STRUCTURAL STUDIES ON DIHYDROFOLATE REDUCTASE

预计的工作部分涉及继续进行中的研究 二氢叶酸还原酶（DHFR）结构与催化机理 来自屎链球菌、干酪乳杆菌和牛肝。 的 对S.粪便将完成。 停流 将使用同位素标记底物的分光光度法和NMR 进一步研究催化机理。 NMR也将用于获得 细菌催化位点侧链运动信息 DHFR。 该项目的另一部分涉及制定方法， 在来自患者的肿瘤组织中寻找DHFR的变体形式 在甲氨蝶呤治疗期间或之后复发。 从病人的 肿瘤细胞（白血病细胞或肺转移）将用于 在培养物中选择合适的哺乳动物细胞（例如，3T3细胞）。 文化 将筛选转化的细胞，以产生变异的人 DHFR或扩增的正常人DHFR，通过在 甲氨蝶呤 将检查存活菌落是否存在 人类DNA和在这项测试中呈阳性的DNA将用于第二次检测， 转染 将研究次级转化体的DHFR水平， DHFR的动力学性质。 复发患者的肿瘤细胞将 还检查甲氨蝶呤转运的改变。

Projected work deals in part with the continuation of ongoing studies on the structure and catalytic mechanism of dihydrofolate reductase (DHFR) from Streptococcus faecium, Lactobacillus casei and bovine liver. The sequencing of isoenzyme 1 from S. faecium will be completed. Stopped flow spectrophotometry and NMR of isotopically labeled substrates will be used to further study the catalytic mechanism. NMR will also be used to obtain information about sidechain motion at the catalytic site of bacterial DHFR. Another part of the project involves the development of methods to search for variant forms of DHFR in neoplastic tissue from patients relapsing on or after methotrexate therapy. DNA extracted from patients' tumor cells (leukemic cells, or pulmonary metastases) will be used to transfect suitable mammalian cells in culture (e.g., 3T3 cells). Cultures will be screened for cells transformed so as to produce the variant human DHFR or amplified normal human DHFR by growth in the presence of methotrexate. Surviving colonies will be examined for the presence of human DNA and those positive in this test will be used for secondary transfection. Secondary transformants will be studied for DHFR levels and kinetic properties of DHFR. Neoplastic cells from relapsed patients will also be examined for altered methotrexate transport.