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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}
}