Kucera R, Paulus H
Exp Cell Res. 1986 Dec;167(2):417-28. doi: 10.1016/0014-4827(86)90182-5.
Two different approaches were used to define the intracellular localization in mouse L929 cells of two deoxyribonucleotide biosynthetic enzymes: ribonucleoside diphosphate reductase (EC1.17.4.1) and thymidylate synthase (EC2.1.1.45). The first involved treatment with saponins, which render the plasma membrane permeable to proteins without disrupting intracellular organelles. Under conditions where nuclear DNA synthesis and the activity of the nuclear enzyme NMN adenylyltransferase were unaffected, the entire cellular complements of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, and of ribonucleotide reductase and thymidylate synthase were released at the same rate and with similar dependence on saponin concentration. The second approach involved centrifugal enucleation of cells treated with cytochalasin B (CB) and measurement of the distribution of enzyme activities in the resulting cytoplast and karyoplast fractions. Whereas most NMN adenylyltransferase activity remained with the karyoplasts, glucose-6-phosphate dehydrogenase, ribonucleotide reductase, and thymidylate synthase were almost exclusively associated with the enucleated cytoplasts. These results indicate that, under conditions where nuclear DNA synthesis is apparently unperturbed, the intracellular distribution of the deoxyribonucleotide biosynthetic enzymes studied is the same as that of glucose-6-phosphate dehydrogenase, a typical cytosol enzyme, and clearly differs from that of NMN adenylyltransferase, a nuclear enzyme.
采用两种不同的方法来确定小鼠L929细胞中两种脱氧核糖核苷酸生物合成酶的细胞内定位：核糖核苷二磷酸还原酶（EC1.17.4.1）和胸苷酸合成酶（EC2.1.1.45）。第一种方法是用皂苷处理，皂苷可使质膜对蛋白质具有通透性，而不会破坏细胞内细胞器。在核DNA合成和核酶NMN腺苷酸转移酶的活性不受影响的条件下，胞质酶葡萄糖-6-磷酸脱氢酶、核糖核苷酸还原酶和胸苷酸合成酶的整个细胞成分以相同的速率释放，并且对皂苷浓度的依赖性相似。第二种方法是对用细胞松弛素B（CB）处理的细胞进行离心去核，并测量所得胞质体和核质体部分中酶活性的分布。虽然大部分NMN腺苷酸转移酶活性仍留在核质体中，但葡萄糖-6-磷酸脱氢酶、核糖核苷酸还原酶和胸苷酸合成酶几乎完全与去核后的胞质体相关。这些结果表明，在核DNA合成明显未受干扰的条件下，所研究的脱氧核糖核苷酸生物合成酶的细胞内分布与典型的胞质酶葡萄糖-6-磷酸脱氢酶相同，并且明显不同于核酶NMN腺苷酸转移酶的分布。
