Nagata K, Ogino M, Shimada M, Miyata M, Gonzalez F J, Yamazoe Y
Faculty of Pharmaceutical Sciences, Tohoku University, Sendai, Aoba-ku, 980-8578, Japan.
Arch Biochem Biophys. 1999 Feb 15;362(2):242-53. doi: 10.1006/abbi.1998.1030.
A P450 gene (P450/6betaB) of the CYP3A subfamily was isolated from a rat genomic library. Nucleotide sequencing of the exons revealed a high similarity with P450PCN1 cDNA (Gonzalez et al. (1985), J. Biol. Chem. 260, 7345-7441), but differed in 41 nucleotides, resulting in 11 changes and 2 deletions of amino acid residues. The P450/6betaB spanned about 30 kbp and consisted of 13 exons, and was in exon number and size identical with CYP3A2 gene except in the 6th exon, which was shorter than that of CYP3A2. 6beta-B mRNA, which may be transcribed from P450/6betaB, was detected on Northern blotting and by reverse transcription-polymerase chain reaction (RT-PCR). Profiles of the developmental change and induction by a treatment with several chemicals were very similar to those of P450PCN1 mRNA reported previously. P450PCN1 mRNA and gene, however, were not detected by PCR in rats. To determine whether P450/6betaB encodes an active protein, a cDNA was isolated and expressed. Expression of 6beta-B cDNA in COS-1 cells was carried out and revealed that the recombinant protein comigrated with purified P4506beta-4 previously identified as CYP3A1. The recombinant 6beta-B protein showed similar turnover rate and regioselectivity for testosterone with purified P4506beta-4 by the simultaneous addition of NADPH-cytochrome P450 reductase and cytochrome b5. These data suggest that P450/6betaB encodes an active P450 form corresponding to CYP3A1 and P450PCN1 reported previously does not exist in rats.
从大鼠基因组文库中分离出CYP3A亚家族的一个P450基因（P450/6βB）。外显子的核苷酸测序显示与P450PCN1 cDNA高度相似（冈萨雷斯等人，（1985年），《生物化学杂志》260，7345 - 7441），但在41个核苷酸上存在差异，导致11个氨基酸残基改变和2个缺失。P450/6βB跨度约30 kbp，由13个外显子组成，除第6个外显子比CYP3A2的短外，其外显子数量和大小与CYP3A2基因相同。在Northern印迹和逆转录 - 聚合酶链反应（RT - PCR）中检测到可能由P450/6βB转录而来的6β - B mRNA。发育变化和几种化学物质处理诱导的图谱与先前报道的P450PCN1 mRNA的图谱非常相似。然而，在大鼠中通过PCR未检测到P450PCN1 mRNA和基因。为了确定P450/6βB是否编码一种活性蛋白，分离并表达了一个cDNA。在COS - 1细胞中进行了6β - B cDNA的表达，结果显示重组蛋白与先前鉴定为CYP3A1的纯化P4506β - 4迁移率相同。通过同时添加NADPH - 细胞色素P450还原酶和细胞色素b5，重组6β - B蛋白与纯化的P4506β - 4对睾酮显示出相似的周转速率和区域选择性。这些数据表明P450/6βB编码一种与先前报道的CYP3A1相对应的活性P450形式，并且大鼠中不存在P450PCN1。
