STUDIES ON NORMAL AND LEUKEMIC LEUKOCYTES. IV. TETRAHYDROFOLATE-DEPENDENT ENZYME SYSTEMS AND DIHYDROFOLIC REDUCTASE*

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Abstract
Leukocytes have been isolated from blood by a procedure consisting of removal of platelets by centrifugation, removal of the majority of erythrocytes by sedimentation in the presence of dextran, and selective lysis of the remaining erythrocytes in a hypotonic medium. Lysis of the isolated leukocytes was accomplished by high-speed homo-genization, repeated freezing and thawing, or treatment with acetone at low temperature. Levels of the following enzymes have been measured in normal and leukemic leukocytes formate-activating enzyme, N5, N10-methylene tetrahydrofolic dehydrogenase, serine hydroxymethylase, cyclohydrolase, dihydrofolic reductase, glucose-6-phosphate dehydrogenase, and lactic dehydrogenase. The level of dihydrofolic reductase is elevated about 30 times in chronic myelocycitc and acute leukemic cells as compared with normal and chronic lymphocytic leukemic cells. Formate-activating enzyme and dihydrofolic reductase have been partially purified from leukocytes. The following properties of these enzymes were determine: pH optimum, substrates and co-factor specificity, metal ion requirements, Michaelis constants, and sensitivity to inhib-tors. Dihydrofolic reauctase is inhibited by aminopterin and ametho-pterin at 10-8 M.