Effect of Thiolactomycin on Lipid Synthesis in Higher Plants

The effect of thiolactomycin (TLM), an inhibitor of type IIfatty acid synthase, on lipid synthesis in greening tissueswas examined. Pulse-chase experiments with Na[1-¹⁴C]acetatefor greening Avena leaves showed that continuous administrationof TLM (100µg/ml) decisively reduced phosphatidylcholine(PC) synthesis from acetate and blocked the subsequent conversionof PC to monogalactocyldiacylglycerol (MGDG), whereas temporaladministration of TLM (100 µ/ml) reduced PC synthesisfrom acetate by only 50% and did not block the conversion ofPC to MGDG. In the reduced PC synthesis, the ratio of oleicto palmitic acid decreased at earlier stages of greening, reflectingmore suppression of oleic acid synthesis. In later greeningstages the modulated fatty acid composition recovered to thenormal composition. In further steps, the fatty acid compositionwas not affected by TLM throughout the greening stages. Greeningof either etiolated Avena leaves or etiolated Brassica cotyledonsin the presence of TLM led to a marked decrease in the contentsof MGDG, digalactosyldiacylglycerol (DGDG) and phosphatidylglycerol(PG), but only a small change in the fatty acid compositionof their lipids. The only inhibition characteristic of TLM wasthe desaturation of palmitic to 3-trans-hexadecenoic acid inAvena leaf PG. These results suggest the presence of a mechanismby which the modulated fatty acid composition of lipids is normalizedin the flow of the synthesis. Electron microscopic observationsshowed that Avena chloroplasts developed into round forms ratherthan normal ellipse forms and the thylakoid membranes of Brassicachloroplasts were abnormally swollen everywhere in the presenceof TLM. Photosynthetic oxygen evolution in both tissues wasnot inhibited. (Received December 26, 1986; Accepted April 24, 1987)     

Development of Lipid Synthesis from CO2 in Avena Leaves during Greening of Etiolated Seedlings

The development of the lipid synthesizing system in Avena leafsections was examined in connection with carbon fixation duringthe greening of etiolated seedlings under light. During theinitial 2 h illumination there was a low level of CO₂ fixationby PEP carboxylation, but its products, malate and citrate,did not serve as a carbon source for lipid synthesis, althoughlipid synthesis from acetate had already been established. Withthe initiation of Calvin cycle activity after the initial 2h illumination, lipid synthesis began, with CO₂ fixed by RuBPcarboxylation serving exclusively as the carbon source. Fattyacid synthesis in the leaves during the initial 3 h illumination,unlike the fatty acid synthesis thereafter, was insensitiveto thiolactomycin, an inhibitor of type II fatty acid synthetasecontained in the plastids, and was not dependent on light, incontrast to light-dependent activity in greened leaves. The distribution of ¹⁴C incorporated into lipid molecules fromNaH¹⁴CO₃ showed an equal ratio of ¹⁴C in fatty acid, glyceroland choline moieties of labeled phosphatidylcholine, but a denserradioactivity in the galactose moiety than in the residual moietyof mono- and di-galactosyldiacylglycerols. This suggests a regulatedsupply of glycerol, choline and fatty acid moieties for phosphatidylcholinesynthesis, and an excess supply of galactose to diacylglycerolmoiety for galactosyldiacylglycerol synthesis in Avena leaves. (Received October 31, 1984; Accepted January 25, 1985)     

Lipid composition and metabolism in megakaryocytes at different stages of maturation

The lipid composition and metabolism of isolated guinea pig megakaryocyte subgroups at various stages of maturation were investigated. Three groups were studied: 1) 67% of megakaryocytes in Group A were immature; 2) Group B was heterogeneous and contained both immature and mature subgroups of megakaryocytes; 3) 92% of megakaryocytes in Group C were mature. Lipid composition was determined by thin-layer chromatography, lipid-phosphorus, and gas-liquid chromatography. Cholesterol, ceramide, and de novo fatty acid synthesis were evaluated with [14C]acetate. [14C]Glycerol was used to assess de novo phospholipid synthesis. 14C-Labeled fatty acids were used to evaluate fatty acid uptake. The phospholipid and cholesterol content was found to be four times greater in mature megakaryocytes than that in immature megakaryocytes, which paralleled the protein content and volume of mature and immature cells. The cholesterol-phospholipid ratio was similar and there were no differences in the phospholipid species in the three groups. Phospholipid and cholesterol synthesis were established in immature megakaryocytes and persisted at about the same level in mature megakaryocytes. The uptake of arachidonic and palmitic acids also occurred primarily in immature cells, while the de novo synthesis of palmitic acid occurs predominantly in mature megakaryocytes. There was an inverse relationship between the uptake of exogenous palmitic acid and fatty acid synthesis, but the uptake of palmitic acid primarily inhibited fatty acid synthesis in mature megakaryocytes. There were differences in the acylation of phospholipid species with arachidonic acid in megakaryocytes at different stages of maturation since the acylation of phosphatidylcholine occurred primarily in immature megakaryocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seasonal changes in activity of the enzyme system producing cis-3-hexenal and n-hexanal from linolenic and linoleic acids in tea leaves

The enzyme system producing cis-3-hexenal, a precursor of cis-3-hexenol(leaf alcohol) and trans-2-hexenal (leaf aldehyde), from linolenicacid showed high activity in summer and no activity in winterin tea (Thea sinensis) leaves and isolated chloroplasts. Theenzyme system producing n-hexanal from linoleic acid also showedsimilar seasonal changes in activity. These changes were closelyrelated to temperature and solar radiation. Enzyme activitycould not be induced after the leaves had been cut and was notaccompanied by de novo protein synthesis. (Received July 9, 1976; )     

Effect of prostaglandins on fatty acid metabolism in lung

This is the first report of the effect of prostaglandins on the biochemical pathways for fatty acid synthesis. PGE₂ and PGF_2α inhibited fatty acid elongation in a lung microsomal fraction. Neither prostaglandin affected the de novo, or soluble, system for fatty acid synthesis (i.e. acetyl CoA carboxylase or fatty acid synthetase). The results also suggest that the initial inhibition of fatty acid synthesis leads to a decrease in free fatty acids available for esterification into phospholipids. The site and possible mechanisms of inhibition are discussed.     

Abscisic Acid Stimulation of the Formation of an Ageing-specific Nuclease in Avena Leaves

In excised Avena leaves, depending on the duration of treatment,abscisic acid (10^–5 M) had two distinctly different effectson the level of individual nucleases. In short-term experiments(3-h treatment, abscisic acid increased the level of a relativelypurine (guanine)-specific ribonuclease, in comparison with thewater control. Accumulation of the abscisie acid-induced ribonuclease,however, levelled off rapidly during incubation and the amountof the enzyme approached a plateau in about 6 h. As the accumulationof this ribonuclease became retarded, abscisic acid induceda striking increase in the level of another nuclease, an enzymenon-specific in relation to the sugar moiety but exhibitinga relative adenine specificity. This latter nuclease also wasshown to accumulate slowly in intact Avena leaves during ‘natural’senescence. The Avena leaves contain, in small concentrations,a chromatographic variant of the sugar non-specific nuclease.This minor variant, despite its identical enzymological properties,was found to be physiologically different from the main componentin that its concentration did not depend on the age of the tissuesand was not affected by abscisic acid.     

Etomoxir mediates differential metabolic channeling of fatty acid and glycerol precursors into cardiolipin in H9c2 cells

We examined the effect of etomoxir treatment on de novo cardiolipin (CL) biosynthesis in H9c2 cardiac myoblast cells. Etomoxir treatment did not affect the activities of the CL biosynthetic and remodeling enzymes but caused a reduction in [1-14C]palmitic acid or [1-14C]oleic acid incorporation into CL. The mechanism was a decrease in fatty acid flux through the de novo pathway of CL biosynthesis via a redirection of lipid synthesis toward 1,2-diacyl-sn-glycerol utilizing reactions mediated by a 35% increase (P < 0.05) in membrane phosphatidate phosphohydrolase activity. In contrast, etomoxir treatment increased [1,3-3H]glycerol incorporation into CL. The mechanism was a 33% increase (P < 0.05) in glycerol kinase activity, which produced an increased glycerol flux through the de novo pathway of CL biosynthesis. Etomoxir treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thereby channeling both glycerol and fatty acid away from 1,2,3-triacyl-sn-glycerol utilization toward phosphatidylcholine and phosphatidylethanolamine biosynthesis. In contrast, etomoxir inhibited myo-[3H]inositol incorporation into phosphatidylinositol and the mechanism was an inhibition in inositol uptake. Etomoxir did not affect [3H]serine uptake but resulted in an increased formation of phosphatidylethanolamine derived from phosphatidylserine. The results indicate that etomoxir treatment has diverse effects on de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir mediates a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL.     

Fatty Acids in Buckwheat are Growth Inhibitors

Four fatty acids, palmitic, stearic, arachidic and behenic acids,were identified (using gas chromatography-mass spectrometry)from buckwheat seedlings. These fatty acids at a concentrationof 250 ppm caused a slight but significant inhibition in growthof rice seedlings. Fagopyrum cymosum, fatty acid, growth inhibitor     

Regulation of fetal lung disaturated phosphatidylcholine synthesis by de novo palmitate supply

Lung surfactant disaturated phosphatidylcholine (PC) is highly dependent on the supply of palmitate as a source of fatty acid. The purpose of this study was to investigate the importance of de novo fatty acid synthesis in the regulation of disaturated PC production during late prenatal lung development. Choline incorporation into disaturated PC and the rate of de novo fatty acid synthesis was determined by the relative incorporation of [14C]choline and 3H2O, respectively, in 20-day-old fetal rat lung explants and in 18-day-old explants which were cultured 2 days. Addition of exogenous palmitate (0.15 mM) increased (26%) choline incorporation into disaturated PC but did not inhibit de novo fatty acid synthesis, as classically seen in other lipogenic tissue. Even in the presence of exogenous palmitate, de novo synthesis accounted for 87% of the acyl groups for disaturated PC. Inhibition of fatty acid synthesis by agaric acid or levo-hydroxycitrate decreased the rate of choline incorporation into disaturated PC. When explants were subjected to both exogenous palmitate and 60% inhibition of de novo synthesis, disaturated PC synthesis was below control values and 75% of disaturated PC acyl moieties were still provided by de novo synthesis. These data show that surfactant disaturated PC synthesis is highly dependent on the supply of palmitate from de novo fatty acid synthesis.     

Fatty Acid Transport and Utilization for the Developing Brain

Abstract: To determine the transport and utilization of dietary saturated, monounsaturated, and n-6 and n-3 polyunsaturated fatty acids for the developing brain and other organs, artificially reared rat pups were fed a rat milk substitute containing the perdeuterated (each 97 atom% deuterium) fatty acids, i.e., palmitic, stearic, oleic, linoleic, and linolenic, from day 7 after birth to day 14 as previously described. Fatty acids in lipid extracts of the liver, lung, kidney, and brain were analyzed by gas chromatography-mass spectrometry to determine their content of each of the deuterated fatty acids. The uptake and metabolism of perdeuterated fatty acid lead to the appearance of three distinct groups of isotopomers: the intact perdeuterated, the newly synthesized (with recycled deuterium), and the natural unlabeled fatty acid. The quantification of these isotopomers permits the estimation of uptake and de novo synthesis of these fatty acids. Intact perdeuterated palmitic, stearic, and oleic acids from the diet were found in liver, lung, and kidney, but not in brain. By contrast, perdeuterated linoleic acid was found in all these organs. Isotopomers of fatty acid from de novo synthesis were observed in palmitic, oleic, and stearic acids in all tissues. The highest enrichment of isotopomers with recycled deuterium was found in the brain. The data indicate that, during the brain growth spurt and the prelude to myelination, the major saturated and monounsaturated fatty acids in brain lipids are exclusively produced locally by de novo biosynthesis. Consequently, the n-6 and n-3 polyunsaturated fatty acids must be transported and delivered to the brain by highly specific mechanisms.     

Hormonal Control of Nuclease Level in Excised Avena Leaf Tissues1

In excised Avena leaves, kinetin and benzyladenine decreased,while abscisic acid and benzimidazole increased the over-allnuclease level. Significant effects were observed as early as2 h after treatment. Not all the nucleases of the Avena leafwere affected by the growth regulators. Changes in over-allnuclease activity were accounted for almost entirely by changesin the amount of a relatively purine-specific endo-ribonuclease,which produces 2',3'-cyclic phosphates as breakdown products.Slight changes induced by the growth regulators were also detectedin the amount of a sugar non-specific endo-nuclease which produces5'-nucleotides and has a relative specificity for adenylic acid.The level of an alkaline phosphodiesterase, an exo-nucleasewhich produces 5'-nucleotides, was not affected by any of thegrowth regulators tested. Gibberellic acid and indol-3yl-aceticacid did not influence the level of Avena nucleases.     

Progressive hypoxia inhibits the de novo synthesis of galactosylceramide in cultured oligodendrocytes

Neonatal rat oligodendrocyte (OLG) cultures exposed to 6 h of gradual, progressive hypoxia in a GasPak (BBL, Becton Dickinson) apparatus were not injured or metabolically impaired, but instead showed a specific inhibition of de novo synthesis (measured by [3H]palmitic acid labeling) of the major myelin component galactosylceramide (GalCer). De novo synthesis of the 2-hydroxy fatty acid GalCer (HFA-GalCer) species, which requires O2 for its synthesis, was most severely inhibited (by 65%), while non-hydroxy GalCer species (NFA-GalCer) were less affected. The synthesis of membrane glycerophospholipids and sphingomyelin was unaffected by hypoxia. Treatment of OLG with 12 nM oligomycin, an inhibitor of mitochondrial ATP synthesis, resulted in an inhibition (by 50-60%) of synthesis of all GalCer species. [3H]Palmitate labeling of NFA-ceramide, the ungalactosylated precursor of NFA-GalCer species, increased in both hypoxia and oligomycin treatments, suggesting that the conversion of newly synthesized ceramide to GalCer was blocked. Newly synthesized HFA-ceramide did not accumulate in OLG, but the small labeled HFA-ceramide pool present during hypoxia was not converted into HFA-GalCer. Pulse-chase studies indicated that NFA- and HFA-ceramides labeled during these treatments were available for galactosylation and could be converted into GalCer upon reoxygenation. [3H]Galactose labeling of NFA-GalCer species was enhanced 2-fold in hypoxia, in contrast to the inhibition seen with [3H]palmitic acid labeling. Thus, while de novo GalCer synthesis was blocked in hypoxia, galactosylation of pre-existing ceramide pools was actually enhanced. Our evidence suggests that hypoxia results in a reversible inhibition of transport of newly synthesized ceramide from its site of synthesis to its site of galactosylation, but causes an increase in galactosylation of subcellular pools of pre-existing ceramide.     

Albumin and fatty acid effects on the stimulated production of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) by human polymorphonuclear leukocytes.

Production of platelet-activating factor (PAF) during opsonized zymosan stimulation of human polymorphonuclear leukocytes is dependent on the concentration of extracellular albumin and on the presence of exogenous fatty acids. Fatty acid-free albumin caused a concentration-dependent increase in PAF synthesis up to 5% albumin concentrations (w/v) where the amount of PAF produced was three- to four-fold higher than in controls containing no albumin. The addition of free fatty acids, particularly arachidonic acid and palmitic acid, to 5% fatty acid-free albumin media caused a concentration-dependent decrease in PAF synthesis. A 50% inhibition of PAF synthesis was observed at an arachidonic acid concentration of 120 microM and at a palmitic acid concentration of 100 microM. The inhibition of PAF production by palmitic acid was also dependent on the concentration of extracellular albumin. In 0.5% fatty acid-free albumin media, a palmitic acid concentration of 40 microM produced a 50% inhibition in PAF synthesis. The addition of palmitic acid did not affect the release of endogenous arachidonic acid during stimulation. In contrast, the addition of stearic acid up to 120 microM in 5% fatty acid-free albumin media had no effect on PAF production. The different inhibitory effects of palmitic acid and stearic acid on PAF production may be related to differences in intracellular utilization of these two fatty acids during cell stimulation.     

De novo fatty acid synthesis by freshly isolated alveolar type II epithelial cells

Fatty acid synthesis was studied in freshly isolated type II pneumocytes from rabbits by 3H2O and (U-14C)-labeled glucose, lactate and pyruvate incorporation and the activity of acetyl-CoA carboxylase. The rate of lactate incorporation into fatty acids was 3-fold greater than glucose incorporation; lactate incorporation into the glycerol portion of lipids was very low but glucose incorporation into this fraction was approximately equal to incorporation into fatty acids. The highest rate of de novo fatty acid synthesis (3H2O incorporation) required both glucose and lactate. Under these circumstances lactate provided 81.5% of the acetyl units while glucose provided 5.6%. Incubations with glucose plus pyruvate had a significantly lower rate of fatty acid synthesis than glucose plus lactate. The availability of exogenous palmitate decreased de novo fatty acid synthesis by 80% in the isolated cells. In a cell-free supernatant, acetyl-CoA carboxylase activity was almost completely inhibited by palmitoyl-CoA; citrate blunted this inhibition. These data indicate that the type II pneumocyte is capable of a high rate of de novo fatty acid synthesis and that lactate is a preferred source of acetyl units. The type II pneumocyte can rapidly decrease the rate of fatty acid synthesis, probably by allosteric inhibition of acetyl-CoA carboxylase, if exogenous fatty acids are available.     

An Inhibitor of Mitochondrial Oxidations in Extracts Prepared from Senescing Leaves

Aqueous extracts were prepared from leaves of Ricinus Communis,L. by centrifuging homogenates at 18000 g for 20 min. Extractsprepared from yellow or brown leaves inhibited the oxidationof succinate by mitochondria prepared from Ricinus endosperm.Extracts became less inhibitory when dialysed or treated withbovine serum albumen. The inhibitory properties of the extractsare attributed to their fatty acid content.     

Inhibitory effect of undecanoic acid on the biosynthesis of long-chain fatty acids in Mortierella isabellina

Summary Following growth on n-alkanes, undecanoic acid in high concentrations completely inhibits the acylation of fatty acids formed during the terminal oxidation so that the intracellular fatty acid pattern is composed exclusively of components from the de novo synthesis. An inhibitory effect of undecanoic acid stems presumably from the effect it has on the long-chain acyl-coenzyme A synthetase I, whereas the corresponding long-chain acyl-coenzyme A synthetase II, which is bound to specific cell organelles remains untouched by this inhibition. The strongly reduced growth, even following glucose oxidation, probably comes from the effect of C₁₁-acid on specific intramitochondrial situated enzymes.Prof. Dr. H. G. Schlegel dedicated to his 60th birthday     

Effect of Peeling on IAA-induced Growth in Avena Coleoptiles

The act of peeling removes the epidermis exclusively from Avenacoleoptiles. Peeling inhibits IAA-induced growth, by inhibitingthe growth of segments incubated in the presence of IAA, andpromoting that of those incubated in water. The magnitude ofthe inhibition of IAA-induced growth is proportional to theamount of epidermis removed. It is shown that neither lateralswelling, wounding, anaerobiosis, nor exposure to supraoptimalconcentrations of IAA cause the inhibition. It is concludedthat in Avena coleoptiles the epidermis regulates the rate ofexpansion of the underlying parenchyma cells and is the principaltarget of IAA-action. Avena sativa L., oat, coleoptile, indol-3-ylacetic acid, auxin, extension growth     

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 inhibition of phospholipid synthesis in escherichia coli by phenethyl alcohol.

The kinetics of lipid metabolism during phenethyl alcohol treatment of Escherichia coli were examined. Phenethyl alcohol at a non-bacteriostatic concentration reduces the accumulation of [32-P] phosphate into phospholipids and alters the phospholipid composition of the cell membrane. The changes in phospholipid composition are a result of the inhibitory effect of phenethyl alcohol on the rates of synthesis of the individual phospholipids. The inhibition in the rate of phosphatidylethanolamine synthesis by phenethyl alcohol was twice the inhibition in the rate of phosphatidyglycerol synthesis. The de novo rate of cardiolipin synthesis was only slightly inhibited. However, net cardiolipin accumulation increased during phenethyl alcohol treatment due to a more rapid turnover of phosphatidylglycerol to cardiolipin. Phenethyl alcohol also altered the fatty acid composition of the cell as a result of its inhibitory effect on the rate of individual fatty acid synthesis. However, the inhibition of phospholipid synthesis was not reversed by fatty acid supplementation of phenethyl alcohol treated cells. This result indicates that phenethyl alcohol does not inhibit phospholipid synthesis solely at the level of fatty acid synthesis.     

Comparison of Glycolipids and Plastids in Callus Cells and Leaves of Alnus and Betula

The fine structure of plastids and fatty acid composition ofglycolipids (e.g. monogalactosyl diacylglycerides, MGDG; digalactosyldiacylglycerides, DGDG) in callus cells of Alnus glutinosa,A. incana and Betula pendula cultured in light was comparedwith that in intact leaves. The tissues were qualitatively verysimilar but a rather high amount oflignoceric acid (24:0) wascharacteristic for the callus of A. incana. This fatty acidwas found only in trace amount in other tissues. Linolenic (18:3)and palmitic (16:0) acids are the most abundant (25–65%and 17–27% respectively) fatty acids in all tissues studied.The proportion of 18:1 and 18:2 was much higher in the calluscompared with corresponding intact leaves, which are especiallyrich (48–65%) in 18:3. In callus cultures a higher proportion(17–19%) of linoleic acid (18:2) is found in both Alnusspecies than in the two callus strains of Betula (9–12%). All leaf and callus samples contained esterified steryl glycosidesand two cerebrosidelike spots in thin-layer chromatography,but they were more prominent in callus cultures than in leaves.The callus cells have plastids with rather well developed thylakoidswhich explains the similarity of the main glycolipid components(MGDG and DGDG) to that of leaves. (Received April 23, 1984; Accepted August 17, 1984)