title: Formation of murine macrophage-derived 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG7) is catalyzed by leukotriene C4 synthase.
abstract: 5-Oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG(7)), a biologically active glutathione (GSH) adduct of the eicosanoid 5-oxo-eicosatrienoic acid (5-oxoETE), is the major metabolite formed within the murine peritoneal macrophage. The conjugation of GSH to electrophilic 5-oxoETE in vitro was found to be catalyzed by both soluble glutathione S-transferase and membrane-bound leukotriene C(4) (LTC(4)) synthase. The cytosolic glutathione S-transferase-catalyzed products were not biologically active; however, the adduct formed from recombinant LTC(4) synthase had identical mass spectrometric properties and biological activity to the macrophage-derived FOG(7). The biosynthesis of FOG(7) in the macrophage was inhibited by MK-886, a known inhibitor of LTC(4) synthase, suggesting that this nuclear membrane-bound enzyme might be responsible for GSH conjugation to 5-oxoETE in the intact cell. Subcellular fractionation revealed that the microsomal fraction from the murine macrophage contained the enzyme responsible for FOG(7) biosynthesis. Western blot analysis confirmed the presence of LTC(4) synthase in the microsomal fraction that did not catalyze conjugation of GSH to 1-chloro-2,4-dinitrobenzene, indicating an absence of microsomal glutathione S- transferase activity. These results suggest that LTC(4) synthase, thought to be specific for the conjugation of GSH to LTA(4), can also recognize 5-oxoETE as an electrophilic substrate.
authors.affiliation: Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Hevko
authors.forename: Robert C
authors.initials: RC
authors.lastname: Murphy
mesh_terms: D058927:5-Lipoxygenase-Activating Proteins; D000199:Actins / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000096:biosynthesis*; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004137:Dinitrochlorobenzene / Q000378:metabolism; D005434:Flow Cytometry; D005978:Glutathione / Q000096:biosynthesis* / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007477:Ions; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D008264:Macrophages / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D008861:Microsomes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D002722:Chlorobenzenes
chemical_list: D058927:5-Lipoxygenase-Activating Proteins; D000199:Actins; D016718:Arachidonic Acid; D002118:Calcium; D002352:Carrier Proteins; D004137:Dinitrochlorobenzene; D005978:Glutathione; D005982:Glutathione Transferase; D007211:Indoles; D007477:Ions; D016859:Lipoxygenase Inhibitors; D008565:Membrane Proteins; D011994:Recombinant Proteins; C548592:ALOX5AP protein, human; C548593:Alox5ap protein, mouse; C414562:FOG7 chemotactic; C060893:MK-886; D002722:Chlorobenzenes; C041116:leukotriene-C4 synthase