Kikuta Y, Kusunose E, Ito M, Kusunose M
Department of Applied Biological Science, Fukuyama University, Hiroshima, Fukuyama, 7290292, Japan.
Arch Biochem Biophys. 1999 Sep 15;369(2):193-6. doi: 10.1006/abbi.1999.1271.
CYP4F1 was discovered by Chen and Hardwick (Arch. Biochem. Biophys. 300, 18-23, 1993) as a new CYP4 cytochrome P450 (P450) preferentially expressed in rat hepatomas. However, the catalytic function of this P450 remained poorly defined. We have purified recombinant CYP4F1 protein to a specific content of 12 nmol of P450/mg of protein from transfected yeast cells by chromatography of solubilized microsomes on an amino-n-hexyl Sepharose 4B column, followed by sequential HPLC on a DEAE column and two hydroxylapatite columns. The purified P450 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 53 kDa. The enzyme catalyzed the omega-hydroxylation of leukotriene B(4) with a K(m) of 134 microM and a V(max) of 6.5 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b(5). In addition, 6-trans-LTB(4), lipoxin A(4), prostaglandin A(1), and several hydroxyeicosatetraenoic acids (HETEs) were also omega-hydroxylated. Of several eicosanoids examined, 8-HETE was the most efficient substrate, with a K(m) of 18.6 microM and a V(max) of 15.8 nmol/min/nmol of P450. In contrast, no activity was detected toward lipoxin B(4), laurate, palmitate, arachidonate, and benzphetamine. The results suggest that CYP4F1 participates in the hepatic inactivation of several bioactive eicosanoids.
1993年，陈和哈德威克（《生物化学与生物物理学报》300卷，第18 - 23页）发现CYP4F1是一种新的细胞色素P450（P450），在大鼠肝癌中优先表达。然而，这种P450的催化功能仍不清楚。我们通过将溶解的微粒体在氨基正己基琼脂糖4B柱上进行层析，然后依次在DEAE柱和两根羟基磷灰石柱上进行高效液相色谱，从转染的酵母细胞中纯化出重组CYP4F1蛋白，使其P450的比含量达到12 nmol/mg蛋白。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳判断，纯化后的P450是均一的，表观分子量为53 kDa。在兔肝NADPH - P450还原酶和细胞色素b5存在的情况下，该酶催化白三烯B4的ω - 羟化反应，米氏常数（K(m)）为134 μM，最大反应速度（V(max)）为6.5 nmol/min/nmol P450。此外，6 - 反式 - LTB4、脂氧素A4、前列腺素A1和几种羟基二十碳四烯酸（HETEs）也能被ω - 羟化。在所检测的几种类花生酸中，8 - HETE是最有效的底物，K(m)为18.6 μM，V(max)为15.8 nmol/min/nmol P450。相比之下，未检测到对脂氧素B4、月桂酸、棕榈酸、花生四烯酸和苄非他明的活性。结果表明，CYP4F1参与了几种生物活性类花生酸的肝脏失活过程。
