid in the seed oil. A substantial increase in erucic acid content would significantly reduce processing costs and could increase market prospects of HEAR oil. It has been proposed that erucic acid content in rapeseed is limited because of insufficient fatty acid elongation, lack of insertion of erucic acid into the central sn-2 position of the triaclyglycerol backbone and due to competitive desaturation of the precursor oleic acid (18:1) to linoleic acid (18:2). The objective of the present study was to increase erucic content of HEAR winter rapeseed through over expression of the rapeseed fatty acid elongase gene (fae1) in combination with expression of the lysophosphatidic acid acyltransferase gene from Limnanthes douglasii (Ld-LPAAT), which enables insertion of erucic acid into the sn-2 glycerol position. Furthermore, mutant alleles for low contents of polyunsaturated fatty acids (18:2 + 18:3) were combined with the transgenic material. Selected transgenic lines showed up to 63% erucic acid in the seed oil in comparison to a mean of 54% erucic acid of segregating non-transgenic HEAR plants. Amongst 220 F(2) plants derived from the cross between a transgenic HEAR line and a non-transgenic HEAR line with a low content of polyunsaturated fatty acids, recombinant F(2) plants were identified with an erucic acid content of up to 72% and a polyunsaturated fatty acid content as low as 6%. Regression analysis revealed that a reduction of 10% in polyunsaturated fatty acids content led to a 6.5% increase in erucic acid content. Results from selected F(2) plants were confirmed in the next generation by analysing F(4) seeds harvested from five F(3) plants per selected F(2) plant. F(3) lines contained up to 72% erucic acid and as little as 4% polyunsaturated fatty acids content in the seed oil. The 72% erucic acid content of rapeseed oil achieved in the present study represents a major breakthrough in breeding high erucic acid rapeseed.

Toll-like receptor 2-mediated dendritic cell activation by a Porphyromonas gingivalis synthetic lipopeptide

A PG1828 gene-encoded triacylated lipoprotein was previously isolated from a Porphyromonas gingivalis lipopolysaccharide preparation as a Toll-like receptor (TLR) 2 agonist and its lipopeptide derivatives were synthesized based on the chemical structure. In the present study, granulocyte-macrophage colony stimulating factor-differentiated bone marrow-derived dendritic cells (BMDDCs) were stimulated separately with the P. gingivalis synthetic lipopeptide N-palmitoyl-S-[2-pentadecanoyloxy, 3-palmitoyloxy-(2R)-propyl]-l-Cys-Asn-Ser-Gln-Ala-Lys (PGTP2-RL) and its glyceryl stereoisomer (PGTP2-SL). Only PGTP2-RL activated BMDDCs from wild-type mice to secrete tumour necrosis factor-alpha, interleukin (IL)-6, IL-10 and IL-12p40, whilst PGTP2-RL-induced cytokine production was eliminated in TLR2 knockout (-/-) BMDDCs. BMDDCs from wild-type mice but not TLR2-/- mice responded to PGTP2-RL as well as Pam(3)CSK(4) by increasing the expression of maturation markers, including CD80 (B7-1), CD86 (B7-2), CD40, CD275 (B7RP-1/inducible T-cell co-stimulatory ligand) and major histocompatibility complex class II. Taken together, these results indicate that the fatty acid residue at the glycerol position in the P. gingivalis lipopeptide plays a pivotal role in TLR2-mediated dendritic cell activation.

MS/MS Approach for Characterization of the Fatty Acid Distribution on Mycobacterial Phosphatidyl-myo-inositol Mannosides

Phosphatidyl-myo-inositol mannosides (PIM) are not only important structural components of the mycobacterial envelope but also are major non-peptidic antigens of the host innate and acquired immune responses. Indeed, they are ligands of TLR-2 and they activate CD1-restricted T lymphocytes. In addition, PIM constitute the basic structure of the lipidic anchor of two lipoglycans, lipomannans and lipoarabinomannans, which are important immunomodulators in the course of tuberculosis. The fatty acyl substituents present on PIM molecules play a crucial role for both their physical properties and biological activities. PIM contain four acylation sites, two on the glycerol, one on a mannose, and one on the myo-inositol units. We propose here an analytical procedure, based on mass spectrometry, to determine the structure of the fatty acids present on each of these different acylation sites. We show that the nature of the fatty acids located on both positions of glycerol can be deduced from IRMPD analysis of negative precursor ions from native PIM species, while the fatty acids located on myo-inositol and mannose units can be identified by MALDI-TOF CID MS of protonated and cationized molecular ions. Thus, the combination of MS/MS data obtained from positive and negative pseudomolecular ions generated by ESI or MALDI appears as a powerful approach for the structural characterization of the PIM acyl form structure.

Correlation between chemical structure and biological activities of Porphyromonas gingivalis synthetic lipopeptide derivatives

We recently separated a PG1828-encoded triacylated lipoprotein (Pg-LP), composed of two palmitoyl and one pentadecanoyl groups at the N-terminal of glycerocysteine from Porphyromonas gingivalis, a periodontopathic bacteria, and found that Pg-LP exhibited definite biological activities through Toll-like receptor (TLR) 2. In the present study, we synthesized 12 different Pg-LP N-terminal peptide moieties (PGTP) using four combinations of glyceryl (R and S) and cysteinyl (l and d) stereoisomers, and three different acyl group regioisomers, N-pentadecanoyl derivative (PGTP1), S-glycero 2-pentadecanoyl derivative (PGTP2) and S-glycero 3-pentadecanoyl derivative (PGTP3). All the PGTP compounds (RL, SL, SD, RD) tested showed TLR2-dependent cell activation. The activating capacities of the PGTP-R compounds were more potent than those of the PGTP-S compounds, whereas there were no differences between the PGTP-L and -D compounds. Furthermore, the production of interleukin (IL)-6 following stimulation with the PGTP1-RL, PGTP2-RL and PGTP3-RL compounds was impaired in peritoneal macrophages from TLR2 knock-out (KO), but not those from TLR1 KO or TLR6 KO mice. These results suggest that P. gingivalis triacylated lipopeptides are capable of activating host cells in a TLR2-dependent and TLR1-/TLR6-independent manner, and the fatty acid residue at the glycerol position in the PGTP molecule plays an important role in recognition by TLR2.

Letter Regarding Article by Winkler et al, “Platelet-Activating Factor Acetylhydrolase Activity Indicates Angiographic Coronary Artery Disease Independently of Systemic Inflammation and Other Risk Factors: The Ludwigshafen Risk and Cardiovascular Health Study”

To the Editor: Winkler and colleagues1 have published an epidemiological study on the platelet-activating factor acetylhydrolase (PAF-AH) and coronary artery disease. The authors stated erroneously in the Introduction that PAF-AH releases arachidonic acid, a precursor of eicosanoid production including prostaglandins and leukotrienes. On the basis of this assumption, the authors suggest in the Discussion that PAF-AH possesses proinflammatory functions within the atherosclerotic plaque because it is preferentially associated with small dense LDL2; the latter may thus contribute to atherogenesis by functioning as a reservoir of the essential substrate (ie, the arachidonate-containing phosphatidylcholine), and the source of crucial enzyme activity necessary for prostaglandin synthesis. In addition, the authors emphasize that this concept would confer an …

Glycerol Copolyesters: Control of Branching and Molecular Weight Using a Lipase Catalyst

Immobilized Lipase B from Candida antartica (Novozyme 435) catalyzed bulk polycondensations at 70 °C for 42 h that resulted in hyperbranched polyesters with octanediol-adipate and glycerol-adipate repeat units. Instead of using organic solvents, the monomers adipic acid (A2), 1,8-octanediol (B2), and glycerol (B‘B2) were combined to form monophasic ternary mixtures. During the first 18 h of a copolymerization with monomer feed ratio (A2 to B2 to B‘B2) 1.0:0.8:0.2 mol/mol, the regioselectivity of Novozyme 435 resulted in linear copolyesters. Extending the reaction to 42 h gave hyperbranched copolymers with dendritic glycerol units. The % regioselectivity for esterifications at the primary glycerol positions ranged from 77 to 82% and was independent of glycerol content in the monomer feed. Variation of glycerol in the monomer feed gave copolymers with degree of branching (DB) from 9 to 58%. In one example, a hyperbranched copolyester with 18 mol % glycerol−adipate units was formed in 90% yield, with Mw 75 6...

Structural characterization of cardiolipin by tandem quadrupole and multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization

We report negative-ion electrospray tandem mass spectrometric methods for structural characterization of cardiolipin (CL), a four-acyl-chain phospholipid containing two distinct phosphatidyl moieties, of which structural assignment of the fatty acid residues attached to the glycerol backbones performed by low-energy CAD tandem mass spectrometry has not been previously described. The low-energy MS2-spectra of the [M - H]- and [M - 2H]2- ions obtained with ion-trap or with tandem quadrupole instrument combined with ion-trap MS3-spectra or with source CAD product-ion spectra provide complete structural information for CL characterization. The MS2-spectra of the [M - H]- ions contain two sets of prominent fragment ions that comprise a phosphatidic acid, a dehydrated phosphatidylglycerol, and a (phosphatidic acid + 136) anion. The substantial differences in the abundances of the two distinct phosphatidic anions observed in the MS2-spectra of the [M -H]- ions lead to the assignment of the phosphatidyl moieties attached to the 1' or 3' position of central glycerol. Upon further collisional dissociation, the MS3-spectra of the phosphatidic anions provide information to identify the fatty acyl substituents and their position in the glycerol backbone. The MS2-spectra of the [M - 2H]2- ions obtained with TSQ or ITMS contain complementary information to confirm structural assignment. The applications of the above methods in the differentiation of cardiolipin isomers and in the identification of complex cardiolipin species consisting of multiple molecular structures are also demonstrated.

Isolation of a Gene Encoding a 1,2-Diacylglycerol-sn-acetyl-CoA Acetyltransferase from Developing Seeds of Euonymus alatus

1,2-Diacyl-3-acetyl-sn-glycerols (ac-TAG) are unusual triacylglycerols that constitute the major storage lipid in the seeds of Euonymus alatus (Burning Bush). These ac-TAGs have long-chain acyl groups esterified at both the sn-1 and sn-2 positions of glycerol. Cell-free extracts of developing seeds of E. alatus contain both long-chain acyl-CoA and acetyl-CoA sn-1,2-diacylglycerol acyltransferase (DGAT) activity. We have isolated a gene from developing seeds of Euonymus alatus that shows a very high sequence similarity to the members of the DGAT1 gene family (i.e. related to acyl-CoA:cholesterol acyltransferases). This Euonymus DGAT1 gene, when expressed in wild type yeast, results in a 5-fold enhancement of long-chain triacylglycerol (lc-TAG) accumulation, as well as the appearance of low levels of ac-TAG. Hydrogenated ac-TAG molecular species were identified by gas chromatography-mass spectrometry. Microsomes isolated from this transformed yeast show diacylglycerol:acetyl-CoA acetyltransferase activity, which is about 40-fold higher than that measured in microsomes prepared from yeast transformed with the empty vector or with the Arabidopsis thaliana DGAT1 gene. The specific activity of this microsomal acetyltransferase activity is of the same order of magnitude as the microsomal long-chain DGAT activities measured for yeast lines transformed with the empty vector or either the Arabidopsis or Euonymus DGAT1 genes. Despite this, ac-TAG accumulation in yeast transformed with the Euonymus DGAT1 gene was very low (0.26% of lc-TAG), whereas lc-TAG accumulation was enhanced. Possible reasons for this anomaly are discussed. Expression of the Euonymus DGAT1-like gene in yeast lines where endogenous TAG synthesis has been deleted confirmed that the gene product has both long-chain acyl- and acetyltransferase activity.