STRUCTURAL STUDIES ON DIHYDROFOLATE REDUCTASE

预计的工作部分涉及继续正在进行的关于 二氢叶酸还原酶(DHFR)的结构及其催化机理 来自粪链球菌、干酪乳杆菌和牛肝。这个 粪链球菌同工酶1的测序工作将完成。停流 将使用同位素标记底物的分光光度和核磁共振技术 以进一步研究其催化机理。核磁共振也将被用来获得 细菌催化部位侧链运动的信息 DHFR。该项目的另一部分涉及开发方法以 寻找患者肿瘤组织中DHFR的变异型 甲氨蝶呤治疗时或治疗后复发。从患者体内提取DNA 肿瘤细胞(白血病细胞，或肺转移瘤)将用于 将合适的哺乳动物细胞(如3T3细胞)转入培养中。文化 将对转化的细胞进行筛选，以产生变种人类 Dhfr或扩增的正常人dhfr在存在 甲氨蝶呤。幸存的殖民地将被检查是否存在 人类DNA和那些在这项测试中呈阳性的人将被用于第二次 转染法。将研究二次转化体的DHFR水平和 DHFR的动力学性质。复发患者的肿瘤细胞将 也要检查甲氨蝶呤的转运是否改变。

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.