Characterization of cloned human leukocyte 5-lipoxygenase expressed in mammalian cells.

摘要: 5-Lipoxygenase has been expressed in a mammalian osteosarcoma cell line transfected with the cloned cDNA for human leukocyte 5-lipoxygenase. Two clonal lines derived from cells enzymatic activity. When incubated arachidonic acid (100 microM), major 5-lipoxygenase products of 10,000 X g supernatants these were 5-hydroxyeicosatetraenoic (5-HETE), and nonenzymatic hydrolysis leukotriene (LT)A4. The ratio 5-HETE to LT (between 6:1 9:1) was similar that observed supernatants. Furthermore, incubation 5-hydroperoxyeicosatetraenoic (5-HPETE) (75 microM) resulted synthesis LTA4 products. Control produced no or 5-HPETE. Maximal activity enzyme required Ca2+, ATP, two cellular stimulatory factors prepared leukocytes. Immunoblot analysis clones revealed an immunoreactive 80,000-dalton band indistinguishable Therefore, functional exhibited characteristics identical those intact challenged ionophore A 23187, formed. If added along ionophore, synthesized LTA4. These results verify used transfect encodes studies offer independent evidence this single protein possesses both synthase activity, as reported previously purification data.