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Title: Synthesis of 8,9-leukotriene A{sub 4} by murine 8-lipoxygenase

Abstract

Arachidonate 8-lipoxygenase was identified in phorbol ester induced mouse skin. We expressed the enzyme in an Escherichia coli system using pET-15b carrying an N-terminal histidine-tag sequence. The enzyme, purified by nickel-nitrilotriacetate affinity chromatography, showed specific activity of about 0.1 {mu}mol/min/mg of protein with arachidonic acid as a substrate. When metabolites of arachidonic acid were reduced and analyzed by reverse-phase HPLC, 8-hydroxy derivative was a major product as measured by absorbance at 235 nm. In addition, three polar compounds (I, II, and III) were detected by measuring absorbance at 270 nm. These compounds were also produced when the enzyme was incubated with 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid. Neither heat-inactivated enzyme nor mutated enzyme produced these compounds, suggesting that they are enzymatically generated. Ultraviolet spectra of these compounds showed typical triplet peaks around 270 nm, indicating that they have a triene structure. Molecular weight of these compounds was determined to be 336 by liquid chromatography-mass spectrometry, indicating that they carry two hydroxyl groups. Compounds I and III were generated even under anaerobic condition, indicating that oxygenation reaction was not required for their generation from 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid. By analogy to the reactions of 5-lipoxygenase pathway where leukotriene A{sub 4} is generated, it is suggested that 8-hydroperoxyeicosa-5,9,11,14-tetraenoicmore » acid is converted by the 8-lipoxygenase to 8,9-epoxyeicosa-5,10,12,14-tetraenoic acid which degrades to compounds I and III by non-enzymatic reaction. In contrast, compound II was not generated under anaerobic condition, indicating that it was produced by oxygenation reaction. Taken together, 8-lipoxygenase catalyzes both dehydration reaction to yield 8,9-epoxy derivative and oxygenation reaction presumably at 15-position of 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid.« less

Authors:
 [1];  [1];  [2];  [1];  [3];  [4]
  1. Department of Molecular Pharmacology, Kanazawa University Graduate School of Medical Science, Kanazawa 920-8640 (Japan)
  2. Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-1197 (Japan)
  3. Research Institute, Ono Pharmaceutical Co., Osaka 618-8585 (Japan)
  4. Department of Molecular Pharmacology, Kanazawa University Graduate School of Medical Science, Kanazawa 920-8640 (Japan). E-mail: yoshimot@med.kanazawa-u.ac.jp
Publication Date:
OSTI Identifier:
20793191
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 338; Journal Issue: 1; Other Information: DOI: 10.1016/j.bbrc.2005.08.099; PII: S0006-291X(05)01832-2; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ARACHIDONIC ACID; DEHYDRATION; ENZYMES; EPOXIDES; ESCHERICHIA COLI; HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY; HISTIDINE; MASS SPECTROSCOPY; MICE; MOLECULAR STRUCTURE; NICKEL; PHORBOL ESTERS; POLAR COMPOUNDS; SKIN; ULTRAVIOLET SPECTRA

Citation Formats

Kawajiri, Hiroo, Piao, Yingshi, Takahashi, Yoshitaka, Murakami, Takashi, Hamanaka, Nobuyuki, and Yoshimoto, Tanihiro. Synthesis of 8,9-leukotriene A{sub 4} by murine 8-lipoxygenase. United States: N. p., 2005. Web. doi:10.1016/J.BBRC.2005.0.
Kawajiri, Hiroo, Piao, Yingshi, Takahashi, Yoshitaka, Murakami, Takashi, Hamanaka, Nobuyuki, & Yoshimoto, Tanihiro. Synthesis of 8,9-leukotriene A{sub 4} by murine 8-lipoxygenase. United States. doi:10.1016/J.BBRC.2005.0.
Kawajiri, Hiroo, Piao, Yingshi, Takahashi, Yoshitaka, Murakami, Takashi, Hamanaka, Nobuyuki, and Yoshimoto, Tanihiro. Fri . "Synthesis of 8,9-leukotriene A{sub 4} by murine 8-lipoxygenase". United States. doi:10.1016/J.BBRC.2005.0.
@article{osti_20793191,
title = {Synthesis of 8,9-leukotriene A{sub 4} by murine 8-lipoxygenase},
author = {Kawajiri, Hiroo and Piao, Yingshi and Takahashi, Yoshitaka and Murakami, Takashi and Hamanaka, Nobuyuki and Yoshimoto, Tanihiro},
abstractNote = {Arachidonate 8-lipoxygenase was identified in phorbol ester induced mouse skin. We expressed the enzyme in an Escherichia coli system using pET-15b carrying an N-terminal histidine-tag sequence. The enzyme, purified by nickel-nitrilotriacetate affinity chromatography, showed specific activity of about 0.1 {mu}mol/min/mg of protein with arachidonic acid as a substrate. When metabolites of arachidonic acid were reduced and analyzed by reverse-phase HPLC, 8-hydroxy derivative was a major product as measured by absorbance at 235 nm. In addition, three polar compounds (I, II, and III) were detected by measuring absorbance at 270 nm. These compounds were also produced when the enzyme was incubated with 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid. Neither heat-inactivated enzyme nor mutated enzyme produced these compounds, suggesting that they are enzymatically generated. Ultraviolet spectra of these compounds showed typical triplet peaks around 270 nm, indicating that they have a triene structure. Molecular weight of these compounds was determined to be 336 by liquid chromatography-mass spectrometry, indicating that they carry two hydroxyl groups. Compounds I and III were generated even under anaerobic condition, indicating that oxygenation reaction was not required for their generation from 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid. By analogy to the reactions of 5-lipoxygenase pathway where leukotriene A{sub 4} is generated, it is suggested that 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid is converted by the 8-lipoxygenase to 8,9-epoxyeicosa-5,10,12,14-tetraenoic acid which degrades to compounds I and III by non-enzymatic reaction. In contrast, compound II was not generated under anaerobic condition, indicating that it was produced by oxygenation reaction. Taken together, 8-lipoxygenase catalyzes both dehydration reaction to yield 8,9-epoxy derivative and oxygenation reaction presumably at 15-position of 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid.},
doi = {10.1016/J.BBRC.2005.0},
journal = {Biochemical and Biophysical Research Communications},
number = 1,
volume = 338,
place = {United States},
year = {Fri Dec 09 00:00:00 EST 2005},
month = {Fri Dec 09 00:00:00 EST 2005}
}
  • Leukotriene C{sub 4} is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C{sub 4} synthase (LTC{sub 4}S) participate in its biosynthesis. We report evidence that LTC{sub 4}S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC{sub 4}S. FLAP bound to the N-terminal part/first hydrophobic region of LTC{sub 4}S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC{sub 4}S. Fluorescent FLAP- and LTC{sub 4}S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC{sub 4}S co-localized withmore » a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC{sub 4}S. Direct interaction of 5-LO and LTC{sub 4}S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC{sub 4}S.« less
  • When arachidonic acid is added to lysates of rat polymorphonuclear leukocytes, it is oxidized to (5S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HPETE). The 5-HPETE then partitions between reduction to the 5-hydroxyeicosanoid and conversion to leukotriene A4 (LTA4). Both steps in the formation of LTA4 are catalyzed by the enzyme 5-lipoxygenase. When (/sup 3/H)arachidonic acid and unlabeled 5-HPETE were incubated together with 5-lipoxygenase, approximately 20% of the arachidonic acid oxidized at low enzyme concentrations was converted to LTA4 without reduction of the specific radioactivity of the LTA4 by the unlabeled 5-HPETE. A significant fraction of the (/sup 3/H)-5-HPETE intermediate that is formed from arachidonic acidmore » must therefore be converted directly to LTA4 without dissociation of the intermediate from the enzyme. This result predicts that even in the presence of high levels of peroxidase activity, which will trap any free 5-HPETE by reduction, the minimum efficiency of conversion of 5-HPETE to LTA4 will be approximately 20%, and this prediction was confirmed. 5-HPETE was found to be a competitive substrate relative to arachidonic acid, so that it is likely that the two substrates share a common active site.« less
  • The first microporous solids incorporating two octahedrally coordinated transition elements, the phosphates (TMA)[sub 2](NH[sub 4])[sub 2][Fe[sub 2]Mo[sub 12]O[sub 30](H[sub 2]PO[sub 4])[sub 6](HOP[sub 4])[sub 2]][center dot]11H[sub 2]O (1) and (TMA)[sub 2]Na[sub 4][Fe[sub 3]Mo[sub 12]O[sub 30](H[sub x]PO[sub 4])[sub 8]][center dot]16H[sub 2]O (2) (TMA = (CH[sub 3])[sub 4]N[sup +]), have been hydrothermally synthesized and characterized by single-crystal X-ray diffraction and water absorption isotherms. Phosphate 1 is prepared in 94% yield from Na[sub 2]MoO[sub 4], Mo, FeCl[sub 3], (NH[sub 4])[sub 2]HPO[sub 4], (TMA)OH, H[sub 3]PO[sub 4], and H[sub 2]O in a mole ratio of 5:1:1:2:7:16:150 at 200[degrees]C for 64 h, while 2 is synthesizedmore » in 62% yield by the reaction of Na[sub 2]MoO[sub 4], Mo, FeCl[sub 3], (TMA)OH, H[sub 3]PO[sub 4] and H[sub 2]O in a mole ratio of 5:1:1:8:18:250 at 200[degrees]C for 3 days. Orange crystals of 1 are rhombohedral. Both structures are based on Fe[Mo[sub 6]O[sub 15](H[sub x]PO[sub 4])[sub 2]] units which are connected via their phosphate groups to additional Fe[sup 3+] ions to give three-dimensional frameworks. Both compounds display structures that can be rationalized on the basis of regions of hydrophobic and hydrophilic interactions. The interconnected voids and channels in the ferric molybdenum phosphate frameworks are filled with a mixture of charged-compensating cations and water of solvation. Reversible water absorption isotherms indicate that both compounds are microporous with internal void volumes of about 15 and 25 vol % for 1 and 2, respectively. 23 refs., 10 figs., 3 tabs.« less
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  • An alcoholysis exchange between tris(hydroxymethyl)ethane (THME-H{sub 3}) or tris(hydroxymethyl)propane (THMP-H{sub 3}) and group IV metal isopropoxides yields compounds of the general formula (THMR){sub 2}M{sub 4}(OCHMe{sub 2}){sub 10}[M = Ti (R = E, 1; P, 2); Zr (R = E, 3; P, 4)]. 1 and 2 are formed in toluene, at ambient glovebox temperatures, and adopt a typical fused-M{sub 3}O{sub 12} structure where each titanium atom is surrounded by six oxygens in a slightly distorted face-shared bioctahedral arrangement. All of the oxygens of the central core are from the THMR ligand, present as {mu}-O and {mu}{sub 3}-O oxygen bridges. Generation ofmore » 3 or 4 requires heating in toluene at reflux temperatures. The zirconium atoms of 3 possess an extremely distorted edge-shared bioctahedral geometry where the central core consists of a Zr{sub 4}O{sub 8} ring (eight oxygens: six from THME ligands and two from isopropoxide ligands). Each of the zirconium atoms is six-coordinated with four bridging oxygens and two terminal isopropoxide ligands. Spincast deposited films generated from toluene solutions of 1 and 3 indicate that increased uniformity of the films and decreased hydrolysis occur in comparison to the cases of Ti(OCHMe{sub 2}){sub 4}, 5, and [Zr(OCHMe{sub 2}){sub 4}{center_dot}HOCHMe{sub 2}]{sub 2}, 6, respectively.« less