Kikuta Y, Kusunose E, Sumimoto H, Mizukami Y, Takeshige K, Sakaki T, Yabusaki Y, Kusunose M
Department of Food Science and Technology, Fukuyama University, Hiroshima, Fukuyama, 7290292, Japan.
Arch Biochem Biophys. 1998 Jul 15;355(2):201-5. doi: 10.1006/abbi.1998.0724.
Recombinant human neutrophil leukotriene B4 (LTB4) omega-hydroxylase (cytochrome P450 4F3) has been purified to a specific content of 14. 8 nmol of P450/mg of protein from yeast cells. The purified enzyme was homogenous as judged from the SDS-PAGE, with an apparent molecular weight of 55 kDa. The enzyme catalyzed the omega-hydroxylation of LTB4 with a Km of 0.64 microM and Vmax of 34 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b5. Furthermore, various eicosanoids such as 20-hydroxy-LTB4, 6-trans-LTB4, lipoxin A4, lipoxin B4, 5-HETE and 12-HETE, and 12-hydroxy-stearate and 12-hydroxy-oleate were efficiently omega-hydroxylated, although their Km values were much higher than that of LTB4. In contrast, no activity was detected toward laurate, palmitate, arachidonate, 15-HETE, prostaglandin A1, and prostaglandin E1, all of which are excellent substrates for the CYP4A fatty acid omega-hydroxylases. This is the first time human neutrophil LTB4 omega-hydroxylase has been isolated in a highly purified state and characterized especially with respect to its substrate specificity.
重组人中性粒细胞白三烯B4（LTB4）ω-羟化酶（细胞色素P450 4F3）已从酵母细胞中纯化至特定含量，即每毫克蛋白质含14.8 nmol的P450。从SDS-PAGE判断，纯化后的酶是均质的，表观分子量为55 kDa。在兔肝NADPH-P450还原酶和细胞色素b5存在的情况下，该酶催化LTB4的ω-羟化反应，Km为0.64 μM，Vmax为34 nmol/分钟/ nmol的P450。此外，各种类二十烷酸，如20-羟基-LTB4、6-反式-LTB4、脂氧素A4、脂氧素B4、5-羟二十碳四烯酸（5-HETE）和12-羟二十碳四烯酸（12-HETE），以及12-羟基硬脂酸和12-羟基油酸都能被有效地ω-羟化，尽管它们的Km值比LTB4的Km值高得多。相比之下，对月桂酸、棕榈酸、花生四烯酸、15-HETE、前列腺素A1和前列腺素E1未检测到活性，所有这些都是CYP4A脂肪酸ω-羟化酶的优良底物。这是首次将人中性粒细胞LTB4 ω-羟化酶以高度纯化的状态分离出来，并对其底物特异性进行了特别表征。
