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