L-acetylcarnitine, a substrate for chloroplast fatty acid synthesis

Abstract. H¹⁴CO₃ was not incorporated into fatty acids by isolated pea leaf chloroplasts, which, therefore, do not possess a self-contained pathway for the synthesis of fatty acids from early intermediates of the Calvin cycle. Citrate, pyruvate, acetate and L-acetylcarnitine were all shown to act as sources of acetyl groups for fatty acid synthesis by pea leaf chloroplasts. L-acetylcarnitine was the best substrate, being incorporated into fatty acids at rates that were at least five-fold higher than those achieved with the other substrates. Citrate was incorporated into fatty acids at the lowest rate, followed by pyruvate, with acetate being incorporated at the second highest rate of all. When the isolated chloroplasts were ruptured, an inhibition of L-acetylcarnitine incorporation into fatty acids was noted, whilst acetate incorporation remained unaffected. L-acetylcarnitine also increased the ratio of monoenoic: saturated fatty acids synthesized, compared with a 1:1 ratio observed when citrate, pyruvate and acetate were supplied as substrates. It is suggested that L-carnitine and carnitine acyltransferases play a central role in plant acyl CoA metabolism by facilitating the transfer of activated acyl groups across membranes (acyl CoA barriers).     

High rates of [1-14C]acetate incorporation into the lipid of isolated spinach chloroplasts.

Spinach chloroplasts, isolated by techniques yielding preparations with high O2- evolving activity, showed rates of light-dependent acetate incorporation into lipids 3-4 fold higher than any previously reported. Incorporation rates as high as 500 nmol of acetate/h per mg of chlorophyll were measured in buffered sorbitol solutions containing only NaHCO3 and [1-14C]acetate, and as high as 800 nmol/h per mg of chlorophyll when 0.13 mM-Triton X-100 was also included in the reaction media. The fatty acids synthesized were predominantly oleic (70-80% of the total fatty acid radioactivity) and palmitic (20-25%) with only minor amounts (1-5%) of linoleic acid. Linolenic acid synthesis was not detected in the system in vitro. Free fatty acids accounted for 70-90% of the radioactivity incorporated and the remainder was shared fairly evenly between 1,2-diacylglycerols and polar lipids. Oleic acid constituted 80-90% of the free fatty acids synthesized, but the diacylglycerols and polar lipids contained slightly more palmitic acid than oleic acid. Triton X-100 stimulated the synthesis of diacylglycerols 3-6 fold, but stimulated free fatty acid synthesis only 1-1.5-fold. Added glycerol 1-phosphate stimulated both the synthesis of diacylglycerols and palmitic acid relative to oleic acid, but did not increase acetate incorporation into total chloroplast lipids. CoA and ATP, when added separately, stimulated acetate incorporation into chloroplast lipids to variable extents and had no effect on the types of lipid synthesized, but when added together resulted in 34% of the incorporated acetate appearing in long-chain acyl-CoA. Pyruvate was a much less effective precursor of chloroplast fatty acids than was acetate.     

Control of plastidic glycolipid synthesis and its relation to chlorophyll formation

Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv “Halo”) and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [¹⁴C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [¹⁴C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [¹⁴C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [¹⁴C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat-bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [¹⁴C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity.     

Lipid biosynthesis by intact mesophyll and bundle sheath chloroplasts from maize

The two dimorphic forms of chloroplast isolated from maize leaves utilized acetate for fatty acid biosynthesis and had similar requirements for cofactors. The oleate:palmitate ratio of the fatty acid products was lower for bundle sheath chloroplasts as was acetate incorporation into total fatty acids. Galactose from UDP-galactose was incorporated into galactolipids by both morphological forms to give monogalactosyl diacylglycerol and digalactosyl diacylglycerol in the ratio of 4:1.     

On the control of long-chain-fatty acid synthesis in isolated intact spinach (Spinacia oleracea) chloroplasts

1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510-512] had higher rates of HCO(3) (-)-dependent oxygen evolution (up to 369mumol/h per mg of chlorophyll) and higher rates of [1-(14)C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H(14)CO(3) (-) in assays permitting high rats of photosynthesis. Incorporation of H(14)CO(3) (-) into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3-4 times in excess of that required to account for rates of [1-(14)C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloroplasts. 3. Rates of long-chain-fatty acid synthesis from [1-(14)C]acetate in the highly active chloroplast preparations, compared with those used previously, were less dependent on added cofactors, but showed a greater response to light. The effects of added CoA plus ATP, Triton X-100 and sn-glycerol 3-phosphate on the products of [1-(14)C]acetate incorporation were similar to those reported for less active chloroplast preparations. 4. Endogenous [(14)C]acetyl-CoA plus [(14)C]malonyl-CoA was maintained at a constant low level even when fatty acid synthesis was limited by low HCO(3) (-) concentrations. Endogenous [(14)C]acyl-(acyl-carrier protein) concentrations increased with increasing HCO(3) (-) concentration and higher rates of fatty acid synthesis, but were slightly lower in the presence of Triton X-100. It is proposed that rates of long-chain-fatty acid synthesis in isolated chloroplasts at saturating [1-(14)C]acetate concentrations and optimal HCO(3) (-) concentrations may be primarily controlled by rates of removal of the products of the fatty acid synthetase.     

Lipid biosynthesis in adult Acyrthosiphon pisum: Effect of age and symbiont population

The incorporation of [1-¹⁴C]acetate into total lipids in 1-day-old adult pea aphids is 3.3-fold higher than in 20-, 22-, and 24-day old adults. The polar lipid fraction was the main lipid class synthesized at any age and contained primarily eighteen carbon fatty acids. While the relative mass of 18:0, 18:1 and 18:2 decreased in older aphids, the relative amount of label incorporated into these fatty acids remained constant. Myristic acid was the main fatty acid of the triacylglycerol fraction, and the relative amount of radioactivity incorporated into this fatty acid decreased in older aphids. Twenty-day-old aphids had 60% fewer mycetocytes than did 1-day-old insects. We conclude that symbionts within the mycetocytes do not appear to be involved in the synthesis of linoleic acid, while their role in the synthesis of myristic acid is less clear.     

Fatty acid synthesis in pea root plastids is inhibited by the action of long-chain acyl- coenzyme as on metabolite transporters.

The uptake in vitro of glucose (Glc)-6-phosphate (Glc-6-P) into plastids from the roots of 10- to 14-d-old pea (Pisum sativum L. cv Puget) plants was inhibited by oleoyl-coenzyme A (CoA) concentrations in the low micromolar range (1--2 microM). The IC(50) (the concentration of inhibitor that reduces enzyme activity by 50%) for the inhibition of Glc-6-P uptake was approximately 750 nM; inhibition was reversed by recombinant rapeseed (Brassica napus) acyl-CoA binding protein. In the presence of ATP (3 mM) and CoASH (coenzyme A; 0.3 mM), Glc-6-P uptake was inhibited by 60%, due to long-chain acyl-CoA synthesis, presumably from endogenous sources of fatty acids present in the preparations. Addition of oleoyl-CoA (1 microM) decreased carbon flux from Glc-6-P into the synthesis of starch and through the oxidative pentose phosphate (OPP) pathway by up to 73% and 40%, respectively. The incorporation of carbon from Glc-6-P into fatty acids was not detected under any conditions. Oleoyl-CoA inhibited the incorporation of acetate into fatty acids by 67%, a decrease similar to that when ATP was excluded from incubations. The oleoyl-CoA-dependent inhibition of fatty acid synthesis was attributable to a direct inhibition of the adenine nucleotide translocator by oleoyl-CoA, which indirectly reduced fatty acid synthesis by ATP deprivation. The Glc-6-P-dependent stimulation of acetate incorporation into fatty acids was reversed by the addition of oleoyl-CoA.     

Effects of the selective herbicide fluazifop on fatty acid synthesis in pea (Pisum sativum) and barley (Hordeum vulgare).

Concentrations of fluazifop-butyl sprayed on intact plants caused large decreases in the incorporation of radioactivity from [1-14C]acetate into lipids of barley (Hordeum vulgare) leaves and stems, but did not affect leaves or stems of pea (Pisum sativum). Labelling of all acyl lipids, but not pigments, was reduced. The effects of the active acid form, fluazifop, were also determined in leaf pieces and chloroplasts. Concentrations of (R,S)-fluazifop up to 100 microM had no affect upon quality or quantity of fatty acids produced from [1-14C]acetate in pea. In barley, however, 100 microM-(R,S)-fluazifop caused 89% (leaf) or 100% (chloroplasts) inhibition in labelling of fatty acids from [1-14C]acetate. Lower concentrations of fluazifop (less than 25 microM) caused incomplete inhibition and significant decreases in the proportion of C18 fatty acids synthesized, particularly by isolated chloroplasts. Synthesis of fatty acids from [2-14C]malonate was also inhibited (59%) in barley leaf tissue by 100 microM-(R,S)-fluazifop. The labelling pattern of products showed that elongation reactions were unaffected by the herbicide, but synthesis de novo was specifically diminished. By using resolved stereoisomers, it was found that the (R) isomer was the form which inhibited fatty acid synthesis, a finding that is in agreement with its herbicidal activity. These results suggest that inhibition of fatty acid synthesis de novo forms the basis for the selective mode of action of fluazifop.     

The effects of specific lipogenic substrates and metabolic inhibitors on de Novo fatty acid synthesis in isolated hepatocytes from chow-fed female rats

The effects of glucose (10 mm), glycerol (3 mm), and lactate/pyruvate (10 mm) on the incorporation of ³H from ³H₂O into fatty acids were studied in isolated hepatocytes prepared from chow-fed female rats. Lactate/pyruvate markedly increased lipogenic rates, while glucose and glycerol did not significantly affect rates of lipogenesis. In cells incubated with lactate/pyruvate plus glycerol, the increase in ³H incorporation was greater than observed with lactate/pyruvate alone. In hepatocytes isolated from 24-h starved rats, lactate/pyruvate again increased de novo fatty acid synthesis to a greater extent than either glucose or glycerol. Glycerol significantly increased lipogenesis compared to the endogenous rates and when incubated with lactate/pyruvate produced an increase above lactate/pyruvate alone. (?)-Hydroxycitrate, a potent inhibitor of ATP-citrate lyase (EC 4.1.3.8), and agaric acid, an inhibitor of tricarboxylate anion translocation, were studied in hepatocytes to determine their effects on lipogenesis by measuring ³H₂O, [1-¹⁴C]acetate, and [2-¹⁴C]lactate incorporation into fatty acids. ³H incorporation into fatty acids was markedly inhibited by both inhibitors with agaric acid (60 μm) producing the greater inhibition. (?)-Hydroxycitrate (2 mm) increased acetate incorporation into fatty acids from [1-¹⁴C]acetate and agaric acid produced a strong inhibitory effect. Combined effects of (?)-hydroxycitrate and agaric acid on lipogenesis from [1-¹⁴C]acetate showed an inhibitory response to a lesser extent than with agaric acid alone. With substrate concentrations of acetate present, there was no significant increase in rates of lipogenesis from [1-¹⁴C]acetate and the increase previously observed with (?)-hydroxycitrate alone was minimized. Agaric acid significantly inhibited fatty acid synthesis from acetate in the presence of exogenous substrate, but the effect was decreased in comparison to rates with only endogenous substrate present. With [2-¹⁴C]lactate as the lipogenic precursor, agaric acid and (?)-hydroxycitrate strongly inhibited fatty acid synthesis. However, agaric acid despite its lower concentration (60 μm vs 2 mm) was twice as effective as (?)-hydroxycitrate. A similar pattern was observed when substrate concentrations of lactate/pyruvate (10 mm) were added to the incubations. When (?)-hydroxycitrate and agaric acid were simultaneously incubated in the presence of endogenous substrate, there was an additive effect of the inhibitors on decreasing fatty acid synthesis. Results are discussed in relation to the origin of substrate for hepatic lipogenesis and whether specific metabolites increase lipogenic rates.     

Changes in Arachidonic Acid Levels and Formation and in Lipid Synthesis in the Human Neuroblastoma SK-N-BE During Retinoic Acid-Induced Differentiation

Abstract: We observed that retinoic acid, which differentiates the human neuroblastoma SK-N-BE into mature neurons, induced an elevation in levels of polyunsaturated fatty acids, especially arachidonic acid (20:4 n-6). This effect was not induced by phorbol myristate acetate, another differentiating agent. We then explored the effects of retinoic acid on the formation of arachidonic acid and of docosahexaenoic acid from precursors and on the de novo lipid synthesis from acetate at various stages of differentiation, which was assessed by morphological (cell number and neurite outgrowth) and biochemical (protein content and thymidine incorporation) criteria. At 3 days of incubation with retinoic acid, in the n-6 series, total conversion of linoleic acid, especially to 20:3 n-6, was elevated, in association with preferential incorporation of acetate into phospholipids; in contrast, at 8 days, synthesis of 20-carbon polyunsaturated fatty acids declined, in association with enhanced incorporation in triglycerides. In the n-3 series, eicosapentaenoic acid was converted to docosahexaenoic acid in SK-N-BE, but the conversion was not affected by retinoic acid. During the early stage of neuronal differentiation, therefore, enhanced production of 20-carbon polyunsaturated fatty acids from their precursors occurred, and newly formed fatty acids were preferentially incorporated in phospholipids, possibly in association with membrane deposition. When differentiation was completed, arachidonic acid formation and incorporation of acetate in phospholipids and cholesterol declined with enhanced labeling of storage lipids.     

The regulation of glyceride synthesis in isolated white-fat cells. The effects of acetate, pyruvate, lactate, palmitate, electron-acceptors, uncoupling agents and oligomycin

1. The incorporation of 5mm-[U-(14)C]glucose into glyceride fatty acids by fat cells from normal rats incubated in the presence of 20munits of insulin/ml was increased by acetate, pyruvate, palmitate, NNN'N'-tetramethyl-p-phenylenediamine, phenazine methosulphate, dinitrophenol, tetrachlorotrifluoromethyl benzimidazole and oligomycin. Lactate did not stimulate glucose incorporation into fatty acids. The effects of these agents were concentration-dependent. 2. In the presence of 5mm-glucose+insulin, [U-(14)C]acetate, [U-(14)C]pyruvate and [U-(14)C]lactate were incorporated into fatty acids in a concentration-dependent manner, thereby further increasing the total rate of fatty acid synthesis. 3. NNN'N'-tetramethyl-p-phenylenediamine decreased the incorporation of [U-(14)C]pyruvate into fatty acids in normal cells and increased the incorporation of [U-(14)C]lactate into fatty acids. 4. In fact cells from 72h-starved rats the stimulatory effects of NNN'N'-tetramethyl-p-phenylenediamine upon glucose and lactate incorporation into fatty acids were totally and partially abolished respectively whereas the stimulatory effects of acetate upon glucose incorporation were retained. 5. Combinations of the optimum concentrations of the substances that stimulate glucose incorporation into fatty acids were tested and compared. The effects of acetate+NNN'N'-tetramethyl-p-phenylenediamine and acetate+palmitate upon normal cells were additive. The effects of NNN'N'-tetramethyl-p-phenylenediamine+palmitate were not additive. It was found that total fatty acid synthesis in the presence of glucose was most effectively increased by raising the concentration of pyruvate in the incubation system. 6. The significance of these results in supporting the proposal that fatty acid synthesis from glucose in adipose tissue is a ;self-limiting process' is discussed.     

Lipid biosynthesis by isolated plastids from greening pea, Pisum sativum

Isolated etioplasts from 8-day-old dark-grown pea seedlings incorporated [1-(14)C]acetate into lipid at a relatively low rate. Plastids from seedlings that had been illuminated for at least 2 hr showed an enhanced incorporation provided the plastids were illuminated during incubation with the labeled acetate. Dark incubation or the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased the acetate-incorporating activity of the developing chloroplasts to the level observed with etioplasts. Light had a marked effect on the type of fatty acid into which acetate was incorporated by the developing chloroplasts. Unsaturated fatty acids (mostly oleic acid) accounted for 60-80% of the incorporated label if the plastids were illuminated, but in the dark or in the presence of DCMU the unsaturated acids accounted for only 0-15% of the label incorporated into lipid. The effect of ATP on incorporation was dependent on the maturity of the chloroplasts; mature pea chloroplasts were inhibited by ATP, whereas in developing plastids there was a slight stimulation by ATP. Inhibition of acetate incorporation into lipid by DCMU appears to be due to inhibition of noncyclic phosphorylation. Incorporation was restored by reduced 2,3,5,6-tetramethylphenylenediamine, which restored phosphorylation, but not by reduced N,N,N',N'-tetramethylphenylenediamine.     

Metabolism of tween-Fatty Acid esters by cultured soybean cells : kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous Fatty Acid synthesis

Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-¹⁴C]-fatty acids and in cells treated with [1-¹⁴C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-¹⁴C]acetate or Tween-[1-¹⁴C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-¹⁴C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1-¹⁴C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism.     

Fatty Acid Specificity and Selectivity of the Chloroplast sn-Glycerol 3-Phosphate Acyltransferase of the Chilling Sensitive Plant, Amaranthus lividus

Chilling sensitivity of plants is strongly correlated with the presence of high levels of a species of chloroplast phosphatidylglycerol that contains two saturated fatty acids. The most straightforward synthetic pathway for this lipid would require the primary acylation of sn-glycerol 3-phosphate (G3P) with a saturated fatty acid (palmitic acid) rather than with oleic acid, an unsaturated acid. This selective incorporation would differ markedly from the reported properties of the chloroplast G3P acyltransferases of pea and spinach, two chilling resistant plants and thus we have studied the chloroplast G3P acyltransferase of Amaranthus lividus, a chilling sensitive plant. In contrast to our results and those of others (M. Frentzen et al. 1983 Eur J Biochem 129: 629-636 and previous work) with the pea and spinach enzymes, the amaranthus chloroplast G3P acyltranferase did not select oleic acid donors from a mixture of oleic and palmitic acid donors (either coenzyme A or acyl carrier protein thioesters). Instead the fatty acid composition of the synthesized 1-acyl G3P faithfully reflected the composition of the acyl donor mixture. However, the amaranthus enzyme did strongly select against incorporation of stearic acid. The properties of the amaranthus G3P acyltransferase are consistent with this enzyme having the major role in synthesis of the disaturated phosphatidylglycerol species.     

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.     

Malate- and pyruvate-dependent Fatty Acid synthesis in leucoplasts from developing castor endosperm

Leucoplasts were isolated from the endosperm of developing castor (Ricinis communis) endosperm using a discontinuous Percoll gradient. The rate of fatty acid synthesis was highest when malate was the precursor, at 155 nanomoles acetyl-CoA equivalents per milligram protein per hour. Pyruvate and acetate also were precursors of fatty acid synthesis, but the rates were approximately 4.5 and 120 times less, respectively, than when malate was the precursor. When acetate was supplied to leucoplasts, exogenous ATP, NADH, and NADPH were required to obtain maximal rates of fatty acid synthesis. In contrast, the incorporation of malate and pyruvate into fatty acids did not require a supply of exogenous reductant. Further, the incorporation of radiolabel into fatty acids by leucoplasts supplied with radiolabeled malate, pyruvate, or acetate was reduced upon coincubation with cold pyruvate or malate. The data suggest that malate and pyruvate may be good in vivo sources of carbon for fatty acid synthesis and that, in these preparations, leucoplast fatty acid synthesis may be limited by activity at or downstream of the acetyl-CoA carboxylase reaction.     

Acyl-CoA dependent acylation of phospholipids in the chloroplast envelope

Acyl-CoAs are substrates for acyl lipid synthesis in the endoplasmic reticulum. In addition, they may also be substrates for lipid acylation in other membranes. In order to assess whether lipid acylation may have a role in plastid lipid metabolism, we have studied the incorporation of radiolabelled fatty acids from acyl-CoAs into lipids in isolated, intact pea chloroplasts. The labelled lipids were phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol and free fatty acids. With oleoyl-CoA, the fatty acid was incorporated preferably into the sn-2 position of PC and the acylation activity mainly occurred in fractions enriched in inner chloroplast envelope. Added lysoPC stimulated the activity. With palmitoyl-CoA, the fatty acid was incorporated primarily into the sn-1 position of PG and the reaction occurred at the surface of the chloroplasts. As chloroplast-synthesized PG generally contains 16C fatty acids in the sn-2 position, we propose that the acylation of PG studied represents activities present in a domain of the endoplasmic reticulum or an endoplasmic reticulum-derived fraction that is associated with chloroplasts and maintains this association during isolation. This domain or fraction contains a discreet population of lipid metabolizing activities, different from that of bulk endoplasmic reticulum, as shown by that with isolated endoplasmic reticulum, acyl-CoAs strongly labelled phosphatidic acid and phosphatidylethanolamine, lipids that were never labelled in the isolated chloroplasts.     

Inhibition of plant acetyl-coenzyme A carboxylase by the herbicides sethoxydim and haloxyfop

Incorporation of [14C]acetate or [14C]pyruvate into fatty acids in isolated corn seedling chloroplasts was inhibited 90% or greater by 10 microM sethoxydim or 1 microM haloxyfop. At these concentrations, neither sethoxydim nor haloxyfop inhibited [14C]acetate incorporation into fatty acids in isolated pea chloroplasts. Sethoxydim (10 microM) and haloxyfop (1 microM) did not inhibit incorporation of [14C]malonyl-CoA into fatty acids in cell free extracts from corn tissue cultures. Acetyl coenzyme A carboxylase (EC 6.4.1.2) from corn seedling chloroplasts was inhibited by both sethoxydim and haloxyfop, with I50 values of 2.9 and 0.5 microM, respectively. This enzyme in pea was not inhibited by 10 microM sethoxydim or 1 microM haloxyfop.     

De novo synthesis and elongation of fatty acids by subcellar fractions of monkey aorta

Subcellular fractions of aorta of squirrel monkey (Saimiri sciureus) were examined for their ability to synthesize and elongate fatty acids. High-speed supernate (HSS) incorporated substantial quantities of malonyl CoA into fatty acids while acetyl CoA was much less effectively utilized. Acetyl-CoA carboxylase activity exceeded the amount of acetyl CoA incorporated into fatty acids and thus does not account for the low incorporation of this substrate. Microsomes used malonyl CoA and acetyl CoA equally well; mitochondria incorporated either acetyl CoA or acetate. The amounts of substrate incorporated into fatty acids (m micro moles/mg of protein per hr) were 2.3 for HSS, 1.2 for microsomes, and 0.9 for mitochondria. The synthesized fatty acids were separated by gas-liquid chromatography, radioassayed, extracted from the scintillation fluid, and decarboxylated. HSS completely synthesized palmitic and stearic acids from malonyl CoA. Microsomes and mitochondria utilized acetyl CoA to elongate endogenous fatty acids and gave mainly palmitic, stearic, and C(18) and C(20) monoenoic acids, with lesser amounts of other saturated and unsaturated fatty acids. A significant quantity of malonyl CoA was utilized by microsomes to yield a fatty acid tentatively identified as docosapentaenoic. Radioactive fatty acids are incorporated into various lipid classes by the particulate preparations. These studies demonstrate that aortic tissue in a nonhuman primate is able to carry out several processes of fatty acid metabolism and that the aortic synthesis and elongation of fatty acids may play an important role in providing fatty acids for incorporation into aortic lipids.     

Fatty acid biosynthesis by a particulate preparation from germinating pea

1. Fatty acid synthesis was studied in microsomal preparations from germinating pea (Pisum sativum). 2. The preparations synthesized a mixture of saturated fatty acids up to a chain length of C(24) from [(14)C]malonyl-CoA. 3. Whereas hexadecanoic acid was made de novo, octadecanoic acid and icosanoic acid were synthesized by elongation. 4. The products formed during [(14)C]malonyl-CoA incubation were analysed, and unesterified fatty acids and polar lipids were found to be major products. [(14)C]Palmitic acid represented a high percentage of the acyl-carrier protein esters, whereas (14)C-labelled very-long-chain fatty acids were mainly present as unesterified fatty acids. CoA esters were minor products. 5. The addition of exogenous lipids to the incubation system usually resulted in stimulation of [(14)C]malonyl-CoA incorporation into fatty acids. The greatest stimulation was obtained with dipalmitoyl phosphatidylcholine. Both exogenous palmitic acid and dipalmitoyl phosphatidylcholine increased the amount of [(14)C]-stearic acid synthesized, relative to [(14)C]palmitic acid. Addition of stearic acid increased the amount of [(14)C]icosanoic acid formed. 6. [(14)C]Stearic acid was elongated more effectively to icosanoic acid than [(14)C]stearoyl-CoA, and its conversion was not decreased by addition of unlabelled stearoyl-CoA. 7. Incorporation of [(14)C]malonyl-CoA into fatty acids was markedly decreased by iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid). Palmitate elongation was sensitive to arsenite addition, and stearate elongation to the presence of Triton X-100 or fluoride. The action of fluoride was not, apparently, due to chelation. 8. The microsomal preparations differed from soluble fractions from germinating pea in (a) synthesizing very-long-chain fatty acids, (b) not utilizing exogenous palmitate-acyl-carrier protein as a substrate for palmitate elongation and (c) having fatty acid synthesis stimulated by the addition of certain complex lipids.