4] The 5-lipoxygenase and leukotriene forming enzymes

Abstract

Publisher Summary This chapter describes the preparation of a single cell-free enzyme system that can be utilized for the study of the enzymic formation of leukotrienes (LT) B4 and other dihydroxyeicosatetraenoic acids (di-HETE), leukotrienes (LT)C 4 , LTD 4 , and 5-HETE. The sum of these products reflects the total 5-1ipoxygenase activity. The rat basophilic leukemia (RBL-1) cells were used for the preparation. Gentle conditions were used for breaking the cells. The cells were washed with 50 mM sodium phosphate buffer, pH 7, containing 1 mM EDTA, and 0.1% gelatin and resuspended at 5 × 10 r cells/ml in ice cold 35 mM sodium phosphate buffer, pH 7, containing 1 mM EDTA and 0.1% gelatin. The cells were homogenized with a Tekmar Tissuemizer. Thin-layer chromatography system is quite adequate to evaluate 5-1ipoxygenase activity. With no glutathione (GSH) added, the 5-1ipoxygenase activity was expressed in the formation of 5-HETE and 5,12-diHETE. Therefore, the sum of the 5-HETE and di-HETE bands provides the total 5-1ipoxygenase activity. No adequate thin-layer chromatography system is presently available for the separation of LTC 4 and LTD 4 . Although bioassay for slow-reacting substance (LTC 4 , LTD 4 , and LTE 4 ) on the guinea pig ileum has been used for a long time.