STRUCTURE-FUNCTION STUDIES OF DIHYDROFOLATE REDUCTASE

二氢叶酸还原酶的结构功能研究

• 批准号: 3181957
• 负责人: JAMES H. FREISHEIM
• 金额: $ 22.18万
• 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 1993-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3181957
• 关键词: NAD(H) phosphate; X ray crystallography; aminoacid; chemical structure function; cytotoxicity; dihydrofolate reductase; drug metabolism; enzyme inhibitors; enzyme mechanism; enzyme structure; genetic mapping; high performance liquid chromatography; immunogenetics; laboratory rabbit; laboratory rat; methotrexate; myelogenous leukemia; neoplasm /cancer chemotherapy; nucleic acid hybridization; plasmids; protein engineering; proteolysis; thymidylate synthase; transport proteins

This project is concerned with various aspects of the relation of the structure to the function of dihydrofolate reductase (DHFR). The proposed problems to be investigated include: (a) site- directed mutagenesis studies of human DHFR Using recombinant DNA techniques to evaluate the functional role of various NADPH and methotrexate (MTX) or dihydrofolate (FAH2) binding amino acid residues. These studies will involve construction of recombinant plasmids and high level DHFR expression in E. coli, purification and characterization of mutant forms of human DHFR by kinetic, equilibrium binding and x-ray crystallographic studies; (b) mapping of the immunogenic determinants of human DHFR using antibodies to peptides produced by proteolysis or chemical cleavage of intact human DHFR or produced by automated peptide synthesis. These sequence-specific antibodies will also be used to detect MTX-insensitive DHFRs from human acute myelogenous leukemic cells and other tumors; (c) to obtain sequence information on a MTX-insensitive DHFR from L5178Y cells; (d) evaluation of newly synthesized folate antagonists as inhibitors of DHFRs and thymidylate synthase and as cytotoxic agents; (e) molecular characterization of the one-carbon FAH4/MTX transport system in transport-competent and - defective L1210 cells. These experiments include photoaffinity labeling of transport proteins with an MTX analogue under various conditions, a determination of the relatedness of the membrane and cytoplasmic MTX binding components, possible expression of components in transport-defective cells and sequencing of a transport protein in L1210 cells which overproduce this protein.

这个项目涉及到两国关系的各个方面。 二氢叶酸还原酶(DHFR)的结构与功能。 建议调查的问题包括：(A)地盘- 重组人DHFR的定向诱变研究 DNA技术评价不同NADPH的功能作用 和甲氨蝶呤(MTX)或二氢叶酸(FAH2)结合氨基 酸残留物。这些研究将涉及建造 重组质粒的构建及其在大肠杆菌中的高效表达 人突变型基因的纯化与鉴定 动力学结合、平衡结合和x射线结晶学研究 研究；(B)绘制人类免疫原性决定因素图 DHFR使用针对蛋白水解肽的抗体或 完整人DHFR或由其产生的化学切割 自动合成多肽。这些序列特异性抗体 也将用于检测人类对MTX不敏感的DHFRs 急性髓系白血病细胞和其他肿瘤；(C)获得 来自L5178Y的甲氨蝶呤不敏感的DHFR的序列信息 细胞；(D)评价新合成的叶酸拮抗剂 DHFRs和胸苷合成酶的抑制剂以及AS细胞毒性 试剂；(E)一碳的分子表征 FAH4/MTX运输系统中的运输能力和- L1210电池有缺陷。这些实验包括光亲和力 不同条件下MTX类似物标记转运蛋白的研究 条件，膜的关联性的测定 和细胞质MTX结合成分，可能表达 运输缺陷细胞中的成分和一种 L1210细胞中的转运蛋白，从而过量生产该蛋白。