Combalbert J, Fabre I, Fabre G, Dalet I, Derancourt J, Cano J P, Maurel P
INSERM U 278, Faculté de Pharmacie, Montpellier, France.
Drug Metab Dispos. 1989 Mar-Apr;17(2):197-207.
A cytochrome P-450 involved in the metabolism of cyclosporin A (CsA) was isolated and purified to electrophoretic homogeneity from human liver microsomes of renal transplant donors. This cytochrome, designated P-450(CsA), exhibited a type I binding spectrum in the presence of CsA with a Ks(app) of 25 microM, a molecular weight of 52 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a maximal absorbance at 449 nm when reduced in the presence of carbon monoxide. The N-terminal sequence of P-450(CsA), determined by Edman degradation reaction, was 63% homologous with that of the rabbit liver CsA oxidase P-450 3c and 100% homologous with that of the human liver isozyme P-450(HLp/NF), recently identified as the human nifedipine (NF) oxidase. Polyclonal and monoclonal antibodies directed against P-450 3c and P-450(HLp/NF), respectively, recognized native microsomal and highly purified P450(CsA). As observed in the rabbit, human liver microsomes were shown to generate mono- and dihydroxy, as well as dihydroxy and/or monohydroxy N-demethylated, derivatives of CsA. Production of these metabolites was shown to be specifically inhibited by anti-P-450 3c polyclonal antibodies. CsA oxidase, NF oxidase, and erythromycin demethylase were shown to be closely correlated with the level of P-450(CsA) determined from Western blot or enzyme-linked immunosorbent assay. Moreover, these monoxygenase activities and the hepatic level of P-450(CsA) were simultaneously increased in the liver of patients treated for 4 days with 600 mg of rifampicin per day. Finally, NF was shown to be a competitive inhibitor of CsA oxidation and vice versa. We conclude that P-450(CsA) is responsible for most (80%) of CsA oxidase activity in human liver, is encoded by gene P450IIIA3, as is NF oxidase, or a very closely related gene, and is strongly inducible by rifampicin pretreatment.
从肾移植供体的人肝微粒体中分离并纯化出一种参与环孢素A（CsA）代谢的细胞色素P - 450，使其达到电泳纯。这种细胞色素，命名为P - 450（CsA），在存在CsA的情况下呈现I型结合光谱，其表观解离常数（Ks(app)）为25微摩尔，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上的分子量为52 kDa，在一氧化碳存在下还原时在449纳米处有最大吸光度。通过埃德曼降解反应确定的P - 450（CsA）的N端序列与兔肝CsA氧化酶P - 450 3c的N端序列有63%的同源性，与最近被鉴定为人硝苯地平（NF）氧化酶的人肝同工酶P - 450（HLp/NF）的N端序列100%同源。分别针对P - 450 3c和P - 450（HLp/NF）的多克隆抗体和单克隆抗体能够识别天然微粒体和高度纯化的P450（CsA）。正如在兔中观察到的那样，人肝微粒体被证明能生成CsA的单羟基和二羟基衍生物，以及二羟基和/或单羟基N - 去甲基化衍生物。这些代谢产物的生成被证明能被抗P - 450 3c多克隆抗体特异性抑制。CsA氧化酶、NF氧化酶和红霉素脱甲基酶被证明与通过蛋白质印迹法或酶联免疫吸附测定法测定的P - 450（CsA）水平密切相关。此外，在每天用600毫克利福平治疗4天的患者肝脏中，这些单加氧酶活性和P - 450（CsA）的肝脏水平同时升高。最后，NF被证明是CsA氧化的竞争性抑制剂，反之亦然。我们得出结论，P - 450（CsA）负责人类肝脏中大部分（80%）的CsA氧化酶活性，由基因P450IIIA3编码，NF氧化酶也是如此，或者是一个非常密切相关的基因，并且能被利福平预处理强烈诱导。
