STUDIES ON NORMAL AND LEUKEMIC LEUKOCYTES. VI. THYMIDYLATE SYNTHETASE AND DEOXYCYTIDYLATE DEAMINASE*

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Abstract
Thymidylate synthetase, which catalyzes the reaction, deoxyuridylate + N5, N10-methylene tetrahydrofolate [forward arrow] thymidylate + dihydrof olate, has been partially purified from leukocytes. The enzymic reaction is assayed by means of the tetrahydrofolate-and deoxyuridylate-dependent incorporation of HC14HO into thymidylate. There is also a partial requirement for Mg++ in the reaction. Deoxycytidylate can function as a substrate, but only after conversion to deoxyuridylate via a deaminase present in leukocytes. The synthetase has a pH optimum at 6.5 and is inhibited by 5-fluorodeoxyuridylate and by the reaction product, thymidylate. The continual synthesis of thymidylate involves a cyclic process in which the primary synthetase reaction is coupled with dihydrofolic reductase. The latter enzyme regenerates tetrahydrofolate, which is converted ultimately to the N5, N10-methylene derivative. The level of thymidylate synthetase has been determined in the leukocytes of 12 normal subjects and 27 patients with leukemia. In chronic myelocytic leukemia, the mean level was 0.19 m[mu]mole per hour per mg protein, and in four out of seven acute leukemia patients, the level was 0.07 m[mu]moles per hour per mg protein. No activity could be detected in the cells from eight patients with chronc lymphocytic leukemia or from 12 normal subjects.