"STRUCTURE-FUNCTION STUDIES OF DIHYDROFOLATE REDUCTASE"

“二氢叶酸还原酶的结构功能研究”

• 批准号: 3181955
• 负责人: JAMES H. FREISHEIM
• 金额: $ 17.81万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-05-01 至 1988-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3181955
• 关键词: chemical structure function; dihydrofolate reductase; drug metabolism; enzyme structure; high performance liquid chromatography; methotrexate; neoplasm /cancer chemotherapy; pteridines; quinazolines; triazines

This project is concerned with the relation of the structure to the function of dihydrofolate reductase (dihydrofolate+ NADPH + H+ - tetrahydrofolate + NADP+). Using a number of techniques of protein chemistry, the problems to be investigated include: (a) obtaining protein sequence information on a methotrexate (MTX)-insensitive enzyme from MTX-resistant L5178Y cells and from Walker 256 carcinosarcoma cells primarily by HPLC-mapping of derived peptides and amino acid analysis; (b) chemical modification of specific amino acid residues of the protein and measurements of the effect of these modifications on the substrate, cofactor and inhibitor binding and on enzymic activity; (c) equilibrium binding studies of substrate, cofactor and inhibitors using reductases from human, L5178Y and Walker 256 cells and employing fluorescence, circular dichroism and equilibrium dialysis techniques; (d) evaluation of MTX analogues as affinity and photoaffinity labels of dihydrofolate reductases; (e) examination of selected, substituted quinazolines, triazines and pteridines as enzyme inhibitors and a determination of their transport characteristics in MTX-sensitive and -resistant cells.

本项目关注的是结构与 二氢叶酸还原酶的功能（二氢叶酸+ NADPH + H+ -） 四氢叶酸+ NADP+）。 利用蛋白质的一些技术 化学，要研究的问题包括：（a）获得蛋白质 甲氨蝶呤（MTX）不敏感酶的序列信息， MTX抗性L5178 Y细胞和来自步行者256癌肉瘤细胞 主要通过衍生肽的HPLC图谱和氨基酸分析;（B） 蛋白质的特定氨基酸残基的化学修饰， 测量这些改性对基材的影响， 辅因子和抑制剂结合以及对酶活性的影响;（c）平衡 使用还原酶进行底物、辅因子和抑制剂的结合研究， 人、L5178 Y和步行者256细胞，并使用荧光、圆形 二色性和平衡透析技术;（d）MTX的评价 作为二氢叶酸还原酶的亲和和光亲和标记的类似物; (e)选择的取代的喹唑啉、三嗪和 作为酶抑制剂的蝶啶及其转运的测定 MTX敏感和耐药细胞的特征。