STRUCTURAL STUDIES ON DIHYDROFOLATE REDUCTASE

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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.