Ourlin J C, Baader M, Fraser D, Halpert J R, Meyer U A
Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, 4056, Switzerland.
Arch Biochem Biophys. 2000 Jan 15;373(2):375-84. doi: 10.1006/abbi.1999.1566.
CYP3As represent a family of cytochromes P450 involved in the metabolism of both endogenous and exogenous natural and synthetic compounds. Well described in mammals, none have yet been cloned and characterized in avian species. In this paper, we report the cloning and analysis of an avian CYP3A (CYP3A37). Using an RNA differential display approach, an 80-bp phenobarbital-inducible cDNA fragment was amplified from chicken embryo liver. Based on its homology with mammalian CYP3As, this fragment was used to clone a full-length cDNA consisting of 1638 bp encoding a putative protein of 509 amino acids. The sequence shares between 57.4 and 62% identity at the amino acid level with CYP3As of other species. This cDNA was designated CYP3A37 according to the current cytochrome P450 nomenclature. When expressed in COS1 cells, the CYP3A37 cDNA produced a protein of congruent with55 kDa, which was recognized by polyclonal anti-rat CYP3A1 antiserum. In a bacterial expression system, the CYP3A37 cDNA produced a protein capable of steroid 6beta-hydroxylation. At a substrate concentration of 100 microM, progesterone, testosterone, and androstenedione were found to be 6beta-hydroxylated at a rate of 15.4, 11.7, 12.2 nmol/min/nmol P450, respectively. Used as control, the human CYP3A4 gave similar hydroxylation rates. Finally, in both chicken embryo liver and chicken hepatoma cells (LMH), CYP3A37 mRNA was increased after treatment with typical CYP3A inducers, such as metyrapone, phenobarbital, dexamethasone, and pregnenolone 16alpha-carbonitrile, but not rifampicin. CYP2H1, a well-characterized inducible chicken cytochrome P450, also was induced by the same compounds, suggesting similar regulation of CYP3 and CYP2 genes in this species.
细胞色素P450 3A（CYP3A）家族参与内源性和外源性天然及合成化合物的代谢。在哺乳动物中已有充分描述，但尚未在鸟类中克隆和鉴定出该家族成员。本文报道了一种鸟类CYP3A（CYP3A37）的克隆和分析。利用RNA差异显示方法，从鸡胚肝脏中扩增出一个80 bp的苯巴比妥诱导型cDNA片段。基于其与哺乳动物CYP3A的同源性，该片段被用于克隆一个全长1638 bp的cDNA，其编码一个由509个氨基酸组成的假定蛋白。该序列在氨基酸水平上与其他物种的CYP3A具有 (57.4%) 至 (62%) 的同一性。根据目前的细胞色素P450命名法，该cDNA被命名为CYP3A37。当在COS1细胞中表达时，CYP3A37 cDNA产生了一种与55 kDa一致的蛋白，该蛋白能被多克隆抗大鼠CYP3A1抗血清识别。在细菌表达系统中，CYP3A37 cDNA产生了一种能够进行类固醇6β-羟基化的蛋白。在底物浓度为100 μM时，发现孕酮、睾酮和雄烯二酮的6β-羟基化速率分别为15.4、11.7、12.2 nmol/min/nmol P450。作为对照，人CYP3A4的羟基化速率相似。最后，在用典型的CYP3A诱导剂（如甲吡酮、苯巴比妥、地塞米松和孕烯醇酮16α-腈）处理后，鸡胚肝脏和鸡肝癌细胞（LMH）中的CYP3A37 mRNA均增加，但利福平未使其增加。CYP2H1是一种已被充分鉴定的可诱导的鸡细胞色素P450，也被相同的化合物诱导，这表明该物种中CYP3和CYP2基因的调控相似。
