A B S T R A C T Cytidine deaminiase, an enzyme that catalyses the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara-C) and 5-azacytidine (5-azaC), lhas been partially purified from normal and leukemic humiian granulocytes. The purification procedure included lheat precipitation at 700 C, ammoniunm sulfate precipitation, calcium phosphate gel ionl exchange, and Sephadex G-150 gel filtration. The enzymlle has mol xvt 51,000, isoelectric pH of 4.8, and maximlum activity over a broad pH range of 5-9.5. The enzyme is stabilized by the presence of the sulfhydryl reagent, dithiothreitol.Cytidine deaminase from normal human graniulocytes lhas a greater affinity for its physiologic substrate cytidine (Km = 1.1 X 10-5 AM) than for ara-C (8.8 X 10-°MI) or 5-azaC (4.3 X 10' M). Halogenated analogues such as 5-fluorocytidine and 5-bromo-2'-deoxycytidine also exhiibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine anid deoxycytidine. No activity was observed witlh nucleotides or (leoxyniucleotides. The relative maximumlii velocity of the enzyme for cytidine and its nucleoside analogues remaiie(l constanit during purification, inidicating that a single enzynme was responsible for deaminiatioln of these substrates.Tetrahydrouridine (THU) was founid to be a strong competitive inhibitor of partially purified deamiiinase with a KT of 5.4 X 10' M.The X 10'/nmg protein) than chronic myelocytic leukemia (CML) cells (1.40±0.70 X 10' U/mg proteini) or acute myelocytic leukemia (AML) cells (0.19+0.17 X 103 U/ mg protein). To explain these differences in enzyme levels in leukemic versus normal cells, the changes in cytidine deaminase levels associated with maturation of nornmal granulocvtes were studied in niormal human bone marrow. Myeloid precursors obtained from bone marrow aspirates were separated into mature and immature fractions by Ficoll density centrifugation. Deaminase activity in lysates of mature granulocytes was 3.55-14.2 times greater than the activity found in the lysates of immature cells. Decreased enzyme activity was also found in immature myeloid cells from a patient with CML as compared to mature granulocytes from the same patient. These observations support the conclusion that the greater specific activity of cytidine deaminase in normal mature granulocytes as compared to leukemic cells is related to the process of granulocyte maturation ratlher than a specific enzymatic defect in leukemic cells.