Determination of stereochemistry in the fatty acid hydroperoxide products of lipoxygenase catalysis

High-performance liquid chromatography has been found to be an effective method for the determination of absolute configuration in the products of the lipoxygenase-catalyzed oxygenation of polyunsaturated fatty acids. Methyl esters of fatty acid hydroperoxides that had been reduced to the corresponding alcohols were converted into (+)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid esters. Enantiomeric alcohols were converted into diastereomeric esters that were readily resolved by normal-phase HPLC. The instrumental requirements for the technique are an isocratic HPLC and a uv absorbance monitor. The method was found to be effective in the determination of stereochemistry in the products derived from the action of plant lipoxygenases on linoleic acid. In addition, the chromatography of the derivatives obtained from lipoxygenase catalysis on arachidonic acid was found to be effective for the assignment of stereochemistry in those products. A comparison of the chromatography of these derivatives with that for the corresponding menthyloxycarbonyl derivatives demonstrated the superiority of this approach for the resolution of the diastereomeric pairs. The technique was applied to the determination of stereochemistry in the products derived from soybean lipoxygenase isoenzymes under a variety of experimental conditions.     

A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from the moss Physcomitrella patens

A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C(18)-C(22) fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.     

A manganese catalase from Thermomicrobium roseum with peroxidase and catecholase activity

Extremophiles - An enzyme with catechol oxidase activity was identified in Thermomicrobium roseum extracts via solution assays and activity-stained SDS-PAGE. Yet, the genome of T. roseum does not...     

Self-inactivation by 13-hydroperoxylinoleic acid and lipohydroperoxidase activity of the reticulocyte lipoxygenase

1. The self-inactivation of lipoxygenase from rabbit reticulocytes with linoleic acid at 37 degrees C is caused by the product 13-hydroperoxylinoleic acid. This inactivation is promoted by either oxygen or linoleic acid. 2. Lipohydroperoxidase activity was demonstrated with 13-hydroperoxylinoleic acid plus linoleic acid as hydrogen donor under anaerobic conditions at 2 degrees C. The products were 13-hydroxylinoleic acid, oxodienes and compounds of non-diene structure similar to those produced by soybean lipoxygenase-1. 3. 13-Hydroperoxylinoleic acid also changed the absorbance and fluorescence properties of reticulocyte lipoxygenase. The results indicate that one equivalent of 13-hydroperoxylinoleic acid converts the enzyme from the ferrous state into the ferric state as described for soybean lipoxygenase-1. The spectral changes were reversed by sodium borohydride at 2 degrees C, but not at 37 degrees C; it is assumed that the ferric form of reticulocyte lipoxygenase suffers inactivation.     

Overproduction of mycobacterial ribosomal protein S13 induces catalase/peroxidase activity and hypersensitivity to isoniazid in Mycobacterium smegmatis

A Bacillus Calmette Guerlin (BCG) DNA fragment was identified which conferred hypersensitivity to isoniazid (INH) upon Mycobacterium smegmatis (Ms) when present on a multicopy plasmid. The gene cluster present on this fragment contains the genes encoding ribosomal proteins L36 (rpmJ), S13 (rpsM), S11 (rpsK) and S4 (rpsD), as well as the gene encoding initiation factor-1 (infA), an open reading frame of unknown function (ORFX) and a putative promoter region. The rpsM gene, from either BCG or Ms is necessary and sufficient to produce the INH-hypersensitive phenotype in Ms, but the gene cluster has no effect on INH sensitivity when introduced into BCG on a multicopy plasmid. The presence of rpsM on a multicopy plasmid also causes an increase in catalase/peroxidase (Kat/Prx) activity in Ms. The overproduction of S13 may induce a stress response, resulting in increased expression of katG (encoding Kat/Prx) in Ms, thereby causing hypersensitivity to INH     

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.     

Hydroperoxy fatty acid formation in selenium deficient rat platelets: coupling of glutathione peroxidase to the lipoxygenase pathway

After incubation with [1-14C]-arachidonic acid, washed platelets from selenium deficient rats produced a sevenfold greater amount of 12-hydroperoxytetraenoic acid than platelets from control animals. When stimulated with either arachidonic acid or t-butyl-hydroperoxide, antimycin-A1 treated platelets from the deficient rats also converted markedly lower amounts of [1-14C]-glucose to [14C]-CO2 than platelets from control rats. These results indicate a significant role for platelet selenium-dependent glutathione peroxidase in the enzymatic reduction of platelet-produced hydroperoxides.     

A new fungal peroxidase with alkaline-tolerant,chloride-enhancing activity and dye decolorization capacity

A new fungal peroxidase (Pspd) from Perenniporia subacida was purified by ammonium sulfate precipitation, DEAE-cellulose DE52 anionic exchange and Sepharose GL-6B chromatography, resulting in a high specific activity of 9.138 U mg⁻¹, 3.622-fold higher than that of crude enzyme at the same level. Polyacrylamide gel electrophoresis and UV–vis adsorption spectrum analysis showed that the purified enzyme is a heme-containing monomer with a molecular mass of 43.0 kDa. Optimal peroxidase activity was obtained at pH 5.5 and 30 °C when using 100.0 mM n-propanol as substrate, and under these conditions, the catalytic efficiency (k_cat/K_m) is 1.57 s⁻¹ μM⁻¹. Pspd was inhibited by l-cysteine, dithiothreitol, EDTA and sodium azide, but stimulated by Mn²⁺, Na⁺, Mg²⁺ and K⁺. The enzyme is stable over a broad pH range of 7.0–8.5 after incubation for 72 h, which indicated that the enzyme is lasting alkaline-tolerant. It was worth noting that the chloride at relatively low concentrations can enhance the peroxidase activity, with concomitant increase in substrate affinity. Additionally, Pspd performed high decolorization capability toward structurally various dyes and the capability was independent of the oxidizing mediators, with 75.31% of Neutral Red (50.0 mg L⁻¹) being decolorized by 1.5 U mL⁻¹ pure enzyme after incubation for 72 h. These properties demonstrated that Pspd has potentials for textile dyes decolorization applications.     

Thioredoxin-linked lipid hydroperoxide peroxidase activity of human serum albumin in the presence of palmitoyl coenzyme A

Human Serum Albumin (HSA) exerted a significant lipid peroxidase activity with the use of a thiol-reducing equivalent such as dithiothreitol (DTT). Carboxyl group-modified HSA (CM-HSA) showed a 10-fold stronger lipid peroxidase activity (1.6 nmol/min/mg) than that of HSA (0.17 nmol/min/mg). Instead of DTT, thioredoxin (Trx) also supported reducing equivalent to the reduction of lipid hydroperoxide by CM-HSA. Contrast to CM-HSA, HSA did not reduce lipid peroxide with the use of Trx. In the presence of palmitoyl coenzyme A (palmitoyl-CoA) however, HSA used Trx as an electron donor to the reduction of lipid hydroperoxide. The Trx-linked peroxidase activity of HSA sharply increased with elongation in the carbon chain of the acyl moiety of acyl-CoA, showing an optimum activity in the presence of palmitoyl-CoA. Fluorescence study indicates the conformational changes of HSA induced by palmitoyl-CoA. Together, these data suggest that palmitoyl-CoA-bound HSA has a capability to remove lipid peroxide with the use of electrons given by Trx system.     

Inhibitory effect of phospholipid hydroperoxide glutathione peroxidase on the activity of lipoxygenases and cyclooxygenases

The partially purified phospholipid hydroperoxide glutathione peroxidase (PHGPx) from A431 cells was used to systematically compare the inhibitory effect on the enzyme activity of various lipoxygenases and cyclooxygenases. Under the standard assay system, platelet 12-lipoxygenase, 15-lipoxygenase, and cyclooxygenase-2 were the most sensitive to the inhibition by PHGPx. 5-Lipoxygenase and cyclooxygenase-1 were less sensitive to the inhibition by PHGPx than platelet 12-lipoxygenase and cyclooxygenase-2, respectively, and the difference was approximately 10-fold. Reduction of 12(S)-hydroperoxyeicosatetraenoic acid to 12(S)-hydroxyeicosatetraenoic acid by PHGPx was observed in the presence of glutathione (GSH), and the inhibitory effect of PHGPx on 12-lipoxygenase-catalyzed arachidonate metabolism was reversed by the addition of exogenous lipid hydroperoxide. The results indicate that PHGPx directly reduced lipid hydroperoxides and then down-regulated the activity of arachidonate oxygenases. Moreover, a high-level expression of PHGPx mRNA and its 12-lipoxygenase-inhibitory activity was observed in cancer cells and endothelial cells, and these results suggest that PHGPx may play a significant role in the regulation of reactive oxygen species formation in these cells.     

Pitfalls in the use of arachidonic acid oxidation products to assign lipoxygenase activity in cancer cells

Arachidonic acid (AA) reaction with cyclooxygenase (COX) and lipoxygenases (LOX) yield eicosanoids that can mediate prostate cancer proliferation and enhance both tumour vascularization and metastasis. Increasingly measurement of eicosanoids with liquid chromatography is employed to implicate LOX activity in different biological systems and in particular link LOX activity to the progression of cancer in experimental models. This study demonstrates that simply identifying patterns of eicosanoid regio-isomerism is insufficient to designate LOX activity in prostate cancer cells and the analysis must include complete stereochemical assignment of the various isomers in order to validate the assignment of LOX activity.     

Effect of 1,10-phenanthroline on peroxidase and catalase activity and chlorophyll, sugar, and ascorbic acid contents

Changes in the peroxidase (EC 1.11.1.7) and catalase (EC 1.11.1.6) activities, and total chlorophyll, soluble sugars, and ascorbic acid contents of leek leaves treated with the herbicide 1,10-phenanthroline (Phe) in concentrations 0.5, 2.5, 5.0, 7.5, and 10.0 mM have been determined. Plants treated with Phe were characterised by a higher activity of peroxidase and a lower activity of catalase and lower contents of chlorophyll, soluble sugars, and ascorbic acid as compared to non-treated plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of catalase inhibitors on the ultrastructure and peroxidase activity of proliferating microbodies

Summary The relationship between the formation of microbodies and catalase synthesis in the hepatic cells of male rats was examined with conventional electron microscopy and with the peroxidase staining technic for demonstrating catalase. Daily intraperitoneal injections of ethyl--p-chlorophenoxyisobutyrate (CPIB) for 5 days caused a profound increase in microbody numbers without markedly affecting the appearance of the matrix material and all microbodies retained peroxidase activity. A single injection 5 days before sacrifice of 3-amino-1,2,4-triazole (AT), an inhibitor of catalase activity but not catalase synthesis, did not affect their numbers, appearance of matrix material or peroxidase staining. Twice daily injection for 5 days of allylisopropylacetamide (AIA), an inhibitor of catalase synthesis, also did not affect microbody numbers but lowered the electron-density of the microbody matrix and abolished peroxidase staining. After combined administration of these drugs, the number of hepatic microbodies increased but they did not contain peroxidase activity. The results suggest strongly that microbody proliferation is dependent not on catalase synthesis but on synthesis of non-enzymatic protein.This study was supported by research grant HD-01337 from the Institute of Child Health and Human Development, United States Public Health Service. The authors thank Mrs. Judith Henrickson, and Mr. Gerald Haiden for technical assistance. Dr. Legg is at present on leave from the Department of Anatomy, Monash University, Melbourne, Australia.     

Different effects of Triton X-100, deoxycholate, and fatty acids on the kinetics of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase

The effects of Triton X-100, deoxycholate, and fatty acids were studied on the two steps of the ping-pong reaction catalyzed by Se-dependent glutathione peroxidases. The study was carried out by analyzing the single progression curves where the specific glutathione oxidation was monitored using glutathione reductase and NADPH. While the "classic" glutathione peroxidase was inhibited only by Triton, the newly discovered "phospholipid hydroperoxide glutathione peroxidase" was inhibited by deoxycholate and by unsaturated fatty acids. The kinetic analysis showed that in the case of glutathione peroxidase only the interaction of the lipophilic peroxidic substrate was hampered by Triton, indicating that the enzyme is not active at the interface. Phospholipid hydroperoxide glutathione peroxidase activity measured with linoleic acid hydroperoxide as substrate, on the other hand, was not stimulated by the Triton concentrations which have been shown to stimulate the activity on phospholipid hydroperoxides. Furthermore a slight inhibition was apparent at high Triton concentrations and the effect could be attributed to a surface dilution of the substrate. Deoxycholate and unsaturated fatty acids were not inhibitory on glutathione peroxidase but inhibited both steps of the peroxidic reaction of phospholipid hydroperoxide glutathione peroxidase, in the presence of either amphiphilic or hydrophilic substrates. This inhibition pattern suggests an interaction of anionic detergents with the active site of this enzyme. These results are in agreement with the different roles played by these peroxidases in the control of lipid peroxide concentrations in the cells. While glutathione peroxidase reduces the peroxides in the water phase (mainly hydrogen peroxide), the new peroxidase reduces the amphyphilic peroxides, possibly at the water-lipid interface.     

A novel plastidial lipoxygenase of maize (Zea mays) ZmLOX6 encodes for a fatty acid hydroperoxide lyase and is uniquely regulated by phytohormones and pathogen infection

Lipoxygenases (LOXs) are members of a large enzyme family that catalyze oxygenation of free polyunsaturated fatty acids into diverse hydroperoxide compounds, collectively called oxylipins. Although LOXs have been well studied in dicot species, reports of the genes encoding these enzymes are scarce for monocots, especially maize. Herein, we reported the cloning, characterization and molecular functional analysis of a novel maize LOX gene, ZmLOX6. The ZmLOX6 nucleotide sequence encodes a deduced translation product of 892 amino acids. Phylogenetic analysis showed that ZmLOX6 is distantly related to previously reported 9- or 13-LOXs from maize and other plant species, including rice and Arabidopsis. Although sequence prediction suggested cytoplasmic localization of this protein, ZmLOX6 protein has been reportedly isolated from mesophyll cell chloroplasts, emphasizing the unique features of this protein. Plastidial localization was confirmed by chloroplast uptake experiments with the in vitro translated protein. Analysis of recombinant protein revealed that ZmLOX6 has lost fatty acid hydroperoxide forming activity but 13-LOX-derived fatty acid hydroperoxides were cleaved into odd-chain ω-oxo fatty acids and as yet not identified C5-compound. In line with its reported abundance in mesophyll cells, ZmLOX6 was predominantly expressed in leaf tissue. Northern blot analysis demonstrated that ZmLOX6 was induced by jasmonic acid, but repressed by abscisic acid, salicylic acid and ethylene and was not responsive to wounding or insects. Further, this gene was strongly induced by the fungal pathogen Cochliobolus carbonum during compatible interactions, suggesting that ZmLOX6 may contribute to susceptibility to this pathogen. The potential involvement of ZmLOX6 in maize interactions with pathogens is discussed.     

Differential impact of low temperature on fatty acid unsaturation and lipoxygenase activity in figleaf gourd and cucumber roots

Previous studies show that low temperature strongly induces suberin layers in the roots of chilling-sensitive cucumber plants, while in contrast, low temperature produces a much weaker induction of suberin layers in the roots of the chilling-tolerant figleaf gourd [S.H. Lee, G.C. Chung, S. Steudle, Gating of aquaporins by low temperature in roots of chilling-sensitive cucumber and -tolerant figleaf gourd, J. Exp. Bot. 56 (2005) 985-995; S.H. Lee, G.C. Chung, E. Steudle, Low temperature and mechanical stresses differently gate aquaporins of root cortical cells of chilling-sensitive cucumber and figleaf gourd, Plant Cell Environ. (2005) in press; S.J. Ahn, Y.J. Im, G.C. Chung, B.H. Cho, S.R. Suh, Physiological responses of grafted-cucumber leaves and rootstock roots affected by low root temperature, Scientia Hort. 81 (1999) 397-408]. Here, the effect of low temperature on fatty acid unsaturation and lipoxygenase activity was examined in cucumber and figleaf gourd. The double bond index demonstrated that membrane lipid unsaturation shows hyperbolic saturation curve in figleaf gourd roots while a biphasic response in cucumber roots to low temperature. In figleaf gourd, the hyperbolic response in the double bond index was primarily due to accumulation of linolenic acid. Chilling stress also significantly induced lipoxygenase activity in figleaf gourd roots. These results suggest that the degree of unsaturation of root plasma membrane lipids correlates positively with chilling-tolerance. Therefore, studies that compare the effects of chilling on cucumber and figleaf gourd may provide broad insight into stress response mechanisms in chilling-sensitive and chilling-tolerant plants. Furthermore, these studies may provide important information regarding the relationship between lipid unsaturation and lipoxygenase function/activity, and between lipoxygenase activity and water channeling during the response to chilling stress. The possible roles of these processes in chilling tolerance are discussed.     

Induction of plasminogen activator by 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore: Suppression by inhibitors of fatty acid lipoxygenase

HeLa cells incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and rat basophilic leukemia (RBL-1) cells incubated with calcium ionophore, showed increased levels of the protease plasminogen activator. These treatments have previously been shown to stimulate the cellular metabolism of arachidonic acid. The induction of plasminogen activator in both cell types was inhibited in a dose-dependent manner by 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, two compounds known to inhibit arachidonate metabolism via lipoxygenases. In contrast, indomethacin, which selectively inhibits arachidonate metabolism via cyclooxygenase, was inactive. The levels of four enzyme markers in HeLa cells were unchanged by treatment with TPA plus the lipoxygenase inhibitors, indicating that the inhibitors did not exert their effects on plasminogen activator via general cell toxicity. HeLa cells preincubated with [³H]arachidonate and subsequently challenged with TPA produced small amounts of material with the chromatographic mobilities and resistance to indomethacin expected of hydroxylated fatty acids derived via lipoxygenase. RBL-1 cells have been shown previously to produce leukotrienes and other lipoxygenase metabolites when treated with calcium ionophore. Plasminogen activator in HeLa cells was stimulated by up to 2.5-fold by incubation with 0.5–2 μg/ml 5-hydroxyeicosatetraenoic acid. Our results suggest that the induction of plasminogen activator in HeLa and RBL-1 cells is not mediated by prostaglandins or thromboxanes, but may be mediated or modulated by arachidonate metabolites derived via a lipoxygenase pathway.     

Phospholipid hydroperoxide glutathione peroxidase activity and vitamin E level in the liver microsomal membrane: effects of age and dietary alpha-linolenic acid deficiency

Age and diet-induced variations of phospholipid hydroperoxide glutathione peroxidase (PHGPx) activity and alpha-tocopherol concentration in the liver microsomal membrane were studied in male Wistar rats fed a semipurified diet either balanced in n-6 and n-3 polyunsaturated fatty acids (PUFA) (Control) or deprived of alpha-linolenic acid, i.e. n-3 PUFA (Deficient) over two generations. The animals were studied at the age of 6 months (adult) or 24 months (old). Both PHGPx activity and vitamin E level were significantly higher in 24-month old rats as compared to 6-month old rats. By contrast, the thiobarbituric acid reactive substances (TBARS) following stimulated in vitro peroxidation of membrane lipids were markedly lower (P < 0.01) with aging. The fatty acid composition of microsomal membrane phospholipids (PL) was also considerably modified by age. In particular, the levels of arachidonic acid and total n-6 PUFA were lower (P < 0.001) whereas n-3 PUFA levels were higher (P < 0.001) in most PL main classes. The alpha-linolenic acid deficiency markedly influenced these age-related changes. The higher PHGPx activity in the old rats as compared to the adult rats was only significant in those fed the control diet. In the 6-month old rats (but not in the 24-month old rats), the deficient diet led to a higher membrane vitamin E level and to lower TBARS production than the control diet. The results suggest that the nature of dietary PUFA may influence the age-related variations in this pair of membrane antioxidants and also in the fatty acid composition of microsomes.     

Glutathione peroxidase activity modulates fatty acid profiles of plasma and breast milk in Chinese women.

Since little is known about the effect of selenium on the fatty acid profiles (FAP) of human breast milk, the purpose of this study was to measure the effect of habitual dietary selenium (Se) intake on this profile in plasma and breast milk. Subjects were lactating women from three locations in China where habitual selenium intakes are extremely low (Xichang), adequate (Beijing), or extremely high (Enshi). Plasma and milk samples were obtained within seven days of parturition (early samples) or within eighteen months postpartum (mature samples) and analyzed for selenium concentration, glutathione peroxidase (Gpx) activity and FAP. Plasma and milk selenium concentrations were significantly lower in the samples from women from Xichang and significantly higher in those from Enshi when compared to those from Beijing. Plasma Gpx activity, however, was higher in samples from Beijing than Xichang or Enshi. In contrast, the early breast milk samples had similar Gpx activity regardless of location. The mature samples, however, followed the same trend as plasma with the samples obtained from the women in Beijing having the highest activity. Of the unsaturated fatty acids examined, the concentration of linoleic acid, 18:2(n-6), in both plasma and milk was greater in the samples from Beijing when compared to those from Xichang or Enshi. Thus dietary selenium appears to influence the fatty acid composition in human breast milk, but influences Gpx activity only in mature milk samples.