Bhattacharyya K K, Brake P B, Eltom S E, Otto S A, Jefcoate C R
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706, USA.
J Biol Chem. 1995 May 12;270(19):11595-602. doi: 10.1074/jbc.270.19.11595.
Antibodies against a novel adrenocorticotropic hormone-inducible cytochrome P450 (P450RAP), responsible for polycyclic aromatic hydrocarbon metabolism in rat adrenal microsomes (Otto, S., Bhattacharyya, K.K., and Jefcoate, C.R. (1992) Endocrinology 131, 3067-3076), identified a cDNA clone encoding a partial cytochrome P450 sequence from a rat adrenal cDNA library. Rescreening a second cDNA library yielded several clones up to 5.0 kilobases (kb) encoding a 1629-base pair open reading frame. The deduced amino acid sequence (543 residues) matched completely with five peptides cleaved from P450RAP. The amino acid sequence of P450RAP is 92% identical to a 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible CYP1B1, cloned from mouse C3H10T1/2 (10T1/2) embryo fibroblast cells (Savas, U., Bhattacharyya, K. K., Christou, M., Alexander, D.L., and Jefcoate, C. R. (1994) J. Biol. Chem. 269, 14905-14911), which shows nearly the same characteristics in polycyclic aromatic hydrocarbon metabolism. The available 5'- and 3'-noncoding regions show, respectively, 93 and 83% sequence identity. We conclude that P450RAP protein is encoded by a rat CYP1B1 gene orthologous to the mouse CYP1B1 gene. Alignment of rat CYP1B1 amino acid sequences with rat CYP1A1 (39% identical) indicated eight regions of high identity for each (60-78%), interspersed by extensive regions of less than 30% similarity. The CYP1B1 cDNAs hybridize a 5.2-kb mRNA in rat adrenals, consistent with the length of the longest clones and the mRNA recognized in 10T1/2 cells. CYP1B1 mRNA was elevated by a 2-day adrenocorticotropic hormone treatment but much less than CYP11A1 (cytochrome P450 side chain cleavage) mRNA (2-fold versus 4-fold). The lower levels of the 5.2-kb mRNA in other steroidogenic cells (ovary) was consistent with the amount of immunodetectable CYP1B1 protein and, unlike the adrenal, expression in the ovary was stimulated 5-fold by beta-naphthoflavone, an aryl hydrocarbon receptor agonist, in parallel with CYP1A1 induction. In several other tissues (liver > lung > uterus >> kidney), CYP1B1 mRNA and protein were constitutively undetectable but highly induced by beta-naphthoflavone, although at much lower levels than CYP1A1. Rat CYP1B1, therefore, exhibits regulation through hormonal signaling and the aryl hydrocarbon receptor in a cell-specific manner.
针对一种新型促肾上腺皮质激素诱导的细胞色素P450（P450RAP）的抗体，该酶负责大鼠肾上腺微粒体中的多环芳烃代谢（奥托，S.，巴塔查里亚，K.K.，和杰夫科特，C.R.（1992年）《内分泌学》131卷，3067 - 3076页），从大鼠肾上腺cDNA文库中鉴定出一个编码部分细胞色素P450序列的cDNA克隆。对第二个cDNA文库进行再次筛选，得到了几个长达5.0千碱基（kb）的克隆，它们编码一个1629个碱基对的开放阅读框。推导的氨基酸序列（543个残基）与从P450RAP切割得到的五个肽段完全匹配。P450RAP的氨基酸序列与从小鼠C3H10T1/2（10T1/2）胚胎成纤维细胞中克隆的2,3,7,8 - 四氯二苯并 - p - 二恶英诱导的CYP1B1有92%的同一性（萨瓦斯，U.，巴塔查里亚，K.K.，克里斯托，M.，亚历山大，D.L.，和杰夫科特，C.R.（1994年）《生物化学杂志》269卷，14905 - 14911页），其在多环芳烃代谢中表现出几乎相同的特征。可用的5' - 和3' - 非编码区分别显示出93%和83%的序列同一性。我们得出结论，P450RAP蛋白由与小鼠CYP1B1基因直系同源的大鼠CYP1B1基因编码。大鼠CYP1B1氨基酸序列与大鼠CYP1A1（39%同一性）的比对表明，二者各自有八个高度同源区域（60 - 78%），其间穿插着相似度低于30%的广泛区域。CYP1B1 cDNA在大鼠肾上腺中与一个5.2 - kb的mRNA杂交，这与最长克隆的长度以及在10T1/2细胞中识别的mRNA长度一致。促肾上腺皮质激素处理2天后，CYP1B1 mRNA水平升高，但远低于CYP11A1（细胞色素P450侧链裂解酶）mRNA（分别为2倍和4倍）。其他类固醇生成细胞（卵巢）中5.2 - kb mRNA水平较低，这与免疫可检测到的CYP1B1蛋白量一致，并且与肾上腺不同，卵巢中的表达受到芳烃受体激动剂β - 萘黄酮的刺激，与CYP1A1诱导平行增加了5倍。在其他几个组织（肝脏＞肺＞子宫＞＞肾脏）中，CYP1B1 mRNA和蛋白在基础状态下无法检测到，但受到β - 萘黄酮的高度诱导，尽管其水平远低于CYP1A1。因此，大鼠CYP1B1以细胞特异性方式通过激素信号和芳烃受体进行调控。
