Renaud J P, Cullin C, Pompon D, Beaune P, Mansuy D
Centre National de la Recherche Scientifique, Unité de Recherche Associée 400, Paris, France.
Eur J Biochem. 1990 Dec 27;194(3):889-96. doi: 10.1111/j.1432-1033.1990.tb19483.x.
Cytochrome P-450 (P450) NF, a member of the P450 IIIA subfamily, is the major contributor to the oxidation of the calcium-channel blocker nifedipine in human liver microsomes. A cDNA clone designated NF25 encoding for human P450 NF was isolated from a bacteriophage lambda gt11 expression library [Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S. & Guengerich, F. P. (1986) Proc. Natl Acad. Sci. USA 83, 8064-8068]. We have expressed NF25 cDNA in Saccharomyces cerevisiae using an expression vector constructed from pYeDP1/8-2 [Cullin, C. & Pompon, D. (1988) Gene 65, 203-217]. Yeast transformed with the plasmid containing the NF25 sequence (pVNF25) showed a ferrous-CO spectrum typical of cytochrome P-450. Microsomal preparations contained a protein with an apparent molecular mass identical to that of P450-5 (a form isolated from human liver indistinguishable from P450 NF) that was not present in microsomes from control yeast (transformed with pYeDP1/8-2 alone), as revealed by immunoblotting with anti-P450-5 antibodies. On the other hand, antibodies raised in rabbits against human liver P450 IIC8-10 and rat liver P450 IA1 and P450 IIE1 did not recognize yeast-expressed P450 NF25. The P450 NF25 content in microsomes was about 90 pmol/mg protein. Microsomal, yeast-expressed P450 NF25 exhibited a high affinity for different substrates including macrolide antibiotics, dihydroergotamine and miconazole as shown by difference visible spectroscopy. Microsomal suspensions containing P450 NF25 were also able to catalyze several oxidation reactions that were expected from the activities of the protein isolated from human liver, including nifedipine 1,4-oxidation, quinidine 3-hydroxylation and N-oxygenation, and N-demethylation of the macrolide antibiotics erythromycin and troleandomycin. The yeast endogenous NADPH-cytochrome P-450 reductase thus couples efficiently with the heterologous P450 NF25 though its level is far lower than that of its ortholog in human liver. Indeed addition of rabbit liver NADPH-cytochrome P-450 reductase increased the oxidation rates. Rabbit liver cytochrome b5 also caused a marked enhancement of catalytic activities, as had been noted previously for this particular P450 enzyme in a reconstituted system involving the protein purified from human liver. Furthermore, the level of the yeast endogenous cytochrome P-450 (lanosterol 14-demethylase) has been found to be negligible compared to the heterologously expressed cytochrome P-450 (30 times less). Thus, yeast microsomes containing P450 NF25 constitute by themselves a good functional model for studying the binding capacities and catalytic activities of this individual form of human hepatic cytochrome P-450.
细胞色素P-450（P450）NF是P450 IIIA亚家族的成员，是人类肝微粒体中钙通道阻滞剂硝苯地平氧化反应的主要贡献者。从噬菌体λgt11表达文库中分离出一个编码人类P450 NF的cDNA克隆，命名为NF25[博内，P.H.，乌本豪尔，D.R.，博尔克，R.W.，劳埃德，R.S.和根杰里希，F.P.（1986年）《美国国家科学院院刊》83，8064 - 8068]。我们使用由pYeDP1/8 - 2构建的表达载体[卡兰，C.和庞蓬，D.（1988年）《基因》65，203 - 217]在酿酒酵母中表达了NF25 cDNA。用含有NF25序列的质粒（pVNF25）转化的酵母呈现出细胞色素P - 450典型的亚铁 - 一氧化碳光谱。微粒体制剂中含有一种表观分子量与P450 - 5（一种从人肝脏分离出的与P450 NF无法区分的形式）相同的蛋白质，用抗P450 - 5抗体进行免疫印迹分析表明，对照酵母（仅用pYeDP1/8 - 2转化）的微粒体中不存在这种蛋白质。另一方面，用兔抗人肝脏P450 IIC8 - 10以及大鼠肝脏P450 IA1和P450 IIE1产生的抗体不能识别酵母表达的P450 NF25。微粒体中P450 NF25的含量约为90 pmol/mg蛋白质。通过差示可见光谱法表明，酵母表达的微粒体P450 NF25对包括大环内酯类抗生素、二氢麦角胺和咪康唑在内的不同底物具有高亲和力。含有P450 NF25的微粒体悬浮液也能够催化一些从人肝脏分离的该蛋白质活性所预期的氧化反应，包括硝苯地平1,4 - 氧化、奎尼丁3 - 羟基化和N - 氧化，以及大环内酯类抗生素红霉素和醋竹桃霉素的N - 去甲基化反应。酵母内源性NADPH - 细胞色素P - 450还原酶尽管其水平远低于人肝脏中的同源物，但仍能有效地与异源P450 NF25偶联。实际上，添加兔肝脏NADPH - 细胞色素P - 450还原酶可提高氧化速率。兔肝脏细胞色素b5也显著增强了催化活性，正如之前在涉及从人肝脏纯化的该特定P450酶的重组系统中所观察到的那样。此外，与异源表达的细胞色素P - 450相比（少30倍），已发现酵母内源性细胞色素P - 450（羊毛甾醇14 - 去甲基酶）的水平可忽略不计。因此，含有P450 NF25的酵母微粒体自身构成了一个用于研究这种人肝细胞色素P - 450个体形式的结合能力和催化活性的良好功能模型。
