Habteyesus A, Nordenskjöld A, Bohman C, Eriksson S
Department of Biochemistry I, Karolinska Institutet, Stockholm, Sweden.
Biochem Pharmacol. 1991 Oct 9;42(9):1829-36. doi: 10.1016/0006-2952(91)90522-7.
Three key enzymes in the anabolic phosphorylation of deoxyribonucleosides and deoxyribonucleoside analogs were purified i.e. cytoplasmic thymidine kinase (TK1), mitochondrial thymidine kinase (TK2) and cytoplasmic deoxycytidine kinase (dCK) from human, mouse and monkey liver and spleen. Their subunit structure and substrate specificities were compared. Extensive purification of TK1 and dCK from mouse spleen and TK2 from mouse and monkey livers revealed major polypeptide bands of 25, 30 and 28 kD, respectively, on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which are very similar to the subunit molecular weights of the corresponding human enzymes. Affinity purified polyclonal antibodies against human dCK also cross-reacted with 30 kD bands in extracts from both mouse and monkey spleen. Thus, the molecular weights of the subunits of these three enzymes appeared to be very similar in all three species. TK1 and TK2 from these different sources appeared to have similar substrate specificities against several deoxyribonucleoside analogs. However, mouse dCK differed significantly from monkey and human dCK in its capacity to phosphorylate dAdo and 2',3'-dideoxycytidine (ddCyd) with a Vmax approximately 10-fold lower than that of the two latter enzymes. The Km and Vmax values for dCyd and arabinocytosine appeared to be very similar with the enzymes from all three species. The fact that mouse dCK shows low activity with dAdo and ddCyd explains differences reported previously in the metabolism of dAdo and ddCyd in mouse compared to that in human lymphocytes. These results argue against the use of mice as model systems for human deoxynucleoside metabolism.
从人、小鼠和猴的肝脏及脾脏中纯化出了脱氧核糖核苷和脱氧核糖核苷类似物合成代谢磷酸化过程中的三种关键酶，即细胞质胸苷激酶（TK1）、线粒体胸苷激酶（TK2）和细胞质脱氧胞苷激酶（dCK）。比较了它们的亚基结构和底物特异性。从小鼠脾脏中对TK1和dCK以及从小鼠和猴肝脏中对TK2进行了广泛纯化，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上分别显示出主要的25、30和28 kD多肽条带，这与相应人类酶的亚基分子量非常相似。针对人dCK的亲和纯化多克隆抗体也与小鼠和猴脾脏提取物中的30 kD条带发生交叉反应。因此，这三种酶的亚基分子量在所有三个物种中似乎非常相似。来自这些不同来源的TK1和TK2对几种脱氧核糖核苷类似物似乎具有相似的底物特异性。然而，小鼠dCK在磷酸化脱氧腺苷（dAdo）和2'，3'-二脱氧胞苷（ddCyd）的能力上与猴和人dCK有显著差异，其最大反应速度（Vmax）比后两种酶低约10倍。对于脱氧胞苷（dCyd）和阿糖胞苷，所有三个物种的酶的米氏常数（Km）和最大反应速度（Vmax）值似乎非常相似。小鼠dCK对dAdo和ddCyd活性较低这一事实解释了先前报道的小鼠与人类淋巴细胞中dAdo和ddCyd代谢的差异。这些结果反对将小鼠用作人类脱氧核苷代谢的模型系统。
