Kloepper-Sams P J, Park S S, Gelboin H V, Stegeman J J
Arch Biochem Biophys. 1987 Feb 15;253(1):268-78. doi: 10.1016/0003-9861(87)90660-6.
Monoclonal antibody (MAb) 1-12-3 generated against liver cytochrome P-450E (P-450E), an aryl hydrocarbon hydroxylase of the marine fish Stenotomus chrysops (scup), reacted only with P-450E when tested in immunoblot analysis with five P-450 fractions from scup liver. This and six other MAbs against P-450E recognized purified P-450E, as well as a single band in beta-naphthoflavone (BNF)-induced scup microsomes that comigrated with authentic P-450E. Like MAb 1-12-3, polyclonal anti-P-450E reacted with P-450E but not with other scup P-450 fractions and reacted strongly with a band coincident to P-450E in BNF-treated scup microsomes. However, the polyclonal antibody (PAb) also faintly recognized additional microsomal proteins. MAb 1-12-3 recognized P-450E induced by 3,3',4,4',5,5'-hexachlorobiphenyl and by polychlorinated biphenyl mixtures in scup, and a single band induced by BNF or 3-methylcholanthrene (MC) in microsomes of other teleosts, including two trout species, killifish and winter flounder. The content of the P-450E counterpart in these fish and also in untreated scup coincided with induced ethoxyresorufin O-deethylase (EROD) activity. Induced EROD activity in scup and trout was strongly inhibited by MAb 1-12-3, further demonstrating the relationship between P-450E and induced P-450E in trout. MAb 1-12-3, two other MAbs, and anti-P-450E PAb recognized a band comigrating with P-450c in BNF-induced rat microsomes. MAb 1-12-3 also recognized purified rat P-450c. MAb 1-12-3 and anti-P-450E PAb recognized a second band of lower molecular weight than P-450c in BNF rat microsomes which may correspond to P-450d, the MC- and isosafrole-inducible rat isozyme. The results firmly establish the identity of scup P-450E, the relationship of BNF-induced P-450 in other teleosts with P-450E, and the immunochemical relationship of P-450E with rat P-450c. Furthermore, results with untreated fish suggest that effects of environmental chemicals may be detected by immunoblotting with monoclonal anti-P-450E.
针对海洋鱼类条纹鲈（Stenotomus chrysops）的芳烃羟化酶——肝细胞色素P-450E（P-450E）产生的单克隆抗体（MAb）1-12-3，在用条纹鲈肝脏的5种P-450组分进行免疫印迹分析时，仅与P-450E发生反应。这种抗体以及其他6种针对P-450E的单克隆抗体识别纯化的P-450E，以及在β-萘黄酮（BNF）诱导的条纹鲈微粒体中与真实P-450E共迁移的一条带。与单克隆抗体1-12-3一样，多克隆抗P-450E与P-450E发生反应，但不与条纹鲈的其他P-450组分发生反应，并且与BNF处理的条纹鲈微粒体中与P-450E一致的条带强烈反应。然而，多克隆抗体（PAb）也微弱地识别其他微粒体蛋白。单克隆抗体1-12-3识别条纹鲈中由3,3',4,4',5,5'-六氯联苯和多氯联苯混合物诱导产生的P-450E，以及在其他硬骨鱼的微粒体中由BNF或3-甲基胆蒽（MC）诱导产生的一条带，这些硬骨鱼包括两种鳟鱼、食蚊鱼和冬比目鱼。这些鱼类以及未处理的条纹鲈中P-450E对应物的含量与诱导的乙氧异吩恶唑酮O-脱乙基酶（EROD）活性一致。条纹鲈和鳟鱼中诱导的EROD活性被单克隆抗体1-12-3强烈抑制，进一步证明了P-450E与鳟鱼中诱导的P-450E之间的关系。单克隆抗体1-12-3、另外两种单克隆抗体以及抗P-450E PAb识别在BNF诱导大鼠微粒体中与P-450c共迁移的一条带。单克隆抗体1-12-3也识别纯化的大鼠P-450c。单克隆抗体1-12-3和抗P-450E PAb在BNF大鼠微粒体中识别一条分子量低于P-450c的第二条带，其可能对应于P-450d，即MC和异黄樟素诱导的大鼠同工酶。这些结果确凿地确定了条纹鲈P-450E的身份、其他硬骨鱼中BNF诱导的P-450与P-450E的关系，以及P-’450E与大鼠P-450c的免疫化学关系。此外，未处理鱼类的结果表明，环境化学物质的影响可以通过用单克隆抗P-450E进行免疫印迹来检测。
