Qian Ming-rong, Chen Jing, Liu Yao, Yu Lu-shan, Chen Shu-qing, Zeng Su
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
Yao Xue Xue Bao. 2011 Feb;46(2):207-12.
CYP2D6 is an important drug-metabolizing enzyme. The polymorphism of CYP2D6 leads to metabolism difference and the different reactions of drugs in the individuals and different races are normal phenomenon in clinical medication. CYP2D610 is an important subtype in Asian people and 51.3% Chinese are classified with this subtype. To obtain recombinant active CYP2D61/CYP2D610 in baculovirus system by optimizing coexpression with CYPOR, and detect their activity to catalyze dextromethorphan, three recombinants pFastBac-CYP2D61, pFastBac-CYP2D610 and pFastBac-CYPOR were constructed and transformed into DH10Bac cell to obtain the recombinant Bacmid-CYPOR, Bacmid-CYP2D61 and Bacmid-CYP2D610. And then the recombinant CYP2D61 and CYP2D610 virus were obtained by transfecting Sf9. Then homogenate protein activity was determined with dextromethorphan as substrate. The multiple of infection (MOI) and its ratio of recombinant CYP2D6 virus to CYPOR virus were adjusted by detecting the activity of the homogenate protein. The Km and Vmax are 26.67 +/- 2.71 micromol x L(-1) (n=3) and 666.7 +/- 56.78 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D61 to catalyze dextromethaphan. The Km and Vmax are 111.36 +/- 10.89 micromol x L(-1) (n=3) and 222.2 +/- 20.12 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D610 to catalyze dextromethorphan. There is significant difference between CYP2D61 and CYP2D610 for Vmax and Km (P < 0.01). The clearance ratio of CYP2D61 is 25.0 and the clearance ratio of CYP2D610 is 2.0. The expressed CYP2D61 and CYP2D6*10 are useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro, which are benefit to understand individual metabolism difference.
细胞色素P450 2D6（CYP2D6）是一种重要的药物代谢酶。CYP2D6的基因多态性导致代谢差异，药物在个体和不同种族中产生不同反应是临床用药中的正常现象。CYP2D610是亚洲人群中的一种重要亚型，51.3%的中国人属于该亚型。通过优化与细胞色素P450还原酶（CYPOR）的共表达，在杆状病毒系统中获得重组活性CYP2D61/CYP2D610，并检测其催化右美沙芬的活性，构建了三种重组体pFastBac-CYP2D61、pFastBac-CYP2D610和pFastBac-CYPOR，并将其转化到DH10Bac细胞中以获得重组杆粒-CYPOR、杆粒-CYP2D61和杆粒-CYP2D610。然后通过转染草地贪夜蛾细胞（Sf9）获得重组CYP2D61和CYP2D610病毒。接着以右美沙芬为底物测定匀浆蛋白活性。通过检测匀浆蛋白活性来调整重组CYP2D6病毒与CYPOR病毒的感染复数（MOI）及其比例。CYP2D61催化右美沙芬的米氏常数（Km）和最大反应速度（Vmax）分别为26.67±2.71 μmol·L⁻¹（n = 3）和666.7±56.78 pmol·nmol⁻¹（CYP2D6）·min⁻¹（n = 3）。CYP2D610催化右美沙芬的Km和Vmax分别为111.36±10.89 μmol·L⁻¹（n = 3）和222.2±20.12 pmol·nmol⁻¹（CYP2D6）·min⁻¹（n = 3）。CYP2D61和CYP2D610在Vmax和Km方面存在显著差异（P < 0.01）。CYP2D61的清除率为25.0，CYP2D610的清除率为2.0。所表达的CYP2D61和CYP2D6*10是体外筛选多种外源性和内源性物质代谢谱的有用工具，有助于了解个体代谢差异。
