Powell P K, Wolf I, Lasker J M
Department of Biochemistry, Mount Sinai School of Medicine, New York 10029, USA.
Arch Biochem Biophys. 1996 Nov 1;335(1):219-26. doi: 10.1006/abbi.1996.0501.
Human liver microsomes are capable of oxidizing lauric acid (laurate), a model medium-chain fatty acid, at both the omega- and omega-1 positions to form 12- and 11-hydroxylaurate, respectively. These laurate hydroxylation reactions are apparently catalyzed by distinct P450 enzymes. While the P450 responsible for microsomal laurate omega-1 hydroxylation in human liver has been identified as CYP2E1, the enzyme catalyzing omega-hydroxylation remains poorly defined. To that end, we employed conventional purification and immunochemical techniques to characterize the major hepatic laurate omega-hydroxylase in humans. Western blotting with rat CYP4A1 antibodies was used to monitor a cross-reactive P450 protein (M(r) = 52 kDa) during its isolation from human liver microsomes. The purified enzyme (7.4 nmol P450/mg protein) had an NH2-terminal amino acid sequence identical to that predicted from the human CYP4A11 cDNA over the first 20 residues found. Upon reconstitution with P450 reductase and cytochrome b5, CYP4A11 proved to be a potent laurate omega-hydroxylase, exhibiting a turnover rate of 45.7 nmol 12-hydroxylaurate formed/min/nmol P450 (12-fold greater than intact microsomes), while catalyzing the omega-1 hydroxylation reaction at much lower rates (5.4 nmol 11-hydroxylaurate formed/min/nmol P450). Analysis of the laurate omega-hydroxylation reaction in human liver microsomes revealed kinetic parameters (a lone Km of 48.9 microM with a VMAX of 3.72 nmol 12-hydroxylaurate formed/min/nmol P450) consistent with catalysis by CYP4A11. In fact, incubation of human liver microsomes with antibodies raised to CYP4A11 resulted in nearly 85% inhibition of laurate omega-hydroxylase activity while omega-1 hydroxylase activity remained unaffected. Furthermore, a strong correlation (r = 0.89; P < 0.001) was found between immunochemically determined CYP4A11 content and laurate omega-hydroxylase activity in liver samples from 11 different subjects. From the foregoing, it appears that CYP4A11 is the principle laurate omega-hydroxylating enzyme expressed in human liver.
人肝微粒体能够在ω位和ω-1位氧化月桂酸（月桂酸盐），月桂酸是一种典型的中链脂肪酸，分别生成12-羟基月桂酸和11-羟基月桂酸。这些月桂酸羟基化反应显然是由不同的细胞色素P450酶催化的。虽然已确定负责催化人肝微粒体月桂酸ω-1羟基化的细胞色素P450为CYP2E1，但催化ω-羟基化的酶仍不清楚。为此，我们采用传统的纯化和免疫化学技术来鉴定人肝脏中主要的月桂酸ω-羟化酶。用大鼠CYP4A1抗体进行蛋白质免疫印迹，以监测从人肝微粒体中分离出的一种交叉反应性细胞色素P450蛋白（相对分子质量=52 kDa）。纯化后的酶（7.4 nmol细胞色素P450/毫克蛋白）的氨基末端氨基酸序列，在前20个残基上与人CYP4A11 cDNA预测的序列相同。用细胞色素P450还原酶和细胞色素b5重组后，CYP4A11被证明是一种高效的月桂酸ω-羟化酶，其转换率为45.7 nmol 12-羟基月桂酸生成/分钟/ nmol细胞色素P450（比完整的微粒体高12倍），而催化ω-1羟基化反应的速率则低得多（5.4 nmol 11-羟基月桂酸生成/分钟/ nmol细胞色素P450）。对人肝微粒体中月桂酸ω-羟基化反应的分析显示，其动力学参数（唯一的Km为48.9 μM，VMAX为3.72 nmol 12-羟基月桂酸生成/分钟/ nmol细胞色素P450）与CYP4A11的催化作用一致。事实上，用人肝脏微粒体与针对CYP4A11产生的抗体一起温育，导致月桂酸ω-羟化酶活性受到近85%的抑制，而ω-1羟化酶活性不受影响。此外，在来自11个不同受试者的肝脏样本中，免疫化学测定的CYP4A11含量与月桂酸ω-羟化酶活性之间存在很强的相关性（r = 0.89；P < 0.001）。综上所述，CYP4A11似乎是在人肝脏中表达的主要月桂酸ω-羟化酶。
