Evaluation of a novel photoactive and biotinylated dehydroepiandrosterone analog.
Evaluation of a novel photoactive and biotinylated dehydroepiandrosterone analog.
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新型光活性和生物素化脱氢表雄酮类似物的评估。
DOI：
10.1016/j.mce.2010.07.002
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发表时间：
2010
影响因子：
4.1
通讯作者：
Dillon,JosephS
中科院分区：
文献类型：
--
作者：
Liu,Dongmin;O'Leary,Brianne;Iruthayanathan,Mary;Love-Homan,Laurie;Perez-Hernandez,Nury;Olivo,HoracioF;Dillon,JosephS
为了表征脱氢表雄酮 (DHEA) 的细胞表面受体，我们合成了含有生物素和二苯甲酮基团的 DHEA 类似物 (DHEA-BP-Bt)。 DHEA-BP-Bt 在与 [3H]DHEA 竞争与牛主动脉内皮细胞 (BAEC) 溶解质膜的结合方面与 DHEA 等效。此外，预缀合亲和素并固定在琼脂糖上的 DHEA-BP-Bt 也抑制 [3H]DHEA 的质膜结合。此外，DHEA-BP-Bt 激活内皮一氧化氮合酶，与 DHEA 类似。共聚焦显微照片显示，光照射后，DHEA-BP-Bt 以 DHEA 可抑制的方式结合到 BAEC 细胞表面的位点上。最后，当活细胞用通过 SDS-PAGE 分离的类似物和质膜蛋白进行紫外线照射时或在配体印迹分析中，DHEA-BP-Bt 特异性结合约 55kDa 和 80kDa 的蛋白质。这些数据证实了光活性、生物素化 DHEA 类似物的成功合成，该类似物能够交联并识别质膜 DHEA 结合位点，这将使我们能够进一步纯化该受体。
To characterize the cell surface receptor for dehydroepiandrosterone (DHEA), we synthesized a DHEA analog containing biotin and benzophenone groups (DHEA–BP–Bt). DHEA–BP–Bt was equipotent with DHEA in competing with [3H]DHEA for binding to solubilized plasma membranes of bovine aortic endothelial cells (BAEC). Additionally, DHEA–BP–Bt pre-conjugated to avidin and immobilized on agarose, also inhibited plasma membrane binding of [3H]DHEA. Furthermore, DHEA–BP–Bt activated endothelial nitric oxide synthase, similar to DHEA. Confocal micrographs showed that, upon photoirradiation, DHEA–BP–Bt bound to sites on the cell surface of BAEC in a DHEA inhibitable manner. Finally, DHEA–BP–Bt bound specifically to proteins of approximately 55kDa and 80kDa, either when live cells were UV irradiated with the analog and plasma membrane proteins separated by SDS-PAGE or in a ligand blot analysis. These data confirm the successful synthesis of a photoactive, biotinylated DHEA analog which is capable of cross-linking to and identifying plasma membrane DHEA binding sites and which will allow us to further purify this receptor.