Chain Fatty Acid Synthesis Research Articles

Chloroacetamide Mode of Action, II: Inhibition of Very Long Chain Fatty Acid Synthesis in Higher Plants

Abstract In short-term -experiments [14C]-labelled malonic acid, stearic acid and acetate have been incorporated into leaf fatty acids of seedlings of Cucumis sativus, Hordeum vulgare and Zea mays. The pattern of labelled fatty acids changed markedly by treatment with the chloroacetamide herbicides metazachlor, metolachlor or butachlor. During a 2-h incubation time, 1 μᴍ chloroacetamide specifically inhibited up to 100% the formation of the saturated very long chain fatty acids (V LCFAs) with a carbon number of 20, 22 and 24. In cucumber and barley a 50% inhibition of VLCFA formation is achieved with 10 to 100 nM metazachlor representing the most sensitive effect of inhibitors on fatty acid elongation reported as yet. Sensitivity of fatty acid elongation depends on the amide structure present in the com pound and on its stereochemistry. Inhibition of oleic acid incorporation correlates with growth inhibition by chloroacetam ides of the intact cell (comp. Pestic. Sei. 52, 381-387, 1998). The present study extends this correlation to inhibition of VLCFA synthesis in higher plants. Obviously the primary mode of action of chloroacetam ides and related herbicidal substances is involved in the enzymic four-step fatty acid elongation system.

The "Spot 14" gene resides on the telomeric end of the 11q13 amplicon and is expressed in lipogenic breast cancers: implications for control of tumor metabolism.

Enhanced long chain fatty acid synthesis may occur in breast cancer, where it is necessary for tumor growth and predicts a poor prognosis. "Spot 14" (S14) is a carbohydrate- and thyroid hormone-inducible nuclear protein specific to liver, adipose, and lactating mammary tissues that functions to activate genes encoding the enzymes of fatty acid synthesis. Amplification of chromosome region 11q13, where the S14 gene (THRSP) resides, also predicts a poor prognosis in breast tumors. We localized the S14 gene between markers D11S906 and D11S937, at the telomeric end of the amplified region at 11q13, and found that it was amplified and expressed in breast cancer-derived cell lines. Moreover, concordant expression of S14 and a key lipogenic enzyme (acetyl-CoA carboxylase) in a panel of primary breast cancer specimens strongly supported a role for S14 as a determinant of tumor lipid metabolism. S14 expression provides a pathophysiological link between two prognostic indicators in breast cancer: enhanced lipogenesis and 11q13 amplification.

Effects of pebulate and pebulate sulphoxide on very long chain fatty acid biosynthesis

The effects of the thiocarbamate herbicide, pebulate ( S-propylbutylethylthiocarbamate) and its sulphoxide were investigated in vivo. The sulphoxide caused greater inhibition of very long chain fatty acid (VLCFA) synthesis from [1- 14C]acetate than pebulate, suggesting that the former was the physiologically-active form of the herbicide. In contrast, to fatty acid elongation, de novo synthesis was insensitive to pebulate or its sulphoxide in vivo. The greater sensitivity of elongation to the sulphoxide was confirmed by in vitro assays which measured stearoyl-CoA or arachidoyl-CoA elongation with [2- 14C]malonyl-CoA. The results confirm suggestions that thiocarbamates are oxidised to their sulphoxide derivatives for full herbicidal activity and it is the latter which inhibit VLCFA and, hence, surface wax synthesis.

The Aspergillus parasiticus polyketide synthase gene pksA, a homolog of Aspergillus nidulans wA, is required for aflatoxin B1 biosynthesis.

Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced by Aspergillus parasiticus and Aspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway in A. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipient A. parasiticus genome at a specific locus, designated pksA. Sequence analysis suggest that pksA is a homolog of the Aspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conserved beta-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of the pksA product. No beta-ketoacyl reductase and enoyl reductase domains were found, suggesting that pksA does not encode catalytic activities for processing beta-carbon similar to those required for long chain fatty acid synthesis. The pksA gene is located in the aflatoxin pathway gene cluster and is linked to the nor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose that pksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.

Selective inhibition of fatty acid oxidation in colonocytes by ibuprofen: a cause of colitis?

Ibuprofen is associated with initiation or exacerbation of ulcerative colitis. As ibuprofen selectively inhibited fatty acid oxidation in the liver or caused mitochondrial damage in intestinal cells, its effect on substrate oxidation by isolated colonocytes of man and rat was examined. Ibuprofen dose dependently (2.0-7.5 mmol/l) and selectively inhibited 14CO2 production from labelled n-butyrate in colonocytes from the proximal and distal human colon (n = 12, p = < 0.001). Glucose oxidation was either unaltered or increased. Because short chain fatty acid oxidation is the main source of acetyl-CoA for long chain fatty acid synthesis, the inhibition of prostaglandin synthesis by ibuprofen in the colonic mucosa could also occur at this level. Because the concentrations of ibuprofen that can be attained in the human colon are not known, conclusions drawn from current dosages are tentative. The inhibition of fatty acid oxidation by ibuprofen may be biochemically implicated in the initiation and exacerbation of ulcerative colitis, manifestation of which would depend on the ibuprofen concentrations reached in the colon.

Pattern and Regulation of Acetyl-CoA Carboxylase Gene Expression1–3

Acetyl-CoA carboxylase is the rate-limiting enzyme in the biogenesis of long chain fatty acids. There is a single copy of the gene for acetyl-CoA carboxylase per haploid chromosome set. The gene contains two promoters whose primary transcripts are differentially spliced resulting in multiple forms of acetyl-CoA carboxylase mRNA. These mRNA species are different in the 5'-untranslated region, but contain the same coding region. Generation of different forms of the mRNA is tissue specific and controlled by physiological conditions. Two promoters contain an extensive array of cis-elements that perceive changes in the cellular environment signalling repression and induction of long chain fatty acid synthesis. The ability of the gene to respond to various lipogenic signals and the presence of the same coding sequence in all acetyl-CoA carboxylase mRNA species suggest that the biosynthesis of fatty acids required for multiple functions in the cells is primarily regulated at the gene level.

Interaction of thiocarbamate herbicides with fatty acid synthesis in germinating peas and their microsomal fractions

The thiocarbamate herbicides, diallate and triallate, inhibit very long chain fatty acid synthesis in germinating peas without affecting the de novo formation of palmitate and stearate. This result explains the herbicides' ability to interfere with cutin and suberin formation. When the herbicides were tested directly with a microsomal fraction from pea seeds, they inhibited the labelling of all fatty acids from [ 14C]malonyl-CoA and did not selectively reduce elongation. On the other hand, pre-treating seeds before the isolation of microsomes yielded preparations which lacked the ability to synthesise very long chain fatty acids but not palmitate and stearate. The latter result was not due to prevention of the translation of the elongation enzymes as shown by experiments where exogenous precursors were used for elongation. We conclude, from our experiments, that the thiocarbamates are converted to more active metabolites (possibly sulphoxides) and it is these metabolites which are responsible for the selective inhibition of fatty acid elongation and, hence, interfere with the formation of surface coverings.

Adrenoleukodystrophy: biochemical procedures in diagnosis, prevention and treatment.

The childhood form of adrenoleukodystrophy is an X-linked recessive disorder which is characterized biochemically by elevated concentrations of saturated very long chain fatty acids in tissues and plasma and impaired very long chain fatty acid oxidation in fibroblasts and leukocytes from adrenoleukodystrophy patients. The most consistently observed increase is that in hexacosanoic acid (C26:0); thus, measurement of plasma C26:0 concentration by gas-liquid chromatography provides a rapid, sensitive method of diagnosis. Prenatal diagnosis of adrenoleukodystrophy can be made by measurement of C26:0 concentrations in amniocytes and chorionic villus cells. Heterozygote (carrier) detection has also been accomplished by biochemical measurement of C26:0 in plasma and skin fibroblasts. In a study of over 200 obligate heterozygotes, greater than 90% showed abnormal concentrations of C26:0. Hybridization studies using the cloned DNA fragment St14 detects polymorphisms in the distal end of the long arm of the X chromosome (Xq27-28) and six informative kindreds have shown co-segregation of adrenoleukodystrophy and the St14 marker through 65 meioses. Thus, such studies can supplement very long chain fatty acid concentrations in heterozygote detection. Therapeutic interventions for adrenoleukodystrophy, such as dietary restriction of very long chain fatty acids, administration of clofibrate or carnitine, immunosuppression and adrenal hormone replacement, have not been successful. Recently, a modification of the very long chain fatty acid-restricted diet has been employed in which this diet is supplemented with synthetic glycerol trioleate. The rationale for this diet is that decreased very long chain fatty acid synthesis by fibroblasts from patients with adrenoleukodystrophy was observed when oleic acid was added to the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Elongation of acyl-CoAs by microsomes from etiolated leek seedlings

Long chain fatty acid synthesis was studied using etiolated leek seedling microsomes. In the presence of ATP, [2- 14C]malonyl-CoA was incorporated into fatty acids of C 16C 26. The omission of ATP, even in the presence of acetyl-CoA, led to a complete loss of activity, which was restored by addition of exogeneous acyl-CoAs. Comparison of acyl-CoA (C 12C 24) elongation showed that stearoyl-CoA, in the presence of [2- 14C]malonyl-CoA, was the more efficient precursor leading to the formation of fatty acids having a chain length of C 20C 26. [1- 14C]C 16CoA and [1- 14C]C 18CoA were elongated in the presence of malonyl-CoA, without degradation of the acyl chain. The time-course and the malonyl-CoA concentration curves showed that [1- 14C]C 18CoA was a better primer than [1- 14C]C 16CoA. Acyl-CoA elongation was also studied over the concentration range 4.5–45 μM [1- 14C]C 18CoA. Comparison of the radioactivity incorporated into the fatty acids formed using [2- 14C]malonyl-CoA in the presence of C 18CoA, on the one hand, and [1- 14C]C 18CoA in the presence of malonyl-CoA, on the other, demonstrated clearly that the acyl chain of the acyl-CoA was elongated by malonyl-CoA.

Effect of thyroid hormones and cyclic AMP on some lipogenic enzymes of the fat cell.

Thyroid hormones exert two types of effect on the lipogenic pathway of the fat cell of the young rat: one which is independent of cyclic AMP, and another which is under cyclic nucleotide control: Thyroidectomy leads to an enhanced conversion of glucose to glucose 6‐phosphate by the hexokinase system. The activity of hexokinase I is increased twofold in the fat cell from hypothyroid rats. The oxidation of glucose 6‐phosphate by the pentose phosphate pathway is also increased after thyroidectomy. The V of both glucose 6‐phosphate and gluconate‐6‐phosphate dehydrogenases is doubled (56 mU × min−1× mg−1 to 114 mU × min−1× mg−1) whereas the Km, is unchanged (19 μM). The activities of ATP citrate lyase and of acetyl‐CoA carboxylase are higher in the adipocytes from hypothyroid rats. The activity of fatty acid synthetase is also stimulated, but to a lesser extent. The apparent Km of acetyl‐CoA carboxylase is 0.14 mM and 0.06 mM and the V is 17.8 nmol xmin−1 xmg‐′ and 27.8 nmolxmin−1 xmg protein−1 in the adipocytes from normal and hypothyroid rats respectively. In contrast, for ATP citrate lyase the V only is doubled (62 mU × min−1× mg−1 to 111 mUxmin−1xmg−1). When fat cells from normal or hypothyroid rat were incubated in the presence of methylxanthines, dibutyryladenosine 3′,5′‐monophosphate, isoproterenol or epinephrine the activities of the hexokinase system and of glucose‐6‐phosphate and gluconate‐6‐phosphate dehydrogenases remained unchanged. In contrast, dibutyryl‐ adenosine 3′,5′‐monophosphate inhibited, by 60 ‐ 80%, the activities of ATP citrate lyase and acetyl‐CoA carboxylase in both cell types. In adipocytes prepared from normal rats, isoproterenol and epinephrine inhibited the activities of these two latter enzymes by 30 ‐ 60% while isobutylmethylxanthine potentiated their effect. In contrast, with fat cells from hypothyroid rats, isoproterenol and epinephrine had no effect upon the activities of ATP citrate lyase and acetyl‐CoA carboxylase, whereas a combination of epinephrine and ibobutylmethylxanthine was clearly inhibitory. In conclusion, thyroid hormones regulate lipogenesis in the fat cell of the young rat firstly because they control the accumulation of the substrates of this pathway, glucose 6‐phosphate and NADPH, by changing the concentrations of the hexokinase system and of the rate‐limiting enzymes of the pentose phosphate pathway. Secondly, because they have a specific effect on the amount of the enzymes (ATP citrate lyase and acetyl‐CoA carboxylase) which channel acetyl‐CoA towards long chain fatty acid synthesis; and thirdly because they allow, in response to the lipolytic hormones, the accumulation of cyclic AMP which is responsible for the inactivation of these two latter enzymes.

Effect of glucose concentration in the growth medium on the synthesis of fatty acids by the yeast Candida bogoriensis

The yeast Candida bogoriensis produced large quantities of an extracellular glycolipid, the diacetyl sophoroside of 13-hydroxydocosanoic acid, when grown on a standard glucose rich medium (3% glucose, 0.15% yeast extract), but not when grown on a low glucose medium (0.5% glucose, 0.4% yeast extract) (A. J. Cutler and R. J. Light. 1979. J. Biol. Chem. 254: 1944–1950). Glucose levels also affected the quantity and distribution of the free fatty acid and triglyceride fractions synthesized by this organism. Cells grown on the low glucose medium contained palmitate and stearate as the major fatty acids in these two fractions, and a 3-h incubation with [1-14C]acetate led primarily to the labeling of these two acids. Cells grown on the standard enriched glucose medium contained relatively less stearate and more behenate than the low glucose grown cells, and the incorporation of [1-14C]acetate into stearate was decreased, while that into behenate was increased.Supplementation of low glucose grown cells with glucose led to a rapid stimulation of fatty acid synthesis, primarily palmitate and stearate in the free fatty acid fraction and stearate in the triglyceride fraction. Total triglyceride began to increase a few hours after supplementation, but synthesis of the extracellular glycolipid, and hence 13-hydroxydocosanoic acid, did not occur until 12–24 h after supplementation. The stimulation by glucose of long chain fatty acid synthesis in C. bogoriensis was therefore a process distinct from the glucose stimulation of palmitate and stearate synthesis, though the two events may be causally related.

Development of the capacity of mouse mammary glands for medium chain fatty acid synthesis during pregnancy and lactation

The time-course for appearance of fatty acid synthetase and thioesterase II, enzymes required for the synthesis of medium chain (C 8-C 12) fatty acids by the mammary gland, has been studied in the mouse and compared with that in the rat. The development of high levels of fatty acid synthetase in the mouse mammary gland during the early days of lactation coincided with an observed increase in the overall lipogenic capacity of the gland, assessed by measuring the incorporation of radioactive acetate into fat in tissue slices. Both the level of thioesterase II in the gland and the proportion of medium chain fatty acids synthesized from acetate by tissue slices began to increase in late gestation but did not reach a maximum until lactation was well established. In this regard the mouse is distinctly different from the rat, which establishes maximum levels of thioesterase II in the mammary gland prior to parturition. The significance of this difference as it relates to mammary gland development is discussed.

Hormonal regulation of medium chain fatty acid synthesis by mouse mammary gland explants

AbstractExplants of pregnant mouse mammary tissue were cultured in media supplemented with various hormones. During the last few hours of culture, explants were labeled with [14C] acetate. Fatty acid synthesis by the tissue was analyzed using reverse phase thin layer chromatography, and incorporation of radioactivity into the medium chain fatty acid (MCFA) fraction was calculated as total MCFA per mg tissue fresh weight and as a percentage of the total fatty acid radioactivity (%MCFA). After 48 hr of culture, explants had an elevated %MCFA synthesis only when exposed to media containing insulin, cortisol and prolactin, confirming previous reports. Increasing doses of prolactin (maximal response at 300 mg/ml) caused a coordinate rise in both %MCFA synthesis and total MCFA synthesis per mg fresh weight. Epithelial cells isolated from explants after culture for 48 hr with insulin, cortisol and prolactin showed an elevated %MCFA synthesis compared to cells from explants cultured with insulin and cortisol alone, indicating that hormonal stimulation of mammary explants causes a change in the chain length of fatty acids synthesized by mammary epithelial cells. The specificity of the explant response to prolactin was tested with other hormones: FSH and calcitonin had no effect, whereas bovine growth hormone showed activity only at high concentration. Progesterone and 17β‐estradiol also had no effect. The analysis of MCFA synthesis provides another means of (a) assessing hormonal action upon mammary tissue, and (b) evaluating the biological activity of prolactin.

Asynchronous appearance of two enzymes concerned with medium chain fatty acid synthesis in developing rat mammary gland.

Asynchronous appearance of two enzymes concerned with medium chain fatty acid synthesis in developing rat mammary gland.

Effect of thiocarbamate herbicides on fatty acid synthesis by potato

S-ethyldipropylthiocarbamate (EPTC), S-(2,3-dichloroallyl)diisopropylthiocarbamate (diallate) and S-(2,3,3-trichloroallyl)diisopropylthiocarbamate (triallate) inhibited the formation of very long chain fatty acids by aged potato discs. Incorporation of acetate-[ 14C] into total fatty acids was inhibited 24% by EPTC, 50% by triallate and 55% by diallate at 10 −4 M. The relative sensitivity of very long chain fatty acid synthesis to thiocarbamates in potato tuber provides further evidence that these herbicides reduce cuticular wax by inhibiting fatty acid elongation.

Long chain fatty acid synthesis in developing castor bean seeds IV. The synthetic system in proplastids

Long chain fatty acid synthesis in developing castor bean seeds IV. The synthetic system in proplastids

Long chain fatty acid synthesis in developing castor bean seeds II. Operation of the path from sucrose to long chain fatty acids in a subcellular particulate system

The path of LFA synthesis from acetate in developing castor bean seeds was associated with subcellular 10,000; particles. Further fractionation of these particles by a stepwise density gradient method showed the high possibility that the site of LFA synthesis is the proplastid. A study on cofactor requirements when [l-C]acetate predominantly incorporated into LFAa indicated that synthesis would be achieved by acetyl-CoA carboxylation, malonyl-ACP condensation. ATP, GoA, HCOjand Mg-H or Mn++ were essential for synthesis from acetate by the 10,000 g particulate system. Results of inhibitor experiment suggested that the supply of ATP to the LFA synthesizing system is brought about by mitochondrial oxidative phosphorylation, when acetate is the sole precursor for U A synthesis in this system. The NADPH generating system was contained in the patides, although the addition of NADP+ and G-6-P increased synthesis. NADH markedly stimulated LFA synthesis from acetate. The primary role of NADH seems to be as a direct reductant in both steps involving the reduction and oxidative desaturation of fatty acid chains; particularly, in the former step, although NADH partially contributes to the supply of ATP as a respiratory substrate. It is unlikely that NADH works as a hydrogen donor to NADP+. LFA synthesis by the castor bean particulate system was not stimulated by light, thus differing from that by leaf chloroplasts.

Long chain fatty acid synthesis in developing castor bean seeds I. The operation of the path from acetate to long chain fatty acids in a subcellular particulate system

Long chain fatty acid synthesis in developing castor bean seeds I. The operation of the path from acetate to long chain fatty acids in a subcellular particulate system

Fat Metabolism in Higher Plants XXXVI: Long Chain Fatty Acid Synthesis in Germinating Peas

A low lipid, high starch containing tissue, namely cotyledons of germinating pea seedlings was examined for its capacity to synthesize fatty acid. Intact tissue slices readily incorporate acetate-(14)C into fatty acids from C(16) to C(24). Although crude homogenates synthesize primarily 16:0 and 18:0 from malonyl CoA, subsequent fractionation into a 10,000g pellet, a 10(5)g pellet and supernatant (soluble synthetase) revealed that the 10(5)g pellet readily synthesizes C(16) to C(28) fatty acids whereas the 10,000g and the supernatant synthesize primarily C(16) and C(18). All systems require acyl carrier protein (ACP), TPNH, DPNH if malonyl CoA is the substrate and ACP, Mg(2+), CO(2), ATP, TPNH, and DPNH if acetyl CoA is the substrate. The cotyledons of germinating pea seedlings appear to have a soluble synthetase and 10,000g particles for the synthesis of C(16) and C(18) fatty acid, and 10(5)g particles which specifically synthesize the very long chain fatty acid from malonyl CoA, presumably via malonyl ACP.

Acyl Carrier Protein: STRUCTURAL REQUIREMENTS FOR FUNCTION IN FATTY ACID BIOSYNTHESIS

Abstract The mechanism of the reactivity of the enzymes of fatty acid synthesis from Escherichia coli with thioester substrates of acyl carrier protein has been investigated by structural modification of this protein. Carboxypeptidase A treatment of acyl carrier protein releases the carboxyl-terminal residue, alanine, and 0.3 residue each of histidine and glutamine. Carboxypeptidase A-treated acyl carrier protein remains fully active as assayed in the malonyl-coenzyme A CO2-exchange reaction of fatty acid synthesis. Limited acid hydrolysis of acyl carrier protein at pH 2.0, 100°, results in the preferential cleavage and release of aspartic acid residues and rapidly inactivates the protein. After a single aspartic acid residue has been released, 80% of acyl carrier protein activity has been lost. Treatment of acyl carrier protein for 80 min at pH 2.0, 100°, results in the release of 0.39 mole of aspartic acid per mole of acyl carrier protein. Following this treatment a single peptide was isolated in large yield. This peptide comprised three-fourths of the molecule and contained the intact carboxyl-terminal end of acyl carrier protein. The amino terminus of this peptide was serine, and the peptide was inactive in the malonyl-CoA CO2-exchange reaction. After tryptic hydrolysis of acyl carrier protein a peptide containing 42 amino acid residues was isolated. This peptide contained the intact prosthetic group. It had aminoterminal glutamic acid and the carboxyl-terminal sequence (Leu,Val)-Met-Ala-Lys, and was unable to replace acyl carrier protein, either in the malonyl-CoA CO2-exchange reaction or in long chain fatty acid synthesis. Malonyl tryptic peptide was unable to replace malonyl acyl carrier protein in the β-ketoacyl acyl carrier protein synthetase reaction, thus explaining the inability of this peptide to function in long chain fatty acid synthesis. Acetoacetyl tryptic peptide was reduced slowly in the presence of β-ketoacyl acyl carrier protein reductase with a Michaelis constant of 1.7 x 10-3 m and a maximum velocity of 0.21 µmole of reduced triphosphopyridine nucleotide oxidized per min per mg, compared to values of 6.6 x 10-5 m and 1.36 µmoles of TPNH oxidized per min per mg for acetoacetyl acyl carrier protein. This difference suggests that the site which confers the high reactivity of the acyl carrier protein substrate is missing in the tryptic peptide. The existence of such a site is indicated by the fact that free acyl carrier protein competitively inhibited (Ki = 2.2 x 10-4 m) the reduction of acetoacetyl acyl carrier protein catalyzed by β-ketoacyl acyl carrier protein reductase. The tryptic peptide at concentrations up to 5 x 10-4 m did not inhibit the reduction of acetoacetyl acyl carrier protein. These experiments suggest that acyl carrier protein interacts with β-ketoacyl acyl carrier protein reductase at a site different from the prosthetic group-active site interaction and that this site has been altered or lost upon tryptic digestion of acyl carrier protein. Further primary structure of acyl carrier protein is presented.