Miranda C L, Wang J L, Henderson M C, Buhler D R
Marine/Freshwater Biomedical Center, Oregon State University, Corvallis 97331.
Biochim Biophys Acta. 1990 Feb 9;1037(2):155-60. doi: 10.1016/0167-4838(90)90161-8.
Immunoglobulin G fractions (IgGs), isolated from rabbits immunized against hepatic cytochrome P-450 isozymes were used to investigate the immunochemical homology among trout P-450s and between trout and rat P-450s. The antigens used for immunization were five constitutive trout P-450s (LMC1 to LMC5), one beta-naphthoflavone (BNF)-inducible trout P-450 (LM4b), and one phenobarbital-induced rat P4500IIB1 (PB-B). In the enzyme-linked immunosorbent assay (ELISA), strong cross-reactivity was observed between anti-LMC2 IgG and P-450 LMC1, and between anti-LMC3 IgG and P-450 LMC4. There was little or no cross-reactivity of anti-LMC5 IgG with other trout P-450s. Trout P-450 LM4b was not recognized by any of the antibodies against constitutive trout P-450s. Antibodies to P-450 LMC1 and P450 LMC2 cross-reacted strongly with rat P450IIB1 and with proteins of PB-induced rat liver microsomes. Rat P450IA1 (BNF-B) did not cross-react with anti-LMC1 or anti-LMC2 IgG. These cross-reactions were essentially confirmed by immunoblot (Western blot) analysis. Western blots of PB-induced rat liver microsomes probed with anti LMC1 revealed two major immunoreactive proteins in the P-450 region, one of which co-migrated with rat P450IIB1. P450IIB1 itself cross-reacted strongly with anti-LMC1 IgG. In control rats, a single protein band cross-reacted poorly with anti-LMC1 IgG. Antibodies to LMC1 and LMC2 did not cross-react with rat P450IA1 in Western blots. The antigenic epitopes in rat P450IIB1 recognized by anti-LMC1 IgG and anti-LMC2 IgG are probably not located at or near the active site of the enzyme since these antibodies did not inhibit benzphetamine N-demethylase activity of P450IIB1 or of PB-induced rat liver microsomes. In general, our results demonstrate: (1) the presence of a significant homology between LMC1 and LMC2, and between constitutive trout P-450 (LMC1) and PB-induced rat P-450 (P450IIB1); and (2) distant homology between constitutive trout P-450s and constitutive rat P-450s or BNF-induced rat P-450s.
从免疫兔肝脏细胞色素P-450同工酶中分离出的免疫球蛋白G组分（IgGs），用于研究虹鳟鱼P-450之间以及虹鳟鱼与大鼠P-450之间的免疫化学同源性。用于免疫的抗原是五种组成型虹鳟鱼P-450（LMC1至LMC5）、一种β-萘黄酮（BNF）诱导型虹鳟鱼P-450（LM4b）和一种苯巴比妥诱导型大鼠P4500IIB1（PB-B）。在酶联免疫吸附测定（ELISA）中，观察到抗LMC2 IgG与P-450 LMC1之间以及抗LMC3 IgG与P-450 LMC4之间有强烈的交叉反应。抗LMC5 IgG与其他虹鳟鱼P-450几乎没有或没有交叉反应。任何针对组成型虹鳟鱼P-450的抗体都不能识别虹鳟鱼P-450 LM4b。针对P-450 LMC1和P450 LMC2的抗体与大鼠P450IIB1以及PB诱导的大鼠肝微粒体蛋白有强烈的交叉反应。大鼠P450IA1（BNF-B）与抗LMC1或抗LMC2 IgG没有交叉反应。这些交叉反应通过免疫印迹（Western印迹）分析得到了基本证实。用抗LMC1探测PB诱导的大鼠肝微粒体的Western印迹显示，在P-450区域有两种主要的免疫反应性蛋白，其中一种与大鼠P450IIB1共迁移。P450IIB1本身与抗LMC1 IgG有强烈的交叉反应。在对照大鼠中，单一的蛋白条带与抗LMC1 IgG的交叉反应较弱。在Western印迹中，针对LMC1和LMC2的抗体与大鼠P450IA1没有交叉反应。抗LMC1 IgG和抗LMC2 IgG识别的大鼠P450IIB1中的抗原表位可能不在该酶的活性位点或其附近，因为这些抗体不抑制P450IIB1或PB诱导的大鼠肝微粒体的苄非他明N-脱甲基酶活性。总的来说，我们的结果表明：（1）LMC1和LMC2之间以及组成型虹鳟鱼P-450（LMC1）与PB诱导的大鼠P-450（P450IIB1）之间存在显著的同源性；（2）组成型虹鳟鱼P-450与组成型大鼠P-450或BNF诱导的大鼠P-450之间存在较远的同源性。
