Use of formycin B as a general substrate for measuring facilitated nucleoside transport in mammalian cells.
Use of formycin B as a general substrate for measuring facilitated nucleoside transport in mammalian cells.
复制标题
使用福霉素 B 作为测量哺乳动物细胞中促进核苷转运的通用底物。
DOI：
10.1016/0167-4889(89)90177-8
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Woffendin,C
中科院分区：
文献类型：
--
作者：
Plagemann,PG;Woffendin,C
甲霉素B是一种肌苷的C-核苷类似物，不被人红细胞和小鼠P388白血病细胞分解，仅在这些细胞中非常低效地被磷酸化。这种相对惰性使得可以测量其进入和离开细胞的运输，而不会因代谢转化而复杂化。我们用快速动力学技术测量了甲霉素B在这些细胞中的零传递和平衡交换通量。甲霉素B在两种细胞中的米氏常数和最大转运速度与先前报道的尿苷和胸苷相似。然而，与尿苷为底物相比，甲霉素B对载底物和空载体的人红细胞的差别迁移率较小。其他核苷根据其与载体的亲和力而抑制Formycin B的内流，但不受嘌呤的影响。硝基苄基硫代肌苷和潘生丁对甲霉素B内流的抑制作用与以尿苷为底物的抑制作用相同(IC50分别为10和30 nM)。福尔霉素B在两种类型的细胞中积累，浓度比培养液中的浓度高30-40%。这种集中积累不是由于活跃的运输、代谢或分配到膜脂中所致。它似乎反映了甲霉素B与细胞内成分的结合，但并不显著干扰其转运的测量。
Formycin B, a C-nucleoside analog of inosine, is not catabolized by human erythrocytes and mouse P388 leukemia cells and is only very inefficiently phosphorylated in these cells. This relative inertness allows the measurement of its transport into and out of the cells uncomplicated by metabolic conversions. We have measured the zero-transand equilibrium exchange flux of formycin B in these cells by rapid kinetic techniques. The Michaelis-Menten constants and maximum velocities for formycin B transport in both types of cell were similar to those previously reported for uridine and thymidine. Nevertheless, the differential mobility of the substrate-loaded and empty carrier of human erythrocytes was less for formycin B than uridine as substrate. Formycin B influx was inhibited by other nucleosides in accordance with their affinities for the carrier, but unaffected by purines. The inhibition of formycin B influx by nitrobenzylthioinosine and dipyridamole was also identical to that observed with uridine as substrate (IC50= 10 and 30 nM, respectively). Formycin B accumulated in both types of cell to 30–40% higher concentrations than were present in the medium. This concentrative accumulation was not due to active transport, metabolism or partitioning into membrane lipids. It seems to reflect binding of formycin B to intracellular components, but does not interfere significantly with measurements of its transport.