Effect of BASF 13-338, a Substituted Pyridazinone, on Lipid Metabolism in Leaf Tissue of Spinach, Pea, Linseed, and Wheat

A substituted pyridazinone (BASF 13-338) inhibited photosynthesis in spinach (Spinacia oleracea, Hybrid 102 Arthur Yates Ltd.) leaf discs and reduced the incorporation of [1-¹⁴C]acetate into trienoic acids of diacylgalactosylglycerol while causing radioactivity to accumulate in diacylgalac-tosylglycerol dienoic acids. Although BASF 13-338 inhibited photosynthesis in isolated spinach chloroplasts, it did not prevent dienoate desaturation. In discs, the labeling of fatty acids was affected by the inhibitor only in diacylgalactosylglycerol. Very little radioactivity was incorporated into trienes of phosphatidylcholine and the proportion of the label recovered in the fatty acids of phosphatidylcholine was not changed by BASF 13-338. The herbicides caused an increase in the proportion of the lipid ¹⁴C incorporated into diacylgalactosylglycerol and a decrease in labeling of phosphatidylcholine, whereas the proportion of ¹⁴C recovered in other lipids remained unchanged. Similar results were obtained with pea (Pisum sativum cv. Victory Freeze), linseed (Linum usitatissimum cv. Punjab), and wheat (Triticum aestivum cv. Karamu). With these species, a greater proportion of the label was incorporated into phosphatidylcholine and less into diacylgalactosylglycerol than with spinach. The data indicate that trienoate synthesis uses diacylgalactosylglycerol as substrate. BASF 13-338 appears to act at that step, and seems to cause in spinach a shift in polyenoate synthesis from the pathway involving microsomal phosphatidylcholine to the pathway operating inside the chloroplast.     

Lipids and lipid metabolism in the brown alga, Fucus serratus

The lipids of the brown alga Fucus serratus were isolated, identified and quantified. The major acyl lipids were the three glycosylglycerides, diacylgalactosylglycerol, diacyldigalactosylglycerol and diacylsulphoquinovosylglycerol. These represent over 70% of the total acyl lipids. The fatty acid compositions of the major lipids were examined and most showed rather distinctive fatty acid contents. For example, diacylgalactosylglycerol was enriched in n-3 polyunsaturated fatty acids while phosphatidylcholine and phosphatidylethanolamine had very high levels of arachidonate. Phosphatidylglycerol contained the unusual trans-Δ3-hexadecenoic acid. The labelling of lipids and fatty acids from [¹⁴C]acetate was examined and the distribution of label between individual components as a function of the incubation period and in algae collected at different times of the year is reported. Algae collected in the winter incorporated much more radioactivity into non-esterified fatty acids when compared to algae collected in the summer. All algae could label myristate, palmitate, stearate and oleate at high rates. Longer incubation times allowed the labelling of polyunsaturated fatty acids such as linoleic acid.     

The rat retina is a useful in vivo model to study membrane lipid synthesis: Rates of biosynthesis of neutral glycerides and phospholipids

The phospholipid composition was studied in the whole rat retina, as well as in its subcellular fractions. A relative enrichment of phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine was observed in rod outer segments (ROS) in comparison with entire retina: nuclear-photoreceptor inner segmentssynaptic bodies (P₁) and synaptosomal-mitochondrial (P₂) fractions. Phosphatidylcholine was the predominant phospholipid class found in all subcellular fractions analyzed. The microsomal fraction was relatively enriched in phosphatidic acid and in phosphatidylinositol. In addition, the rat eye has been used as an in vivo system to study membrane lipid synthesis. After intravitreal injections of [2-³H]glycerol a rapid labeling of retinal glycerolipids took place. Up to 120 min after injection only the glycerol backbone of lipids was labeled. Phosphatidic acid and diacylglycerol displayed rapid rates of synthesis and breakdown. Fastest rates of labeling were attained by phosphatidylcholine followed by phosphatidylinositol. Differences were found when in vitro labeling by [2-³H]glycerol was compared with intravitreal injections. Labeling of phospholipids of subcellular fractions by intravitreally injected [2-³H]glycerol showed that most of the label accumulated in microsomal phosphatidylcholine and phosphatidylinositol. Diacylglycerols and phosphatidylethanolamine also took up 10 and 20% respectively of the precursor. It is concluded that the rat eye is a useful experimental model to study synthesis and metabolism of membrane lipids in the retina.     

Phosphatidylglycerol synthesis in spinach chloroplasts: characterization of the newly synthesized molecule

Intact chloroplasts from spinach (Spinacia oleracea L., hybrid 424) readily incorporate [¹⁴C]glycerol-3-phosphate and [¹⁴C]acetate into diacylglycerol, monoacylglycerol, diacylglycrol, free fatty acids (only when acetate is the precursor), phosphatidic acid, phosphatidylcholine, and most notably phosphatidylglycerol. The fraction of phosphatidylglycerol synthesized is greatly increased by the presence of manganese chloride in the reaction mixture. Glycerol-3-phosphate-labeled phosphatidylglycerol is equally labeled in the two glycerol moieties of the molecule. Acetate-labeled phosphatidylglycerol is equally labeled in both acyl groups. Position one contains primarily oleate, linoleate and small amounts of palmitate. Position two contains primarily palmitate. No radioactive trans-Δ³-hexadecenoate was detected. The labeling patterns indicate that the radioactive phosphatidylglycerol is the product of de novo chloroplast lipid biosynthesis and furthermore, phosphatidylglycerol may be a substrate for fatty acid desaturation.     

The role of chloroplasts and microsomal fractions in polar-lipid synthesis from [1-14C]acetate by cell-free preparations from spinach (Spinacia oleracea) leaves.

1. Isolated spinach (Spinacia oleracea) chloroplasts were incapable of accumulating polar lipids when incubated with [1-14C]acetate in a cofactor-free medium. When CoA, ATP and glycerol 3-phosphate were added to incubation media, the accumulated products were non-esterified fatty acids, acyl-CoA and 1,2-diacylglycerol, all intermediates of lipid metabolism. 2. Chloroplast acyl-CoA was used to synthesize phosphatidylcholine only when a microsomal fraction was added back to the incubation medium. 3. The 1,2-diacylglycerol synthesized by isolated chloroplasts was converted almost quantitatively into diacylgalactosylglycerol when exogenous UDP-galactose was available. 4. Stereospecific analyses of the isolated lipids suggested that the diacylglycerol synthesized by isolated chloroplasts may be an important precursor for the synthesis in vivo of diacylgalactosylglycerol and phosphatidylglycerol but was unlikely to be a precursor of phosphatidylcholine. 5. A scheme for plant-lipid biosynthesis is presented that integrates the functions of chloroplasts, the cytoplasm and the endoplasmic reticulum.     

Fatty acid and glycerolipid synthesis in abscised daffodil flowers after acetate feeding

The coronae of Narcissus pseudonarcissus flowers incorporated [1-¹⁴C]acetate almost exclusively into the fatty acid moieties of glycerolipids. After a 4 h incubation, the newly synthesized acids were: stearate plus palmitate (50%); oleate (17%); linoleate (32%); and linolenate (0.5%). Phosphatidylcholine and diacylglycerol were the principal labelled lipids. In pulse experiments these acids were further desaturated, with time, to an appreciable extent and, concurrently, transferred essentially from phosphatidylcholine to diacylglycerol, diacylgalactosylglycerol, and diacylgalabiosylglycerol. The labelling of diacylgalactosylglycerol and diacylgalabiosylglycerol paralleled the appearance of linolenate. The distribution of labelled acids in phosphatidylcholine, diacylgalactosylglycerol, and diacylgalabiosylglycerol was very different. The results were compared with those obtained in vitro with isolated coronae chromoplasts and discussed in relation to current schemes of fatty acid and glycerolipid synthesis in plant cells.     

On the mechanism of the phospholipase C-mediated attenuation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells

The effect of phospholipase C treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with phosphatidylcholine-specific Clostridium welchii phospholipase C reduced the pool size of phosphatidylcholine compared with controls whereas the pool size of cardiolipin and phosphatidylglycerol were unaffected. Pulse labeling experiments with [1,3-³H]glycerol and pulse-chase labeling experiments with [1,3-³H]glycerol were performed in cells incubated or pre-incubated in the absence or presence of phospholipase C. In all experiments, radioactivity incorporated into cardiolipin and phosphatidylglycerol were reduced in phospholipase C-treated cells with time compared with controls indicating attenuated de novo biosynthesis of these phospholipids. Addition of 1,2-dioctanoyl-sn-glycerol, a cell permeable 1,2-diacyl-sn-glycerol analog, to cells mimicked the inhibitory effect of phospholipase C on cardiolipin and phosphatidylglycerol biosynthesis from [1,3-³H]glycerol indicating the involvement of 1,2-diacyl-sn-glycerol. The mechanism for the reduction in cardiolipin and phosphatidylglycerol biosynthesis in phospholipase C-treated cells appeared to be a decrease in the activities of phosphatidic acid:cytidine-5triphosphate cytidylyltransferase and phosphatidylglycerolphosphate synthase, mediated by elevated 1,2-diacyl-sn-glycerol levels. Upon removal of phospholipase C from the incubation medium, phosphatidylcholine biosynthesis from [methyl-³H]choline was markedly stimulated. These data suggest that de novo phosphatidylglycerol and cardiolipin biosynthesis may be regulated by 1,2-diacyl-sn-glycerol and support the notion that phosphatidylglycerol and cardiolipin biosynthesis may be coordinated with phosphatidylcholine biosynthesis in H9c2 cardiac myoblast cells.     

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [¹⁴C]-bicarbonate or [¹⁴C]-acetate. [¹⁴C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [¹⁴C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [¹⁴C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C₂₀ fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C₁₈ fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C₂₀ fatty acids.     

Effects of water stress on lipid metabolism in cotton leaves

After 2, 10 and 24 hr labelling with [1-¹⁴C] acetate, radioactivity incorporated into the lipids of cotton leaves is mainly found in phosphatidylcholine, phosphatidylglycerol and neutral lipids. Galactolipids are slowly synthesized and after 24 hr, account for only 10% of the total radioactivity. Under water stress, a marked decrease of precursor incorporation into leaf lipids occurs, particularly in phosphatidylcholine and galactolipids. Relative incorporation into neutral lipids, on the contrary, increases. Water deficits provoke an inhibition of the fatty acid desaturation, resulting in a sharp decrease of linoleic and linolenic acid biosynthesis. The decrease in unsaturated fatty acid biosynthesis occurs in all lipid classes, but is most pronounced in the galactolipid fractions. In the drought-resistant cotton variety (Mocosinho), the variations in lipid and fatty acid metabolism under water stress are less pronounced than in the drought-sensitive variety (Reba), and this attests a greater stability of the membrane system.     

Metabolic regulation in the senescing tobacco leaf: I. Changes in pattern of p incorporation into leaf disc metabolites

Changes in phosphate metabolism were explored in discs from tobacco (Nicotiana tabacum) leaves of three contrasting types: green leaves which were fully expanded and attached to the plant, leaves which had yellowed following excision and dark starvation, and leaves which had yellowed while attached to the plant. 2,4-Dinitrophenol at 10⁻⁵m stimulated the respiration rate of discs from green and yellow-detached leaves only slightly, but markedly stimulated that of discs from yellow-attached leaves. Following a 10-minute uptake period the incorporation of ³²P-orthophosphate into phosphate esters and lipids of discs from yellow-detached leaves was resistant to 2,4-dinitrophenol, whereas in discs from green and yellow-attached leaves it was inhibited by 2,4-dinitrophenol. Incorporation into a salt-soluble fraction containing unidentified nucleotide material showed converse behavior in that it was stimulated by 2,4-dinitrophenol in discs from green and yellow-attached leaves; in discs from yellow-detached leaves it was resistant to 2,4-dinitrophenol. In discs from yellow-detached and yellow-attached leaves there was a shift in the labeling pattern of phosphate esters toward increased label in hexose phosphates at the expense of adenine nucleotides, 3-phosphoglycerate, and phosphoenolpyruvate. It is concluded that incorporation into phosphate esters in discs from yellow-detached leaves is by substrate level phosphorylation coupled to enhanced aerobic glycolysis. In discs from yellow-attached leaves, on the other hand, incorporation depends on oxidation phosphorylation, and it is suggested that the shift in labeling pattern is caused by senescence-induced changes in activity of glycolytic enzymes.     

Phosphatidylglycerol synthesis in pea chloroplasts: pathway and localization

Isolated intact pea chloroplasts synthesized phosphatidylglycerol from either [¹⁴C]acetate or [¹⁴C]glycerol 3-phosphate. Both time-course and pulse-chase labeling studies demonstrated a precursor-product relationship between newly synthesized phosphatidic acid and newly synthesized phosphatidylglycerol.

The synthesis both of CDP-diacylglycerol from exogenous phosphatidic acid and CTP, and of phosphatidylglycerol from exogenous CDP-diacylglycerol and glycerol 3-phosphate, could be assayed in fractions obtained from disrupted chloroplasts. Moreover, the enzymes catalyzing these reactions were localized in the inner envelope membrane. Exogenous phosphatidic acid was incorporated into phosphatidylglycerol, but only following its incorporation into CDP-diacylglycerol. Finally, radio-active phosphatidic acid synthesized in the envelope membranes from [¹⁴C]palmitoyl-ACP and 1-oleoyl-glycerol 3-phosphate was sequentially incorporated into labeled CDP-diacylglycerol and phosphatidylglycerol upon the addition of appropriate substrates and cofactors. Thus, we have demonstrated that (a) the synthesis of phosphatidylglycerol in chloroplasts occurs by the pathway: phosphatidic acid → CDP-diacylglycerol →→ phosphatidylglycerol, and (b) phosphatidylglycerol synthesis is located in the inner envelope membrane.

     

Effect of Substituted Pyridazinones on Chloroplast Structure and Lipid Metabolism in Greening Barley Leaves

The effect of the substituted pyridazinone herbicides, Sandoz9785 and Sandoz 6706, on lipid metabolism was studied in greeningbarley leaves. The herbicides had no effect on chlorophyll formationbut caused an altered chloroplast morphology during greening.In leaves supplied with {¹⁴C} acetate, Sandoz 9785 decreasedincorporation of radioactivity into linolenate while Sandoz6706 decreased incorporation into both linolenate and trans-3-hexadecenoate.Decreased linolenate labelling was accompanied by an accumulationof {¹⁴C}linolenate in diacylgalactosylglycerol. {¹⁴C}Palmitateaccumulated in phosphatidylglycerol when synthesis of trans-3-hexadecenoatewas inhibited. The results are discussed in relation to thefunction of acyl lipids in fatty acid desaturation and the roleof lipids in chloroplast morphology. Key words: Chloroplast structure, Lipid synthesis, Substituted pyridazinones, Fatty acid desaturation     

Termination of nutrient import and development of vein loading capacity in albino tobacco leaves

The sink-source conversion in developing leaves of tobacco (Nicotiana tabacum L.) was studied to determine whether import termination is caused by the onset of export or is related to achievement of positive carbon balance. Albino shoots were grown in vitro and grafted to detopped stems of green tobacco plants. Termination of import was studied by providing mature leaves of the stock plant with ¹⁴CO₂ and detecting the presence of labeled nutrient in developing albino leaves by whole-leaf autoradiography. In albino leaves, import terminated progressively in the basipetal direction at the same stage of development as in leaves of green shoots. Starch was not present in the plastids of mesophyll cells of mature albino leaves but starch was synthesized when discs were cut from these leaves and incubated on 3 millimolar sucrose. Import ceased progressively in developing green leaves even when photosynthesis was prevented by darkening. It was concluded that cessation of import does not require achievement of positive carbon balance and is not the direct result of export initiation.

To determine whether vein loading capacity develops in albino leaves, discs were cut from mature leaves and floated on [¹⁴C]sucrose solution. Uptake of label into the veins was detected by autoradiography and this uptake was sensitive to the phloem loading inhibitor p-chloromercuribenzenesulfonic acid. However, the amount of label taken up by veins in albino leaves was less than that taken up by veins of mature green leaves.

     

Positional distribution of fatty acids in lipids of the marine diatom Phaeodactylum tricornutum

The composition of fatty acids and lipids in the marine diatom, Phaeodactylum tricornutum was determined. The Lipids consisted of monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulphoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphtidylinositol, triacylglycerol and minor unidentified ones. At the early stationary phase of growth, the total fatty acids were mainly 20:5, 16:1, 16:0 and 16:3. 20:5 was distributed in polar lipids, particularly in monogalactosyldiacylglycerol, phosphatidylcholine and phosphatidylglycerol. This fatty acid was exclusively located at the sn-1 position of the glycerol moiety in all polar lipids except for phosphatidylcholine. In phosphatidylcholine 20:5 was distributed at both the sn-1 and sn-2 positions. 16:3 was concentrated at the sn2 position of monogalactosyldiacylglycerol and trans-16:1 (n-13) was dominant at the sn-2 position of phosphatidylglycerol. C₁₈ fatty acids, the minor fatty acids in P. tricornutum, were confined to the sn-2 position of phosphatidylcholine.     

Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves

In barley, glycine betaine is a metabolic end product accumulated by wilted leaves; betaine accumulation involves acceleration of de novo synthesis from serine, via ethanolamine, N-methylethanolamines, choline, and betaine aldehyde (Hanson, Scott 1980 Plant Physiol 66: 342-348). Because in animals and microorganisms the N-methylation of ethanolamine involves phosphatide intermediates, and because in barley, wilting markedly increases the rate of methylation of ethanolamine to choline, the labeling of phosphatides was followed after supplying [¹⁴C]ethanolamine to attached leaf blades of turgid and wilted barley plants. The kinetics of labeling of phosphatidylcholine and betaine showed that phosphatidylcholine became labeled 2.5-fold faster in wilted than in turgid leaves, and that after short incubations, phosphatidylcholine was always more heavily labeled than betaine. In pulse-chase experiments with wilted leaves, label from [¹⁴C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [¹⁴C]monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine.     

Carbon tetrachloride target lipids in the rat liver microsomes. Effect of cystamine administration on their pattern of labeling by 14CCl4

¹⁴C from ¹⁴CCl₄ irreversibly binds to lipids from the smooth (SER) and rough (RER) endoplasmic reticulum. Most of the label is associated with the phospholipid fraction (> 95%). Prior cystamine administration decreased the extent of that binding but does not change its pattern of distribution. About the half of the label in phospholipids is in the phosphatidylcholine fraction; the other half is distributed similarly among lysophosphatidylcholine, sphingomyelin and phosphatidylethanolamine, while only a very minor fraction is associated with diphosphatidyl glycerol. No differences were found in the pattern of labeling of phospholipids in SER and RER.     

Plastid differentiation, acyl lipid, and Fatty Acid changes in developing green maize leaves

Plastid differentiation, acyl lipid, and fatty acid composition have been followed in successive 2-cm sections from the base (youngest tissue) to the tip (oldest tissue) of green Zea mays (maize) leaves grown under a normal diurnal light regime. Although the youngest cells (0-4 cm from the leaf base) had only proplastids with one or two grana, they contained chlorophylls a and b, monogalactosyldiglyceride, digalactosyldiglyceride, sulfolipid, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. In the more mature sections, the plastids increased in size 5-fold, and differentiation into mesophyll and bundle-shealth chloroplasts had occurred. Concomitantly, the levels of all the lipids increased with the exception of phosphatidylcholine and phosphatidylethanolamine which decreased. With increasing cell maturity, the percentage of linolenic acid increased in all the individual acyl lipids, but palmitic acid remained constant in phosphatidylcholine, phosphatidylethanolamine, and sulfolipid. The Δ^3t-hexadecenoic acid was only detectable in the phosphatidylglycerol of the most mature maize tissue.     

Differential responses of a range of photosynthetic tissues to a substituted pyridazinone,sandoz 9785. Specific effects on fatty acid desaturation

The effect of a substituted pyridazinone (4-chloro-5(dimethylamino)-2-phenyl-3(2H)pyridazinone; Sandoz 9785; BASF 13-338) on the formation of fatty acids from radiolabelled precursors has been studied in a number of angiosperms, bryophytes and algae. The labelling of [¹⁴C]linolenic acid was decreased by the herbicide in leaves of barley and rye grass and in cucumber cotyledons regardless of whether [¹⁴C]acetate,[¹⁴C]oleate or [¹⁴C]linoleate was used as precursor. A commensurate increase in the labelling of [¹⁴C]linoleic acid was also observed in these species. In contrast, the pattern of fatty acid labelling in maize, pea and spinach leaves was unaffected by 0.1 mM Sandoz 9785. More generalized inhibition of the incorporation of radioactivity from [¹⁴C]acetate into the fatty acids of bryophytes and algae was seen. Sandoz 9785 did not alter the distribution of radioactivity in different lipid classes of higher plant leaves, nor did it change the proportions of radioactive fatty ac ids in phosphatidylcholine. In contrast to phosphatidylcholine, which never contained more than trace amounts of [¹⁴C]linolenate, diacylgalactosylglycerol contained high levels of the radioactive acid. The relative labelling of linolenate was severely reduced in diacylgalactosylglycerol by Sandoz 9785 in sensitive angiosperms. Uptake studies, in which [³H]Sandoz 9785 was employed demonstrated that the uptake of Sandoz 9785 was reflection of water uptake. Following its uptake, Sandoz 9785 was rapidly converted into other compounds in pea but only gradually metabolized in cucumber and ryegrass. The results are interpreted as showing, firstly, that the different sensitivity of higher plants to Sandoz 9785 is due to variations both in uptake and in metabolism. Secondly, Sandoz 9785 specifically inhibits the desaturation of linoleate to linolenate and, thirdly, diacylgalactosylglycerol plays a role in this conversion.     

Labelling of glycerolipids in the cotyledons of developing oilseeds by [1-14C]acetate and [2-3H]glycerol

1. 3-sn-Phosphatidylcholine was identified as the major lipid in cotyledons from the developing seeds of soya bean, linseed and safflower when tissue was steamed before lipid extraction. The proportion of oleate in this lipid decreased markedly and that of the polyunsaturated C₁₈ fatty acids increased when detached developing cotyledons were incubated for up to 3h. Similar but less pronounced changes occurred in diacylglycerol, which had a fatty acid composition resembling that of the 3-sn-phosphatidylcholine from cotyledons of the same species. 2. [1-¹⁴C]Acetate supplied to detached cotyledons was incorporated into the acyl moieties of mainly 3-sn-phosphatidylcholine, 1,2-diacylglycerol and triacylglycerol. Initially label was predominantly in oleate, but subsequently entered at accelerating rates the linoleoyl moieties of the above lipids in soya-bean and safflower cotyledons and the linoleoyl and linolenyl moieties of these lipids in linseed cotyledons. In pulse–chase experiments label was rapidly lost from the oleate of 3-sn-phosphatidylcholine and accumulated in the linoleoyl and linolenoyl moieties of this phospholipid and of the di- and tri-acylglycerols. 3. [2-³H]Glycerol was incorporated into the glycerol moieties of mainly 3-sn-phosphatidylcholine and di- and tri-acylglycerols of developing linseed and soya-bean cotyledons. The label entered the phospholipid and diacylglycerol at rates essentially linear with time from the moment the substrate was supplied, and entered the triacylglycerol at an accelerating rate. With linseed cotyledons the labelled glycerol was incorporated initially mainly into species of 3-sn-phosphatidylcholine and diacylglycerol that contained oleate, but accumulated with time in more highly unsaturated species. In pulse–chase experiments with linseed cotyledons, label was lost from both 3-sn-phosphatidylcholine and diacylglycerol, preferentially from the dioleoyl species, and accumulated in triacylglycerol, mainly in species containing two molecules of linolenate. 4. The results suggest a rapid turnover of 3-sn-phosphatidylcholine during triacylglycerol accumulation in developing oilseeds, and are consistent with the operation of a biosynthetic route whereby oleate initially esterified to the phospholipid is first desaturated, then polyunsaturated fatty acids transferred to triacylglycerol, via diacylglycerol. The possible role of oleoyl phosphatidylcholine as a substrate for oleate desaturation is discussed.     

Fatty Acid Incorporation in Normal and Degenerating Rat Sciatic Nerve In Vivo

Abstract: Labeled palmitic acid ([16-¹⁴C]palmitate) (0).5 μCi) was injected into rat sciatic nerves in vivo to characterize thc incorporation of this fatty acid into complex peripheral nerve lipids after time lapses of 1 min to 2 weeks. For the first 30 min after intraneural injection, the label was concentrated in nerve diglycerides. Thereafter, the relative diglyccride label declined rapidly, and phospholipid radioactivity rose steadily. After 120 min, phospholipids contained over 70% of the total lipid radioactivity. Among the phospholipids, phosphatidylcholine had the largest percentage of total phospholipid label, and acylation of lysophosphatidylcholine accounted for approximately 75% of this label. With time, there was conversion of [16-¹⁴C]palmitate to other long-chain fatty acids by elongation and desaturation. Phosphatidic acid was labeled also, suggesting the operation of the de novo biosynthetic mechanism. However, the specific radioactivity of 1,2-diacylglycerol was much higher than that of phosphatidic acid, suggesting phosphorylation of diglycerides by diglyceride kinase. After nerve section and survival of 2 h to 50 days, the injection of [16-¹⁴C]palmitate into the degenerating distal segment revealed an overall decline of phospholipid labeling and a commensurate increase of triglyceride radioactivity. Phosphatidylcholine in degenerating nerve contained a larger percentage of the fatty acid label than that in normal nerve. Almost all of the labeling was due to acylation of lysophosphatidylcholine, implying a much smaller contribution of the de novo pathway. Phosphatidylethanolamine and phosphatidylserine showed a relative loss of radioactivity. The changes were apparent at 1 day, but not at 2 h, suggesting loss of homeostatic control, presumably by interruption of axonal flow. An incidental observation was the stimulation of phosphatidylcholine biosynthesis by acylation of lysophosphatidylcholine in the contralateral unoperated sciatic nerve.