Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells.
Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells.
复制标题
来自培养的 Novikoff 肝癌细胞的硝基苄基硫代肌苷结合多肽的光亲和标记。
DOI：
10.1042/bj2360665
复制
发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Paterson,AR
中科院分区：
文献类型：
--
作者：
Gati,WP;Belt,JA;Jakobs,ES;Young,JD;Jarvis,SM;Paterson,AR
硝基苄基硫代肌苷（NBMPR）与某些动物细胞质膜的位点特异性结合导致核苷易化扩散的抑制。本研究表明，Novikoff 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。平衡结合实验表明，在这些细胞的膜富集部分中存在NBMPR的高亲和力位点。在尿苷或双嘧达莫的存在下，在这些网站的NBMPR的特异性结合被抑制。当Novikoff UA膜通过使用光亲和技术用[3 H]NBMPR共价标记时，特异性结合的放射性仅掺入多肽中，表观Mr为72，000 - 80，000，通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳测定。这种多肽的共价标记被废除在过量的硝基苄基硫代鸟苷（NBTGR）的存在下，并在腺苷，尿苷或双嘧达莫的存在下减少。Novikoff UA细胞中NBMPR结合多肽的表观Mr显著高于其他细胞类型中相应多肽的报告值（Mr 45，000 - 66，000）。当来自S49小鼠淋巴瘤细胞的膜富集制剂被光标记并与标记的NovikoffUA膜富集制剂混合时，凝胶电泳从两种制剂中分离出NBMPR结合多肽。
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.