PURIFICATION AND PROPERTIES OF CYTIDINE DEAMINASE FROM NORMAL AND LEUKEMIC GRANULOCYTES
PURIFICATION AND PROPERTIES OF CYTIDINE DEAMINASE FROM NORMAL AND LEUKEMIC GRANULOCYTES
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DOI：
10.1172/jci107633
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发表时间：
1974-01-01
影响因子：
15.9
通讯作者：
EVANS, WH
中科院分区：
文献类型：
--
作者：
CHABNER, BA;JOHNS, DG;EVANS, WH
胞苷脱氨酶是一种催化胞苷及其核苷类似物脱氨的酶，包括抗肿瘤药物胞嘧啶阿拉伯糖（ara-C）和5-氮杂胞苷（5-azaC），已从正常和白血病人粒细胞中部分纯化。纯化过程包括70℃热沉淀、硫酸铵沉淀、磷酸钙凝胶离子交换、Sephadex G-150凝胶过滤。该酶的摩尔质量为51,000，等电pH为4.8，在5-9.5的广泛pH范围内具有最大活性。该酶通过巯基试剂二硫代苏糖醇的存在而稳定。正常人粒细胞胞苷脱氨酶对其生理底物胞苷（Km= 1.1 × 10−5M）的亲和力高于对ara-C （8.8 × 10−5M）或5-azaC （4.3 × 10−4M）的亲和力。卤代类似物，如5-氟胞苷和5-溴-2 ' -脱氧胞苷也表现出底物活性，其最大速度大于生理底物胞苷和脱氧胞苷。在核苷酸或脱氧核苷酸中未观察到活性。在纯化过程中，胞苷及其核苷类似物的酶的相对最大速度保持不变，表明一种酶负责这些底物的脱氨。四氢吡啶（THU）是一种较强的部分纯化脱氨酶竞争性抑制剂，其ki值为5.4 × 10−8M。从正常细胞和白血病细胞中部分纯化的胞苷脱氨酶制剂的生化特性在等电pH值、分子量、底物和抑制剂动力学参数方面进行了比较，没有发现差异。正常循环粒细胞胞苷脱氨酶浓度（3.52±1.86 × 103/mg蛋白）明显高于慢性髓细胞白血病（CML）细胞（1.40±0.70 × 103U/mg蛋白）或急性髓细胞白血病（AML）细胞（0.19±0.17 × 103U/mg蛋白）。为了解释白血病细胞与正常细胞中酶水平的差异，在正常人骨髓中研究了与正常粒细胞成熟相关的胞苷脱氨酶水平的变化。从骨髓抽吸液中获得的髓系前体通过Ficoll密度离心分离成成熟和未成熟的部分。成熟粒细胞裂解物的脱氨酶活性是未成熟粒细胞裂解物活性的3.55 ~ 14.2倍。与来自同一患者的成熟粒细胞相比，来自CML患者的未成熟髓细胞也发现酶活性降低。这些观察结果支持这样的结论，即与白血病细胞相比，正常成熟粒细胞中胞苷脱氨酶的特异性活性更高，这与粒细胞成熟过程有关，而不是白血病细胞中的特异性酶缺陷。
Cytidine deaminase, 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), has been partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70°C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G-150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum 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 granulocytes has a greater affinity for its physiologic substrate cytidine (Km= 1.1 × 10−5M) than for ara-C (8.8 × 10−5M) or 5-azaC (4.3 × 10−4M). Halogenated analogues such as 5-fluorocytidine and 5-bromo-2′-deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates.Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with aKiof 5.4 × 10−8M.The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, molecular weight, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52±1.86 × 103/mg protein) than chronic myelocytic leukemia (CML) cells (1.40±0.70 × 103U/mg protein) or acute myelocytic leukemia (AML) cells (0.19±0.17 × 103U/mg protein). To explain these differences in enzyme levels in leukemic versus normal cells, the changes in cytidine deaminase levels associated with maturation of normal granulocytes were studied in normal 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 rather than a specific enzymatic defect in leukemic cells.