IMMUNOPURIFICATION AND CHARACTERIZATION OF CYTOCHROME P-450

细胞色素P-450代谢多种药物和致癌物。这个 这种酶的多种形式表现出独特而广泛的底物和 反应特异性。这个项目的重点是识别， 结构-功能关系的表征和阐明 这些同工酶。特异性细胞色素P-450的单抗 是这些研究中必不可少的工具。几种细胞色素P-450具有 在一步免疫吸附程序中得到基本纯化，使用 琼脂糖单抗与大鼠肝细胞色素P-450的主要结合形式 3-甲基胆蒽和苯巴比妥(MC-P-450和PB-P-450， )。当与溶解的组织微生物体混合时， 免疫吸附剂结合多肽，这些多肽在pH值为3.0时很容易解吸。 细胞色素P-450用单抗1-7-1和1-31-2- C57B1/6和DBA/2小鼠、豚鼠和仓鼠以及大鼠的肝脏 阿龙。这种基于不同单抗的免疫吸附实验揭示了 不同组织中同色素酶P-450之间的同源性 菌株和物种。用此法分离细胞色素P-450 通过肽图谱和氨基末端氨基酸对其结构进行了分析 分析。这些系统色素P-450的不同程度的同源性是 找到了。MC和PB诱导大鼠肝细胞色素P-450的分离 表现出少量但可检测到的酶活性。更高水平的 用一种新的抗原交换方法制备了具有活性的细胞色素P-450 用哪种失活变性的P-450交换天然细胞色素P-450 结合到免疫吸附剂上。

The cytochromes P-450 metabolize a variety of drugs and carcinogens. The multiple forms of this enzyme display unique yet broad, substrate and reaction specificity. The focus of this project is the identification, characterization, and elucidation of structure-function relationships of these isozymes. Monoclonal antibodies (MAbs) to specific cytochromes P-450 are an essential tool in these studies. Several cytochromes P-450 have been substantially purified in a one-step immunoadsorption procedure using Sepharose bound MAbs to the major forms of rat liver cytochrome P-450 induced by 3-methylcholanthrene and phenobarbital (MC-P-450 and PB-P-450, respectively). When mixed with solubilized tissue microsomes, the immunoadsorbents bind polypeptides which are readily desorbed at pH 3.0. Cytochromes P-450 have been purified with MAbs 1-7-1 and 1-31-2- from livers of C57B1/6 and DBA/2 mice, guinea pigs and hamsters, and from rat lung. Such immunoadsorption experiments based on different MAbs reveal eptitope relatedness between sytochromes P-450 in different tissues, strains, and species. The cytochromes P-450 isolated by this procedure were analyzed structurally by peptide mapping and NH2-terminal amino acid analysis. Varying degrees of homology of these sytochromes P-450 were found. The hepatic cytochromes P-450 isolated from MC- and PB induced rats exhibit small yet detectable level of enzyme activity. A higher level of active cytochrome P-450 was prepared by a novel antigen-exchange method in which inactive denatured P-450 was exchanged for native cytochrome P-450 bound to the immunoadsorbents.