Sen A, Arinç E
Department of Chemistry, Pamukkale University, Denizli, Turkey.
Comp Biochem Physiol C Toxicol Pharmacol. 2000 Jul;126(3):235-44. doi: 10.1016/s0742-8413(00)00117-1.
CYP1A is known to play important roles in the metabolism, detoxification and bioactivation of carcinogens and other xenobiotics in animals including fish. In our laboratory, CYP1A1 was obtained in a highly purified form with a specific content of 15-17 nmol P450 per mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens). Purified mullet CYP1A1 showed a very high substrate specificities for 7-ethoxyresorufin and 7-methoxyresorufin in a reconstituted system containing purified fish P450 reductase and lipid. In addition, effects of each individual components of the reconstituted system, i.e., CYP1A1 and P450 reductase on 7-methoxyresorufin O-demethylase (MROD) activity were studied. 7-ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by alpha-naphthoflavone (ANF). At 0.5 and 2.5 microM. ANF inhibited EROD activity by 90 and 98%, respectively. Mullet CYP1A1 did not catalyze monooxygenations of other substrates such as aniline, ethylmorphine, N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in fish such as trout and scup showed strong cross-reactivity with the purified mullet CYP1A1. In addition, anti-L. saliens liver CYP1A1 produced in our laboratory inhibited both the EROD and MROD activities catalyzed by L. saliens liver microsomes but stronger inhibition was observed with EROD activity. On the other hand, anti-mullet CYP1A1 antibodies showed very weak cross-reactivity with two proteins (presumably CYP1A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated rat liver microsomal EROD activity was weakly inhibited by the anti-L. saliens liver CYP1A1. These results strongly suggested that the purified mullet CYP1A1 is structurally, functionally and immunochemically similar to the CYP1A1 homologues purified from other teleost species but functionally and immunochemically distinct from mammalian CYP1A1.
已知细胞色素P450 1A（CYP1A）在包括鱼类在内的动物体内致癌物和其他外源性物质的代谢、解毒及生物活化过程中发挥重要作用。在我们实验室中，从野生鲻鱼（Liza saliens）肝脏微粒体中获得了高纯度的CYP1A1，其比含量为每毫克蛋白质含15 - 17 nmol细胞色素P450。在含有纯化的鱼类细胞色素P450还原酶和脂质的重组系统中，纯化的鲻鱼CYP1A1对7 - 乙氧基异吩恶唑酮和7 - 甲氧基异吩恶唑酮表现出非常高的底物特异性。此外，研究了重组系统中各个组分，即CYP1A1和细胞色素P450还原酶对7 - 甲氧基异吩恶唑酮O - 脱甲基酶（MROD）活性的影响。7 - 乙氧基异吩恶唑酮O - 脱乙基酶（EROD）活性受到α - 萘黄酮（ANF）的强烈抑制。在0.5和2.5 microM浓度下，ANF分别抑制EROD活性90%和98%。鲻鱼CYP1A1不催化其他底物如苯胺、乙基吗啡、N - 亚硝基二甲胺和对硝基苯酚的单加氧反应。针对鱼类如鳟鱼和鲷鱼中CYP1A1同源物产生的抗体与纯化的鲻鱼CYP1A1表现出强烈的交叉反应性。此外，我们实验室制备的抗鲻鱼肝CYP1A1抑制了鲻鱼肝微粒体催化的EROD和MROD活性，但对EROD活性的抑制更强。另一方面，抗鲻鱼CYP1A1抗体与经3 - 甲基胆蒽（3MC）处理的大鼠肝脏微粒体中的两种蛋白质（可能是CYP1A1和CYP1A2）表现出非常弱的交叉反应性。此外，经3MC处理的大鼠肝脏微粒体EROD活性受到抗鲻鱼肝CYP1A1的微弱抑制。这些结果有力地表明，纯化的鲻鱼CYP1A1在结构、功能和免疫化学上与从其他硬骨鱼物种纯化的CYP1A1同源物相似，但在功能和免疫化学上与哺乳动物CYP1A1不同。
