Expression of human liver cytochrome P450 IIIA4 in yeast. A functional model for the hepatic enzyme.
Expression of human liver cytochrome P450 IIIA4 in yeast. A functional model for the hepatic enzyme.
复制标题
人肝细胞色素 P450 IIIA4 在酵母中的表达。
DOI：
10.1111/j.1432-1033.1990.tb19483.x
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
D. Mansuy
中科院分区：
文献类型：
--
作者：
J. Renaud;C. Cullin;D. Pompon;P. Beaune;D. Mansuy
细胞色素P-450（P450）NF是P450 IIIA亚家族的成员，是人肝微粒体中钙通道阻滞剂硝苯地平氧化的主要贡献者。从噬菌体λ gt 11表达文库中分离编码人P450 NF的命名为NF 25的cDNA克隆[Beaune，P.H.，Umbenhauer，D. R.，博尔克河W.，劳埃德河S. & Guengerich，F. P.（1986）Proc. Natl Acad. Sci. USA 83，8064-8068]。我们已经使用由pYeDP 1/8-2构建的表达载体在酿酒酵母中表达了NF 25 cDNA [Cullin，C. & Pompon，D.（1988）Gene 65，203-217]。用含有NF 25序列的质粒（pVNF 25）转化的酵母显示出典型的细胞色素P-450的亚铁-CO光谱。微粒体制备物中含有一种蛋白质，其表观分子量与P450-5（从人肝脏中分离的一种形式，与P450 NF无法区分）相同，而对照酵母（仅用pYeDP 1/8-2转化）的微粒体中不存在这种蛋白质，这通过抗P450 -5抗体的免疫印迹法显示。另一方面，在兔中产生的抗人肝P450 IIC 8 -10和大鼠肝P450 IA 1和P450 IIE 1的抗体不识别酵母表达的P450 NF 25。微粒体中的P450 NF 25含量约为90 pmol/mg蛋白。微粒体，酵母表达的P450 NF 25表现出不同的底物，包括大环内酯类抗生素，二氢麦角胺和咪康唑的高亲和力，所示的差异可见光谱。含有P450 NF 25的微粒体悬浮液也能够催化几种氧化反应，这些反应是从人肝脏分离的蛋白质的活性中预期的，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-脱甲基。因此，酵母内源性NADPH-细胞色素P-450还原酶与异源性P450 NF 25有效偶联，尽管其水平远低于其在人肝脏中的直向同源物。事实上，添加兔肝NADPH-细胞色素P-450还原酶增加了氧化速率。兔肝细胞色素b5也引起了催化活性的显着增强，如前所述，在涉及从人肝纯化的蛋白质的重建系统中，这种特定的P450酶。此外，已经发现酵母内源性细胞色素P-450（羊毛甾醇14-脱甲基酶）的水平与异源表达的细胞色素P-450（低30倍）相比可以忽略不计。因此，酵母微粒体含有P450 NF 25本身构成了一个很好的功能模型，用于研究这种个人形式的人肝细胞色素P-450的结合能力和催化活性。
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.
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影响因子：
3.6
作者：
F. Guengerich;D. Müller-Enoch;I. Blair
通讯作者：
F. Guengerich;D. Müller-Enoch;I. Blair
多个物种的诱导型肝细胞色素 P-450 及其与大鼠糖皮质激素诱导型细胞色素 P-450 相关的 mRNA 的演示。
影响因子：
3.6
作者：
Wrighton,SA;Schuetz,EG;Watkins,PB;Maurel,P;Barwick,J;Bailey,BS;Hartle,HT;Young,B;Guzelian,P
通讯作者：
Guzelian,P
人肝微粒体中六种细胞色素 P-450 同工酶的纯化和表征。
影响因子：
2.9
作者：
Wang,PP;Beaune,P;Kaminsky,LS;Dannan,GA;Kadlubar,FF;Larrey,D;Guengerich,FP
通讯作者：
Guengerich,FP
与人细胞色素 P-450 硝苯地平氧化酶相关的 cDNA 克隆的分离和序列测定。
DOI：
10.1073/pnas.83.21.8064
发表时间：
1986
影响因子：
11.1
作者：
Beaune,PH;Umbenhauer,DR;Bork,RW;Lloyd,RS;Guengerich,FP
通讯作者：
Guengerich,FP
人肝细胞色素 P-450 硝苯地平氧化酶相关 mRNA 种类的表征及其催化活性的调节。
DOI：
--
发表时间：
1989
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Bork,RW;Muto,T;Beaune,PH;Srivastava,PK;Lloyd,RS;Guengerich,FP
通讯作者：
Guengerich,FP