A cryptic, microsomal-type arachidonate 12-lipoxygenase is tonically inactivated by oxidation-reduction conditions in cultured epithelial cells.
Cultured ovine tracheal epithelial cells converted arachidonic acid to prostaglandin E2 (PGE2), but microsome-containing subcellular fractions prepared from these cells under calcium-free conditions converted arachidonic acid to PGE2 and to 12-hydroxyeicosatetraenoic acid (12-HETE) at a high rate (2-4 nmol/mg of protein/15 min). Identification of the membrane-bound 12-HETE-forming activity as a 12-lipoxygenase included 12S-stereospecificity of product formation and trapping of 12-hydroperoxyeicosatetraenoic acid as a reaction product. The 12-lipoxygenase activity was extracted from cell membranes only with detergent (1% Triton X-100), and the activity (membrane-bound or detergent-solubilized) was completely inactivated by mixing with the cytosol-containing subcellular fraction. The inhibitory effect of the cytosolic fraction was reversed by treating the cytosol with GSH-depleting agents (2-cyclohexene-1-one or N-ethylmaleimide) or by mixing it with lipid hydroperoxide (13-hydroperoxyoctadecadienoic acid) at a concentration that had little direct effect on enzyme activity. Inhibition of 12-lipoxygenase activity could also be achieved by treatment of enzyme preparations with GSH at levels (0.1-10 mM) found in epithelial cell cytosol. In addition, treatment of cultured epithelial cells with a GSH-depleting agent (buthionine sulfoximine) and lipid hydroperoxide restored cellular 12-lipoxygenase activity. Little or no detectable 12-lipoxygenase activity was found in freshly isolated ovine tracheal epithelial cells, but the cytosolic 12-lipoxygenase found in freshly isolated bovine tracheal epithelial cells was relatively insensitive to regulation by GSH or lipid hydroperoxide. These observations indicate that a 12-lipoxygenase is expressed in a cryptic, microsomal-type form in primary-culture epithelial cells and that this form of the enzyme may be selectively regulated by changes in cellular oxidation-reduction conditions dependent on cytosolic levels of GSH versus lipid hydroperoxide.
Shornick
L P
LP
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Holtzman
M J
MJ
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D002478:Cells, Cultured; D003512:Cyclohexanones / Q000494:pharmacology; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism; D004848:Epithelium / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007700:Kinetics; D008041:Linoleic Acids / Q000494:pharmacology; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; D008861:Microsomes / Q000201:enzymology*; D010084:Oxidation-Reduction; D012756:Sheep; D013237:Stereoisomerism; D013347:Subcellular Fractions / Q000201:enzymology; D014132:Trachea / Q000201:enzymology*; D014461:Ultracentrifugation
D003512:Cyclohexanones; D016861:Cyclooxygenase Inhibitors; D006893:Hydroxyeicosatetraenoic Acids; D008041:Linoleic Acids; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; C038649:13-hydroperoxy-9,11-octadecadienoic acid; C028215:2-cyclohexen-1-one; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase; D005978:Glutathione; D015232:Dinoprostone; D005033:Ethylmaleimide