Abramovitz M, Wong E, Cox M E, Richardson C D, Li C, Vickers P J
Department of Molecular Biology, Merck Frosst Centre for Therapeutic Research, Québec, Canada.
Eur J Biochem. 1993 Jul 1;215(1):105-11. doi: 10.1111/j.1432-1033.1993.tb18012.x.
5-Lipoxygenase (5-LO) and its activating protein (FLAP) are both required for cellular leukotriene (LT) synthesis, with 5-LO catalyzing both the synthesis of (5S)-5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) from arachidonic acid and the subsequent synthesis of LTA4 from 5-HPETE. We have previously expressed both human 5-LO and human FLAP to high levels in Spodoptera frugiperda (Sf9) insect cells, using recombinant baculoviruses. To study the mechanism by which FLAP activates 5-LO, we compared cellular 5-LO activity in Sf9 cells expressing this enzyme to that in Sf9 cells coexpressing FLAP and 5-LO. In this system, FLAP stimulates the utilization of arachidonic acid by 5-LO as a substrate, and increases the efficiency with which 5-LO converts 5-HPETE to LTA4. LT synthesis in cells coexpressing FLAP and 5-LO is inhibited by 3-[1-(p-chlorophenyl)-5-isopropyl-3-tert-butylthio-1H-indol-2-yl]-2,2- dimethyl-propanoic acid (MK-886), an LT biosynthesis inhibitor which specifically binds to FLAP. These studies in Sf9 cells, together with our recent demonstration that FLAP specifically binds arachidonic acid, suggests that FLAP activates 5-LO by acting as an arachidonic acid transfer protein.
5-脂氧合酶（5-LO）及其激活蛋白（FLAP）是细胞白三烯（LT）合成所必需的，5-LO催化花生四烯酸合成（5S）-5-氢过氧-6,8,11,14-二十碳四烯酸（5-HPETE）以及随后由5-HPETE合成白三烯A4（LTA4）。我们之前使用重组杆状病毒在草地贪夜蛾（Sf9）昆虫细胞中高水平表达了人5-LO和人FLAP。为了研究FLAP激活5-LO的机制，我们将表达该酶的Sf9细胞中的细胞5-LO活性与共表达FLAP和5-LO的Sf9细胞中的活性进行了比较。在这个系统中，FLAP刺激5-LO将花生四烯酸作为底物利用，并提高5-LO将5-HPETE转化为LTA4的效率。共表达FLAP和5-LO的细胞中的LT合成受到3-[1-（对氯苯基）-5-异丙基-3-叔丁基硫代-1H-吲哚-2-基]-2,2-二甲基丙酸（MK-886）的抑制，MK-886是一种特异性结合FLAP的LT生物合成抑制剂。这些在Sf9细胞中的研究，以及我们最近证明FLAP特异性结合花生四烯酸的结果，表明FLAP通过作为花生四烯酸转运蛋白来激活5-LO。
