The utilisation of fatty-acid substrates in triacylglycerol biosynthesis by tissue-slices of developing safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) cotyledons

Developing cotyledons of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) readily utilised exogenously supplied ¹⁴C-labelled fatty-acid substrates for the synthesis of triacylglycerols. The other major radioactive lipids were phosphatidylcholine and diacylglycerol. In safflower cotyledons, [¹⁴C]oleate was rapidly transferred to position 2 of sn-phosphatidylcholine and concomitant with this was the appearance of radioactive linoleate. The linoleate was further utilised in the synthesis of diacyl- and triacyl-glycerol via the reactions of the so-called Kennedy pathway. Supplying [¹⁴C]linoleate, however, resulted in a more rapid labelling of the diacylglycerols than from [¹⁴C]oleate. In contrast, sunflower cotyledons readily utilised both labelled acyl substrates for rapid diacylglycerol formation as well as incorporation into position 2 of sn-phosphatidylcholine. In both species, however, [¹⁴C]palmitate largely entered sn-phosphatidylcholine at position 1 during triacylglycerol synthesis. The results support our previous in-vitro observations with isolated microsomal membrane preparations that (i) the entry of oleate into position 2 of sn-phosphatidylcholine, via acyl exchange, for desaturation to linoleate is of major importance in regulating the level of polyunsaturated fatty acids available for triacylglycerol formation and (ii) Palmitate is largely excluded from position 2 of sn-phosphatidylcholine and enters this phospholipid at position 1 probably via the equilibration with diacylglycerol. Specie differences appear to exist between safflower and sunflower in relation to the relative importance of acyl exchange and the interconversion of diacylglycerol with phosphatidylcholine as mechanisms for the entry of oleate into the phospholipid for desaturation.Abbreviations FW fresh weight - TLC thin-layer chromatography     

Oil synthesis in vitro in microsomal membranes from developing cotyledons of Linum usitatissimum L.

Microsomal preparations from developing linseed (Linum usitatissimum L.) cotyledons catalyzed i) acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine, ii) acylation of sn-glycerol 3-phosphate to yield phosphatidic acid, and iii) the utilisation of phosphatidic acid in the production of diacylglycerol and triacylglycerol. Selectivity studies for C₁₈ acyl species of acyl-CoA indicated a bias for the channelling of oleate to phosphatidylcholine for, presumably, its desaturation, and the utilisation of the polyunsaturated fatty-acid products in the acyl-CoA pool for phosphatidic acid and subsequent triacylglycerol synthesis. The microsomal preparations were capable of returning glycerol backbone with associated acyl components to phosphatidylcholine from diacylglycerol where it may be further enriched with polyunsaturated C₁₈ acids by desaturation. The acyl quality in linolenate-rich oilseeds appears to be under similar control to that found in linoleate-rich species. Present address: To whom the correspondence should be addressed     

The regulation of the fatty-acid composition of the triacylglycerols in microsomal preparations from avocado mesocarp and the developing cotyledons of safflower

The utilisation of [¹⁴C]glycerol 3-phosphate and [¹⁴C]linoleoyl-CoA in the synthesis of triacylglycerol has been studied in the microsomal preparations of developing cotyledons of safflower seed. The results confirm that the glycerol backbone, which flows towards triacylglycerol from phosphatidic acid through the Kennedy pathway, can enter phosphatidylcholine from diacylglycerol. The equilibration between diacylglycerol and phosphatidylcholine offers a mechanism for the return of oleate to phosphatidylcholine for desaturation to linoleate. We have established that the oleate entering position 1 of sn-phosphatidylcholine from diacylglycerol is desaturated in situ to linoleate. The results indicate that the diacylglycerol phosphatidylcholine interconvertion coupled to the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C₁₈-polyunsaturated fatty acids and hence these enzymes are of major importance in regulating the acyl quality of the accumulating triacylglycerols. Microsomal preparations from avocado mesocarp, however, did not have detectable acyl exchange between acyl-CoA and phosphatidylcholine or diacylglycerol phosphatidylcholine interconversion despite the high activity of the enzymes of the Kennedy pathway. A scheme is presented which incorporates many of the observations on triacylglycerol synthesis and provides a working model for the regulation of acyl quality in linoleate-rich vegetable oils.Abbreviation BSA bovine serum albumin     

Formation of phospholipids from sn-glycerol-3-phosphate and free fatty acids or their derivatives by homogenates of soybean cotyledons

Cell-free homogenates of soybean cotyledons contain a sn-glycerol-3-phosphate acyltransferase system which incorporated [U-¹⁴C]-sn-glycerol-3-phosphate into 5 labelled lipids when incubated with palmitic acid in the presence of ATP and CoA. In decreasing order of incorporation of label, the lipids were: lysophosphatidic acid, monoacylglycerol, phosphatidic acid, diacylglycerol and triacylglycerol. The substrate specificity of the acyltransferase system was investigated with the fatty acids shown in order of decreasing rates of reaction; palmitate > stearate > oleate > linoleate > linolenate > laurate. Making these acids more soluble as triethanolamine salts or as polyoxyethylene sorbitan esters did not greatly enhance these rates of reaction. Activity was found in a 10000 g pellet containing plastids, mitochondria and glyoxysomes and also in the lipid layer; the activity in these particulate fractions was enhanced by the addition of cytosol which itself had little activity when gentle methods of cell disruption were used. During cotyledon development the total acyltransferase activity increased, although its specific activity slowly declined due to more rapid synthesis of other proteins. During germination total activity decreased but there was a transient increase in specific activity due to more rapid degradation of other proteins.     

Studies on Phospholipid-synthesizing Enzyme Activities during the Growth of Etiolated Cucumber Cotyledons

The enzymatic incorporation of sn-glycerol 3-phosphate into lipid by extracts of cucumber (Cucumis sativus) cotyledons showed an absolute requirement for ATP (saturation 2 mM). The incorporation was stimulated 4-fold by 0.2 mM oleate. Ethyldiaminetetraacetate stimulated the incorporation at concentrations below 1 mM and inhibited at higher concentrations. Mg²⁺ did not affect the reaction. Triton X-100 and Cutscum inhibited the reaction, while a third detergent, Span 80, was stimulatory. p-Mercuribenzoate was inhibitory. The enzymatic reaction has a pH optimum in the range of 8.8 to 9.6. The Michaelis constant was 112 μM for sn-glycerol 3-phosphate. The major amount of product was phosphatidic acid, the remainder was diacylglycerol, monoacylglycerol, and an unknown phospholipid.     

Coupling of ethanol metabolism to lipid biosynthesis: labelling of the glycerol moieties of sn-glycerol-3-phosphate,a phosphatidic acid and a phosphatidylcholine in liver of rats given [1,1-2H2]ethanol

The mechanism behind ethanol-induced fatty liver was investigated by administration of [1,1-²H₂]ethanol to rats and analysis of intermediates in lipid biosynthesis. Phosphatidic acid and phosphatidylcholine were isolated by chromatography on a lipophilic anion exchanger and molecular species were isolated by high-performance liquid chromatography in a non-aqueous system. The glycerol moieties of palmitoyl-linoleoylphosphatidic acid, the corresponding phosphatidylcholine and free sn-glycerol-3-phosphate were analysed by GC/MS of methyl ester t-butyldimethylsilyl derivatives. The deuterium labelling in the glycerol moiety of the phosphatidic acid was 2–3-times higher than in free sn-glycerol-3-phosphate, indicating that a specific pool of sn-glycerol-3-phosphate was used for the synthesis of phosphatidic acid in liver. The results indicate that NADH formed during ethanol oxidation is used in the formation of a pool of sn-glycerol-3-phosphate that gives rise to triacylglycerol and possibly fatty liver.     

Triacylglycerol Bioassembly in Microspore-Derived Embryos of Brassica napus L. cv Reston

Erucic acid (22:1) was chosen as a marker to study triacylglycerol (TAG) biosynthesis in a Brassica napus L. cv Reston microspore-derived (MD) embryo culture system. TAGs accumulating during embryo development exhibited changes in acyl composition similar to those observed in developing zygotic embryos of the same cv, particularly with respect to erucic and eicosenoic acids. However, MD embryos showed a much higher rate of incorporation of ¹⁴C-erucoyl moieties into TAGs in vitro than zygotic embryos. Homogenates of early-late cotyledonary stage MD embryos (14-29 days in culture) were assessed for the ability to incorporate 22:1 and 18:1 (oleoyl) moieties into glycerolipids. In the presence of [1-¹⁴C]22:1-coenzyme A (CoA) and various acyl acceptors, including glycerol-3-phosphate (G-3-P), radiolabeled erucoyl moieties were rapidly incorporated into the TAG fraction, but virtually excluded from other Kennedy Pathway intermediates as well as complex polar lipids. This pattern of erucoyl incorporation was unchanged during time course experiments or upon incubation of homogenates with chemicals known to inhibit Kennedy Pathway enzymes. In marked contrast, parallel experiments conducted using [1-¹⁴C]18:1-CoA and G-3-P indicated that ¹⁴C oleoyl moieties were incorporated into lyso-phosphatidic acids, phosphatidic acids, diacylglycerols, and TAGs of the Kennedy Pathway, as well as other complex polar lipids, such as phosphatidylcholines and phosphatidylethanolamines. When supplied with l-[2-³H(N)]G-3-P and [1-¹⁴C]22:1-CoA, the radiolabeled TAG pool contained both isotopes, indicating G-3-P to be a true acceptor of erucoyl moieties. Radio-high-performance liquid chromatography, argentation thin-layer chromatography/gas chromatography-mass spectrometry, and stereospecific analyses of radiolabeled TAGs indicated that 22:1 was selectively incorporated into the sn-3 position by a highly active diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), while oleoyl moieties were inserted into the sn-1 and sn-2 positions. In the presence of sn-1,2-dierucin and [1-¹⁴C]22:1-CoA, homogenates and microsomal preparations were able to produce radiolabeled trierucin, a TAG not found endogenously in this species. A 105,000g pellet fraction contained 22:1-CoA:DGAT exhibiting the highest specific activity. The rate of 22:1-CoA:DGAT activity in vitro could more than account for the maximal rate of TAG biosynthesis observed in vivo during embryo development. In double label experiments, G-3-P was shown to stimulate the conversion of [³H]phosphatidylcholines to [³H]diacylglycerols, which subsequently acted as acceptors for ¹⁴C erucoyl moieties. In vitro, 22:1 moieties did not enter the sn-1 position of TAGs by a postsynthetic modification or transacylation of preformed TAGs.     

Rapid temperature-induced changes in the fatty acid composition of certain lipids in developing linseed and soya-bean cotyledons.

A change in ambient temperature caused marked alterations, over a 24h period, in the proportions of the unsaturated C18 fatty acids in 3-sn-phosphatidylcholine and 1,2-diacyglycerols during the development of soya-bean and linseed cotyledons. The molar proportion of oleate increased when the temperature was increased whereas that of linoleate or linolenate, depending on the species, increased when the temperature was lowered. Concomitant changes in the composition of 3-sn-phosphatidylethanolamine and triacylglycerols were small.     

The regulation of triacylglycerol biosynthesis in cocoa (Theobroma cacao) L.

Developing cocoa cotyledons accumulate initially an unsaturated oil which is particularly rich in oleate and linoleate. However, as maturation proceeds, the characteristic high stearate levels appear in the storage triacylglycerols. In the early stages of maturation, tissue slices of developing cotyledons (105 days post anthesis, dpa) readily accumulate radioactivity from [¹⁴C]acetate into the diacylglycerols and label predominantly palmitate and oleate. In older tissues (130 dpa), by contrast, the triacylglycerols are extensively labelled and, at the same time, there is an increase in the percentage labelling of stearate. Thus, the synthesis of triacylglycerol and the production of stearate are co-ordinated during development. The relative labelling of the phospholipids (particularly phosphatidylcholine) was rather low at both stages of development which contrasts with oil seeds that accumulate a polyunsaturated oil (e.g. safflower). Microsomal membrane preparations from the developing cotyledons readily utilised an equimolar [¹⁴C]acyl-CoA substrate (consisting of palmitate, stearate and oleate) and glycerol 3-phosphate to form phosphatidate, diacylglycerol and triacylglycerol. Analysis of the [¹⁴C]acyl constituents at the sn-1 and sn-2 positions of phosphatidate and diacylglycerol revealed that the first acylase enzyme (glycerol 3-phosphate acyltransferase) selectively utilised palmitate over stearate and excluded oleate, whereas the second acylase (lysophosphatidate acyltransferase) was highly selective for the unsaturated acyl-CoA. On the other hand, the third acylase (diacylglycerol acyltransferase) exhibited an almost equal selectivity for palmitate and stearate. Thus, stearate is preferentially enriched at position sn-3 of triacylglycerol at 120–130 dpa because of the relatively higher selectivity of the diacylglycerol acyltransferase for this fatty acid compared with those of the other two acylation enzymes.Abbreviation dpa days post anthesis We are grateful to Drs. G. Pettipher (Cadbury-Schweppes, Reading, UK), M. End and P. Hadley (Department of Horticulture, University of Reading) for the supply of cocoa pods and to the Agricultural and Food Research Council for financial support. We also wish to thank Dr. S. Stymne (Swedish University of Agricultural Sciences, Uppsala, Sweden) for a generous gift of acyl-CoA substrates.     

Precursor supply and hepatic enzyme activities as regulators of triacylglycerol synthesis in isolated hepatocytes and perfused liver

The relative significance of alterations in precursor supply and enzyme activities for the rate of triacylglycerol synthesis was studied in isolated hepatocytes and perfused livers. Precursor availability was varied in vitro by changing the fatty acid concentration in the incubation medium or adding ethanol to the perfusion medium in order to increase the cellular glycerol 3-phosphate concentration. The rate of glycerolipid synthesis in hepatocytes, measured in terms of the label incorporated into the various lipid classes from tritiated glycerol, was strongly dependent on the fatty acid concentration up to 2 mm of oleate (fatty acid/albumin molar ratio $\text{7}\text{1}$). Ethanol in vitro increased the incorporation of labeled oleate into phosphatidic acid and diacylglycerol in the isolated perfused liver, but its effect on the incorporation into triacylglycerol was small. Ethanol in vitro increased the label incorporation into both diacylglycerol and triacylglycerol in the livers from cortisol-treated rats. Although cortisol treatment increased the soluble phosphatidate phosphohydrolase activity 4.4-fold in the hepatocytes, it had no effect on the rate of triacylglycerol synthesis, whereas fasting increased this rate about 3-fold, although only a moderate concomitant increase in soluble phosphatidate phosphohydrolase activity was observed. Neither cortisol treatment nor fasting affected the microsomal glycerol-3-phoshate acyltransferase activity. The results demonstrate that substrate availability can override enzyme modulations in the regulation of triacylglycerol synthesis and that phosphatidate phosphohydrolase is not the main regulator of triacylglycerol synthesis.     

Changes in Soybean (Glycine max [L.] Merr.) Glycerolipids in Response to Water Stress

Soybean (Glycine max [L.] Merr.) plants with the first trifoliate leaf fully expanded were exposed to 4 and 8 days of water stress. Leaf water potentials dropped from −0.6 megapascal to −1.7 megapascals after 4 days of stress; then to −3.1 megapascals after 8 days without water. All of the plants recovered when rewatered. The effects of short-term drought stress on triacylglycerol, diacylglycerol, phospholipid, and galactolipid metabolism in the first trifoliate leaves was determined. Leaf triacylglycerol and diacylglycerol content increased 2-fold during the first 4 days of stress and returned to control levels 3 days after rewatering. The polar lipid fraction, which contained phospholipids and galactolipids, changed little during this time. The linolenic acid (18:3) content of the triacylglycerol and diacylglycerol increased 25% during stress and the polar lipid 18:3 content decreased 15%. The pattern of glycerolipid labeling, after applying [2-¹⁴C]acetate to intact leaves was altered by water stress. After 4 days of water stress the radioactivity of phosphatidic acid + phosphatidylinositol, phosphatidylcholine, triacylglycerol, and diacylglycerol increased between 4 and 9% (compared to control plans) while radioactivity of phosphatidylethanolamine, monogalactosyldiglyceride, and digalactosyldiglyceride decreased 2 to 11%. These data indicated that increased levels of triacylglycerol and diacylglycerol observed during water stress were attributed to de novo synthesis rather than breakdown or reutilization of existing glycerolipids and fatty acids.     

Lipids containing very long chain monounsaturated acyl moieties in seeds of lunaria annua

Lipids in developing as well as mature seeds of Lunaria annua are mainly composed of triacylglycerols which contain almost exclusively nervonoyl (24:1), erucoyl (22:1) and oleoyl (18:1) moieties. Maturation of the seeds proceeds with successive reduction in the relative proportions of phospholipids and glycolipids as well as linoleoyl (18:2) and linolenoyl (18:3) moieties in the total lipids. Concomitantly, the most predominant fraction of triacylglycerols, which contain nervonoyl and erucoyl moieties at the sn-1,3 positions and oleoyl moieties at the sn-2 position, are accumulated.     

The active site and the phospholipid activation of rat liver lysosomal lipase are not stereospecific

The stereochemical specificity of lysosomal lipase of rat liver was investigated using enantiomeric triacylglycerol analogs, sn-1-alkyl-2,3-diacylglycerol and sn-3-alkyl-1,2-diacylglycerol as substrates. Lysosomal lipase utilized both substrates with equal rates. The dependence of the activity of lysosomal lipase on the stereoconfiguration of activating acidic phospholipid was also studied. Our results showed that both sn-3-phospholipids (diphosphatidylglycerol, phosphatidylserine) and sn-1-phospholipids (bis(monoacylglycero)phosphate (BMP) were efficient activators of this enzyme and thus the stereochemical configuration of the activating phospholipid is not important. Accordingly, the rat liver lysosomal lipase lacks stereospecificity with respect to both the triacylglycerol substrate and the acidic phospholipid activator.     

In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase

Seed oils of flax (Linum usitatissimum L.) and many other plant species contain substantial amounts of polyunsaturated fatty acids (PUFAs). Phosphatidylcholine (PC) is the major site for PUFA synthesis. The exact mechanisms of how these PUFAs are channeled from PC into triacylglycerol (TAG) needs to be further explored. By using in vivo and in vitro approaches, we demonstrated that the PC deacylation reaction catalyzed by the reverse action of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) can transfer PUFAs on PC directly into the acyl-CoA pool, making these PUFAs available for the diacylglycerol acyltransferase (DGAT)-catalyzed reaction for TAG production. Two types of yeast mutants were generated for in vivo and in vitro experiments, respectively. Both mutants provide a null background with no endogenous TAG forming capacity and an extremely low LPCAT activity. In vivo experiments showed that co-expressing flax DGAT1-1 and LPCAT1 in the yeast quintuple mutant significantly increased 18-carbon PUFAs in TAG with a concomitant decrease of 18-carbon PUFAs in phospholipid. We further showed that after incubation of sn-2-[¹⁴C]acyl-PC, formation of [¹⁴C]TAG was only possible with yeast microsomes containing both LPCAT1 and DGAT1-1. Moreover, the specific activity of overall LPCAT1 and DGAT1-1 coupling process exhibited a preference for transferring ¹⁴C-labeled linoleoyl or linolenoyl than oleoyl moieties from the sn-2 position of PC to TAG. Together, our data support the hypothesis of biochemical coupling of the LPCAT1-catalyzed reverse reaction with the DGAT1-1-catalyzed reaction for incorporating PUFAs into TAG. This process represents a potential route for enriching TAG in PUFA content during seed development in flax.     

Analysis of Acyl Fluxes through Multiple Pathways of Triacylglycerol Synthesis in Developing Soybean Embryos

The reactions leading to triacylglycerol (TAG) synthesis in oilseeds have been well characterized. However, quantitative analyses of acyl group and glycerol backbone fluxes that comprise extraplastidic phospholipid and TAG synthesis, including acyl editing and phosphatidylcholine-diacylglycerol interconversion, are lacking. To investigate these fluxes, we rapidly labeled developing soybean (Glycine max) embryos with [¹⁴C]acetate and [¹⁴C]glycerol. Cultured intact embryos that mimic in planta growth were used. The initial kinetics of newly synthesized acyl chain and glycerol backbone incorporation into phosphatidylcholine (PC), 1,2-sn-diacylglycerol (DAG), and TAG were analyzed along with their initial labeled molecular species and positional distributions. Almost 60% of the newly synthesized fatty acids first enter glycerolipids through PC acyl editing, largely at the sn-2 position. This flux, mostly of oleate, was over three times the flux of nascent [¹⁴C]fatty acids incorporated into the sn-1 and sn-2 positions of DAG through glycerol-3-phosphate acylation. Furthermore, the total flux for PC acyl editing, which includes both nascent and preexisting fatty acids, was estimated to be 1.5 to 5 times the flux of fatty acid synthesis. Thus, recycled acyl groups (16:0, 18:1, 18:2, and 18:3) in the acyl-coenzyme A pool provide most of the acyl chains for de novo glycerol-3-phosphate acylation. Our results also show kinetically distinct DAG pools. DAG used for TAG synthesis is mostly derived from PC, whereas de novo synthesized DAG is mostly used for PC synthesis. In addition, two kinetically distinct sn-3 acylations of DAG were observed, providing TAG molecular species enriched in saturated or polyunsaturated fatty acids.     

Overexpression of rat long chain acyl-coa synthetase 1 alters fatty acid metabolism in rat primary hepatocytes

Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 mum [1-(14)C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, beta-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and beta-oxidation. The increase in [1,2,3-(3)H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [(14)C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [(14)C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.     

Mitochondrial glycerol phosphate acyltransferase directs the incorporation of exogenous fatty acids into triacylglycerol

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [(14)C]oleate ( approximately 3 microm), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [(14)C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 microm oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.     

Properties of sonicated vesicles of three synthetic phospholipids

The following synthetic phospholipids were prepared, and the structures that were formed by ultrasonic irradiation in aqueous solution were studied: 1,2-di(10-bromo stearoyl)-3-sn-phosphatidylcholine (DBrPC), 1,2-di(10-methyl stearoyl)-3-sn-phosphatidylcholine (DMePC), and 1-palmitoyl-2-oleyl-3-sn-phosphatidylcholine (POPC). Uniform populations of small, unilamellar vesicles were obtained in all cases by gel filtration on Sepharose 4B. Hydrodynamic and trapped volume measurements show that POPC is nearly identical in size and shape to vesicles of egg phosphatidylcholine whereas DBrPC and DMePC appear to have a non-spherical shape. Fluorescence depolarization measurements show that vesicles from all three lipids are in the liquid crystalline state between 5 and 50°C.The partial specific volume of DMePC is larger than that of egg PC, whereas the partial specific volume of DBrPC is considerably lower; these lipids should therefore be useful in studies requiring the separation of vesicle populations. POPC, being virtually identical in size, shape and bilayer fluidity to egg PC, should be an excellent model of a ‘natural’ lecithin with a defined fatty acid composition.     

Activities of acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) in microsomal preparations of developing sunflower and safflower seeds

The last step in triacylglycerols (TAG) biosynthesis in oil seeds, the acylation of diacylglycerols (DAG), is catalysed by two types of enzymes: the acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). The relative contribution of these enzymes in the synthesis of TAG has not yet been defined in any plant tissue. In the presented work, microsomal preparations were obtained from sunflower and safflower seeds at different stages of development and used in DGAT and PDAT enzyme assays. The ratio between PDAT and DGAT activity differed dramatically between the two different species. DGAT activities were measured with two different acyl acceptors and assay methods using two different acyl-CoAs, and in all cases the ratio of PDAT to DGAT activity was significantly higher in safflower than sunflower. The sunflower DGAT, measured by both methods, showed significant higher activity with 18:2-CoA than with 18:1-CoA, whereas the opposite specificity was seen with the safflower enzyme. The specificities of PDAT on the other hand, were similar in both species with 18:2-phosphatidylcholine being a better acyl donor than 18:1-PC and with acyl groups at the sn-2 position utilised about fourfold the rate of the sn-1 position. No DAG:DAG transacylase activity could be detected in the microsomal preparations.     

Acyl specificity in triacylglycerol synthesis by mammary adenocarcinoma R3230AC in Fischer rats

Activities and acyl specificities of both sn-glycero-3-phosphate and diacylglycerol acyltransferases in microsomal fractions isolated frorn homogenates of the mammary adenocardinoma R3230AC carried by Fischer rats were compared to those from normal mammary glands of lactating Fischer rats. Although the neoplasm exhibited lower activities for these two enzyme reactions, the specificities for acyl-CoAs as donors were quite similar to those found in the normal tissue counterpart. Long-chain acyl-CoAs were preferred substrates for the sn-glycero-3-phosphate acyltransferase reaction while acyltransferase with diacylglycerol as acceptor showed much less preference. With both normal and neoplastic tissues, the products of each reaction were the same i.e., phosphatides with sn-glycero-3-phosphate and triacylglycerol with diacylglycerol as acyl acceptors, respectively. All results support the concept of a non-random distribution of fatty acids in the triacylglycerol of this mammary adenocarcinoma in virgin rats which is the same as that from the normal tissue in lactating animals.