Inhibition of plant fatty acid synthesis by nitroimidazoles.

1. The effect of the addition of a number of nitroimidazoles was tested on fatty acid synthesis by germinating pea seeds, isolated lettuce chloroplasts and a soluble fraction from pea seeds. 2. All the compounds tested had a marked inhibition on stearate desaturation by lettuce chloroplasts and on the synthesis of very-long-chain fatty acids by pea seeds. 3. In contrast, the effect of the drugs on total fatty acid synthesis from [14C]acetate in chloroplasts was related to the compound's electron reduction potentials. 4. Of the compounds used, only metronidazole had a marked inhibition on palmitate elongation in the systems tested. 5. The mechanism of inhibition of plant fatty acid synthesis by nitroimidazoles is discussed and the possible relevance of these findings to their neurotoxicity is suggested.     

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.     

Products of fatty acid synthesis by a particulate fraction from germinating pea (Pisum sativum L.).

The synthesis of lipids and acyl thioesters was studied in microsomal preparations from germinating pea (Pisum sativum cv. Feltham First) seeds. Under conditions of maximal synthesis (in the presence of exogenous acyl-carrier protein) acyl-acyl-carrier proteins accounted for about half the total incorporation from [14C]malonyl-CoA. Decreasing the concentrations of exogenous acyl-carrier protein lowered the overall synthesis of fatty acids by decreasing, almost exclusively, the radioactivity associated with acyl-acyl-carrier proteins. A time-course experiment showed that acyl-acyl-carrier proteins accumulated most of the radioactive label at the beginning of the incubation but, eventually, the amount of radioactivity in that fraction decreased, while a simultaneous increase in the acyl-CoA and lipid fractions was noticed. Addition of exogenous CoA (1 mM) produced a decrease of total incorporation, but an increase in the radioactivity incorporated into acyl-CoA. The microsomal preparations synthesized saturated fatty acids up to C20, including significant proportions of pentadecanoic acid and heptadecanoic acid. Synthesis of these 'odd-chain' fatty acids only took place in the microsomal fraction. In contrast, when the 18,000g supernatant (containing the microsomal and soluble fractions) was incubated with [14C]malonyl-CoA, the radioactive fatty acid and acyl classes closely resembled the patterns produced by germinating in the presence of [14C]acetate in vivo. The results are discussed in relation to the role of acyl thioesters in the biosynthesis of plant lipids.     

The role of Coenzyme A in lipid synthesis by a particulate fraction from germinating peas

The effect of CoA on fatty acid synthesis by the microsomal fraction from germinating pea (Pisum sativum) was examined. Increasing concentrations of CoA progressively decreased total fatty acid synthesis from [¹⁴C]malonyl-CoA. However, the synthesis of very long chain fatty acids was relatively unaffected so that their proportion in the reaction products increased. Other CoA-esters also decreased total fatty acid synthesis while increasing the relative accumulation of radioactivity in very long chain fatty acids. The addition of CoA also altered the distribution of newly synthesized fatty acids in different lipid fractions. Complex lipid labelling was relatively increased while that of acyl-acyl carrier proteins was decreased. Very long chain fatty acids accumulated in lipids rather than thioesters. The role of CoA in controlling fatty acid synthesis in the pea microsomal fraction is discussed.     

The elongation of exogenous fatty acids and the control of phospholipid acyl chain length in Micrococcus cryophilus.

The synthesis of fatty acids de novo from acetate and the elongation of exogenous satuated fatty acids (C12-C18) by the psychrophilic bacterium Micrococcus cryophilus (A.T.C.C. 15174) grown at 1 or 20 degrees C was investigated. M. cryophilus normally contains only C16 and C18 acyl chains in its phospholipids, and the C18/C16 ratio is altered by changes in growth temperature. The bacterium was shown to regulate strictly its phospholipid acyl chain length and to be capable of directly elongating myristate and palmitate, and possibly laurate, to a mixture of C16 and C18 acyl chains. Retroconversion of stearate into palmitate also occurred. Fatty acid elongation could be distinguished from fatty acid synthesis de novo by the greater sensitivity of fatty acid elongation to inhibition by NaAsO2 under conditions when the supply of ATP and reduced nicotinamide nucleotides was not limiting. It is suggested that phospholipid acyl chain length may be controlled by a membrane-bound elongase enzyme, which interconverts C16 and C18 fatty acids via a C14 intermediate; the activity of the enzyme could be regulated by membrane lipid fluidity.     

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.     

The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids.

1. The range of fatty acids formed by preparations of ultrasonically ruptured avocado mesocarp plastids was dependent on the substrate. Whereas [1-14C]palmitate and [14C]oleate were the major products obtained from [-14C]acetate and [1-14C]acetyl-CoA, the principal product from [2-14C]malonyl-CoA was [14-C]stearate. 2. Ultracentrifugation of the ruptured plastids at 105000g gave a supernatant that formed mainly stearate from [2-14C]malonyl-CoA and to a lesser extent from [1-14C]acetate. The incorporation of [1-14C]acetate into stearate by this fraction was inhibited by avidin. 3. The 105000g precipitate of the disrupted plastids incorporated [1-14C]acetate into a mixture of fatty acids that contained largely [14C]plamitate and [14C]oleate. The formation of [14C]palmitate and [14C]oleate by disrupted plastids was unaffected by avidin. 4. The soluble fatty acid synthetase was precipitated from the 105000g supernatant in the 35-65%-saturated-(NH4)2SO4 fraction and showed an absolute requirement for acyl-carrier protein. 5. Both fractions synthesized fatty acids de novo.     

The fatty acid composition of lipids of neuronal and glial nuclear fractions isolated from 15-day-old rabbit cerebral cortex

A neuronal nuclear fraction (N1) and a glial nuclear fraction (N2) have been isolated from 15-day-old rabbit cerebral cortex using the Thompson procedure. More than 56% of the homogenate DNA was recovered in the two nuclear fractions, with N1 being the larger by about eightfold. Fractions N1 and N2 had very similar phospholipid distributions, with phosphatidylinositol being a larger component than phosphatidylserine. Fatty acid analyses demonstrated that phosphatidylethanolamine and phosphatidylinositol, individually, had similar fatty acid profiles in fractions N1 and N2, and also in nuclear and microsomal fractions derived from homogenates of nerve cell bodies isolated from cortex of 15-day-old rabbits. In contrast, the nuclear phosphatidylcholines had lower levels of palmitate and higher levels of arachidonate than did microsomal phosphatidylcholines. Molecular species analyses indicated that monoenes (41 mol%), tetraenes (20 mol%), and saturates (13 mol%, composed chiefly of palmitate) were the principal classes of N1 phosphatidylcholines, while the diacyl species of phosphatidylethanolamine of this fraction were characterized by high levels of tetraenes (44 mol%), pentaenes (17 mol%), and hexaenes + polyenes (24 mol%). The neutral glycerides of fraction N1 occurred collectively at a level of 0.05 mol/mol phospholipid. Prominent fatty acids of diacylglycerols included palmitate (31%), oleate (20%), arachidonate (14%), and stearate (13%). Triacylglycerols showed a similar pattern but with relatively high levels of linoleate (11%), while monoacylglycerols consisted almost entirely of palmitate (33%), stearate (35%), and oleate (24%).     

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 acid biosynthesis in Erlich cells. The mechanism of short term control by exogenous free fatty acids.

We have examined the mechanism by which extracellular free fatty acids regulate fatty acid biosynthesis in Ehrlich ascites tumor cells. De novo biosynthesis in intact cells was inhibited by stearate greater than oleate greater than palmitate greater than linoleate. The amount of citrate and long chain acyl-CoA in the cells was not changed appreciably by the addition of free fatty acids to the incubation medium, indicating than free fatty acids do not regulate fatty acid biosynthesis by changing the total intracellular content of these metabolites. By measuring the incorporation of labeled free fatty acids into acyl-CoA, however, it was determined that the fatty acid composition of the acyl-CoA poolwas changed dramatically to reflect the composition of the exogenous free fatty acids. The relative inhibitory effects of different free fatty acids appear to depend on the ability of their acyl-CoA derivatives to regulate acyl-CoA carboxylase activity. The acyl-CoA concentration needed to produce 50% inhibition of purified Ehrlich cell carboxylase was found to be 0.68 mum for stearoyl-CoA, 1.6 mum for oleoyl-CoA, 2.2 mum for palmitoyl-CoA, 23 mum for myristoyl-CoA, 30 mum for lauroyl-CoA, and 37 mum for linoleoyl-CoA. In contrast to their effects on de novo synthesis, all of the free fatty acids added except stearate stimulated chain elongation in intact cells. Microsomal chain elongation, the major system for elongation in Ehrlich cells, also was regulated by the composition of the cellular acyl-CoA pool. Lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA were good substrates for elongation by isolated microsomes; oleoyl-CoA, and linoleoyl-CoA were intermediate; and stearoyl-CoA was a very poor substrate. We conclude that free fatty acids regulate fatty acid biosynthesis by changing the composition of the cellular acyl-CoA pool. These changes control the rate of malonyl-CoA production and, because of the acyl-CoA substrate specificity of the microsomal elongation system, modulate the amount of malonyl-CoA used for chain elongation.     

Comparison of the acylation of sn-glycerol 3-phosphate and membrane-bound lipid in the microsomal fraction from rabbit brain throughout maturation.

1. Age-related changes in the specific activity of palmitoyl-CoA synthetase, sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15) and the esterification of [3H]palmitate into endogenous lipid in the microsomal fraction from rabbit brain have been determined throughout development. 2. The increased specific activity of sn-glycerol 3-phosphate acyltransferase at the onset of myelination (rising in parallel with other lipogenic enzymes) is consistent with a direct role of the acyltransferase in promoting the accumulation of cerebral lipid. In adult brain microsomes, although the specific activity was low, the total activity was only 20% lower than during active myelination. 3. Palmitoyl-CoA, synthesized by the palmitoyl-CoA synthetase in the microsomal membrane, was the preferred substrate for the esterification of sn-glycerol 3-phosphate. There was no evidence for a pool of palmitoyl-CoA formed from palmitate. 4. The esterification of [3H]palmitate into membrane-bound lipid remained high throughout development and may be part of an acyl-exchange cycle via lysophospholipids. [3H]palmitate was incorporated into both neutral lipids and phospholipids, while phosphatidic acid was the major product of sn-[1(3)-3H]-glycerol-3-phosphate esterification. 5. The microsomal fraction contained a pool of unesterified fatty acid, which was activated and esterified into sn-glycerol 3-phosphate.     

Modulation of palmitate-induced endoplasmic reticulum stress and apoptosis in pancreatic β-cells by stearoyl-CoA desaturase and Elovl6

Induction of endoplasmic reticulum (ER) stress and apoptosis by elevated exogenous saturated fatty acids (FAs) plays a role in the pathogenesis of β-cell dysfunction and loss of islet mass in type 2 diabetes. Regulation of monounsaturated FA (MUFA) synthesis through FA desaturases and elongases may alter the susceptibility of β-cells to saturated FA-induced ER stress and apoptosis. Herein, stearoyl-CoA desaturase (SCD)1 and SCD2 mRNA expression were shown to be induced in islets from prediabetic hyperinsulinemic Zucker diabetic fatty (ZDF) rats, whereas SCD1, SCD2, and fatty acid elongase 6 (Elovl6) mRNA levels were markedly reduced in diabetic ZDF rat islets. Knockdown of SCD in INS-1 β-cells decreased desaturation of palmitate to MUFA, lowered FA partitioning into complex neutral lipids, and increased palmitate-induced ER stress and apoptosis. Overexpression of SCD2 increased desaturation of palmitate to MUFA and attenuated palmitate-induced ER stress and apoptosis. Knockdown of Elovl6 limited palmitate elongation to stearate, increasing palmitoleate production and attenuating palmitate-induced ER stress and apoptosis, whereas overexpression of Elovl6 increased palmitate elongation to stearate and palmitate-induced ER stress and apoptosis. Overall, these data support the hypothesis that enhanced MUFA synthesis via upregulation of SCD2 activity can protect β-cells from elevated saturated FAs, as occurs in prediabetic states. Overt type 2 diabetes is associated with diminished islet expression of SCD and Elovl6, and this can disrupt desaturation of saturated FAs to MUFAs, rendering β-cells more susceptible to saturated FA-induced ER stress and apoptosis.     

The effect of hypolipidemic drugs on plant lipid metabolism

The effect of hypolipidemic drugs, WY14643 and DH990, on plant lipid metabolism has been studied. The total incorporation of [14C]acetate into lipids was inhibited by addition of both drugs to aged potato (Solanum tuberosum) tuber discs, spinach (Spinacia oleracea) leaves, and spinach chloroplasts, while the incorporation in Chlorella vulgaris cells was affected only by DH990. Moreover, DH990 inhibited the incorporation of 14C-labeled fatty acids into phosphatidylcholine and phosphatidylethanolamine of potato discs, and decreased the incorporation into phosphatidylglycerol of Chlorella cells. DH990 inhibited the formation of polyunsaturated fatty acids in potato discs, Chlorella cells, and spinach leaves, whereas WY14643 had no effect on the formation of these fatty acids. Stearoyl-ACP desaturase from safflower (Carthamus tinctorius) seeds was very sensitive to both drugs, especially DH990, which completely blocked the activity at 2 mM levels. When safflower lysophospholipid acyltransferases were solubilized by detergent treatment, only DH990 inhibited the incorporation of [14C]oleoyl-CoA into lysophosphatidylcholine or lysophosphatidylethanolamine. Both drugs inhibited fatty acid synthesis from [14C]malonyl-CoA in the microsomal fraction from safflower seeds, but only DH990 inhibited FAS activity in the soluble fraction; both drugs inhibited severely the formation of stearic acid. Both acetyl-CoA carboxylase and acetyl-CoA synthetase were sensitive to both drugs.     

alpha-Hydroxylation of newly synthesised fatty acids by a soluble fraction from germinating pea.

A soluble fraction from germinating pea (Pisum sativum) seeds alpha-hydroxylated newly-synthesised fatty acids to form alpha-hydroxypalmitic and alpha-hydroxystearic acids. In contrast to fatty acid synthesis from [14C] malonyl CoA, alpha-hydroxylation was inhibited by exogenous phospholipids. alpha-Hydroxylation was optimal at pH 8, required reduced pyridine nucleotides and was inhibited by EDTA and imidazole.     

Triacylglycerol synthesis in goat mammary gland. The effect of ATP, Mg2+ and glycerol 3-phosphate on the esterification of fatty acids synthesized de novo.

Goat mammary-gland microsomal fraction by itself induces synthesis of medium-chain-length fatty acids by goat mammary fatty acid synthetase and incorporates short- and medium-chain fatty acids into triacylglycerol. Addition of ATP in the absence or presence of Mg2+ totally inhibits triacylglycerol synthesis from short- and medium-chain fatty acids, and severely inhibits synthesis de novo of medium-chain fatty acids. The inhibition by ATP of fatty acid synthesis and triacylglycerol synthesis de novo can be relieved by glycerol 3-phosphate. The effect of ATP could not be mimicked by the non-hydrolysable ATP analogue, adenosine 5'-[beta,gamma-methylene]triphosphate and could not be shown to be caused by inhibition of the diacylglycerol acyltransferase by a phosphorylation reaction. Possible explanations for the mechanism of the inhibition by ATP are discussed, and a hypothetical model for its action is outlined.     

Palmitate activation and esterification in microsomal fractions of rat liver.

Palmitoyl-CoA synthetase activity in the microsomal fraction of rat liver was measured directly by palmitoyl-CoA production, and indirectly by converting the palmitoyl-CoA into palmitoylcarnitine under optimum conditions. Even in the latter system, palmitoyl-CoA accumulated. The rate of palmitoyl-CoA hydrolysis and the inhibition of palmitoyl-CoA synthetase by palmitoyl-CoA were each estimated to be less than 10% of the maximum rate of palmitoyl-CoA production. The concentration of palmitoyl-CoA present in the assay systems used for measuring palmitate esterification to glycerol phosphate and the activity of palmitoyl-CoA synthetase by using the carnitine-linked determination were measured. These concentrations were not altered by the addition of glycerol phosphate, or of carnitine plus carnitine palmitoyltransferase. The relationship between the activity of palmitoyl-CoA synthetase and the rate of glycerolipid synthesis was investigated. The latter activity was measured by using palmitoyl-CoA generated from palmitate, palmitoyl--AMP or palmitoylcarnitine. It is concluded that, at optimum substrate concentrations, the activity of glycerol phosphate acyltransferase is rate-limiting in the synthesis of phosphatidate by rat liver microsomal fractions. The implications of these results in the measurement of palmitoyl-CoA synthetase and in the control of glycerolipid synthesis are discussed.     

Studies on the mechanism of action of ACTH on triglyceride synthesis in fat cells.

It was found that ACTH greatly reduced lipogenesis in fat cells in the presence of calcium ion, but not in the absence of calcium ion. Of the enzymes involved in triglyceride synthesis from fatty acid in lipid micelle membranes, only acyl-CoA synthetase was inhibited by calcium ion, the apparent Ki value of calcium ion being 4.2 X 10(-4) M. The Km values of the enzyme for palmitate and ATP were 2.0 X 10(-4) M and 2.5 X 10(-4) M, respectively and calcium ion caused non-competitive inhibition with both palmitate and ATP. The acyl-CoA synthetase activity of lipid micelle membranes was inhibited by treatment with phospholipase A or C, but not by treatment with phospholipase D. The mechanism of inhibition of triglyceride synthesis by ACTH is discussed on the basis of these results.     

Activities of 3-hydroxyl-3-methylglutaryl-CoA reductase and acetyl-CoA carboxylase and the rate of mevalonic acid, squalene, sterol and fatty acid biosynthesis from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in rat liver: effects of Triton WR 1339, starvation and cholesterol diet

The effects of Triton WR 1339, starvation and cholesterol diet on the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and on the rates of mevalonic acid (MVA) biosynthesis from acetyl-CoA and malonyl-CoA in the soluble (140 000 g) and microsomal fractions of rat liver, on the rate of incorporation of these substrates into squalene, cholesterol and lanosterol in the rat liver postmitochondrial fraction and on the rate of fatty acid biosynthesis was studied. The administration of Triton WR 1339 (200 mg per 100 g of body weight twice) stimulated the activity of HMG-CoA reductase and MVA biosynthesis from acetyl-CoA and malonyl-CoA in the intact and solubilized microsomal fractions and had no effect on these parameters in the soluble fraction. Starvation for 36 hrs did not cause inhibition of the reductase activity or MVA biosynthesis from both substrates in the soluble fraction. Alimentary cholesterol significantly increased the activity of HMG-CoA reductase, had no effect on the rate of MVA biosynthesis from acetyl-CoA and stimulated the malonyl-CoA incorporation in to MVA in the soluble fraction. Starvation an alimentary cholesterol inhibited the HMG-CoA reductase activity and MVA biosynthesis from both substrates in the solubilized microsomal fraction. Triton WR 1339 stimulated 4--19-fold the lipid formation in the total unsaponified fraction and its components i.e. squalene, lanosterol, cholesterol, from acetyl-CoA and only insignificantly (1,2--1,7-fold) increased malonyl-CoA incorporation into these compounds. Starvation and alimentary cholesterol repressed lanosterol and cholesterol biosynthesis from acetyl-CoA, decreased malonyl-CoA incorporation into these sterols and had no influence on squalene biosynthesis from the two substrates. Triton WR 1339 and starvation inhibited the acetyl-CoA carboxylase activity, unaffected by alimentary cholesterol. No significant changes in the rate of fatty acid biosynthesis from the substrates were observed. The data obtained provide evidence for the existence of autonomic pathways of MVA biosynthesis localized in the soluble and microsomal fractions of rat liver. The pathway of MVA biosynthesis in the soluble fraction is less sensitive to regulatory factors. Sterol biosynthesis from malonyl-CoA is also more resistant to regulatory effects than sterol biosynthesis from acetyl-CoA. This suggests that HMG-CoA reductase localized in the soluble fraction takes part in MVA and sterol biosynthesis from malonyl-CoA.     

Lipid biosynthesis in developing and germinating soybean cotyledons: The formation of palmitate and stearate by chopped tissue and supernatant preparations

Chopped tissue from developing soybean cotyledons incorporated [1-¹⁴C]acetate into palmitate, stearate, oleate, and linoleate, but with germinating cotyledons much less [1-¹⁴C]acetate was incorporated and the principal labeled products were palmitate, stearate, and oleate. When supernatant fractions from developing cotyledons were incubated with [1-¹⁴C]acetate or [2-¹⁴C]malonate the principal labeled products were palmitate and stearate. Supernatant fractions from germinating seed incorporated [2-¹⁴C]malonate into palmitate and also into short chain fatty acids including decanoate, laurate, and myristate. Supernatants from developing cotyledons required acyl carrier protein (ACP), ATP, CoA, and reduced pyridine nucleotides for maximal rates of incorporation of either [1-¹⁴C]acetate or [2-¹⁴C]malonate into palmitate and stearate. The de novo fatty acid synthetase which converts acetyl- and malonyl-ACP's to palmityl ACP was active in supernatant fractions from both young and old developing cotyledons. The elongation system, converting palmityl ACP to stearyl ACP, was more active in supernatants from younger than from older developing cotyledons. In experiments with chopped tissue the elongation system appeared equally active throughout the development process. These results are consistent with the view that the de novo and elongation systems are separate entities and that the elongation system in older cotyledons is less stable to the methods used to prepare supernatant fractions.     

The importance of the stearoyl-CoA desaturase system in octadecenoate metabolism in the Morris hepatoma 7288C

The sources of octadecenoic acid (18:1) and the importance of the stearoyl-CoA desaturase system in maintaining elevated levels of this fatty acid in the Morris hepatoma 7288C have been investigated. Sterculic acid, an inhibitor of the stearoyl-CoA desaturase system, when added to the culture medium, inhibited the production of monoenoic fatty acids through de novo synthesis by 90% while the production of saturated fatty acids and cholesterol was unaffected. Sterculic acid also inhibited 18:1 formation through desaturation of exogenous stearate (18:0) by 80%. These results indicate that the stearoyl-CoA desaturase system is responsible for most, if not all, of the 18:1 produced within these cells and that an alternate, sterculic acid-insensitive, pathway for 18:1 biosynthesis is not functioning in this cell line. Measurements of fatty acid synthesis, using 3H2O, show that de novo synthesis accounts for approx. 30% of the cellular 16:1 and 18:1 mass, while contributing 63% and 95% of the stearate and palmitate mass, respectively. Cells grown in the presence of sterculic acid displayed a 50% decrease in 18:1 levels while levels of both palmitate and stearate increased. These effects were maximal at 20-30 microM sterculate. Polyunsaturate levels were unaffected. The 50% decrease in 18:1 levels in treated cells could be completely accounted for by the inhibition of de novo 18:1 biosynthesis and the inhibition of exogenous 18:0 desaturation. This enzyme system, although low in activity when measured in this tissue, is responsible for a major portion of the 18:1 observed in these cells.