Steady-state kinetics of the anaerobic reaction of soybean lipoxygenase-1 with linoleic acid and 13-L-hydroperoxylinoleic acid.

The steady-state kinetics of the anaerobic reaction of soybean lipoxygenase-1 with linoleic acid and 13-L-hydroperoxylinoleic acid were studied. Initial rates of the formation of oxodienoic acids**, absorbing at 285 nm, were measured at pH 10. About 50% of the consumed 13-L-hydroperoxylinoleic acid was converted into oxodienoic acids regardless of the initial ratio of the two substrates. A linear inhibition by both linoleic acid and 13-L-hydroperoxylinoleic acid was observed in the concentration range studied, which is on the upper side limited by the concentrations at which micelle- or acid-soap formation starts. A kinetic scheme is proposed based on one active site in lipoxygenase-1 which alternately binds the two substrates. Values for the kinetic constants were calculated by fitting simultaneously the complete set of data to the appropriate rate equation.     

Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase-1.

1. The EPR spectrum at 15 degrees K of soybean lipoxygenase-1 in borate buffer pH 9.0 has been studied in relation to the presence of substrate (linoleic acid), product (13-L-hydroperoxylinoleic acid) and oxygen. 2. The addition of 13-L-hydroperoxylinoleic acid to lipoxygenase-1 at pH 9.0 gives rise to the appearance of EPR lines at g equals 7.5, 6.2, 5.9 and 2.0, and an increased signal at g equals 4.3. 3. In view of the effect of the end product on both the kinetic lag period of the aerobic reaction and the fluorescence of the enzyme, it is concluded that 13-L-hydroperoxylinoleic acid is required for the activation of soybean lipoxygenase-1. Thus it is proposed that the enzyme with iron in the ferric state is the active species. 4. A reaction scheme is presented in which the enzyme alternatingly exists in the ferric and ferrous states for both the aerobic and anaerobic reaction.     

Ionization and phase behavior of fatty acids in water: application of the Gibbs phase rule

The phase behavior of several medium-chain (10- and 12-carbon) and long-chain (18-carbon) fatty acids in water was examined as a function of the ionization state of the carboxyl group. Equilibrium titration curves were generated above and below fatty acid and acid-soap chain melting temperatures and critical micelle concentrations, and the phases formed were characterized by X-ray diffraction, 13C NMR spectroscopy, and phase-contrast and polarized light microscopy. The resulting titration curves were divided into five regions: (i) at pH values less than 7, a two-phase region containing oil or fatty acid crystals and an aqueous phase; (ii) at pH approximately 7, a three-phase region containing oil, lamellar, and aqueous (or fatty acid crystals, 1:1 acid-soap crystals, and aqueous) phases; (iii) between pH 7 and 9, a two-phase region containing a lamellar fatty acid/soap (or crystalline 1:1 acid-soap) phase in an aqueous phase; (iv) at pH approximately 9, a three-phase region containing lamellar fatty acid-soap (or crystalline 1:1 acid-soap), micellar, and aqueous phases; and (v) at pH values greater than 9, a two-phase region containing micellar and aqueous phases. Interpretation of the results using the Gibbs phase rule indicated that, for oleic acid/potassium oleate, the composition of the lamellar fatty acid/soap phase varied from approximately 1:1 to 1:3 un-ionized to ionized fatty acid species. In addition, constant pH regions observed in titration curves were a result of thermodynamic invariance (zero degrees of freedom) rather than buffering capacity. The results provide insights into the physical states of fatty acids in biological systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Lipoxygenase from potato tubers. Partial purification and properties of an enzyme that specifically oxygenates the 9-position of linoleic acid

A lipoxygenase (EC 1.13.1.13) was partially purified from potato tubers and was shown to differ from previously characterized soya-bean lipoxygenases in the positional specificity and pH characteristics of the oxygenation reaction. The potato enzyme converted linoleic acid almost exclusively (95%) into 9-d-hydroperoxyoctadeca-trans-10,cis-12-dienoic acid. The 13-hydroperoxy isomer was only a minor product (5%). Linolenic acid was an equally effective substrate, which was also oxygenated specifically at the 9-position. The enzyme had a pH optimum at 5.5-6.0 and was inactive at pH9.0. A half-maximal velocity was obtained at a linoleic acid concentration of 0.1mm. No inhibition was observed with EDTA (1mm) and cyanide (1mm) or with p-chloromercuribenzoate (0.2mm). Haemoproteins were not involved in the lipoxygenase activity. The molecular weight of the enzyme was estimated from gel filtration to be approx. 10(5). Preliminary evidence suggested that the enzyme oxygenated the n-10 position of fatty acids containing a penta(n-3, n-6)diene structure.     

The regulation of formation of prostaglandins and arachidonoyl-CoA from arachidonic acid in rabbit kidney medulla microsomes by linoleic acid hydroperoxide

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of linoleic acid (LA) and 13-hydroperoxyoctadecadienoic acid (13-HPODE) on the PG and AA-CoA formation from high and low concentrations of AA (60 and 5 microM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 microM [(14)C]-AA in 0.1M Tris-HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl(2) and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. LA (10-50 microM) reduced only PG formation from both 60 and 5 microM AA. 13-HPODE (10-50 microM) also reduced PG formation from 60 and 5 microM AA, but the inhibitory potency was much stronger than that by LA. Furthermore, 13-HPODE had the potential to increase the AA-CoA formation with a decrease in the PG formation from 5 microM AA. These results suggest that 13-HPODE, but not LA, may shift AA away from COX pathway into ACS pathway under low substrate concentration (near physiological concentration of AA).     

Soybean lipoxygenase-1 enzymically forms both (9S)- and (13S)-hydroperoxides from linoleic acid by a pH-dependent mechanism

Soybean lipoxygenase-1 produces a preponderance of two chiral products from linoleic acid, (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid and (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid. The former of these hydroperoxides was generated at all pH values, but in the presence of Tween 20, the latter product did not form at pH values above 8.5. As the pH decreased below 8.5, the proportion of (9S)-hydroperoxide increased linearly until at pH 6 it constituted about 25% of the chiral products attributed to enzymic action. Below pH 6, lipoxygenase activity was barely measurable, and the hydroperoxide product arose mainly from autoxidation and possibly non-enzymic oxygenation of the pentadienyl radical formed by the enzyme. The change in percent enzymically formed 9-hydroperoxide between pH 6.0 and 8.5 paralleled the pH plot of a sodium linoleate/linoleic acid titration. It was concluded that the (9S)-hydroperoxide is formed only from the nonionized carboxylic acid form of linoleic acid. Methyl esterification of linoleic acid blocked the formation of the (9S)-hydroperoxide by lipoxygenase-1, but not the (13S)-hydroperoxide. Since the hydroperoxydiene moieties of the (9S)- and (13S)-hydroperoxides are spatially identical when the molecules are arranged head to tail in opposite orientations, it is suggested that the carboxylic acid form of the substrate can arrange itself at the active site in either orientation, but the carboxylate anion can be positioned only in one orientation. These observations, as well as others in the literature, suggest and active-site model for soybean lipoxygenase-1.     

Influence of acid soap interactions on the solubility of soaps in triglycerides

The dependence of the solubility of sodium octanoate in trioctanoin on the presence of octanoic acid was determined by solubility measurements at 20 °C. The intermolecular forces which cause the formation of acid-soap aggregates were investigated by means of infrared and proton magnetic resonance spectra.     

The origin and structures of dimeric fatty acids from the anaerobic reaction between soya-bean lipoxygenase, linoleic acid and its hydroperoxide

In an anaerobic system soya-bean lipoxygenase catalyses in the presence of linoleic acid and l-13-hydroperoxyoctadeca-cis-9-trans-11-dienoic acid the formation of dimeric fatty acids and of carbonyl compounds. The analogous reaction does not take place when d-9-hydroperoxyoctadeca-trans-10-cis-12-dienoic acid is used instead of the 13-hydroperoxy isomer. Non-oxygenated dimers stem directly from linoleic acid and have C((11))-C((13')) or -C((9')) and C((13))-C((13')) or -C((9')) linkages. Dimers that contain oxygen originate from linoleic acid and linoleic acid hydroperoxide. It is most likely that the oxygen is present in epoxy groups.     

9-LR-linoleyl hydroperoxide, a novel product from the oxygenation of linoleic acid by type-2 lipoxygenases from soybeans and peas.

Type-2 lipoxygenases from soybeans and peas, which have a pH optimum of 6--7 were examined for oxygenation activity at pH 9.0. The reaction velocity was found to be strongly dependent on substrate concentration. At higher substrate concentrations an inhibitory effect was observed, which is connected with the occurrence of a kinetic lag phase. On incubation of linoleic acid at pH 9.0 with either of these enzymes predominantly 9-LR-hydroperoxy-10-trans,12-cis-octadecadienoic acid is formed. The similarity of the product specificity with that of prostaglandin synthetase is discussed in view of the formation of prostaglandin-like substances by soybean lipoxygenase-2 (Bild, G.S., Bhat, S.G., Ramadoss, C.S. and Axelrod, B. (1978) J. Biol. Chem, 253, 21--23).     

Effect of pH on conjugated linoleic acid (CLA) formation of linolenic acid biohydrogenation by ruminal microorganisms

Conventional beliefs surrounding the linolenic acid (LNA; cis-9 cis-12 cis-15 C18:3) biohydrogenation (BH) pathway propose that it converts to stearic acid (SA) without the formation of conjugated linoleic acid (CLA) as intermediate isomers. However, an advanced study (Lee and Jenkins, 2011) verified that LNA BH yields multiple CLAs. This study utilized the stable isotope tracer to investigate the BH intermediates of ¹³C-LNA with different pH conditions (5.5 and 6.5). The ¹³C enrichment was calculated as a ¹³C/¹²C ratio of labeled minus unlabeled. After 24 h, eight CLA isomers were significantly enriched on both pH treatment, this result verifies that these CLAs originated from ¹³C-LNA BH which supports the results of Lee and Jenkins (2011). The enrichment of cis-cis double bond CLAs (cis-9 cis-11 and cis-10 cis-12 CLA) were significantly higher at low pH conditions. Furthermore, the concentration of cis-10 cis-12 CLA at low pH was four times higher than at high pH conditions after a 3 h incubation. These differences support the LNA BH pathways partial switch under different pH conditions, with a strong influence on the cis-cis CLA at low pH. Several mono-, di-, and tri-enoic fatty acid isomers were enriched during 24 h of incubation, but the enrichment was decreased or restricted at low pH treatment. Based on these results, it is proposed that low pH conditions may cause a changed or limited capacity of the isomerization and reduction steps in BH.     

A linoleic acid (8R)-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis. Biosynthesis of (8R)-hydroxylinoleic acid and (7S,8S)-dihydroxylinoleic acid from (8R)-hydroperoxylinoleic acid.

The fungus Gaeumannomyces graminis metabolized linoleic acid extensively to (8R)-hydroperoxylinoleic acid, (8R)-hydroxylinoleic acid, and threo-(7S,8S)-dihydroxylinoleic acid. When G. graminis was incubated with linoleic acid under an atmosphere of oxygen-18, the isotope was incorporated into (8R)-hydroxylinoleic acid and 7,8-dihydroxylinoleic acid. The two hydroxyls of the latter contained either two oxygen-18 or two oxygen-16 atoms, whereas a molecular species that contained both oxygen isotopes was formed in negligible amounts. Glutathione peroxidase inhibited the biosynthesis of 7,8-dihydroxylinoleic acid. These findings demonstrated that the diol was formed from (8R)-hydroperoxylinoleic acid by intramolecular hydroxylation at carbon 7, catalyzed by a hydroperoxide isomerase. The (8R)-dioxygenase appeared to metabolize substrates with a saturated carboxylic side chain and a 9Z-double bond. G. graminis also formed omega 2- and omega 3-hydroxy metabolites of the fatty acids. In addition, linoleic acid was converted to small amounts of nearly (65% R) racemic 10-hydroxy-8,12-octadecadienoic acid by incorporation of atmospheric oxygen. An unstable metabolite, 11-hydroxylinoleic acid, could also be isolated as well as (13R,13S)-hydroxy-(9E,9Z), (11E)-octadecadienoic acids and (9R,9S)-hydroxy-(10E), (12E,12Z)-octadecadienoic acids. In summary, G. graminis contains a prominent linoleic acid (8R)-dioxygenase, which differs from the lipoxygenase family of dioxygenases by catalyzing the formation of a hydroperoxide without affecting the double bonds of the substrate.     

Oxidative metabolism of linoleic acid by human leukocytes

Upon incubation with human leukocytes, [1-14C] linoleic acid is almost exclusively transformed into 13-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) if the linoleic acid concentration is lower than 50 microM. Identification of 13-HODE was done by GLC-MS at the level of its methyl ester, trimethylsilyl ether and by comparison with authentic 13-HODE in two different HPLC systems. Analysis of the products by chiral phase HPLC shows that 13(S)-hydroxy-9Z, 11E-octadecadienoic acid is by far the major metabolite formed by human leukocytes. Comparison of reactions performed with intact or lyzed cells suggests that the formation of 13(S)-HODE by human leukocytes occurs in two steps, a dioxygenation catalyzed by a 15-lipoxygenase and a reduction of intermediate 13-HPODE by a glutathione-dependent peroxidase.     

透性化嗜酸乳杆菌细胞转化亚油酸为共轭亚油酸的反应动力学

本实验旨在研究透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的反应动力学。探讨了细胞浓度、底物浓度、反应体系pH值和温度等因素对生物转化共轭亚油酸反应速度的影响;建立了透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的动力学模型。结果表明,透性化嗜酸乳杆菌细胞有利于共轭亚油酸的生物转化,最适细胞浓度、pH值和反应温度分别为10×1010ufc/mL、4.5和45℃;生物转化共轭亚油酸存在底物抑制现象,当亚油酸的浓度为0.6mg/mL时,反应速度达到最大值17.8μg/(mL·min)。在低亚油酸浓度下,反应初始阶段的反应规律与经典米氏方程相符,而在高亚油酸浓度下,存在底物抑制现象。在最适反应条件下建立了动力学模型,模型基本反映了共轭亚油酸的生物转化特性。     

Factors affecting the formation of 10-hydroxystearic acid from oleic acid by a ruminal strain of Enterococcus faecalis

 A ruminal strain of Enterococcus faecalis was characterised with respect to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydroxy fatty acid was produced after growth had ceased and the carbon source was almost exhausted. Hydroxy fatty acid production was equally rapid whether the inoculum had been grown in the presence of oleic acid or not, and almost complete conversion was achieved when oleic acid was present at a concentration of up to 0.5% (v/v). Incubation under a hydrogen headspace did not result in biohydrogenation of oleic acid. In pH-controlled batch culture the proportion of oleic acid hydrated varied with the pH of incubation, with more hydration at lower pH. Growth was retarded in the presence of 0.1% (v/v) linoleic acid, inhibited by the same concentration of linolenic acid and did not result in the formation of hydrated products from these substrates. If this organism is able to transform oleic acid in the rumen then the only product likely to be formed is 10hydroxystearic acid. Received: 17 July 1995/Received last revision: 24 October 1995/Accepted: 30 October 1995     

Enhanced production of hydroperoxides by immobilized lipoxygenase in the presence of cyclodextrins

The advantages of the presence of cyclodextrins in a reaction catalyzed by immobilized lipoxygenase at neutral pH are reported for the first time. The steady-state rate in the presence of beta-cyclodextrins was seven times higher than in control experiments using the same concentration of linoleic acid; furthermore the percentage of substrate conversion (and product accumulation) obtained in the presence of beta-cyclodextrins was higher than in the control assays. The optimum concentration of free linoleic acid coincided with the critical micellar concentration for linoleic acid at neutral pH. The operational stability of the immobilized enzyme increased in the presence of beta-cyclodextins, while an increase in the percentage of 13-HPOD was also observed.     

Influence of pH and undissociated butyric acid on the production of acetone and butanol in batch cultures of Clostridium acetobutylicum

Summary The kinetics of growth and acid and solvent production are examined in batch fermentation of Clostridium acetobutylicum at pH between 4.5 and 6.0. At the lower pH, growth occurs in two consecutive phases and solvents are the main excreted metabolites. At the higher pH, there is a single growth phase with only acid formation. The influence of the pH can be correlated with a critical role of the concentration of undissociated butyric acid in the medium: cellular growth is inhibited above 0.5 g/l and solvent production starts at an undissociated acid level of 1.5 g/l. Reducing the intracellular acid dissociation by lowering the intracellular pH also favours the production of acetone and butanol.     

Bioconversion of linoleic acid into conjugated linoleic acid by immobilized Lactobacillus reuteri

Lactobacillus reuteri was immobilized on silica gel to evaluate the bioconversion of linoleic acid (LA) into conjugated linoleic acid (CLA), consisting of cis-9,trans-11 and trans-10,cis-12 isomers. The amount of cell to carrier, the reaction time, and the substrate concentration, pH, and temperature for CLA production were optimized at 10 mg of cells/(g of carrier), 1 h, 500 mg/L LA, 10.5, and 55 degrees C, respectively. In the presence of 1.0 mM Cu(2+), CLA production increased by 110%. Under the optimal conditions, the immobilized cells produced 175 mg/L CLA from 500 mg/L LA for 1 h with a productivity of 175 mg/(L.h) and accumulated 5.5 times more CLA than that obtained from bioconversion by free washed cells. The CLA-producing ability of reused cells was investigated over five reuse reactions and was maximal at pH 7.5, 25 degrees C, and 1.0 mM Cu(2+). The total amount of CLA by the combined five reuse reactions was 344 mg of CLA/L reaction volume. This was 8.6 times higher than the amount obtained from reuse reactions by free washed cells.     

Biliary phospholipid secretion is not required for intestinal absorption and plasma status of linoleic acid in mice.

Biliary phospholipids have been hypothesized to be important for essential fatty acid homeostasis. We tested this hypothesis by investigating the intestinal absorption and the status of linoleic acid in mdr2 Pgp-deficient mice which secrete phospholipid-free bile. In mice homozygous (-/-) for disruption of the mdr2 gene and wild-type (+/+) mice, dietary linoleic acid absorption was determined by 72 h balance techniques. After enteral administration, [(13)C]-linoleic acid absorption was determined by measuring [(13)C]-linoleic acid concentrations in feces and in plasma. The status of linoleic acid was determined in plasma and in liver by calculating the molar percentage of linoleic acid and the triene:tetraene ratio. Although plasma concentration of [(13)C]-linoleic acid at 2 h after enteral administration was significantly lower in (-/-) compared to (+/+) mice (P相似文献   

Conversion of linoleic acid into 10-Hydroxy-12(Z)-octadecenoic acid by whole cells of Stenotrophomonas nitritireducens

The conversion of linoleic acid into 10-hydroxy-12(Z)-octadecenoic acid by whole cells of Stenotrophomonas nitritireducens as an isolated bacterium was optimized, and the optimal temperature, pH, and cell and substrate concentrations were 30 degrees C, 7.5, and 20 and 20 g/L, respectively. Under these conditions, whole cells in a bioreactor produced 15 g/L 10-hydroxy-12(Z)-octadecenoic acid in 2 h of reaction time without detectable byproducts. Using 2 g/L linoleic acid, the cells produced 1.92 g/L 10-hydroxy-12(Z)-octadecenoic acid. These are the highest concentration and yield of 10-hydroxy-12(Z)-octadecenoic acid ever reported.