STRUCTURE-FUNCTION STUDIES OF DIHYDROFOLATE REDUCTASE

二氢叶酸还原酶的结构功能研究

• 批准号: 3181960
• 负责人: JAMES H. FREISHEIM
• 金额: $ 23.88万
• 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 1993-02-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3181960
• 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; recombinant DNA; 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.

该项目涉及的关系的各个方面 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─ 使用重组的人DHFR的定向诱变研究 DNA技术评估各种NADPH的功能作用 和氨甲喋呤（MTX）或二氢叶酸（FAH 2）结合氨基 酸残留物 这些研究将涉及建设 重组质粒在E.大肠杆菌， 突变形式的人源化蛋白的纯化和表征 通过动力学、平衡结合和X射线晶体学测定DHFR 研究;（B）人类免疫原性决定簇的作图 使用针对蛋白水解产生的肽的抗体的DHFR，或 化学裂解完整的人DHFR或由 自动化肽合成。 这些序列特异性抗体 也将用于检测来自人类的MTX不敏感的DHF 急性骨髓性白血病细胞和其他肿瘤;（c）获得 来自L5178 Y的MTX不敏感DHFR的序列信息 （d）评价新合成的叶酸拮抗剂作为 DHFR和胸苷酸合成酶的抑制剂， （e）单碳分子的分子表征 FAH 4/MTX运输系统在运输能力和- 缺陷L1210细胞。 这些实验包括光亲和 用MTX类似物在各种条件下标记转运蛋白 条件下，确定膜的相关性 和细胞质MTX结合成分，可能的表达 转运缺陷细胞中的组分和a L1210细胞中的转运蛋白，其过量产生该蛋白。

项目成果

Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines.

人 CCRF-CEM 白血病细胞系中甲氨蝶呤膜转运的分子事件。

• DOI: 10.1016/0065-2571(92)90006-l
• 发表时间: 1992
• 期刊: Advances in enzyme regulation
• 作者: Freisheim,JH;Ratnam,M;McAlinden,TP;Prasad,KM;Williams,FE;Westerhof,GR;Schornagel,JH;Jansen,G
• 通讯作者: Jansen,G

Atypical transient state kinetics of recombinant human dihydrofolate reductase produced by hysteretic behavior. Comparison with dihydrofolate reductases from other sources.

迟滞行为产生的重组人二氢叶酸还原酶的非典型瞬态动力学。

• 发表时间: 1989
• 期刊: The Journal of biological chemistry
• 作者: Appleman,JR;Beard,WA;Delcamp,TJ;Prendergast,NJ;Freisheim,JH;Blakley,RL
• 通讯作者: Blakley,RL

Effects of conversion of phenylalanine-31 to leucine on the function of human dihydrofolate reductase.

苯丙氨酸 31 转化为亮氨酸对人二氢叶酸还原酶功能的影响。

• DOI: 10.1021/bi00437a020
• 发表时间: 1989
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Prendergast,NJ;Appleman,JR;Delcamp,TJ;Blakley,RL;Freisheim,JH
• 通讯作者: Freisheim,JH

Tricyclic 2,4-diaminopyrimidines with broad antifolate activity and the ability to inhibit Pneumocystis carinii growth in cultured human lung fibroblasts in the presence of leucovorin.

三环 2,4-二氨基嘧啶，具有广泛的抗叶酸活性，并且能够在亚叶酸存在的情况下抑制培养的人肺成纤维细胞中卡氏肺囊虫的生长。

• DOI: 10.1016/0006-2952(89)90554-6
• 发表时间: 1989
• 期刊: Biochemical pharmacology
• 影响因子: 5.8
• 作者: Rosowsky,A;Freisheim,JH;Hynes,JB;Queener,SF;Bartlett,M;Smith,JW;Lazarus,H;Modest,EJ
• 通讯作者: Modest,EJ

Further studies on substituted quinazolines and triazines as inhibitors of a methotrexate-insensitive murine dihydrofolate reductase.

取代喹唑啉和三嗪作为甲氨蝶呤不敏感的鼠二氢叶酸还原酶抑制剂的进一步研究。

• DOI: 10.1016/0006-2952(86)90151-6
• 发表时间: 1986
• 期刊: Biochemical pharmacology
• 影响因子: 5.8
• 作者: Dedhar,S;Freisheim,JH;Hynes,JB;Goldie,JH
• 通讯作者: Goldie,JH