Unique molecular species of phosphatidylcholine containing very-long-chain (C24-C38) polyenoic fatty acids in rat brain.

Rat brain has been shown to contain polyenoic very-long-chain fatty acids (VLCFA) belonging to the n-3 and n-6 series with four, five and six double bonds and even-carbon chain lengths from 24 to 38. These fatty acids are almost exclusively located in unusual molecular species of phosphatidylcholine at the sn-1 position of the glycerol backbone, whereas saturated, monoenoic and polyenoic fatty acids with less than 24 carbon atoms are present at the sn-2 position. Polyenoic VLCFA phosphatidylcholine in neonatal rat brain is enriched with n-6 pentaenoic and n-3 hexaenoic VLCFA with up to 36 carbon atoms, whereas the corresponding phospholipid in adult rat brain mainly contains n-6 tetraenoic and n-3 pentaenoic VLCFA with up to 38 carbon atoms. The total amount of polyenoic VLCFA associated with phosphatidylcholine is highest in the brain of immature animals. Polyenoic VLCFA phosphatidylcholine appears to be predominantly confined to nervous tissue in rats, and it is envisaged that this phospholipid is of physiological significance.     

Binding of phospholipids to the phosphatidylinositol transfer protein from bovine brain as studied by steady-state and time-resolved fluorescence spectroscopy

The phosphatidylinositol transfer protein isolated from brain, liver, heart and platelets was found to be present in two subforms which could be distinguished on the basis of the isoelectric points. In this study we have demonstrated that the two subforms isolated from bovine brain are due to the presence of either phosphatidylinositol or phosphatidylcholine in the lipid binding site of the protein. The transfer protein accommodates one phosphatidylinositol molecule in the binding site. The binding site for the sn-2 fatty acyl chain was investigated by incorporating in the transfer protein either phosphatidylinositol or phosphatidylcholine carrying a parinaroyl-chain attached at the sn-2 position. Time-resolved fluorescence spectroscopy revealed that the sn-2 fatty acyl chains for both phospholipids in the lipid-protein complex were completely immobilized (i.e., rotational correlation times of 17.4 ns for phosphatidylcholine and 16.3 ns for phosphatidylinositol). The similarity in correlation times suggests that the sn-2 fatty acyl chains of both phospholipids are accommodated in the same hydrophobic binding site of the protein.     

The lipid binding site of the phosphatidylcholine transfer protein from bovine liver

The phosphatidylcholine transfer protein (PC-TP) from bovine liver has a binding site for phosphatidylcholine (PC). Structural and molecular characteristics of this site were investigated by binding PC-analogues carrying photolabile, fluorescent and short-chain fatty acids. Analysis of the photolabeled PC/PC-TP adduct showed that the hydrophobic peptide segment Val171-Phe-Met-Tyr-Tyr-Phe-Asp177 is part of the lipid binding site for the 2-acyl chain. This site was further studied by binding PC carrying cis-parinaric acid at the sn-2-position. Time resolved fluorescence anisotropy measurements indicated that the 2-acyl chain was immobilized following the rotation of PC-TP. Similar experiments with PC carrying cis-parinaric acid at the sn-1-position demonstrated that the 1-acyl chain was immobilized as well but at a site distinctly different from that of the 2-acyl chain. Binding sites for the 1- and 2-acyl chain were then explored by use of PC-isomers carrying decanoic, lauric and myristic acid at the sn-1- (or sn-2-)-position and oleic acid at the sn-2- (or sn-1-)-position. Incubation with vesicles prepared of these PC-species indicated that binding to PC-TP diminished with decreasing acyl chain length but more so for species with short-chain fatty acids on the sn-2-position than on the sn-1-position. Transfer experiments confirmed that PC-TP discriminates between PC-isomers of apparently equal hydrophobicity favouring the transfer of these species carrying oleic acid at the sn-2-position.     

Utilization of different fatty acids for hepatic and biliary phosphatidylcholine formation and the effect of changes in phosphatidylcholine molecular species on biliary lipid secretion

Biliary cholesterol secretion is ordinarily tightly coupled to phosphatidylcholine (PC) secretion. Bile PCs are distinct in composition and predominantly composed of molecular species with 16:0 in the sn-1 position and 18:2 and 18:1 in the sn-2 position. In an attempt to acutely change the composition of biliary PCs and to assess the effect of a change in PCs on biliary cholesterol secretion, isolated livers were perfused with a variety of single free fatty acids. Rat livers with bile duct cannulas were perfused with a recirculating medium, taurocholate (40 mumol/h), and albumin-bound 16:1, 17:1, 18:1, 20:1, 18:2, 20:4, or 20:5 fatty acids (90 mumol/h) for 2 h. Biliary lipid secretion was measured and bile and liver PC compositions were compared at the start and end of perfusion. Results showed 1) greater utilization of shorter chain than longer chain fatty acids for bile PC formation (16:1 greater than 17:1 greater than 18:2 or 18:1 greater than 20:5, 20:4 or 20:1); 2) no similar pattern of FA utilization for liver PC formation; 3) preferentially greater incorporation of fatty acids into bile PCs compared to liver PCs when perfused fatty acids were used for esterification at both sn-1 and sn-2 positions of PC (to form diunsaturated PCs); and 4) increased biliary secretion of cholesterol relative to PC only when the population of PCs that was newly formed included more hydrophilic molecular species of PC than are present in native bile (that was observed only with perfusion of 16:1). Changes in biliary PC secretion or cholesterol/PC secretion occurred independently of any change in bile salt secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

LPGAT1 controls the stearate/palmitate ratio of phosphatidylethanolamine and phosphatidylcholine in sn-1 specific remodeling

Most mammalian phospholipids contain a saturated fatty acid at the sn-1 carbon atom and an unsaturated fatty acid at the sn-2 carbon atom of the glycerol backbone group. While the sn-2 linked chains undergo extensive remodeling by deacylation and reacylation (Lands cycle), it is not known how the composition of saturated fatty acids is controlled at the sn-1 position. Here, we demonstrate that lysophosphatidylglycerol acyltransferase 1 (LPGAT1) is an sn-1 specific acyltransferase that controls the stearate/palmitate ratio of phosphatidylethanolamine (PE) and phosphatidylcholine. Bacterially expressed murine LPGAT1 transferred saturated acyl-CoAs specifically into the sn-1 position of lysophosphatidylethanolamine (LPE) rather than lysophosphatidylglycerol and preferred stearoyl-CoA over palmitoyl-CoA as the substrate. In addition, genetic ablation of LPGAT1 in mice abolished 1-LPE:stearoyl-CoA acyltransferase activity and caused a shift from stearate to palmitate species in PE, dimethyl-PE, and phosphatidylcholine. Lysophosphatidylglycerol acyltransferase 1 KO mice were leaner and had a shorter life span than their littermate controls. Finally, we show that total lipid synthesis was reduced in isolated hepatocytes of LPGAT1 knockout mice. Thus, we conclude that LPGAT1 is an sn-1 specific LPE acyltransferase that controls the stearate/palmitate homeostasis of PE and the metabolites of the PE methylation pathway and that LPGAT1 plays a central role in the regulation of lipid biosynthesis with implications for body fat content and longevity.     

Properties of the binding sites for the sn-1 and sn-2 acyl chains on the phosphatidylinositol transfer protein from bovine brain

We have studied the properties of the fatty acyl binding sites of the phosphatidylinositol transfer protein (PI-TP) from bovine brain, by measuring the binding and transfer of pyrenylacyl-containing phosphatidylinositol (PyrPI) species and pyrenylacyl-containing phosphatidylcholine (PyrPC) species as a function of the acyl chain length. The PyrPI species carried a pyrene-labeled acyl chain of variable length in the sn-2 position and either palmitic acid [C(16)], palmitoleic acid [C(16:1)], or stearic acid [C(18:1)] in the sn-1 position. Binding and transfer of the PI species increased in the order C(18) less than C(16) less than C(16:1), with a distinct preference for those species that carry a pyrenyloctanoyl [Pyr(8)] or a pyrenyldecanoyl [Pyr(10)] chain. The PyrPC species studied consisted of two sets of positional isomers: one set contained a pyrenylacyl chain of variable length and a C(16) chain, and the other set contained an unlabeled chain of variable length and a Pyr(10) chain. The binding and transfer experiments showed that PI-TP discriminates between positional isomers with a preference for the species with a pyrenylacyl chain in the sn-1 position. This discrimination is interpreted to indicate that separate binding sites exist for the sn-1 and sn-2 acyl chains. From the binding and transfer profiles it is apparent that the binding sites differ in their preference for a particular acyl chain length. The binding and transfer vs chain length profiles were quite similar for C(16)Pyr(x)PC and C(16)Pyr(x)PI species, suggesting that the sn-2 acyl chains of PI and PC share a common binding site in PI-TP.     

Fatty acids of pulmonary surfactant phosphatidylcholine from fetal rabbit lung tissue in culture. Biosynthesis of n-10 monoenoic fatty acids

We have previously reported that fetal rabbit lung tissue in organ culture produces a lamellar body material (pulmonary surfactant) with a lower percentage of disaturated phosphatidylcholine than is typically found in rabbit lung in vivo (Longmuir, K.J., C. Resele-Tiden, and L. Sykes. 1985. Biochim. Biophys. Acta. 833: 135-143). This investigation was conducted to identify all fatty acids present in the lamellar body phosphatidylcholine, and to determine whether the low level of disaturated phosphatidylcholine is due to excessive unsaturated fatty acid at position sn-1, sn-2, or both. Fetal rabbit lung tissue, 23 days gestation, was maintained in culture for 7 days in defined (serum-free) medium. Phospholipids were labeled in culture with [1-14C]acetate or [U-14C]glycerol (to follow de novo fatty acid biosynthesis), or with [1-14C]palmitic acid (to follow incorporation of exogenously supplied fatty acid). Radiolabeled fatty acid methyl esters obtained from lamellar body phosphatidylcholine were first separated by reverse-phase thin-layer chromatography (TLC) into two fractions of 1) 14:0 + 16:1 and 2) 16:0 + 18:1. Complete separation of the individual saturated and monoenoic fatty acids was achieved by silver nitrate TLC of the two fractions. Monoenoic fatty acid double bond position was determined by permanganate-periodate oxidation followed by HPLC of the carboxylic acid phenacyl esters. Lamellar body phosphatidylcholine contained four monoenoic fatty acids: 1) palmitoleic acid, 16:1 cis-9; 2) oleic acid, 18:1 cis-9; 3) cis-vaccenic acid, 18:1 cis-11; and 4) 6-hexadecenoic acid, 16:1 cis-6. In addition, 8-octadecenoic acid, 18:1 cis-8, was found in the fatty acids of the tissue homogenate. The abnormally low disaturated phosphatidylcholine content in lamellar body material was the result of abnormally high levels of monoenoic fatty acid (principally 16:1 cis-9) found at position sn-2. Position sn-1 contained normal levels of saturated fatty acid. The biosynthesis of the unusual n-10 fatty acids was observed from the start of culture throughout the entire 7-day culture period, and was observed in incubations of tissue slices of day 23 fetal rabbit lung. This is the first report of the biosynthesis of n-10 fatty acids (16:1 cis-6 and 18:1 cis-8) in a mammalian tissue other than skin, where these fatty acids are found in the secretory product (sebum) of sebaceous glands.     

Studies on furan fatty acids of salmon roe phospholipids

Mature salmon roe lipids were found to consist of triacylglycerols (63%), phospholipids (30%), sterols (4.2%), steryl esters (0.7%), and other minor components. In the steryl esters and phospholipids, furan fatty acids were detected instead of the triacylglycerols of the testes lipids in male fish. The representative 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid (F6) amounted to 3.8% and 0.6% of the total fatty acids in each fraction, respectively. However, the absolute amount of the acid in the phospholipid was much more than that contained in the steryl esters. The characteristic distribution of the furan acids found in the phospholipids was common to the steryl esters in the liver. Large amounts of furan acids were contained in phosphatidylcholine (PC) rather than in phosphatidylethanolamine. For positional analysis of furan fatty acids in PC, furan-containing species in the molecule were concentrated fourteenfold by using selective hydrogenation and repeated silica gel column chromatography. A series of furan fatty acids in PC was found to be exclusively linked to the sn-1 position. The amount of the acids in the roe exclusively linked to the sn-1 position. The amount of the acids in the roe phospholipids was comparable with that in the testes triacylglycerols. The physiological roles of furan fatty acids are discussed.     

A novel Delta9 acyl-lipid desaturase, DesC2, from cyanobacteria acts on fatty acids esterified to the sn-2 position of glycerolipids

Acyl-lipid desaturases are enzymes that convert a C-C single bond into a C=C double bond in fatty acids that are esterified to membrane-bound glycerolipids. Four types of acyl-lipid desaturase, namely DesA, DesB, DesC, and DesD, acting at the Delta12, Delta15, Delta9, and Delta6 positions of fatty acids respectively, have been characterized in cyanobacteria. These enzymes are specific for fatty acids bound to the sn-1 position of glycerolipids. In the present study, we have cloned two putative genes for a Delta9 desaturase, designated desC1 and desC2, from Nostoc species. The desC1 gene is highly similar to the desC gene that encodes a Delta9 desaturase that acts on C18 fatty acids at the sn-1 position. Homologues of desC2 are found in genomes of cyanobacterial species in which Delta9-desaturated fatty acids are esterified to the sn-2 position. Heterologous expression of the desC2 gene in Synechocystis sp. PCC 6803, in which a saturated fatty acid is found at the sn-2 position, revealed that DesC2 could desaturate this fatty acid at the sn-2 position. These results suggest that the desC2 gene is a novel gene for a Delta9 acyl-lipid desaturase that acts on fatty acids esterified to the sn-2 position of glycerolipids.     

Phosphatidyl choline fatty acid remodeling in the hepatic cell nuclei

This study was performed to determine whether fatty acids incorporated into liver cell nuclei phosphatidylcholine (PtdCho) could be remodeled in the isolated nuclear. For this reason, rat liver cell nuclei were incubated in vitro with [1-14C]20:4n-6-CoA. PtdCho molecular species with the highest specific activity had an unsaturated fatty acid at sn-1 and sn-2 positions (20:4-20:4>18:2-20:4>18:1-20:4). 16:0-20:4 and 18:0-20:4 PtdChos showed a minor specific activity. When labeled nuclei were reincubated in the absence of labeled substrate with the addition of cytosol, ATP and CoA, the specific activity of 20:4-20:4, 18:2-20:4 and 18:1-20:4 species decreased, while that of 16:0-20:4 and 18:0-20:4 increased. In conclusion, the asymmetric fatty acid distribution of saturated fatty acids at sn-1 position, and unsaturated fatty acids at sn-2 position of nuclear PtdCho molecular species was re-established by an acyl-CoA-dependent remodeling process.     

Biosynthesis of diacylglycerols by rat intestinal mucosa in vivo.

The biosynthesis of diacylglycerols was studied in rat intestinal mucosa during in vivo absorption of a low molecular weight fraction fraction of butter oil and of the corresponding medium and long chain fatty acids. The experimental fat solutions were given by stomach tube to the animals after a 24-h fast and mucosal scraping were collected 3 h later. The lipids were isolated and the acylclycerols determined by combined thin-layer chromatography gas-liquid chromatography techniques and stereospecific analyses. Free fatty acid feeding led mainly to sn-1,2-diacyl-glycerols, which contained exogenous and endogenous fatty acids. During triacylglycerol feeding, both sn-1,2-and sn-2,3-diacylglycerols were recovered in significant amounts from the intestinal mucosa. The composition of the sn-2,3-diacylglycerols corresponded to that with exogenous fatty acids but the sn-1,2-diacylglycerols clearly contained both exogenous and endogenous fatty acids. In all cases it was possible to isolate endogenous sn-1,2-diacylglycerols made up largely of species with linoleic and arachidonic acids in the 2 position and palmitic and stearic acids in the 1 position, which apparently were not converted to triacylglycerols. The in vivo reacylation of 2-monoacylglycerols via both sn-1,2- and sn-2,3-diacylglycerols is in agreement with similar findings in vitro with everted sacs of rat intestinal mucosa.     

Metabolism of pyrenyl fatty acids in baby hamster kidney fibroblasts. Effect of the acyl chain length.

Biosynthetic labeling of cellular lipids with a fluorescent pyrenyl fatty acid (PyrxFA) moiety was studied in order to assess the usefulness of this approach in the introduction of fluorescent lipid molecules to living cells for transport and metabolic studies. PyrxFAs containing 4-14 aliphatic carbons were added to the culture medium of baby hamster kidney (BHK) fibroblasts, and their incorporation to various lipid species was monitored by thin layer and high pressure liquid chromatography. The results show that the length of the acyl chain has a remarkable effect on the efficiency of incorporation as well as distribution of the label between lipid species. Accordingly, PyrxFAs can be divided into two groups: the short chain ones including the pyrene butyrate and hexanoate derivatives, which show only modest incorporation to phospholipids and negligible labeling of triglycerides and cholesterol esters, and the long chain PyrxFAs including pyrene octanoate, decanoate, dodecanoate, and myristate derivatives, which incorporate efficiently both to phospholipids, mainly phosphatidylcholine (PC) and -ethanolamine (PE), and neutral lipids, i.e. di- and triglycerides and cholesterol esters. Positional analysis showed that the longer PyrxFAs are esterified preferentially to the sn-1 position of the glycerol moiety of PC and PE while the shorter ones are found exclusively in the sn-2 position indicating that the longer PyrxFAs mimic natural saturated fatty acids whereas the shorter ones may be recognized as polyunsaturated fatty acids by the acylating enzymes. Reverse phase chromatography of PC and PE revealed the presence of a variety of labeled molecular species among which the palmitate and oleate containing species were the major ones. Reverse phase analysis with simultaneous monitoring at the monomer and excimer channels showed the presence of tri- and diglyceride species with either 1 or 2 pyrenyl acyl residues. Analysis of the total cellular fatty acids demonstrated that PyrxFAs are shortened, probably by beta-oxidation in peroxisomes, up to pyrene butyrate. It is concluded that metabolic labeling with PyrxFAs is a promising alternative for studies on intracellular lipid traffic, especially because it allows introduction of fluorescent phospholipid species of very different hydrophobicity into intracellular membrane(s).     

Phosphatidylcholine fluidity and structure affect lecithin:cholesterol acyltransferase activity

The purpose of this study was to test the hypothesis that lipid fluidity regulates lecithin:cholesterol acyltransferase (LCAT) activity. Phosphatidylcholine (PC) species were synthesized that varied in fluidity by changing the number, type (cis vs. trans), or position of the double bonds in 18 or 20 carbon sn-2 fatty acyl chains and recombined with [(3)H]cholesterol and apolipoprotein A-I to form recombinant high density lipoprotein (rHDL) substrate particles. The activity of purified human plasma LCAT decreased with PC sn-2 fatty acyl chains containing trans versus cis double bonds and as double bonds were moved towards the methyl terminus of the sn-2 fatty acyl chain. The decrease in LCAT activity was significantly correlated with a decrease in rHDL fluidity (measured by diphenylhexatriene fluorescence polarization) for PC species containing 18 carbon (r(2) = 0.61, n = 18) and 20 carbon (r(2) = 0.93, n = 5) sn-2 fatty acyl chains. rHDL were also made containing 10% of the 18 carbon sn-2 fatty acyl chain PC species and 90% of an inert PC ether matrix (sn-1 18:1, sn-2 16:0 PC ether) to normalize rHDL fluidity. Even though fluidity was similar among the PC ether-containing rHDL, the order of PC reactivity with LCAT was significantly correlated (r(2) = 0.71) with that of 100% PC rHDL containing the same 18 carbon sn-2 fatty acyl chain species, suggesting that PC structure in the active site of LCAT determines reactivity in the absence of measurable differences in bilayer fluidity. We conclude that PC fluidity and structure are major regulators of LCAT activity when fatty acyl chain length is constant.     

Remodeling of rat hepatocyte phospholipids by selective acyl turnover

Acyl turnover of rat hepatocyte phospholipids and triacylglycerols was assessed by incubating the cells in media containing 40% H2(18)O and measuring the time-dependent incorporation of 18O into ester carbonyls by gas chromatography-mass spectrometry of hydrogenated methyl esters. Incorporation of 18O into 22-carbon acyl groups was low in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine, whereas in phosphatidylethanolamine, it was about the same as in the other acyl groups. Incorporation of 18O into individual molecular species of phosphatidylcholine and phosphatidylethanolamine was determined after phospholipase C hydrolysis, derivatization to dinitrobenzoates, and separation by high-performance liquid chromatography. In most molecular species, acyl groups at the sn-1 and sn-2 positions became 18O-labeled at drastically different rates, indicating remodeling through deacylation-reacylation. Molecular species expected to arise de novo from acylation of glycerophosphate exhibited similar rates of 18O incorporation at the sn-1 and sn-2 positions. The data suggest that hepatocyte phospholipids are continually synthesized, remodeled by deacylation-reacylation at specific turnover rates up to 10-15%/h, and degraded. This acyl turnover probably does not involve the majority of intracellular unesterified fatty acids whose 18O incorporation was found to be very low. In contrast, the oxygens of extracellular unesterified fatty acids were readily exchanged with the media. This exchange was enzyme-catalyzed, possibly by lipases released into the media from damaged cells. Incorporation of 18O into exogenously added fatty acids was also rapid and resulted in enhanced uptake of 18O-labeled fatty acids into cellular lipids, primarily triacylglycerols and phosphatidylcholine, without drastic change of the intracellular free fatty acid pool.     

Phospholipid species containing long and very long polyenoic fatty acids remain with rhodopsin after hexane extraction of photoreceptor membranes

About one-fourth the phosphatidylcholines (PCs) from bovine disk photoreceptor membranes contain very long chain (24-36 carbons) polyunsaturated (4, 5, and 6 double bonds) fatty acids of the n-3 and n-6 series (VLCPUFA). Such fatty acids, exclusively occurring in dipolyunsaturated species, are esterified to the sn-1 position of their glycerol backbone, docosahexaenoate being the major fatty acid at sn-2. Chromatographically, such PCs display a weakly polar character relative to other species, ascribable to their exceedingly large number of carbons. After hexane extraction of lyophilized disks, PC is the major component of the fraction of lipids that remains associated with rhodopsin, followed by phosphatidylserine, while a large proportion of the phosphatidylethanolamine is removed. The fatty acid composition of the hexane-removable and protein-bound lipid fractions markedly differs, the latter being enriched in lipid species containing long-chain and very long chain polyenes. This is observed for all lipid classes except free fatty acids. VLCPUFA-containing PCs are the most highly concentrated species in the rhodopsin-associated lipid fraction. The very long chain polyenes these PCs have at sn-1 may account for their resistance to being separated from the protein. It is hypothesized that their unusually long polyenoic fatty acids could be well suited to partially surround alpha-helical segments of rhodopsin.     

Metabolism of exogenous long-chain fatty acids by spinach leaves

When applied in liquid paraffin to the upper surface of expanding spinach leaves, [1-14C]palmitic acid was efficiently and exclusively incorporated into the sn-1 position of cellular glycerolipids, principally phosphatidylcholine and triacylglycerol. A slow transfer of fatty acids from phosphatidylcholine to chloroplast glycolipids subsequently occurred with the positional specificity of the label remaining intact. Labeled palmitate at the sn-1 position of monogalactosyldiacylglycerol was desaturated to hexadecatrienoate so that 1-[14C]hexadecatrienoyl-2-linolenoyl-3-galactosoylglycerol became the major labeled species of the lipid between 8 and 24 h. There was no evidence of deacylation/reacylation reactions modifying the acyl compositions of spinach leaf glycerolipids for at least 48 h after labeling with [1-14C]palmitic acid; even the partially prokaryotic glycerolipids remained firmly labeled at the sn-1 position. Exogenous [1-14C]stearic acid was also incorporated into the sn-1 position of MGD, presumably by the same mechanism, and was there desaturated to [14C]linolenate. Exogenous [1-14C]oleic acid was initially incorporated equally into both sn-1 and sn-2 positions of phosphatidylcholine, and was desaturated to linoleate at both positions before the label was rapidly transferred to monogalactosyldiacylglycerol. There, desaturation of linoleate to linolenate took place. Galactolipids remained equally labeled at both positions throughout the 6 days of the experiment, but label was concentrated in the 1-saturated-2-[14C]linolenoyl molecular species of phosphatidylcholine as those species with two [14C]linoleoyl residues were drawn off for monogalactolipid synthesis. Glycerolipids synthesised from exogenous [1-14C]acetate by spinach leaves were labeled equally at both the sn-1 and the sn-2 positions. These results are interpreted as providing strong support for the two-pathway scheme of glycerolipid synthesis in plants.     

Conjugated fatty acids accumulate to high levels in phospholipids of metabolically engineered soybean and Arabidopsis seeds

Expression of Delta(12)-oleic acid desaturase-related fatty acid conjugases from Calendula officinalis, Momordica charantia, and Vernicia fordii in seeds of soybean (Glycine max) or an Arabidopsis thaliana fad3/fae1 mutant was accompanied by the accumulation of the conjugated fatty acids calendic acid or alpha-eleostearic acid to amounts as high as 20% of the total fatty acids. Conjugated fatty acids, which are synthesized from phosphatidylcholine (PC)-linked substrates, accumulated in PC and phosphatidylethanolamine, and relative amounts of these fatty acids were higher in PC than in triacylglycerol (TAG) in the transgenic seeds. The highest relative amounts of conjugated fatty acids were detected in PC from seeds of soybean and A. thaliana that expressed the C. officinalis and M. charantia conjugases, where they accounted for nearly 25% of the fatty acids of this lipid class. In these seeds, >85% of the conjugated fatty acids in PC were detected in the sn-2 position, and these fatty acids were also enriched in the sn-2 position of TAG. In marked contrast to the transgenic seeds, conjugated fatty acids composed <1.5% of the fatty acids in PC from seeds of five unrelated species that naturally synthesize a variety of conjugated fatty acid isomers, including seeds that accumulate conjugated fatty acids to >80% of the total fatty acids. These results suggest that soybean and A. thaliana seeds are deficient in their metabolic capacity to selectively catalyze the flux of conjugated fatty acids from their site of synthesis on PC to storage in TAG.     

银杏叶片磷脂酰甘油脂肪酸分子种组成及其位置分布

用高效液相色谱法和酶解的方法检测了银杏叶片磷脂酰甘油(PG)脂肪酸的分子种组成和位置分布,确定银杏叶片PG主要分子种的脂肪酸组成(sn-1/sn-2)是18:3／16:1(3t),18:3／16:0,18:2／16:1(3t),18:2／16:0,18:1／16:1(3t),16:0／16:1(3t),18:1／18:1,18:/16：0和16:0和16:0／16:0。银杏叶片PC脂肪酸组成和位置分布的分析结果表明,C18脂肪酸主要位于sn-l位,16:1(3t)只分布于sn-2位,16:0在sn-1位和sn-2位上均有发现。sn-1位上的不饱和度∑u大于sn-2位上的∑u。     

The effect of sn-2 fatty acid substitution on phospholipase C enzyme activities.

The human monocyte cell line U937 expresses phospholipase A2 and phospholipase C activities and produces eicosanoids. The phospholipase C (PLC) activity exhibits substrate preference for phosphatidyl-choline (PC), rather than phosphatidylinositol or phosphatidylethanolamine. In order to characterize the PLC activity found in these cells, the effects of substitution of the sn-2 fatty acid on this activity were examined. PC substrates with palmitic acid (PC-2P), oleic acid (PC-2O), arachidonic acid (PC-2A) and linoleic acid (PC-2L) at the sn-2 position were used. The sn-1 fatty acid was palmitic acid. PC-2L and PC-2A with the longer-chain less-saturated fatty acids linoleic acid and arachidonic acid esterified at sn-2 were found to be better substrates for PLC activity than PC-2P or PC-2O in these cells. This preference was maintained even when substrate phospholipid was solubilized in non-ionic, anionic, cationic and zwitterionic amphiphiles. Furthermore, when a 500-fold excess of 1,2-diolein or 1,2-dipalmitin was added to the reaction, the specificity of the PLC activity for PC-2A and PC-2L remained unchanged. When similar experiments were performed with phosphatidylinositol as a substrate, we did not observe any effect when the sn-2 position was altered. These data show that the fatty acid constituent at the sn-2 position affects the observed PLC activity when phosphatidylcholine, but not phosphatidylinositol, is used as a substrate by these cells.     

Analysis of lipids in the salivary glands of Amblyomma americanum (L.): Detection of a high level of arachidonic acid

Analysis of lipids in salivary glands of the lone star tick, Amblyomma americanum, demonstrated that arachidonic acid (20:4, n-6) comprises 8% of all fatty acids identified by gas chromatography. The occurrence of arachidonic acid and other C₂₀ polyunsaturated fatty acids in tick salivary glands was confirmed by gas chromatography-mass spectrometry. Arachidonate is located entirely in the phospholipid fraction and is associated exclusively with phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Salivary glands stored and frozen for several months had a similar lipid composition as freshly dissected salivary glands, with the exception of a small amount of free arachidonic acid and an increase in lysophosphatidylcholine. Incubation of salivary gland homogenates with snake venom phospholipase A₂ showed that most saturated fatty acids are esterified in the sn-1 position of PC and PE, with the unsaturated fatty acids in the sn-2 position. Approximately 75% of arachidonic acid is in the sn-2 position of PC and PE, adding support to the hypothesis that arachidonic acid is released into the cytoplasm after activation of a phospholipase A₂ for subsequent metabolism to prostaglandins and/or other eicosanoids. © 1993 Wiley-Liss, Inc.