Harley E R, Paterson A R, Cass C E
Cancer Res. 1982 Apr;42(4):1289-95.
A procedure is described for determining early time courses of nucleoside uptake by cultured cells in suspension. Replicate samples of cell suspensions were exposed to medium containing 3H-nucleosides for brief intervals (sec) ended by addition of nitrobenzylthioinosine, a potent inhibitor of nucleoside transport that terminated nucleoside uptake virtually instantaneously. Time courses of nucleoside uptake were constructed from the cellular content of nucleoside acquired by the replicate samples during graded intervals of exposure to the labeled permeant. Such time courses were definitive of cellular uptake of nucleosides during the first few sec of exposure to permeant and yielded initial rates of uptake of adenosine and 4-amino-7-(beta-d-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin). Defining initial rates of nucleoside uptake as rates of inward transport, relationships between transport rates and extracellular concentrations of these permeants were evaluated in HeLa cells and in two cultured lines of mouse lymphoma L5178Y cells that differ in their abilities to phosphorylate adenosine and tubercidin. Transport rates for these permeants were similar in the two L5178Y cell types and were saturable in the 3 cell lines with Km values between 14 and 38 microM. Adenosine and tubercidin were mutually competitive permeants in L5178Y cells, indicating that they are substrates for the same transport mechanism.
本文描述了一种用于测定悬浮培养细胞摄取核苷的早期时间进程的方法。将细胞悬液的重复样本短暂暴露于含有³H-核苷的培养基中（数秒），然后加入硝基苄硫肌苷终止反应，硝基苄硫肌苷是一种核苷转运的有效抑制剂，能几乎瞬间终止核苷摄取。核苷摄取的时间进程是根据重复样本在分级暴露于标记渗透剂的间隔期间所摄取的核苷细胞含量构建的。这样的时间进程确定了在暴露于渗透剂的最初几秒内细胞对核苷的摄取情况，并得出了腺苷和4-氨基-7-（β-D-呋喃核糖基）吡咯并[2,3-d]嘧啶（杀结核菌素）的初始摄取速率。将核苷摄取的初始速率定义为内向运输速率，在HeLa细胞以及两种小鼠淋巴瘤L5178Y细胞培养系中评估了这些渗透剂的运输速率与细胞外浓度之间的关系，这两种细胞系在磷酸化腺苷和杀结核菌素的能力上有所不同。这两种L5178Y细胞类型中这些渗透剂的运输速率相似，并且在这三种细胞系中均可饱和，Km值在14至38微摩尔之间。在L5178Y细胞中，腺苷和杀结核菌素是相互竞争的渗透剂，表明它们是同一运输机制的底物。
