IMMUNOPURIFICATION AND CHARACTERIZATION OF CYTOCHROME P-450

细胞色素 P-450 的免疫纯化和表征

• 批准号: 3963471
• 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/3963471
• 关键词: aminoacid analyzer; animal age group; antigens; carcinogenesis; chemical structure function; cytochrome P450; detoxification; drug metabolism; enzyme linked immunosorbent assay; enzyme structure; ethanol; hormone metabolism; human tissue; isozymes; liver; lung; methylcholanthrene; microsomes; monoclonal antibody; species difference; testosterone

The cytochromes P-450 metabolize a wide array of compounds, including xenobiotics such as drugs and carcinogens, and endogenous compounds such as steroids. The focus of this project is the identification, characterization, and elucidation of structure-function relationships of the multiple forms of this enzyme. Monoclonal antibodies (MAbs) to rat P-450s are an important tool in these studies. 3-methylcholanthrene (MC)-inducible P0450s have been immunopurified from the livers of rats, C57B1/6 and DBA/2 mice, guinea pigs and hamsters. Their primary structures were compared by amino acid sequence analysis and peptide mapping, and revealed varying degrees of homology. Several previously unidentified amino terminal residues of an immunopurified mouse P-450 were identified, and the sequence of a guinea pig liver P-450 was determined for the first time. A rat lung P-450 was also immunopurified and was indistinguishable from a liver P-450 by several criteria. Using a MAb to ethanol-inducible rat liver P-450, a P-450 has been purified from human liver and was structurally characterized. Since immunopurification typically denatured the P-450s, an alternative approach involving antigen-exchange was developed in which inactive denatured P-450 displaces active P-450 from the immunoadsorbent. Such epitope-specific exchange may be generally applicable to preparation of proteins with retention of activity. The epitopic structure of the P450 surface was mapped by evaluating the binding of MAbs to varius P-450 peptide fragments and by computer-aided alignment of homologous P-450s which are epitopically related. P-450 dependent testosterone metabolism in 3- and 24-month old rats were examined, and hydroxylation patterns as well as content of liver constitutive P-450s varied with age.

细胞色素P-450代谢多种化合物，包括 外来物质，如药物和致癌物质，以及内源化合物，如 类固醇。这个项目的重点是识别， 结构-功能关系的表征和阐明 这种酶的多种形式。抗大鼠的单抗 P-450是这些研究中的重要工具。3-甲基胆蒽 (MC)诱导的P0450已从大鼠肝脏免疫纯化， C57B1/6和DBA/2小鼠、豚鼠和仓鼠。它们的主要结构 通过氨基酸序列分析和肽图谱进行比较，并 显示出不同程度的同源性。几个以前身份不明的人 鉴定了免疫纯化的小鼠P-450的氨基端残基， 并首次测定了豚鼠肝脏P-450的序列 时间到了。大鼠肺P-450也被免疫提纯，无法区分 从几个标准来看，来自肝脏的P-450。用单抗诱导乙醇诱导 大鼠肝脏P-450，一个P-450已从人肝脏中纯化出来，并被 具有结构特征的。因为免疫纯化通常是变性的 P-450，另一种涉及抗原交换的方法是 其中非活性变性的P-450取代活性的P-450 免疫吸附剂。这种特定于表位交换通常可以 适用于制备具有保留活性的蛋白质。这个 通过评估结合，绘制了P450表面的表位结构 单抗与变种P-450多肽片段的比对及计算机辅助比对 表位相关的同源P-450。依赖于P-450 检测了3月龄和24月龄大鼠的睾酮代谢，并 肝脏成分P-450的羟基化模式及含量 随着年龄的增长而变化。