Abstract
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.
Highlights
From the Division ofPulmonary and Critical Care Medicine, Department of Internal Medicine, Washington University Schoolof Medicine, St
The enzyme was arachidonic acid to prostaglandin E2 (PGE2),but mi- described initially in platelets and subsequentlyleiunkocytes, crosome-containing subcellular fractionps repared and the two cellular forms have been distinguished catalytifrom these cells under calcium-free conditions con- cally [1] and structurally[2, 3]
Littleor 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 regulatiobny GSH or lipid hydroperoxide
Summary
Bradford method or by the bicinchoninic acid method Additional experiments using increasing concentrations of detergent (0.01-5% Triton X-100) to extract the membranebound enzyme indicated that the 12-HETE-forming activity was completely extracted from epithelial cell membranes only with 1% Triton X-100 andthat progressive increases in activity were accompanied by corresponding increases in extracted total protein (not shown). Nmol mg nmol/mgjl min was determined by comigration of epithelial cell-derived 12-
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