Monoclonal antibodies distinguish among isozymes of the cytochrome P-450b subfamily.
Monoclonal antibodies distinguish among isozymes of the cytochrome P-450b subfamily.
复制标题
单克隆抗体可区分细胞色素 P-450b 亚家族的同工酶。
DOI：
10.1016/0003-9861(85)90221-8
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发表时间：
1985
影响因子：
3.9
通讯作者：
P. Thomas
中科院分区：
文献类型：
--
作者：
L. Reik;W. Levin;D. Ryan;S. Maines;P. Thomas
多克隆抗体与从Long Evans大鼠中纯化的细胞色素P-450 b和P-450 e以及从Holtzman大鼠中纯化的细胞色素P-450 b的一种变异株（P-450 bH）反应相同。在本研究中，一系列的12种不同的单克隆抗体产生的细胞色素P-450 b已被用来区分这些密切相关的苯巴比妥诱导大鼠肝细胞色素P-450。在免疫印迹和酶联免疫吸附试验中，10种单克隆抗体与细胞色素P-450 b、P-450 e和P-450 bH结合;一种单克隆抗体（B50）识别细胞色素P-450 b和P-450 bH，但不识别细胞色素P-450 e;一种单克隆抗体（B51）对细胞色素P-450 b具有特异性。此外，一种单克隆抗体（BEF 29）与细胞色素P-450 f强烈反应，而另一种抗体（BEA 33）与细胞色素P-450 a弱反应。用这些单克隆抗体检测细胞色素P-450 c、P-450 d和P-450 g-P-450 j均未发现交叉反应。细胞色素P-450 b上的六个空间上不同的表位被鉴定，抗体反应性的差异为三个额外的重叠表位提供了证据。几种单克隆抗体是睾酮和苄非他明代谢的强效抑制剂，在重组系统中由细胞色素P-450 b支持。B50和BE 52不抑制未处理大鼠微粒体对两种底物的代谢，但抑制苯巴比妥处理大鼠微粒体对苄非他明N-去甲基化和睾酮代谢为16α-和16β-羟基睾酮以及雄烯二酮形成67-94%。这些单克隆抗体不抑制睾酮代谢的其他途径。这些单克隆抗体对甲基非他明和睾酮的微粒体代谢的不同抑制反映了细胞色素P-450 b和P-450 e以及其他细胞色素P-450同工酶的含量和诱导。
Polyclonal antibody has been shown previously to react identically with cytochromesP-450b andP-450e purified from Long Evans rats and a strain variant of cytochromeP-450b purified from Holtzman rats (P-450bH). In the present study, an array of 12 different monoclonal antibodies produced against cytochromeP-450b has been used to distinguish among these closely related phenobarbital-inducible rat hepatic cytochromesP-450. In immunoblots and enzyme-linked immunosorbent assays, 10 monoclonal antibodies bind to cytochromesP-450b,P-450e, andP-450bH; one monoclonal antibody (B50) recognizes cytochromesP-450b andP-450bH but not cytochromeP-450e; and one monoclonal antibody (B51) is specific for cytochromeP-450b. In addition, one monoclonal antibody (BEF29) reacts strongly with cytochromeP-450f, and another antibody (BEA33) reacts weakly with cytochromeP-450a. No cross-reactions with cytochromesP-450c,P-450d, andP-450g-P-450j were detected with any of the monoclonal antibodies in these assays. Six spatially distinct epitopes on cytochromeP-450b were identified, and differences in antibody reactivity provided evidence for three additional overlapping epitopes. Several monoclonal antibodies are potent inhibitors of testosterone and benzphetamine metabolism supported by cytochromeP-450b in a reconstituted system. B50 and BE52 do not inhibit metabolism of the two substrates by microsomes from untreated rats, but inhibit benzphetamineN-demethylation and testosterone metabolism to 16α- and 16β-hydroxytestosterone as well as androstenedione formation 67–94% by microsomes from phenobarbital-treated rats. No other pathways of testosterone metabolism are inhibited by these monoclonal antibodies. The differential inhibition of microsomal metabolism of benzphetamine and testosterone by these monoclonal antibodies is a reflection of the content and inducibility of cytochromesP-450b andP-450e as well as other cytochromeP-450 isozymes.