Fagan J B, Pastewka J V, Chalberg S C, Gozukara E, Guengerich F P, Gelboin H V
Arch Biochem Biophys. 1986 Jan;244(1):261-72. doi: 10.1016/0003-9861(86)90116-5.
The mRNAs encoding the major polycyclic aromatic hydrocarbon-induced cytochromes P-450 from rat, P-450BNF/MC-B and P-450ISF/BNF-G, were characterized using three classes of recombinant plasmids: those complementary to (a) only P-450BNF/MC-B mRNA, (b) only P-450ISF/BNF-G mRNA, and (c) both mRNAs. These classes were identified by hybridization-selected translation and immunoprecipitation using six monoclonal and polyclonal antibodies and were later sequenced to confirm their identity and specificity. These findings indicated that the mRNAs encoding these two P-450s have regions that are unique, as well as regions that are homologous. Hybridization-selected translation also showed that the primary in vitro translation products of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs are 55 and 52 kDa, respectively, and have both unique and common structural characteristics that can be distinguished immunologically. By Northern hybridization, the P-450BNF/MC-B mRNA was found to be 2900 bases long, while the P-450ISF/BNF-G mRNA was 2100 bases long. Precursors of 3500 and 5200 bases were detected for P-450BNF/MC-B mRNA, while a 3100-base precursor was detected for P-450ISF/BNF-G mRNA. These two mRNAs were induced by beta-naphthoflavone, isosafrole, and 3-methylcholanthrene, but not by phenobarbital. In untreated rats, the P-450BNF/MC-B mRNA was consistently present at very low levels while the P-450ISF/BNF-G mRNA was present in variable amounts, suggesting that the latter mRNA can be induced by dietary or other environmental factors. The kinetics of induction of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs were measured by dot blot hybridization. P-450BNF/MC-B mRNA increased rapidly, reaching half-maximum by 4 h after treatment with 3-methylcholanthrene, while the P-450ISF/BNF-G mRNA increased more slowly, reaching half-maximum after 12 h. The levels of both mRNAs peaked at 24 h, but decreased thereafter at different rates; P-450BNF/MC-B mRNA dropped by about 20% during the next 24 h, while P-450ISF/BNF-G mRNA dropped by 50 to 70%. These differences in the kinetics of induction and the apparent stabilities of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs, in conjunction with the observed differences in their levels in untreated rats, suggested that these two mRNAs were not coordinately regulated even though they were induced by the same compounds.(ABSTRACT TRUNCATED AT 400 WORDS)
利用三类重组质粒对编码大鼠主要多环芳烃诱导的细胞色素P-450（P-450BNF/MC-B和P-450ISF/BNF-G）的信使核糖核酸（mRNA）进行了特性分析：（a）仅与P-450BNF/MC-B mRNA互补的质粒，（b）仅与P-450ISF/BNF-G mRNA互补的质粒，以及（c）与两种mRNA都互补的质粒。通过杂交选择翻译和使用六种单克隆抗体及多克隆抗体进行免疫沉淀来鉴定这些类别，随后对其进行测序以确认它们的身份和特异性。这些发现表明，编码这两种细胞色素P-450的mRNA既有独特区域，也有同源区域。杂交选择翻译还表明，P-450BNF/MC-B和P-450ISF/BNF-G mRNA的主要体外翻译产物分别为55 kDa和52 kDa，并且具有可通过免疫学区分的独特和共同结构特征。通过Northern杂交发现，P-450BNF/MC-B mRNA长2900个碱基，而P-450ISF/BNF-G mRNA长2100个碱基。检测到P-450BNF/MC-B mRNA的前体为3500和5200个碱基，而P-450ISF/BNF-G mRNA的前体为3100个碱基。这两种mRNA可被β-萘黄酮、异黄樟素和3-甲基胆蒽诱导，但不能被苯巴比妥诱导。在未处理的大鼠中，P-450BNF/MC-B mRNA始终以极低水平存在，而P-450ISF/BNF-G mRNA的含量则各不相同，这表明后者的mRNA可被饮食或其他环境因素诱导。通过斑点印迹杂交测定了P-450BNF/MC-B和P-450ISF/BNF-G mRNA的诱导动力学。P-450BNF/MC-B mRNA迅速增加，在用3-甲基胆蒽处理后4小时达到最大值的一半，而P-450ISF/BNF-G mRNA增加得较慢，在12小时后达到最大值的一半。两种mRNA的水平在24小时达到峰值，但此后以不同速率下降；P-450BNF/MC-B mRNA在接下来的24小时内下降了约20%，而P-450ISF/BNF-G mRNA下降了50%至70%。P-450BNF/MC-B和P-450ISF/BNF-G mRNA在诱导动力学和表观稳定性方面的这些差异，连同在未处理大鼠中观察到的它们水平的差异，表明这两种mRNA即使由相同化合物诱导也不是协同调节的。（摘要截短至400字）
