CHARACTERIZATION AND PURIFICATION OF A LIPOXYGENASE INHIBITOR IN HUMAN EPIDERMOID CARCINOMA A431 CELLS

A lipoxygenase inhibitor in the cytosolic fraction of human epidermoid carcinoma A431 cells was characterized and purified. The cytosolic in hibitor 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 inhibit ory activity recovered when 1 mM GSH was added to the dialysate. The i nhibitory activity of cytosol was also abolished by treatment either w ith 1 mM iodoacetate at 4 degrees C for 1 h or with 0.5 mM H2O2. The p I of the inhibitor was approx. 7.0. In addition to 12-lipoxygenase, th e 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 wa s distinct from selenium-dependent glutathione peroxidase.