We propose to continue our investigations on the mechanisms by which conversion of folate compounds to poly-gama-glutamate forms regulates one-carbon metabolism. Our investigations will proceed along three principal fronts. 1. Folylpoly-gama-glutamate synthetase will be purified from bacterial and mammalian sources and its substrate specificity and kinetic properties examined to determine whether they are responsible for the different types of pterolylpolyglutamates found in different cell lines. Pteroylpolyglutamate substrates and potential analog inhibitors of the synthetase will be synthesized. 2. The effects of products of one-carbon metabolism, such as methionine, thymidine, and adenosine, on the folate requirements, the glutamate chain lengths of endogenous folates, and on folate requiremetns, the glutamate chain lengths of endogenous folates, and on folylpoly-gama-glutamate synthetase levels will be examined in a variety of tumorigenic and nontumorigenic mammalian cells. These studies will investigate whether one-carbon metabolism is controlled by varying the glutamate chain length of endogenous pteroylpolyglutamates and whether differences exist in folate requirements and requirements for products of one-carbon metabolism between normal and malignant cells. 3. Inhibitors of folylpoly-gama-glutamate synthetase will be tested for their ability to cause cellular folate depletion by preventing ghe formation of pteroylpolyglutamates, the forms of the vitamin that are selectively retained by tissues. As pteroyl-monoglutamates are poorer substrates than pteroylpolyglutamates for folate-dependent reactions, the ability of folylpoly-gama-glutamate synthetase inhibitors to increase the exogenous requirements for products of one-carbon metabolism, and to potentiate any differences in nutritional requirements, between tumorigenic and nontumorigenic cells will be studied.
