Identification of the cytochrome P-450 induced by macrolide antibiotics in rat liver as the glucocorticoid responsive cytochrome P-450p.
Identification of the cytochrome P-450 induced by macrolide antibiotics in rat liver as the glucocorticoid responsive cytochrome P-450p.
复制标题
鉴定大鼠肝脏中大环内酯类抗生素诱导的细胞色素 P-450 为糖皮质激素反应性细胞色素 P-450p。
DOI：
10.1021/bi00330a010
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Guzelian,PS
中科院分区：
文献类型：
--
作者：
Wrighton,SA;Maurel,P;Schuetz,EG;Watkins,PB;Young,B;Guzelian,PS
Steven A.放大图片作者：Wrighton，* 帕特里克莫瑞尔，艾琳G. Schuetz，Paul B. Watkins，Beverly Young，and Philip S. Guzelian临床毒理学和环境医学部，弗吉尼亚医学院，里士满，弗吉尼亚23298-0001接收日期：1984年8月6日摘要：我们给大鼠施用三乙酰竹桃霉素（TAO），发现这种大环内酯类抗生素是迄今为止检测的肝微粒体细胞色素P-450（P-450）的最有效诱导剂。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析判断，从TAO处理大鼠制备的肝微粒体中含有大于5.0 nmol P-450/mg蛋白和单一诱导蛋白。该蛋白与P-450诱导的主要形式P-450 p在经16 α-甲腈（PCN）或地塞米松（DEX）处理的大鼠肝微粒体中共同迁移。在用抗P-450 p抗体制备的凝胶上进行免疫印迹，TAO诱导的蛋白质强烈反应为单一条带。（a）未给药大鼠或苯巴比妥、TAO、DEX或PCN给药大鼠制备的肝微粒体中免疫反应性P-450 p的量，（B）这些微粒体催化TAO转化为形成光谱复合物的代谢物的能力，以及（c）乙基吗啡和红霉素脱甲基酶活性之间存在严格的平行性。抗P-450 p抗体特异性阻断微粒体TAO代谢物复合物的形成和乙基吗啡和红霉素脱甲基酶活性。此外，抗P-450 p抗体完全免疫沉淀溶解TAO代谢物复合物制备的洗涤剂处理的肝微粒体从TAO处理的大鼠。最后，我们发现，从TAO处理大鼠的肝微粒体中分离并纯化至均一的P-450的主要形式与纯化的P-450 p无法区分，这是通过分子量、光谱特征、酶活性、结合TAO的能力、肽图谱和氨基末端氨基酸序列来判断的。我们的结论是，除了糖皮质激素，大环内酯类抗生素是P-450 p的特异性诱导剂。肝脏通过增加或减少（或两者）细胞色素P-450的浓度来响应药物或其它外来化合物的存在，细胞色素P-450是微粒体血红素蛋白的多基因家族，其催化许多外来和内源性底物的生物氧化（Snyder & Remmer，1979; Guengerich等人，1982年）。人们习惯将化合物归类为类似于苯巴比妥或3-甲基胆蒽，这两种原型诱导剂增加不同形式的肝细胞色素P-450的肝浓度。最近，我们提出糖皮质激素和抗糖皮质激素如双烯醇酮-16甲腈（PCN）1构成调节另一种独特形式的细胞色素P-450，P-450 p2的“第三类”诱导剂（Heuman等人，1982; Schuetz等人，Schuetz & Guzelian，1984 a）。即使这种简单的分类也是不充分的，因为存在细胞色素P-450的充分表征的形式，其中没有鉴定出诱导剂（Cleveland等人，1977; Cheng和Schenkman，1982; Kamataki等人，1983; Ryan等人，1984）和其它形式，这些形式似乎是由不属于这三组之一的试剂诱导的（Tam-burini等，1984; Past & Cook，1982; Koop等人，1982年）。
Steven A. Wrighton,* Patrick Maurel, Erin G. Schuetz, Paul B. Watkins, Beverly Young, and Philip S. Guzelian Division of Clinical Toxicology and Environmental Medicine, Medical College of Virginia, Richmond, Virginia 23298-0001 Received August 6, 1984 abstract: We administered triacetyloleandomycin (TAO) to rats and found that this macrolide antibiotic is the most efficacious inducer of livermicrosomal cytochrome P-450 (P-450) examinedto date. Liver microsomes prepared from TAO-treated rats contained greater than 5.0 nmol of P-450/mg of protein and a single induced protein as judged by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein comigrated with P-450p, the majorform of P-450 induced inliver microsomes of rats treated with pregnenolone-16a-carbonitrile (PCN) or dexamethasone (DEX). On immunoblots of such gels developed with antibodies toP-450p, the TAO-induced protein reacted strongly as a single band. There was strict parallelism between (a) the amount of immunoreactive P-450p in liver microsomes prepared from untreated rats or from rats treated with phenobarbital, TAO, DEX, or PCN,(b) the ability of these microsomes to catalyze conversion of TAO to a metabolite which forms a spectral complex, and (c) the ethylmorphine and erythromycin demethylase activities. Antibodies toP-450p specifically blocked microsomal TAO metabolite complex formation and ethylmorphine and erythromycin demethylase activities. Moreover, anti-P-450p antibodies completely immunoprecipitated solubilized TAO metabolite complexes prepared by detergent treatment of liver microsomes obtained from TAO-treated rats. Finally, we found that the major form of P-450 isolated from liver microsomes of TAO-treated rats and purified to homogeneity was indistinguishable from purified P-450p as judged by molecular weights, spectral characteristics, enzymatic activities, ability to bind TAO, peptide maps, and amino-terminal amino acid sequences. We concluded that, in addition to glucocorticoids, macrolide antibiotics are specific inducers of P-450p. e liver responds to the presence of drugs or other foreign compounds by increasing or decreasing (or both) the con-centrations of the cytochromes P-450, a multigene familyof microsomal hemoproteins that catalyze the biooxidation of numerous foreign andendogenous substrates (Snyder & Remmer, 1979; Guengerich et al., 1982). It has been cus-tomary to classify compounds as resembling either pheno-barbital or 3-methylcholanthrene, two prototype inducers that increase theliver concentrations of different forms of the hepatic cytochromes P-450. Recently, we proposed that glucocorticoids and anti-glucocorticoids like pregnenolone-16acarbonitrile (PCN) 1 constitute a “third class” of inducers that regulate yet another unique form of cytochrome P-450, P-450p2 (Heuman et al., 1982; Schuetz et al., 1984; Schuetz & Guzelian, 1984a). Even this simple classification is inadequate because there are well-characterized forms of cytochrome P-450 for which no inducer has been identified (Cleveland et al., 1977; Cheng & Schenkman, 1982; Kamataki et al., 1983; Ryan et al., 1984) and other forms that appear to be induced by agents not represented in one of these three groups (Tam-burini et al., 1984; Past & Cook, 1982; Koop et al., 1982).