The incorporation of oleic and linoleic acids and their desaturation products into the glycerolipids of maize leaves

[1-¹⁴C]Oleic and [1-¹⁴C]linoleic acids were rapidly desaturated when incubated with maize leaves from 8-day-old plants and the labeled fatty acids, and their desaturation products, were rapidly incorporated into glycerolipids. Oleic acid was desaturated to linoleate at the rate of 0.7 nmol/100 mg tissue/h and further desaturated to linolenate at about one-third this rate. The rates of linolenate formation were similar when either oleic acid or linoleic acid was the substrate although there was a 2-h lag period when oleic acid was substrate. When radioactive oleic, linoleic, and linolenic acids were substrates, phosphatidylcholine was the most extensively labeled glycerolipid followed by monogalactosyldiacylglycerol. The relative rates of incorporation of label into individual glycerolipids are consistent with a movement of labeled fatty acids from phosphatidylcholine to monogalactosyldiacylglycerol and then to diagalactosyldiacylglycerol. The rates of labeling of phosphatidylcholine oleate and of phosphatidylcholine linoleate are consistent with a precursor-product relationship in that there was a delayed accumulation of phosphatidylcholine linoleate relative to that of phosphatidylcholine oleate and phosphatidylcholine linoleate continued to accumulate while phosphatidylcholine oleate declined. Linoleate formed from oleate was widely distributed in glycerolipids but neither phosphatidylcholine linolenate nor linolenate-containing diacylglycerol was detected at short and intermediate incubation times when either oleic or linoleic acid was substrate. The kinetics of incorporation of linoleate and linolenate into monogalactosyldiacylglycerol suggest a transfer of linoleate from phosphatidylcholine. The initial rate of accumulation of labeled linolenate in monogalactosyldiacylglycerol was very similar to the rate of desaturation of linoleate and it is suggested that desaturation of linoleate occurs while associated with monogalactosyl-diacylglycerol.     

The reverse reaction of cholinephosphotransferase in rat brain microsomes. A new pathway for degradation of phosphatidylcholine

The synthesis of phosphatidylcholine is catalyzed by cholinephosphotransferase (EC 2.7.8.2) which is known to be reversible in liver. The reversibility of cholinephosphotransferase in rat brain in demonstrated in this paper. Labeled microsomes were prepared from young rats which had been given an intracerebral injection of labeled choline or oleate 2 h before killing. During incubation of choline-labeled microsomes with CMP, label was lost from ;choline glycerophospholipids and labeled CDPcholine was produced. The Km for CMP was 0.35 mM and V was 3.3 nmol/min per mg protein. Neither AMP nor UMP could substitute for CMP. Oleate-labeled microsomes were pretreated with e mM diisopropylfluorophosphate (lipase inhibitor). During incubation with CMP, label was lost from choline, and ethanolamine glycerophospholipid and labeled diacylglycerols were produced. When the lipase was not inhibited, labeled oleate was produced. We propose that a principal pathway for degradation of phosphatidylcholine, particularly during brain ischemia, is by reversal of cholinephosphotransferase, followed by hydrolysis of diacylglycerols by the lipase.     

The role of lysophosphatidylcholine in lipid synthesis by developing sunflower (Helianthus annuus L.) seed microsomes

The incorporation of oleate from oleoyl-CoA into lipids by microsomes from developing sunflower (Helianthus annuus L.) seeds has been investigated. Oleate was incorporated mainly into position 2 of phosphatidylcholine or released as free fatty acid. The addition of exogenous 1-acyl-lysophosphatidylcholine increased the incorporation of oleate into position 2 of phosphatidylcholine and decreased the release of free oleate. In the absence of exogenous lysophosphatidylcholine, the incorporation of oleate into phosphatidylcholine was limited by the amount of endogenous acceptor present. DH-990, an inhibitor of acyl-CoA:lysophosphatidylcholine acyltransferase, almost completely inhibited the incorporation of oleate from oleoyl-CoA into phosphatidylcholine at a concentration of 2.5 mM. These results indicate that the incorporation of oleate from oleoyl-CoA into microsomal phosphatidylcholine occurs mainly by the acylation of a 1-acyl-lysophosphatidylcholine acceptor rather than by acyl exchange between oleoyl-CoA and phosphatidylcholine. While the incorporation of oleoyl-CoA was completed within 2 to 5 min, exogenous 1-acyl-lysophosphatidylcholine was incorporated into phosphatidylcholine for up to 30 min. Addition of oleoyl-CoA resulted in an increase in both the rate and magnitude of lysophosphatidylcholine incorporation, which could not be accounted for by a stoichiometric reaction between the two substrates. Evidence is provided that free CoA had an independent stimulatory effect on the incorporation of lysophosphatidylcholine. The implications of this finding are discussed.     

The kinetics of incorporation in vivo of [14C]acetate and [14C]carbon dioxide into the fatty acids of glycerolipids in developing leaves

1. The patterns of incorporation of (14)C into glycerolipid fatty acids of developing maize leaf lamina from supplied [1-(14)C]acetate and from (14)CO(2) during steady-state photosynthesis were similar. Oleate of phosphatidylcholine and palmitate of phosphatidylglycerol attained linear rates of labelling more rapidly than did other fatty acids, particularly the linoleate and linolenate of monogalactosyl diacylglycerol. 2. After the transfer of lamina from labelled to unlabelled acetate, there was a decrease in labelled oleate and linoleate of phosphatidylcholine and a concomitant increase in the amount of radioactivity in the linoleate and linolenate of monogalactosyl diacylglycerol. 3. The rapidly labelled phospholipids, phosphatidylcholine and phosphatidylglycerol, were shown by differential and sucrose-density-gradient centrifugation to be associated with different organelles, the former being mainly in a low-density membrane fraction, probably microsomal, and the latter mainly in chloroplasts. 4. During a 48h period after supplying spinach leaves with [(14)C]acetate, radioactivity was lost from the oleate of phosphatidylcholine present in fractions sedimented at 12000g and 105000g, and accumulated in the linolenate of monogalactosyl diacylglycerol of the chloroplast. 5. It is proposed that the phosphatidylcholine of some non-plastid membranes is intimately involved in the process of oleate desaturation and that this lipid serves as a donor of unsaturated C(18) fatty acids to other lipids, principally monogalactosyl diacylglycerol, of the chloroplasts.     

Fatty acids of Treponema pallidum and Borrelia burgdorferi lipoproteins.

A fundamental ultrastructural feature shared by the spirochetal pathogens Treponema pallidum subsp. pallidum (T. pallidum) and Borrelia burgdorferi, the etiological agents of venereal syphilis and Lyme disease, respectively, is that their most abundant membrane proteins contain covalently attached fatty acids. In this study, we identified the fatty acids covalently bound to lipoproteins of B. burgdorferi and T. pallidum and examined potential acyl donors to these molecules. Palmitate was the predominant fatty acid of both B. burgdorferi and T. pallidum lipoproteins. T. pallidum lipoproteins also contained substantial amounts of stearate, a fatty acid not typically prevalent in prokaryotic lipoproteins. In both spirochetes, the fatty acids of cellular lipids differed from those of their respective lipoproteins. To characterize phospholipids in these organisms, spirochetes were metabolically labeled with [3H]palmitate or [3H]oleate; B. burgdorferi contained only phosphatidylglycerol and phosphatidylcholine, while T. pallidum contained phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Although palmitate predominated in the lipoproteins, there were no apparent differences in the incorporation of these two fatty acids into phospholipids (putative acyl donors). Phospholipase A1 and A2 digestion of phosphatidylcholine from B. burgdorferi and T. pallidum labeled with either [3H]palmitate or [3H]oleate also revealed that neither fatty acid was incorporated preferentially into the 1 and 2 positions (potential acyl donor sites) of the glycerol backbone. The combined findings suggest that fatty acid utilization during lipoprotein synthesis is determined largely by the fatty acid specificities of the lipoprotein acyl transferases. These findings also provide the basis for ongoing efforts to elucidate the relationship between lipoprotein acylation and the physiological functions and inflammatory activities of these molecules.     

Fatty acid biosynthesis in the leaves of barley, wheat and pea.

1. The incorporation of radioactivity from [1-14C]acetate into the leaf lipids of barley, pea and wheat has been studied in pulse-labelling experiments. 2. There was little increase in the total labelling of lipids after the leaves were transferred to non-radioactive medium. However, there was an increase in the relative labelling of unsaturated fatty acids. In addition, there was an increase in the relative labelling of diacylgalactosylglycerol. 3. The principal radioactively labelled acyl lipids were diacylgalactosylglycerol and phosphatidylcholine. Phosphatidylcholine showed a decreasing proportion of [14C]oleate and an increasing amount of [14C]linoleate with time. Diacylgalactosylglycerol also had decreasing amounts of [14C]oleate but, in addition, had an increasing proportion of [14C]linolenate with time. 4. The absence of significant amounts of [14C]linolenate in phosphatidylcholine appeared to exclude a role for this phospholipid in linoleate desaturation. 5. The specific radioactivities of oleate and linoleate in phosphatidylcholine, diacylgalactosylglycerol and diacylgalabiosylglycerol were very similar in any single experiment. It was concluded that these fatty acids can rapidly exchange between the three intact lipids.     

Interaction of a human plasma lipid transfer protein complex with lipid monolayers

The interaction of a purified human plasma lipid transfer complex with cholesteryl ester, triacylglycerol and phosphatidylcholine in binary and ternary lipid monolayers was investigated. The lipid transfer complex, designated LTC, catalyzes the removal of cholesteryl oleate and triacylglycerol from phosphatidylcholine monolayers. Preincubation of LTC with p-chloromercuriphenyl sulfonate inhibits LTC-catalyzed removal of triacylglycerol; cholesteryl ester removal is not affected. The rate of LTC-facilitated removal of cholesteryl oleate from a phosphatidylcholine monolayer depends on the amount of LTC added to the subphase up to 100 μg protein. In addition, the rate of the LTC-catalyzed transfer of cholesteryl oleate to the subphase increases linearly as the amount of cholesteryl oleate in the monolayer increases to 6 mol%. LTC also removes cholesterol from phosphatidylcholine-cholesterol monolayers, albeit at a rate which is 15% of that for removal of cholesteryl oleate. The ability of LTC to facilitate triacylglycerol and cholesteryl ester removal depends on the composition of the monolayer. Phosphatidylcholine supports cholesteryl ester transfer whereas sphingomyelin-cholesteryl ester monolayers are almost refractory to LTC. In contrast, LTC removes triacylglycerol from either a phosphatidylcholine or a sphingomyelin monolayer. The results suggest the existence of at least two lipid transfer proteins, one of which catalyzes the removal of cholesteryl ester and the other triacylglycerol. The role of these proteins as they relate to lipoprotein metabolism is discussed.     

Induction of an endothermic transition in the Arrhenius plot of fatty acid uptake by lipid-depleted ascites tumor cells

Cultured ascites tumor cells and their lipid-depleted variants containing 35-40% less membrane phospholipid and cholesterol were used to study uptake and metabolism of fatty acids complexed to albumin. Uptake of stearate and oleate at 37 degrees C was considerably higher in the lipid-depleted cells, but no significant difference in the affinity constants for stearate uptake of 3.70 microM for the lipid-depleted and 2.50 microM for the control cells was observed. Similar rates of uptake of both cultures were observed at lower temperatures up to 30 degrees C. The drastic increase in stearate uptake above 30 degrees C resulted in an endothermic transition in the Arrhenius plot with an activation energy of 20.8 kJ/mol versus 6.5 kJ/mol for the control cells. Uptake of stearate and oleate of the control cells was only slightly reduced by metabolic inhibitors, which was similar to stearic acid transport in the lipid-depleted variants. However, oleate uptake was substantially decreased in these variants. Incorporated stearate was esterified to about 50% in both cultures, and oleate between 85 and 90%. Mainly triacylglycerols and phospholipids with phosphatidylcholine (41%) and phosphatidylethanolamine (35%) as major polar lipid components, and also lower acylglycerols and cholesterol were found to be labeled. Under lipid-depleted conditions, a pronounced increase in the relative proportion of oleate incorporation into triacylglycerols was determined. It is suggested that fatty acid uptake is controlled by the number of active sites of the putative transport protein, which increases upon lipid depletion as shown from the V values. This increase may result from the segregation of membrane-bound proteins into domains (Haeffner et al. (1986) Cell Mol. Biol. 32, 359-368), which are known to be formed as a consequence of lipid phase separation in the lipid-depleted cells.     

The stimulation of the phospholipase A2-acylation system of synaptic membranes of brain by cyclic nucleotides.

Hydrolysis of phosphatidylcholine by phospholipase A2 of synaptic membranes i n Tris-CHl buffer was stimulated by cyclic AMP, cyclic GMP, cyclic CMP, cyclic UMP and adenosine (0.1 mm). In the presence of 1 mm-NaF and cofactors, the same cyclic nucleotides and adenosine (10 mm) stimulated the incorporation of added oleate into the choline glycerophospholipids of synaptic membranes. Cyclic AMP and noradrenaline stimulated the incorporation of added oleate into position 2 of choline glycerophospholipid. Stimulation of net acylation was increased by preincubation in conditions which stimulated hydrolysis of phosphatidylcholine. Cyclic AMP only slightly stimulated the transfer of oleate from oleoyl-CoA into choline glycerophospholipid. The optimum concentration of CaCl2 for the stimulation of hydrolysis by phospholipase A2 by cyclic AMP was 1 mum. Stimulation of the incorporation of added oleate was maximal in the CaCl2 concentration range 1 mum-1mm. MgCl2 also enhanced stimulations, maximum effects being obtained with concentrations of 10 mum and 0.5 mm for hydrolysis by phospholipase A2 and incorporation of added oleate respectively. ATP enhanced the stimulation of incorporation of oleate but had no effect on the cyclic nucleotide stimulation of hydrolysis of added phosphatidylcholine by phospholipase A2. Adenosine, guanosine, ADP and 5'-AMP (all at 1 mm) inhibited the stimulation of incorporation of oleate by cyclic nucleotides and inhibited the transfer of oleate from oleoyl-CoA to phospholipid. They did not inhibit the stimulation of hydrolysis of added phosphatidylcholine (by phospholipase A2) by cyclic nucleotides, but inhibited the stimulation by noradrenaline, acetylcholine, 5-hydroxytryptamine, dopamine (3,4-dihydroxyphenethylamine) and histamine. Preincubation of synaptic membranes in the water or buffer increased the net activity of phospholipase A2. Preincubation with a mixture of ATP and MgCl2 increased the initial rate of acylation of membrane lipid.     

Oleate metabolism in microsomes from developing leaves ofPisum sativum L.

Microsomes from young leaves of pea,Pisum sativum L., metabolized oleate principally by the reactions mediated by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. Hydrogen peroxide specifically inhibited oleate desaturation and the evidence presented argues for a specific inhibition of the terminal enzyme of the desaturase system, i.e. oleoyl phosphatidylcholine desaturase. Catalase, ascorbic acid, or ascorbate peroxidase, in conjunction with ascorbic acid, stimulated oleate desaturation, possibly by the removal of hydrogen peroxide. Lysophosphatidylcholine was found to be the preferred acceptor for acyl transfer from oleoyl-CoA, which indicates that the transfer of oleoyl moieties was catalyzed predominantly by oleoyl-CoA:lysophosphatidylcholine acyltransferase. Acyl exchange between oleoyl-CoA and phosphatidylcholine, with a possible involvement of phospholipases, was also detected but at much lower rates than acyl transfer. When intact or broken chloroplasts were added to microsomes, which had been preincubated with oleoyl-CoA, some stimulation of the reactions catalyzed by oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase was observed. However, only minor amounts of microsomal linoleoyl phosphatidylcholine were converted to galactolipids containing linolenoyl moieties.Abbreviations FA unesterified fatty acid (s) - PC phosphatidylcholines - 18:1 oleoyl moieties - 18:2 lmoleoyl moieties Dedicated to Professor Helmut K. Mangold, Bundesanstalt für Fettforschung, Münster, on his 60th birthday     

Evidence for an oleoyl phosphatidylcholine desaturase in microsomal preparations from cotyledons of safflower (Carthamus tinctorius) seed.

1. [14C]Oleoyl-CoA was metabolized rapidly and essentially completely by microsomal preparations from developing safflower (Carthamus tinctorius) cotyledons, and most of the [14C]oleate was incorporated into 3-sn-phosphatidylcholine. 2. In aerobic reaction mixtures containing NADH2 the [14C]oleate in 3-sn-phosphatidylcholine was converted into [14C]linoleate without any change in the specific radioactivity of the lipid. Over a 60 min incubation period the extent of conversion of [14C]oleoyl phosphatidylcholine into [14C]linoleoyl phosphatidylcholine was generally greater than 60%. The rate of desaturation of endogenous [14C]oleoyl phosphatidylcholine labelled from [14C]oleoyl-CoA was much greater that of exogenous [14C]dioleoyl phosphatidylcholine the specific radioactivity of the oleoyl moiety of the lipid remained constant, indicating that labelled and unlabelled oleate were desaturated at the same rate. On this assumption an initial rate of desaturation of about 15 nmol of oleate desaturated/min per mumol of 3-sn-phosphatidylcholine was estimated. 4. [14C]Oleate esterified at positions 1 and 2 of both endogenous and exogenous 3-sn-phosphatidylcholine was desaturated. 5. Attempts to demonstrate the presence of an oleoyl-CoA desaturase in safflower microsomal fractions by the appearance of linoleoyl-CoA in reaction mixtures were inconclusive.     

Substrate specificities of the enzymes of the oleate desaturase system from photosynthetic tissue.

In the microsomal fraction from young pea (Pisum sativum L.) leaves, the oleoyl moieties from oleoyl-CoA are principally transferred to the sn-2 position of phosphatidylcholine by oleoyl-CoA:1-acyl-lysophosphatidylcholine acyltransferase. The major product of this acyl transfer is 1-palmitoyl(stearoyl)-2-oleoyl phosphatidylcholine. The 1-palmitoyl(stearoyl)-2-oleoyl phosphatidylcholine is subsequently converted into 1-palmitoyl(stearoyl)-2-linoleoyl phosphatidylcholine by the oleate desaturase complex without equilibrating with the bulk membrane phosphatidylcholine pool. Hence, both the acyl transfer to phosphatidylcholine and the subsequent desaturation of oleoyl moieties occur on the sn-2 position of phosphatidylcholine, and there is also a functional coupling of the acyltransferase and oleate desaturase.     

Metabolism of high density lipoprotein lipids by the rat liver: evidence for participation of hepatic lipase in the uptake of cholesteryl ester.

In order to determine the role of hepatic lipase in the hepatic uptake and metabolism of high density lipoprotein (HDL) triglycerides, cholesteryl esters, and phospholipids, isolated rat livers were perfused with a reconstituted HDL (rHDL) radiolabeled with [3H]triolein and [14C]cholesteryl oleate or palmitoyl-[14C]linoleoyl phosphatidylcholine. A bolus of radiolabeled rHDL was injected into the portal vein and livers were perfused for 5 min using a nonrecirculating perfusion system. Recovery of rHDL triolein in the liver as intact triolein was used to determine the amount of unmetabolized rHDL remaining in the liver. After correcting for the amount of unmetabolized rHDL remaining in the liver, about 30% of the rHDL triolein was hydrolyzed of which 19% was recovered in the liver and 11% in the perfusate. Moreover, about 7% of the rHDL phosphatidylcholine was hydrolyzed to lysophosphatidylcholine, all of which was recovered in the perfusate. Although there was no hydrolysis of rHDL cholesteryl oleate, about 30% of the cholesteryl oleate was taken up by the liver. Preperfusion of the liver with heparin to deplete the liver of hepatic lipase resulted in about a 70% reduction in rHDL triolein hydrolysis and about a 75% reduction in rHDL cholesteryl oleate uptake. Although hepatic lipase hydrolyzes both triglycerides and phosphatidylcholines, elimination of the triolein from rHDL had no effect on the uptake of rHDL cholesteryl oleate, but replacement of the rHDL phosphatidylcholine with a nonhydrolyzable phosphatidylcholine diether resulted in an 87% reduction in cholesteryl oleate uptake. These results indicate that hepatic lipase is necessary for the hepatic uptake of both HDL triglycerides and cholesteryl esters and that the uptake of cholesteryl esters is not dependent on the hydrolysis of HDL triglycerides but is dependent on the hydrolysis of HDL phospholipids.     

Radiolabelling studies of fatty acids in pisum sativum and Vicia faba leaves at different temperatures

Leaves of pea and broad bean plants were incubated with acetate-[¹⁴C] at temperatures varying from 7 to 34°. No significant difference was observed in the distribution of radioactivity between phosphatidylcholine and the galactosylglycerides in pea with different temperatures. However, increasing temperatures increased the labelling of phosphatidylcholine in broad bean leaves, at the expense of polar lipids other than the galactosylglycerides. The incubation temperature had no significant effect on the pattern of labelling of the fatty acids of the major leaf lipids. A correlation was seen in the specific radioactivity of oleate and linoleate in phosphatidylcholine and, especially, in the galactosylglycerides. The data emphasise the rapid equilibration of oleate and linoleate (which probably occurs by transacylation) between the two galactosylglycerides and phosphatidylcholine in leaf tissues.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

Glycerolipid Synthesis in Avena Leaves during Greening of Etiolated Seedlings IV. Effect of Light on Fatty Acid Desaturation

Etiolated Avena sativa L. leaves were fed with [l-¹⁴C]acetatefor 20 h in the dark and labeled fatty acids in glycerolipidswere chased during 24 h in the light (greening condition) orin the dark, to determine the light effect on the fatty aciddesaturation. Oleate decrease in phosphatidylcholine was thesame in the light and in the dark, showing that oleate desaturationis independent of light (or greening). Linoleate desaturationin galactolipids, especially in monogalactosyl diacylglycerol,was enhanced by light and palmitate desaturation to hexadecenoatein phosphatidylglycerol was strictly light-dependent. (Received May 11, 1983; Accepted August 16, 1983)     

Effects of Phosphatidylcholine or Ergosteryl Oleate on Physiological Properties of Saccharomyces sake

Chromatographically homogeneous phosphatidylcholine identified by thin-layer chromatography on silica gel G was isolated from mycelia of Aspergillus oryzae or egg yolk by a combination of alumina and silicic acid column chromatography. Although the 2-position in both Asp. oryzae- and egg yolk-phosphatidylcholine was occupied nearly exclusively by unsaturated fatty acids, significant proportions of unsaturated fatty acids were also found in the 1 -position of Asp. oryzae-phosphatidylcholine. In nitrogen gas-sparging anaerobic culture of Saccharomvces sake Kyokai No. 7, supplementing the basal synthetic medium with phosphatidylcholine enhanced the yeast growth and fermentative activity, whereas adding ergosteryl oleate enhanced alcohol-endurability. Supplementation with both phosphatidylcholine and ergosteryl oleate promoted the yeast growth, fermentative activity and alcohol-endurability of cells.     

Stimulation of phosphatidylcholine synthesis by fatty acids in fetal rabbit type II pneumocytes

After 24 h exposure to 0.1 mM oleate or 0.1 mM palmitate there was a 2- and 1.7-fold increase, respectively, in the incorporation of choline into the lipids of type II pneumocytes. Palmitate increased the labeling of disaturated phosphatidylcholine (PC) from 23.0% of total labeled PC in control cultures to 56.6% and oleate decreased labeling of disaturated PC to 9.4%. The percentage of total cellular radioactivity found in the lipid fraction was also markedly higher in the fatty acid-treated cells (83.3% for oleate and 78.7% for palmitate) than in control cultures (64.0%). Radioactivity in water-soluble choline metabolites was correspondingly lower, with phosphocholine representing more than 95% of the label in both control and experimental cultures. After a 3 h pulse-chase period, oleate and palmitate significantly increased the percentage of total cellular radioactivity in PC and decreased the percentage in phosphocholine. Similar results were obtained by adding melittin (1–2 μg/ml) or phospholipase C (0.05 U/ml) to the culture medium. The stimulation of PC synthesis by fatty acids was demonstrated as early as 1 h after exposure to oleate or palmitate and at all concentrations from 0.025 to 0.25 mM. Cytidylyltransferase activity in total cell homogenates was also enhanced by long-term exposure to fatty acids and short-term addition of fatty acids or phospholipase C and melittin to the culture medium. A similar increase in Cytidylyltransferase activity was found in the 100 000 × g particulate fraction of type II cells exposed to fatty acids, whereas no differences were found between the cytosolic fractions of control and treated cells. These results support the concept that an increase in intracellular level of fatty acids either from an exogenous source or following the activation of endogenous phospholipases regulates PC synthesis in fetal type II pneumocytes.     

Fatty acids reverse the cyclic AMP inhibition of triacylglycerol and phosphatidylcholine synthesis in rat hepatocytes.

The influence of cyclic AMP analogues and fatty acids on glycerolipid biosynthesis in monolayer cultures of rat hepatocytes was investigated. Chlorophenylthio-cyclic AMP and adenosine 3':5'-cyclic phosphorothioate inhibited the rate of triacylglycerol synthesis from [1(3)-3H]glycerol, and phosphatidylcholine synthesis from [Me-3H]-choline. Supplementation of the hepatocytes with palmitate (1 mM) reversed chlorophenylthio-cyclic AMP inhibition of triacylglycerol synthesis. Similarly, cyclic AMP analogue-inhibition of phosphatidylcholine synthesis was abolished when the cells were simultaneously incubated with oleate (3 mM). Reactivation of phosphatidylcholine synthesis in chlorophenylthio-cyclic AMP-supplemented cells with oleate was accompanied by conversion of CTP: phosphocholine cytidylyltransferase into the membrane-bound form, since these cells released the enzyme more slowly after treatment with digitonin. The opposing actions of cyclic AMP and fatty acids are discussed in relation to the regulation of glycerolipid biosynthesis during starvation, diabetes and stress.     

Lipid involvement in oleoyl CoA desaturase activity of Fusarium oxysporum microsomes

Desaturation of oleoyl CoA by the microsomal fraction of Fusarium oxysporum hyphal cells required O2, NADPH, MgCl2, and the addition of either bovine serum albumin or the 105 000g supernatant fraction. In the absence of reduced nucleotide, [14C]oleoyl CoA was rapidly incorporated into phospholipid and triacylglycerol and hydrolyzed to free fatty acids. After addition of NADPH, oleate was desaturated at the normal rate. Analysis of the distribution of [14C]oleate and [14C]linoleate between different lipid classes revealed that phosphatidylcholine and phosphatidylethanolamine were labeled with [14C]linoleate before any other lipid class. These results are consistent with oleoyl phospholipid being a direct intermediate in the desaturation of oleoyl CoA. The preference of the oleoyl-desaturase for NADPH, the relatively high pH optimum of 8.2, and the sensitivity to thenoyltrifluoroacetone inhibition suggest that some components of the microsomal electron-transport chain are common to both the oleoyl desaturase and stearoyl CoA desaturase systems in this fungus.