Epidermal growth factor enhances a microsomal 12-lipoxygenase activity in A431 cells

Wen Chang Chang, Chung Chu Ning, Ming T. Lin, Jin Ding Huang

Research output: Contribution to journalArticle

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Abstract

12-Hydroxyeicosatetraenoic acid (12-HETE) is formed from arachidonic acid either by 12-lipoxygenase or by a cytochrome P450 monooxygenase. 12-Lipoxygenase is generally localized in the soluble cytosolic fraction, and the cytochrome P450 monooxygenase is a microsomal enzyme. In this study, 12-HETE biosynthesis and the regulation of 12-HETE biosynthesis by epidermal growth factor (EGF) in A431 cells were investigated. 12-HETE was biosynthesized from arachidonic acid by the microsomal fraction of A431 cells, but not by the cytosolic fraction. The formation of 12-HETE was inhibited by 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and caffeic acid. Nordihydroguaiaretic acid at 10-4 M and 5,8,11,14-eicosatetraynoic acid at 10-5 M almost completely inhibited its formation. However, the formation of 12-HETE was not affected by the presence of an NADPH-generating system, carbon monoxide, or SKF 525A. The biosynthetic 12-HETE was analyzed by chiral stationary phase high performance liquid chromatography and was highly enriched in (125)-HETE. We therefore concluded that the enzyme responsible for the formation of (12S)-HETE in the microsomes of A431 cells is a 12-lipoxygenase. The microsomal 12-lipoxygenase of A431 cells belongs to the "leukocyte-type" enzyme as determined by substrate specificity and enzyme kinetics studies. The microsomal 12-lipoxygenase oxygenated linoleic acid much faster than the cytosolic platelet 12-lipoxygenase and is a "self-catalyzed inactivation" enzyme. Treatment of cells with 50 ng/ ml EGF significantly induced microsomal 12-lipoxygenase activity. The lag period for the expression of the stimulatory effect of EGF on 12-lipoxygenase activity was -10 h. The stimulatory effect of EGF on 12-lipoxygenase activity was completely blocked by treat-ment with 35 μM cycloheximide, indicating a requirement for de novo protein biosynthesis. Furthermore, the presence of the endogenous inhibitor of 12-lipoxygenase (which masked (12S)-HETE biosynthesis in intact cells) was identified in the cytosolic fraction of A431 cells. The putative inhibitor was enzyme-selective. It inhibited the leukocyte-type 12-lipoxygenase, but not the "platelet-type" enzyme.

Original languageEnglish
Pages (from-to)3657-3666
Number of pages10
JournalJournal of Biological Chemistry
Volume267
Issue number6
Publication statusPublished - Feb 25 1992
Externally publishedYes

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Arachidonate 12-Lipoxygenase
Hydroxyeicosatetraenoic Acids
Epidermal Growth Factor
Biosynthesis
Enzymes
5,8,11,14-Eicosatetraynoic Acid
Masoprocol
Platelets
Mixed Function Oxygenases
Cytochrome P-450 Enzyme System
Leukocytes
Blood Platelets
Proadifen
Lipoxygenase Inhibitors
Enzyme kinetics
High performance liquid chromatography
Protein Biosynthesis
Enzyme Inhibitors
Linoleic Acid
Carbon Monoxide

ASJC Scopus subject areas

  • Biochemistry

Cite this

Epidermal growth factor enhances a microsomal 12-lipoxygenase activity in A431 cells. / Chang, Wen Chang; Ning, Chung Chu; Lin, Ming T.; Huang, Jin Ding.

In: Journal of Biological Chemistry, Vol. 267, No. 6, 25.02.1992, p. 3657-3666.

Research output: Contribution to journalArticle

Chang, Wen Chang ; Ning, Chung Chu ; Lin, Ming T. ; Huang, Jin Ding. / Epidermal growth factor enhances a microsomal 12-lipoxygenase activity in A431 cells. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 6. pp. 3657-3666.
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abstract = "12-Hydroxyeicosatetraenoic acid (12-HETE) is formed from arachidonic acid either by 12-lipoxygenase or by a cytochrome P450 monooxygenase. 12-Lipoxygenase is generally localized in the soluble cytosolic fraction, and the cytochrome P450 monooxygenase is a microsomal enzyme. In this study, 12-HETE biosynthesis and the regulation of 12-HETE biosynthesis by epidermal growth factor (EGF) in A431 cells were investigated. 12-HETE was biosynthesized from arachidonic acid by the microsomal fraction of A431 cells, but not by the cytosolic fraction. The formation of 12-HETE was inhibited by 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and caffeic acid. Nordihydroguaiaretic acid at 10-4 M and 5,8,11,14-eicosatetraynoic acid at 10-5 M almost completely inhibited its formation. However, the formation of 12-HETE was not affected by the presence of an NADPH-generating system, carbon monoxide, or SKF 525A. The biosynthetic 12-HETE was analyzed by chiral stationary phase high performance liquid chromatography and was highly enriched in (125)-HETE. We therefore concluded that the enzyme responsible for the formation of (12S)-HETE in the microsomes of A431 cells is a 12-lipoxygenase. The microsomal 12-lipoxygenase of A431 cells belongs to the {"}leukocyte-type{"} enzyme as determined by substrate specificity and enzyme kinetics studies. The microsomal 12-lipoxygenase oxygenated linoleic acid much faster than the cytosolic platelet 12-lipoxygenase and is a {"}self-catalyzed inactivation{"} enzyme. Treatment of cells with 50 ng/ ml EGF significantly induced microsomal 12-lipoxygenase activity. The lag period for the expression of the stimulatory effect of EGF on 12-lipoxygenase activity was -10 h. The stimulatory effect of EGF on 12-lipoxygenase activity was completely blocked by treat-ment with 35 μM cycloheximide, indicating a requirement for de novo protein biosynthesis. Furthermore, the presence of the endogenous inhibitor of 12-lipoxygenase (which masked (12S)-HETE biosynthesis in intact cells) was identified in the cytosolic fraction of A431 cells. The putative inhibitor was enzyme-selective. It inhibited the leukocyte-type 12-lipoxygenase, but not the {"}platelet-type{"} enzyme.",
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AB - 12-Hydroxyeicosatetraenoic acid (12-HETE) is formed from arachidonic acid either by 12-lipoxygenase or by a cytochrome P450 monooxygenase. 12-Lipoxygenase is generally localized in the soluble cytosolic fraction, and the cytochrome P450 monooxygenase is a microsomal enzyme. In this study, 12-HETE biosynthesis and the regulation of 12-HETE biosynthesis by epidermal growth factor (EGF) in A431 cells were investigated. 12-HETE was biosynthesized from arachidonic acid by the microsomal fraction of A431 cells, but not by the cytosolic fraction. The formation of 12-HETE was inhibited by 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and caffeic acid. Nordihydroguaiaretic acid at 10-4 M and 5,8,11,14-eicosatetraynoic acid at 10-5 M almost completely inhibited its formation. However, the formation of 12-HETE was not affected by the presence of an NADPH-generating system, carbon monoxide, or SKF 525A. The biosynthetic 12-HETE was analyzed by chiral stationary phase high performance liquid chromatography and was highly enriched in (125)-HETE. We therefore concluded that the enzyme responsible for the formation of (12S)-HETE in the microsomes of A431 cells is a 12-lipoxygenase. The microsomal 12-lipoxygenase of A431 cells belongs to the "leukocyte-type" enzyme as determined by substrate specificity and enzyme kinetics studies. The microsomal 12-lipoxygenase oxygenated linoleic acid much faster than the cytosolic platelet 12-lipoxygenase and is a "self-catalyzed inactivation" enzyme. Treatment of cells with 50 ng/ ml EGF significantly induced microsomal 12-lipoxygenase activity. The lag period for the expression of the stimulatory effect of EGF on 12-lipoxygenase activity was -10 h. The stimulatory effect of EGF on 12-lipoxygenase activity was completely blocked by treat-ment with 35 μM cycloheximide, indicating a requirement for de novo protein biosynthesis. Furthermore, the presence of the endogenous inhibitor of 12-lipoxygenase (which masked (12S)-HETE biosynthesis in intact cells) was identified in the cytosolic fraction of A431 cells. The putative inhibitor was enzyme-selective. It inhibited the leukocyte-type 12-lipoxygenase, but not the "platelet-type" enzyme.

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