IMMUNOPURIFICATION AND CHARACTERIZATION OF CYTOCHROME P-450

细胞色素 P-450 的免疫纯化和表征

• 批准号: 4692374
• 负责人: F K FRIEDMAN
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/4692374
• 关键词: aminoacid analyzer; carcinogenesis; chemical structure function; cytochrome P450; drug metabolism; enzyme linked immunosorbent assay; enzyme structure; isozymes; microsomes; monoclonal antibody; physical separation; systematic biology

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.

细胞色素P-450代谢多种药物和致癌物质。 的 这种酶的多种形式显示出独特而广泛的底物， 反应特异性 该项目的重点是识别， 表征，并阐明结构-功能关系 这些同工酶。 特异性细胞色素P-450的单克隆抗体 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基本上纯化， 琼脂糖凝胶结合单克隆抗体对大鼠肝细胞色素P-450的主要形式 由3-甲基胆蒽和苯巴比妥（MC-P-450和PB-P-450， 分别）。 当与溶解的组织微粒体混合时， 免疫吸附剂结合在pH3.0下容易解吸的多肽。 细胞色素P-450已经用来自于 C57 B1/6和DBA/2小鼠、豚鼠和仓鼠以及大鼠的肝脏 肺。 基于不同MAb的这种免疫吸附实验揭示了 不同组织中同色素P-450之间的表位相关性， 菌株和物种。 用此方法分离的细胞色素P-450 通过肽图和NH 2-末端氨基酸分析进行结构分析 分析. 不同程度的同源性，这些sytochromes P-450是 found. MC和PB诱导大鼠肝细胞色素P-450的分离 表现出小但可检测水平的酶活性。 更高水平的 采用一种新的抗原交换法制备活性细胞色素P-450， 将该失活的变性P-450交换为天然细胞色素P-450 与免疫吸附剂结合。