Cytochrome P-450 hPCN3, a novel cytochrome P-450 IIIA gene product that is differentially expressed in adult human liver. cDNA and deduced amino acid sequence and distinct specificities of cDNA-expressed hPCN1 and hPCN3 for the metabolism of steroid hormo
Cytochrome P-450 hPCN3, a novel cytochrome P-450 IIIA gene product that is differentially expressed in adult human liver. cDNA and deduced amino acid sequence and distinct specificities of cDNA-expressed hPCN1 and hPCN3 for the metabolism of steroid hormo
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发表时间：
1989-06
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通讯作者：
T. Aoyama;S. Yamano;D. Waxman;D. Lapenson;U. Meyer;V. Fischer;R. Tyndale;T. Inaba;W. Kalow
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作者：
T. Aoyama;S. Yamano;D. Waxman;D. Lapenson;U. Meyer;V. Fischer;R. Tyndale;T. Inaba;W. Kalow
免疫印迹分析显示，在所研究的40个个体样本中，每个样本都表达人细胞色素P-450 PCN1(hPCN1，MR约为52,000)。在大约10%-20%的肝脏中，也检测到了一种具有较低电泳率(Mr约为52,500)的免疫相关蛋白。发现一个肝脏只表达低迁移率蛋白，命名为hPCN3，从该肝脏提取的RNA用于构建lambda gt11文库。用hPCN1 cDNA探针对文库进行筛选，得到了唯一的全长cDNA，并对其进行了测序，显示其编码hPCN3。推导的氨基酸序列为502个残基，分子质量为57,115道尔顿，与hPCN1的同源性为84%。HPCN3的氨基末端序列与HFLa相同，HFLa是人胎肝中表达的一种主要细胞色素P-450，与几个家族III的细胞色素P-450具有免疫交叉反应。利用痘苗病毒表达系统在Hep G2细胞中表达了hPCN1和hPCN3cDNA，并证明它们编码有活性的酶，两者的特征都是在450 nm处与最大波长的CO结合光谱降低。酶分析表明，两种细胞色素P-450对钙通道阻滞剂硝苯地平的催化氧化活性相似。这两种酶也催化类固醇激素睾酮、孕酮和雄烯二酮的6β-羟基化，尽管hPCN1的表达活性比hPCN3高几倍。这些类固醇的几个次要氧化产物(例如15β-羟基睾酮)约占总代谢物的20%，由hPCN1而不是hPCN3形成，表明hPCN3是一种具有更高区域特异性的类固醇底物单加氧酶催化剂。它们对免疫抑制药物环孢素的催化活性也有明显差异，hPCN1形成两种羟基代谢物(M1和M17)和一种去甲基化代谢物(M21)，而hPCN3只形成一种代谢物(M1)。这些研究证实，hPCN3是一种新发现的细胞色素P-450，它在成人人群中差异表达，并且与hPCN1相比具有重叠的底物特异性，用于类固醇和药物底物的代谢。
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.