Powell P K, Wolf I, Jin R, Lasker J M
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York, USA.
J Pharmacol Exp Ther. 1998 Jun;285(3):1327-36.
20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is a principal arachidonic acid (AA) metabolite formed via P450-dependent oxidation in hepatic and renal microsomes. Although 20-HETE plays an important role in the regulation of cell and/or organ physiology, the P450 enzyme(s) catalyzing its formation in humans remain undefined. In this study, we have characterized AA omega-hydroxylation to 20-HETE by human hepatic microsomes and identified the underlying P450s. Analysis of microsomal AA omega-hydroxylation revealed biphasic kinetics (KM1 and VMAX1 = 23 microM and 5.5 min-1; KM2 and VMAX2 = 144 microM and 18.8 min-1) consistent with catalysis by at least two enzymes. Of the human P450s examined, CYP4A11 and CYP4F2 were both potent AA omega-hydroxylases, exhibiting rates of 15.6 and 6.8 nmol 20-HETE formed/min/nmol P450, respectively. Kinetic parameters of 20-HETE formation by CYP4F2 (KM = 24 microM; VMAX = 7.4 min-1) and CYP4A11 (KM = 228 microM; VMAX = 49.1 min-1) resembled the low and high KM components, respectively, found in liver microsomes. Antibodies to CYP4F2 markedly inhibited (93.4 +/- 6%; n = 5) formation of 20-HETE by hepatic microsomes, whereas antibodies to CYP4A11 were much less inhibitory (13.0 +/- 9%; n = 5). Moreover, a strong correlation (r = 0.78; P < .02) was found between microsomal CYP4F2 content and AA omega-hydroxylation among nine subjects. The correlation (r = 0.76; P < .02) also noted between CYP4A11 content and 20-HETE formation stemmed from the relationship (r = 0.83; P < . 02) between hepatic CYP4A11 and CYP4F2 levels in the subjects. Finally, immunoblot analysis revealed that in addition to liver, both P450s also were expressed in human kidney. Our results indicate that AA omega-hydroxylation in human liver is catalyzed by two enzymes of the CYP4 gene family, namely CYP4F2 and CYP4A11, and that CYP4F2 underlies most 20-HETE formation occurring at relevant AA concentrations.
20-羟基-5,8,11,14-二十碳四烯酸（20-HETE）是一种主要的花生四烯酸（AA）代谢产物，通过肝脏和肾脏微粒体中依赖细胞色素P450的氧化作用形成。尽管20-HETE在细胞和/或器官生理学调节中发挥重要作用，但催化其在人体内形成的细胞色素P450酶仍未明确。在本研究中，我们通过人肝脏微粒体对AA向20-HETE的ω-羟基化进行了表征，并鉴定出了相关的细胞色素P450。微粒体AAω-羟基化分析显示出双相动力学（KM1和VMAX1 = 23μM和5.5 min-1；KM2和VMAX2 = 144μM和18.8 min-1），这与至少两种酶的催化作用一致。在所检测的人细胞色素P450中，CYP4A11和CYP4F2都是有效的AAω-羟基化酶（分别为15.6和6.8 nmol 20-HETE形成/min/nmol细胞色素P450）。CYP4F2（KM = 24μM；VMAX = 7.4 min-1）和CYP4A11（KM = 228μM；VMAX = 49.1 min-1）形成20-HETE的动力学参数分别类似于在肝脏微粒体中发现的低KM和高KM组分。针对CYP4F2的抗体显著抑制（93.4±6%；n = 5）肝脏微粒体形成20-HETE，而针对CYP4A11的抗体抑制作用则小得多（13.0±9%；n = 5）。此外，在9名受试者中，微粒体CYP4F2含量与AAω-羟基化之间存在强相关性（r = 0.78；P <.02）。CYP4A11含量与20-HETE形成之间的相关性（r = 0.76；P <.02）也源于受试者肝脏中CYP4A11和CYP4F2水平之间的关系（r = 0.83；P <.02）。最后，免疫印迹分析表明，除肝脏外，这两种细胞色素P450在人肾脏中也有表达。我们的结果表明，人肝脏中的AAω-羟基化由细胞色素P4基因家族的两种酶，即CYP4F2和CYP4A11催化，并且在相关AA浓度下发生的大多数20-HETE形成是由CYP4F2介导的。
