Gati W P, Belt J A, Jakobs E S, Young J D, Jarvis S M, Paterson A R
Biochem J. 1986 Jun 15;236(3):665-70. doi: 10.1042/bj2360665.
Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.
硝基苄硫基肌苷（NBMPR）与某些动物细胞质膜的位点特异性结合会导致核苷易化扩散受到抑制。本研究表明，诺维科夫UA大鼠肝癌细胞中的核苷转运对位点饱和浓度的NBMPR不敏感。平衡结合实验证明，在这些细胞富含膜的部分中存在NBMPR的高亲和力位点。在尿苷或双嘧达莫存在的情况下，NBMPR在这些位点的特异性结合受到抑制。当使用光亲和技术用[3H]NBMPR对诺维科夫UA细胞膜进行共价标记时，通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳测定，特异性结合的放射性仅掺入表观分子量为72,000 - 80,000的一种或多种多肽中。在过量的硝基苄硫基鸟苷（NBTGR）存在下，该多肽的共价标记被消除，而在腺苷、尿苷或双嘧达莫存在下则减少。诺维科夫UA细胞中NBMPR结合多肽的表观分子量明显高于其他细胞类型中相应多肽的报道值（分子量为45,000 - 66,000）。当用S49小鼠淋巴瘤细胞富含膜的制剂进行光标记并与标记的诺维科夫UA富含膜的制剂混合时，凝胶电泳可分离这两种制剂中的NBMPR结合多肽。
