Chain cleavage and sulfoxidation of thiastearoyl-ACP upon reaction with stearoyl-ACP desaturase

The fatty acid analogues 9- and 10-thiastearate were converted to acyl-ACP derivatives by in vitro enzymatic synthesis and reacted with the reconstituted soluble stearoyl-ACP Delta9 desaturase complex. Electrospray ionization mass spectral analysis of the acyl chains purified from the reaction mixtures showed that 10-thiastearoyl-ACP was converted to the 10-sulfoxide as the sole product. In the presence of (18)O(2), the sulfoxide oxygen was found to be derived exclusively from O(2). This result confirms the ability of the soluble stearoyl-ACP desaturase to catalyze O atom transfer in the presence of the appropriate substrate analogue. Inhibition studies showed that 10-thiastearoyl-ACP was a mixed-type inhibitor of 18:0-ACP, with an apparent K(I) of approximately 10 microM. Comparable reactions of the stearoyl-ACP desaturase complex with 9-thiastearoyl-ACP gave the 9-sulfoxide as approximately 5% of the total products, with the O atom again exclusively derived from O(2). The remaining 95% of the total products arose from an acyl chain cleavage reaction between S-9 and C-10. Matrix-assisted laser desorption ionization time-of-flight mass spectral analysis showed that 9-thiastearoyl-ACP had a mass of 9443 amu while the acyl chain cleavage product had a mass of 9322 amu, corresponding to the calculated mass of 8-mercaptooctanoyl-ACP. Recovery of the acyl chain from the ACP product gave the disulfide of 8-mercaptooctanoate (mass of 349.1 amu), arising from the dimerization of 8-mercaptooctanoate during product workup. Gas chromatography-mass spectral analysis also showed the accumulation of nonanal in sealed reaction vials, accounting for the other product of the acyl chain cleavage reaction. The reactivity at both the 9 and 10 positions of the thia-substituted acyl-ACPs is consistent with the proximity of both positions to the diiron center oxidant in the enzyme-substrate complex. Moreover, the differential reactivity of the 9- and 10-thiastearoyl-ACPs also suggests position-dependent consequences of the reaction within the Delta9D active site. Mechanisms accounting for both sulfoxidation and acyl cleavage reactions by the stearoyl-ACP Delta9 desaturase are proposed.     

Desaturation of fatty acids in seeds of higher plants

Photosynthesizing flax, soybean, and safflower plants were exposed to (14)CO(2) at seed-setting stage for a 1 hr period. Seed was sampled periodically thereafter and the lipids were extracted. A triglyceride-rich fraction was methanolyzed; the resultant methyl esters were analyzed by gas-liquid chromatography and assayed for radioactivity. Of the C(18) unsaturated acids, oleic was the first to acquire radioactivity, which subsequently and successively appeared in linoleic and linolenic acids. The shapes of the radioactivity-time curves provide evidence that consecutive desaturation reactions occur in the seeds of these higher plants.     

Desaturation of oxygenated fatty acids in Lesquerella and other oil seeds

Species of the genus Lesquerella, within the Brassicaceae family, have seed oils containing hydroxy fatty acids. In most Lesquerella species, either lesquerolic (14-hydroxy-eicosa-11-enoic), auricolic (14-hydroxy-eicosa-11,17-dienoic) or densipolic (12-hydroxy-octadeca-9,15-dienoic) acid dominates in the seed oils. Incubations of developing seed from Lesquerella species with 1-¹⁴C-fatty acids were conducted in order to study the biosynthetic pathways of these hydroxylated fatty acids. [¹⁴C]Oleic (octadeca-9-enoic) acid, but not [¹⁴C]linoleic (octadeca-9,12-dienoic) acid, was converted into the hydroxy fatty acid, ricinoleic (12-hydroxy-octadeca-9-enoic) acid, which was rapidly desaturated to densipolic (12-hydroxy-octadeca-9,15-dienoic) acid. In addition, [¹⁴C] ricinoleic acid added to Lesquerella seeds was efficiently desaturated at the 15 carbon. A pathway for the biosynthesis of the various hydroxylated fatty acids in Lesquerella seeds is proposed. The demonstration of desaturation at position 15 of a fatty acid with a hydroxy group at position 12 in Lesquerella prompted a comparison of the substrate recognition of the desaturases from Lesquerella and linseed. It was demonstrated that developing linseed also was able to desaturate ricinoleate at position 15 into densipolic acid. In addition, the linseed 15 desaturase was able to desaturate vernolic (12,13-epoxy-octadeca-9-enoic) acid and safflower microsomal 12 desaturase was able to desaturate 9-hydroxy-stearate. Thus, hydroxy and epoxy groups may substitute for double bonds in substrate recognition for oil-seed 12 and 15 desaturases.Abbreviations GLC gas-liquid chromatography - lysoPC palmitoyl-lysophosphatidylcholine - PC phosphatidylcholine This work was supported by grants from Stifteisen Svensk Oljeväxtforskning, Skanska Lantmännen Foundation, Swedish Farmers Foundation for Agricultural research, The Swedish Natural Science Research Council and The Swedish Council for Forestry and Agricultural Research. Nicki Engeseth was supported by the National Science Foundation under a grant award in 1992.     

The reaction of formaldehyde with unsaturated fatty acids during histological fixation

Synopsis Formaldehyde reacts with unsaturated fatty acids in tissues during histological fixation. The reaction of formaldehyde with oleic acid has been found to given rise to compounds (adducts) with the following structures: Two other compounds were isolated but their nature is still open to doubt.The equilibrium constant for the initial part of the reaction has been approximately estimated as 0·042 at a room temperature of 22°C. The endothermic heat of reaction has been estimated as approximately 12·6 kcal.The occurrence of these adducts in tissues explains why it is that less lipid can be demonstrated histologically in material that has been stored in form-aldehyde for a considerable length of time.     

Long chain fatty acids inhibit and medium chain fatty acids activate mammalian cardiac hexokinase

We investigated the effect of non-esterified fatty acids (FAs) on bovine heart hexokinase (type I: ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). Long chain FAs (C14 to C20) inhibited the enzyme in a way that correlated positively with both the chain length and the degree of unsaturation. Medium chain FA with 12 or less carbons activated hexokinase in a chain length dependent manner with the greater activation shown by laurate. The activation constant of laurate was 91.5 microM with a maximal activation of 60.3%. Oleate caused a maximal decrease in specific activity of 25% with an inhibition constant of 79 microM. Using the fluorescent probe cis-parinarate, we found a saturable binding site with K(d) of 3.5 microM. Oleate competed the fluorescent probe from the protein with a K(d) of 1.4 microM. Medium chain FAs did not compete the probe from HK. The binding of fatty acid to the protein appears to be entropically driven as indicated by an Arrhenius analysis (DeltaS=+231.6 J mol(-1) deg(-1)). The presence of oleate significantly increased the K(ATP)(m) from 0.47 mM to 0.89 mM while the K(glucose)(m) in the presence of the FA (0.026+/-0.003 mM) was not significantly different from the control (0.014+/-0.004 mM). A decrease in V(max) values in the presence of oleate indicated that a mixed allosteric inhibition was operating.     

Fatty acid desaturase 2 (FADS2) but not FADS1 desaturates branched chain and odd chain saturated fatty acids

Branched chain fatty acids (BCFA) and linear chain/normal odd chain fatty acids (n-OCFA) are major fatty acids in human skin lipids, especially sebaceous gland (SG) wax esters. Skin lipids contain variable amounts of monounsaturated BCFA and n-OCFA, in some reports exceeding over 20% of total fatty acids. Fatty acid desaturase 2 (FADS2) codes for a multifunctional enzyme that catalyzes Δ4-, Δ6- and Δ8-desaturation towards ten unsaturated fatty acids but only one saturate, palmitic acid, converting it to 16:1n-10; FADS2 is not active towards 14:0 or 18:0. Here we test the hypothesis that FADS2 also operates on BCFA and n-OCFA. MCF-7 cancer cells stably expressing FADS1 or FADS2 along with empty vector control cells were incubated with anteiso-15:0, iso-16:0, iso-17:0, anteiso-17:0, iso-18:0, or n-17:0. BCFA were Δ6-desaturated by FADS2 as follows: iso-16:0 → iso-6Z-16:1, iso-17:0 → iso-6Z-17:1, anteiso-17:0 → anteiso-6Z-17:1 and iso-18:0 → iso-6Z-18:1. anteiso-15:0 was not desaturated in either FADS1 or FADS2 cells. n-17:0 was converted to both n-6Z-17:1 by FADS2 Δ6-desaturation and n-9Z-17:1 by SCD Δ9-desaturation. We thus establish novel FADS2-coded enzymatic activity towards BCFA and n-OCFA, expanding the number of known FADS2 saturated fatty acid substrates from one to six. Because of the importance of FADS2 in human skin, our results imply that dysfunction in activity of sebaceous FADS2 may play a role in skin abnormalities associated with skin lipids.     

Statistical theory of chain scission and concurrent damage not associated with chain scission in linear polymers

A statistical theory of the parallel accumulation of randomly placed breaks and damage not associated with breaks in linear polymers is presented. Expressions for the weight fraction of polymer in the form of chains that bear n or more sites of nonbreak damage (markers) are developed, and results of numerical computations with them presented. The effects of polydispersity in the undamaged sample are discussed. The relation of the computed quantities to experimental methods used in the study of damage to nucleic acids due to radiation and chemical exposure is discussed.     

Utilization of C20-polyunsaturated fatty acids by a yeast fatty acid desaturase mutant

A study was made of the utilization of C₂₀-polyunsaturated fatty acids by the $\text{S. cerevisiae}$ fatty acid desaturase mutant $\text{olel-1}$, Arachidonic acid, 8,11,14-eicosatrienoic acid, and 5,8,11,14,17-eicosapentaenoic acid were about equally effective in supporting growth with lactate as the carbon source. The relative proportion of these fatty acids in total cell fatty acids was $\text{ca}$. 50%. 5,8,11-eicosatrienoic acid synthesized from oleate was less effective. Very little growth occurred with 11,14,17-eicosatrienoic acid or with 11,14-eicosadienoic acid. These results indicate the usefulness of the yeast mutant as a eucaryotic model for study of membrane systems enriched in specific C₂₀-polyunsaturated fatty acids.     

Desaturation of fatty acids as an adaptive response to shifts in light intensity 1

The comparative study of various unicellular algae, characterised by different carbon chain lengths and different numbers of double bonds per fatty acid (FA) chain, exhibited some similarity in the mechanisms of their response to changes in light conditions, in terms of FA metabolism. In all cases, the optimisation of photosynthetic process resulted in some increase in the relative content of the most unsaturated FA, i.e. C16:3Ω3 and C18:3Ω3 acids in Chlorella cells, C16:4Ω3 and C18:3Ω3 in Dunaliella and Chlamydomonas, C20:5Ω3 in Porphyridium, and C18:2Ω6 in Synechocystis sp. As a rule, these FA were esterified to monogalactosyldiacylglycerols (MGDG), the predominant lipids of thylakoid membranes. Such an increase in the relative content of the polyunsaturated FA usually occurred during the period when the photosynthesis, as well as the biosynthesis of FA de novo, were transiently inhibited following shifts in environmental conditions even at their optimisation. The increase in the relative content of the most unsaturated FA could be performed via desaturation of their immediate precursors. In turn, the deterioration of life conditions (decrease in the light intensity, ageing of cells or cultures, and others) resulted in the accumulation of these precursors. As a result, the cell could change its FA composition without alteration of the whole multistage process but only at the rate of this end reaction of polyunsaturated FA biosynthesis. In the majority of algae, these polyunsaturated acids were Ω3-homologues, regardless of the difference in their structures, but in some cyanobacteria (e.g. Synechocystis) the relative content of Ω6-FA increased. The acceleration of Ω3-FA biosynthesis could be observed, regardless of changes in the total index of unsaturation. This FA desaturation was shown to correlate with the activity of photosystem I (PSI). The specificity of this reaction enables us to assume it to be an adaptive response which provides alterations to lipid-protein interactions in the membrane that may be important for the self-assembly of active chlorophyll-protein complexes for photosynthetic akpparatus.     

Branched chain amino acids as source of specific branched chain volatile fatty acids during the fermentation process of fish sauce

Summary.  The source of the formation of branched chain volatile fatty acids (VFA) in fish sauce was investigated. Certain branched VFA were derived from the degradation of specific amino acids as iso-butyric acid from valine and iso-valeric acid from leucine. Short and long straight chain VFA were significantly higher in the linoleic acid added sample than in the control but did not significantly bring changes to the branched chain VFA. It is suggested that straight chain VFA developed from fish fats. Alanine and isoleucine did not have a clear influence on the production of volatile fatty acids. Received November 23, 2001 Accepted June 20, 2002 Published online December 18, 2002 RID="*" ID="*" Part of this paper was presented in the 7th International Congress on Amino Acids and Proteins in Vienna, Austria from August 6–10, 2001. Authors' address: Norlita G. Sanceda, Ph.D., Institute of Environmental Science for Human Life, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112, Japan, Fax: + 81-3-5978-5805, E-mail: lita@cc.ocha.ac.jp     

Upregulated mRNA expression of desaturase and elongase,two enzymes involved in highly unsaturated fatty acids biosynthesis pathways during follicle maturation in zebrafish

Background  

Although unsaturated fatty acids such as eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (ARA, C20:4n-6), collectively known as the highly unsaturated fatty acids (HUFA), play pivotal roles in vertebrate reproduction, very little is known about their synthesis in the ovary. The zebrafish (Danio rerio) display capability to synthesize all three HUFA via pathways involving desaturation and elongation of two precursors, the linoleic acid (LA, C18:2n-6) and linolenic acid (LNA, C18:3n-3). As a prerequisite to gain full understanding on the importance and regulation of ovarian HUFA synthesis, we described here the mRNA expression pattern of two enzymes; desaturase (fadsd6) and elongase (elovl5), involved in HUFA biosynthesis pathway, in different zebrafish ovarian follicle stages. Concurrently, the fatty acid profile of each follicle stage was also analyzed.     

Effects of polyunsaturated fatty acids and clofibrate on chicken stearoyl-coA desaturase 1 gene expression

In chicken, adiposity is influenced by hepatic stearoyl-CoA desaturase (SCD) 1. This gene is up-regulated by low-fat high-carbohydrate diet and down-regulated by addition of polyunsaturated fatty acids (PUFA). In this study, we present evidence for an inhibition of chicken SCD1 expression by PUFA using reporter gene constructs in transient transfection assays. This inhibition does not involve the peroxisome proliferator-activated receptor pathway, in contrast with what has been observed in rodents. We were able to localise a PUFA as well as an insulin response element within the -372/+125 bp region of the promoter. Sequence analyses of this region allowed identification of several cis-regulatory elements: A sterol regulatory element (SRE) and a juxtaposed NF-Y element which have been shown to be involved in the regulation of mouse SCD genes by PUFA. In addition, we identified an overlapping Sp1/USF motif, which was described to play a role in insulin/glucose and PUFA regulation of fatty synthase, ATP-citrate-lyase, and leptin genes. These data provide the first characterisation of the chicken SCD1 promoter and putative cis-sequences involved in the regulation of this gene by PUFA and insulin.