MECHANISMS OF NUCLEOSIDE TRANSPORT IN MAMMALIAN CELLS

细胞对生理性核苷及其细胞毒类似物的摄取 哺乳动物细胞由核苷特异性转运蛋白介导， 接受广泛的核苷作为底物。 输送机构 在红细胞和许多培养细胞中， 抑制剂对硝基-苄基巯基嘌呤核苷的浓度 （NBMPR）. 这些细胞具有NBMPR的高亲和力结合位点， 抑制剂对这些位点的占据与 核苷转运活性。 我们在步行者256只老鼠中 癌细胞，第二种类型的核苷转运活性， 对这种抑制剂相对不敏感。 这些细胞没有高亲和力 NBMPR和转运的结合位点对蛋白质的敏感性降低了1000倍。 抑制剂. 比较了步行者256细胞和 NBMPR敏感的S49小鼠淋巴瘤细胞的结果显示很少或没有 底物特异性或转运动力学的差异 这两个机制。 然而，两种细胞系中的转运确实不同 对巯基试剂对汞苯磺酸敏感， 步行者256细胞比S49细胞敏感5- 10倍。 检查的四种其他细胞系（L1210、P388和L5178 Y小鼠白血病和L5178 Y小鼠白血病）。 RPMI 6410人淋巴母细胞）均表现出NBMPR敏感性和 - 不敏感的核苷转运活性。 在审查的所有方面 （尿苷转运的动力学常数，底物特异性，和 对巯基试剂的敏感性），L1210中的两种转运机制 细胞与S49和步行者256中表征的活性相似 细胞，分别。 尽管这四种细胞中的NBMPR不敏感活性 细胞系是总转运活性的一小部分（2 - 20%）， 它似乎是NBMPR阻断能力的重要决定因素， 核苷摄取在较长的时期（10至60分钟）。 未来一年的研究将集中在血浆的鉴定上 参与步行者256中两个转运过程的膜蛋白， S49和L1210细胞。 （一）

The uptake of physiological nucleosides and their cytotoxic analogs in mammalian cells are mediated by nucleoside-specific transporters which accept a wide range of nucleosides as substrates. The transport mechanism in erythrocytes and many cultured cells is sensitive to nanomolar concentrations of the inhibitor p-nitro-benzylmercaptopurineriboside (NBMPR). These cells have high-affinity binding sites for NBMPR and occupancy of these sites by the inhibitor is correlated with loss of nucleoside transport activity. We have identified, in Walker 256 rat carcinoma cells, a second type of nucleoside transport activity which was relatively insensitive to this inhibitor. These cells had no high-affinity binding sites for NBMPR and transport was 1000-fold less sensitive to the inhibitor. A comparison of nucleoside transport in Walker 256 cells to that of NBMPR-sensitive S49 mouse lymphoma cells revealed little or no difference in substrate specificity or the kinetics of transport between the two mechanisms. Transport in the two cell lines did, however, differ in sensitivity to the sulfhydryl reagent p-mercuribenzenesulfonate, with Walker 256 cells 5-\to 10-fold more sensitive than S49 cells. Four other cell lines examined (L1210, P388, and L5178Y mouse leukemias and RPMI 6410 human lymphoblasts) all exhibited both NBMPR-sensitive and -insensitive nucleoside transport activity. In all aspects examined (kinetic constants for uridine transport, substrate specificity, and sensitivity to sulfhydryl reagents) the two transport mechanisms in L1210 cells were similar to the activity characterized in S49 and Walker 256 cells, respectively. Although the NBMPR-insensitive activity in these four cell lines was a small fraction (2 to 20%) of the total transport activity, it appeared to be an important determinant in the ability of NBMPR to block nucleoside uptake over prolonged periods (10 to 60 min). Studies in the coming year will focus on the identification of the plasma membrane proteins involved in the two transport processes in Walker 256, S49, and L1210 cells. (A)