Veronese M E, Mackenzie P I, Doecke C J, McManus M E, Miners J O, Birkett D J
Department of Clinical Pharmacology, School of Medicine, Flinders University of South Australia, Adelaide.
Biochem Biophys Res Commun. 1991 Mar 29;175(3):1112-8. doi: 10.1016/0006-291x(91)91680-b.
A human cytochrome P4502C9 cDNA clone has been isolated from a human liver bacteriophage Lambda gt11 library using oligonucleotide probes. Expression of the 1762 base pair cDNA in COS cells demonstrated that the encoded enzyme has a molecular mass of 55 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The expressed enzyme catalysed the methylhydroxylation of tolbutamide with an apparent Km of 131.7 microM, similar to that observed in human liver microsomes. P4502C9 also catalysed the 4-hydroylation of phenytoin, and inhibition experiments demonstrated that phenytoin was a competitive inhibitor of tolbutamide hydroxylation with an apparent Ki of 19.1 microM. Sulphaphenazole was a potent inhibitor of the expressed enzyme with respect to both tolbutamide and phenytoin hydroxylations. These data demonstrate that a single isozyme can catalyse the hydroxylations of both tolbutamide and phenytoin, and suggest that both reactions are mediated by the same isozyme(s) of cytochrome P450 in human liver.
利用寡核苷酸探针从人肝脏噬菌体λgt11文库中分离出了人细胞色素P4502C9 cDNA克隆。在COS细胞中对1762个碱基对的cDNA进行表达，结果表明，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，所编码的酶分子量为55 kDa。表达的酶催化甲苯磺丁脲的甲基羟化反应，表观Km为131.7 μM，与在人肝微粒体中观察到的情况相似。P4502C9还催化苯妥英的4-羟化反应，抑制实验表明苯妥英是甲苯磺丁脲羟化反应的竞争性抑制剂，表观Ki为19.1 μM。磺胺苯吡唑是所表达酶对甲苯磺丁脲和苯妥英羟化反应的强效抑制剂。这些数据表明单一同工酶可催化甲苯磺丁脲和苯妥英的羟化反应，并提示这两种反应均由人肝脏中细胞色素P450的同一同工酶介导。
