Lee S J, Wang-Buhler J L, Cok I, Yu T S, Yang Y H, Miranda C L, Lech J, Buhler D R
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon, 973331, USA.
Arch Biochem Biophys. 1998 Dec 1;360(1):53-61. doi: 10.1006/abbi.1998.0943.
Screening of lambdagt11 and lambdagt22A cDNA libraries of livers from adult females and embryos of rainbow trout (Oncorhynchus mykiss), respectively, using rabbit anti-rainbow trout cytochrome P450 LMC5 polyclonal antibodies showed that there were identical cDNAs of 1802-bp nucleotides with open reading frames coding for proteins containing 518 amino acids (59,206 Da, pI = 6.39). The cDNA was assigned CYP3A27 by the P450 Nomenclature Committee to represent the first CYP3A subfamily member reported for aquatic species. The deduced N-terminal sequence of CYP3A27 was in agreement with 8 of the first 12 confirmed amino acid residues from Edman degradation of LMC5, a P450 previously isolated from juvenile trout liver. In similarity comparisons between species by positional alignment, the deduced amino acid sequence of rainbow trout CYP3A27 was 56.4% identical with dog CYP3A12, 56.0% with monkey CYP3A8, 54.9% with human CYP3A4, 54.7% with rat CYP3A9, and 54.2% with sheep CYP3A24. Marked differences in sex, age, and tissue expression of CYP3A27 in rainbow trout were observed at the mRNA level as shown by Northern blots. The major extrahepatic expression site for CYP3A27 was upper small intestine. Females expressed considerably more CYP3A27 mRNA than male in the fish examined. Southern blot analysis of restriction enzyme-digested rainbow trout genomic DNA demonstrated that multiple CYP3A27-related genes exist in rainbow trout.
分别使用兔抗虹鳟鱼细胞色素P450 LMC5多克隆抗体筛选成年雌性虹鳟鱼（Oncorhynchus mykiss）和胚胎肝脏的λgt11和λgt22A cDNA文库，结果显示存在1802个核苷酸的相同cDNA，其开放阅读框编码含518个氨基酸的蛋白质（59,206 Da，pI = 6.39）。该cDNA被细胞色素P450命名委员会指定为CYP3A27，代表首次报道的水生物种的CYP3A亚家族成员。CYP3A27推导的N端序列与先前从幼体鳟鱼肝脏分离的P450 LMC5经埃德曼降解得到的前12个已确认氨基酸残基中的8个一致。通过位置比对进行物种间相似性比较时，虹鳟鱼CYP3A27推导的氨基酸序列与犬CYP3A12的同一性为56.4%，与猴CYP3A8的同一性为56.0%，与人类CYP3A4的同一性为54.9%，与大鼠CYP3A9的同一性为54.7%，与绵羊CYP3A24的同一性为54.2%。如Northern印迹所示，在mRNA水平观察到虹鳟鱼中CYP3A27在性别、年龄和组织表达上存在显著差异。CYP3A27的主要肝外表达位点是小肠上段。在所检测的鱼中，雌性表达的CYP3A27 mRNA比雄性多得多。对经限制性酶切的虹鳟鱼基因组DNA进行Southern印迹分析表明，虹鳟鱼中存在多个与CYP3A27相关的基因。
