Srivastava P K, Yun C H, Beaune P H, Ged C, Guengerich F P
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Mol Pharmacol. 1991 Jul;40(1):69-79.
Purification and immunoinhibition studies have suggested that the hydroxylations of (S)-mephenytoin and tolbutamide are catalyzed by rather similar forms of human liver cytochrome P-450 (P-450). However, the two activities are not well correlated in vivo; sulfaphenzaole is a selective inhibitor of tolbutamide hydroxylation, and expression of P-450 2C10 cDNA in yeast yields a protein that hydroxylates tolbutamide but not (S)-mephenytoin. The P-450 2C8, 2C9, and 2C10 cDNAs have all been isolated, and their sequences are known to be closely related (greater than 80%). Highly sensitive radiochromatographic assays were set up, and tolbutamide and (S)-mephenytoin hydroxylation activities were monitored during chromatography of human liver microsomal fractions. The two activities could be separated by chromatography, and proteins were purified to near-homogeneity that catalyzed either tolbutamide hydroxylation (P-450TB) or (S)-mephenytoin 4'-hydroxylation (P-450MP) but not both. Approximately 16 and 45% of the primary sequences of P-450TB and P-450MP, respectively, were determined by analysis of the tryptic peptides. The sequences of the P-450TB peptides matched those predicted by the P-450 2C9 and 2C10 cDNAs exactly; the P-450MP peptides showed two mismatches (of 219 residues) with the P-450 2C10 sequence. Proteins with the P-450 2C10 and P-450 2C9 sequences were expressed in Saccharomyces cerevisiae grown under different nutritional conditions, and both were found to be proficient in the hydroxylation of tolbutamide but not (S)-mephenytoin. We conclude, on the basis of this and previous work, that 1) P-450s 2C8, 2C9, and 2C10 all catalyze the hydroxylation of tolbutamide and 2) the protein involved in polymorphic (S)-mephenytoin 4'-hydroxylation is closely related to but distinct from P-450 2C8, 2C9, and 2C10.
纯化和免疫抑制研究表明，（S）-美芬妥英和甲苯磺丁脲的羟基化反应是由人肝细胞色素P-450（P-450）的相当类似的形式催化的。然而，这两种活性在体内并没有很好的相关性；磺胺苯唑是甲苯磺丁脲羟基化反应的选择性抑制剂，P-450 2C10 cDNA在酵母中的表达产生一种能使甲苯磺丁脲羟基化但不能使（S）-美芬妥英羟基化的蛋白质。P-450 2C8、2C9和2C10的cDNA均已被分离出来，并且已知它们的序列密切相关（大于80%）。建立了高灵敏度的放射色谱分析法，并在人肝微粒体组分的色谱分析过程中监测甲苯磺丁脲和（S）-美芬妥英的羟基化活性。这两种活性可以通过色谱法分离，并且纯化得到了接近均一的蛋白质，它们分别催化甲苯磺丁脲的羟基化反应（P-450TB）或（S）-美芬妥英的4'-羟基化反应（P-450MP），但不能同时催化这两种反应。通过对胰蛋白酶肽段的分析，分别确定了P-450TB和P-450MP大约16%和45%的一级序列。P-450TB肽段的序列与P-450 2C9和2C10 cDNA预测的序列完全匹配；P-450MP肽段与P-450 2C10序列有两个错配（共219个残基）。具有P-450 2C10和P-450 2C9序列的蛋白质在不同营养条件下生长的酿酒酵母中表达，并且发现它们都能有效地催化甲苯磺丁脲的羟基化反应，但不能催化（S）-美芬妥英的羟基化反应。基于这项研究和之前的工作，我们得出结论：1）P-450 2C8、2C9和2C10都催化甲苯磺丁脲的羟基化反应；2）参与多态性（S）-美芬妥英4'-羟基化反应的蛋白质与P-450 2C8、2C9和2C10密切相关，但又有所不同。
