"STRUCTURE-FUNCTION STUDIES OF DIHYDROFOLATE REDUCTASE"

这个项目关注的是结构与 二氢叶酸还原酶(二氢叶酸+NADPH+H+-)的功能 四氢叶酸+NADP+)。使用多种蛋白质技术 化学方面，要研究的问题包括：(A)获取蛋白质 甲氨蝶呤(MTX)不敏感酶的序列信息 MTX耐药的L5178Y细胞和Walker 256癌肉瘤细胞 主要通过衍生多肽的高效液相色谱图谱和氨基酸分析；(B) 蛋白质的特定氨基酸残基的化学修饰和 测量这些修饰对衬底的影响， 辅因子和抑制剂结合及对酶活性的影响；(C)平衡 底物、辅因子和抑制剂结合作用的研究 人、L5178Y和Walker 256细胞，采用荧光技术，圆形 二向色性和平衡透析技术；(D)甲氨蝶呤的评价 作为二氢叶酸还原酶亲和标记和光亲和标记的类似物； (E)检查选定的、取代的喹唑啉、三氮唑类和 酶抑制剂的蝶呤类药物及其转运的测定 甲氨蝶呤敏感和耐药细胞的特征。

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.