A lipoxygenase inhibitor in the cytosolic fraction of human epidermoid carcinoma A431 cells was characterized and purified. The cytosolic inhibitor lost the inhibitory activity upon heating at 75 degrees C for 15 min or pretreating with 1 mg/ml trypsin at 37 degrees C for 60 min. Cytosol, after dialysis, lost the inhibitory activity but its inhibitory activity recovered when 1 mM GSH was added to the dialysate. The inhibitory activity of cytosol was also abolished by treatment either with 1 mM iodoacetate at 4 degrees C for 1 h or with 0.5 mM H2O2. The pI of the inhibitor was approx. 7.0. In addition to 12-lipoxygenase, the inhibitor inhibited the activities of 5-lipoxygenase and fatty acid cyclo-oxygenase in a cell-free system. The inhibitor was purified by a series of column chromatographies using CM Sephadex C-50, Sephadex G-100 SF and Mono P columns. A major 22 kDa protein was obtained that was distinct from selenium-dependent glutathione peroxidase.

No abstract

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenium-dependent glutathione peroxidase, can interact with lipophilic substrates, including phospholipid hydroperoxides, fatty acid hydroperoxides and cholesterol hydroperoxides, and can reduce them to hydroxide compounds. It also seems to be a major regulator of lipid oxygenation in human epidermoid carcinoma A431 cells. In order to study the functional role of PHGPx in the regulation of 12-lipoxygenase and cyclooxygenase, cDNA of PHGPx was inserted into pcDNA3.1/His, and a plasmid designated as S4 with the His-tag sequence inserted between PHGPx and its 3′-untranslated region was constructed. A number of stable transfectants of A431 cells that could express the tag-PHGPx were generated using plasmid S4. Using an intact cell assay system, the metabolism of arachidonic acid to prostaglandin E₂ significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line. If the intact cell assay was carried out in the presence of 13-hydroperoxyoctadecadienoic acid as a stimulator of lipid peroxidation, formation of 12-hydroxyeicosatetraenoic acid from arachidonic acid also significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line, indicating that PHGPx could downregulate the 12-lipoxygenase activity in cells. These results support the hypothesis that PHGPx plays a pivotal role in the regulation of arachidonate metabolism in A431 cells.