Aoyama T, Yamano S, Waxman D J, Lapenson D P, Meyer U A, Fischer V, Tyndale R, Inaba T, Kalow W, Gelboin H V
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
J Biol Chem. 1989 Jun 25;264(18):10388-95.
Immunoblotting analysis of human liver microsome preparations revealed that human cytochrome P-450 PCN1 (hPCN1, Mr approximately 52,000) was expressed in each of 40 individual specimens examined. In about 10-20% of the livers, an immunologically related protein having a lower electrophoretic mobility (Mr approximately 52,500) was also detected. A single liver was found that expressed only the lower mobility protein, designated hPCN3, and RNA isolated from this liver was used to construct a lambda gt11 library. The library was screened with an hPCN1 cDNA probe resulting in the isolation of a unique full-length cDNA that was sequenced and shown to encode hPCN3. The deduced amino acid sequence of this cDNA contained 502 residues, a calculated molecular mass of 57,115 daltons, and displayed 84% similarity with hPCN1. The deduced amino-terminal sequence of hPCN3 was identical to that of HFLa, a major cytochrome P-450 expressed in human fetal liver that is immunologically cross-reactive with several family III cytochrome P-450s. hPCN1 and hPCN3 cDNAs were expressed in Hep G2 cells using a vaccinia virus expression system and shown to encode active enzymes, both characterized by reduced CO-binding spectra with lambda max at 450 nm. Enzymatic analysis revealed that both cytochrome P-450s were similarly active in catalyzing oxidation of the calcium channel blocking drug nifedipine. Both enzymes also catalyzed 6 beta-hydroxylation of the steroid hormones testosterone, progesterone, and androstenedione, although hPCN1 exhibited several-fold higher expressed activity than hPCN3. Several minor oxidation products of these steroids (e.g. 15 beta-hydroxytestosterone), comprising up to approximately 20% of the total metabolites, were formed by hPCN1 but not hPCN3, indicating that hPCN3 is a more highly regiospecific monooxygenase catalyst with steroid substrates. Clear differences were also detected in their catalytic activities toward the immunosuppressive drug cyclosporine, with two hydroxylated metabolites (M1 and M17) and one demethylated metabolite (M21) formed by hPCN1 but only one metabolite (M1) formed by hPCN3. These studies establish that hPCN3 is a newly described cytochrome P-450 that is differentially expressed in the adult human population and that has overlapping substrate specificity compared to hPCN1 for metabolism of steroid and drug substrates.
对人肝微粒体制剂进行的免疫印迹分析显示，在所检测的40个个体标本中，人细胞色素P-450 PCN1（hPCN1，分子量约52,000）均有表达。在约10%-20%的肝脏中，还检测到一种电泳迁移率较低（分子量约52,500）的免疫相关蛋白。发现一个肝脏只表达这种迁移率较低的蛋白，命名为hPCN3，从该肝脏分离的RNA用于构建λgt11文库。用hPCN1 cDNA探针筛选该文库，分离出一个独特的全长cDNA，经测序显示其编码hPCN3。该cDNA推导的氨基酸序列包含502个残基，计算分子量为57,115道尔顿，与hPCN1的相似性为84%。hPCN3推导的氨基末端序列与HFLa相同，HFLa是在人胎儿肝脏中表达的一种主要细胞色素P-450，与几种Ⅲ族细胞色素P-450存在免疫交叉反应。hPCN1和hPCN3 cDNA在Hep G2细胞中利用痘苗病毒表达系统进行表达，结果显示它们编码有活性的酶，二者的特征均为一氧化碳结合光谱在450 nm处的λmax降低。酶活性分析表明，两种细胞色素P-450在催化钙通道阻滞剂硝苯地平的氧化反应中活性相似。这两种酶还催化甾体激素睾酮、孕酮和雄烯二酮的6β-羟基化反应，不过hPCN1的表达活性比hPCN3高几倍。这些甾体的几种次要氧化产物（如15β-羟基睾酮）占总代谢产物的比例高达约20%，由hPCN1形成而不由hPCN3形成，这表明hPCN3是一种对甾体底物具有更高区域特异性的单加氧酶催化剂。在它们对免疫抑制药物环孢素的催化活性方面也检测到明显差异，hPCN1形成两种羟基化代谢产物（M1和M17）和一种去甲基化代谢产物（M21），而hPCN3只形成一种代谢产物（M1）。这些研究证实，hPCN3是一种新描述的细胞色素P-450，在成年人群中存在差异表达，并且与hPCN1相比，在甾体和药物底物代谢方面具有重叠的底物特异性。
