Alqawi Omar, Poelarends Gerrit, Konings Wil N, Georges Elias
Institute of Parasitology, McGill University, Macdonald Campus, Ste-Anne de Bellevue, Que, Canada.
Biochem Biophys Res Commun. 2003 Nov 21;311(3):696-701. doi: 10.1016/j.bbrc.2003.10.049.
The Lactococcus lactis multidrug resistance ABC transporter protein LmrA has been shown to confer resistance to structurally and functionally diverse antibiotics and anti-cancer drugs. Using a previously characterized photoreactive drug analogue of Rhodamine 123 (iodo-aryl azido-Rhodamine 123 or IAARh123), direct and specific photoaffinity labeling of LmrA in enriched membrane vesicles could be achieved under non-energized conditions. This photoaffinity labeling of LmrA occurs at a physiologically relevant site as it was inhibited by molar excess of ethidium bromide>Rhodamine 6G>vinblastine>doxorubicin>MK571 (a quinoline-based drug) while colchicine had no effect. The MDR-reversing agents PSC 833 and cyclosporin A were similarly effective in inhibiting IAARh123 photolabeling of LmrA and P-glycoprotein. In-gel digestion with Staphyloccocus aureus V8 protease of IAARh123-photolabeled LmrA revealed several IAARh123 labeled polypeptides, in addition to a 6.8kDa polypeptide that comprises the last two transmembrane domains of LmrA.
乳酸乳球菌多药耐药ABC转运蛋白LmrA已被证明可赋予对结构和功能多样的抗生素及抗癌药物的抗性。使用先前表征的若丹明123的光反应性药物类似物（碘芳基叠氮若丹明123或IAARh123），在非能量化条件下可实现对富集膜囊泡中LmrA的直接和特异性光亲和标记。LmrA的这种光亲和标记发生在生理相关位点，因为它受到摩尔过量的溴化乙锭>若丹明6G>长春花碱>阿霉素>MK571（一种喹啉类药物）的抑制，而秋水仙碱没有影响。多药耐药逆转剂PSC 833和环孢菌素A在抑制IAARh123对LmrA和P-糖蛋白的光标记方面同样有效。用金黄色葡萄球菌V8蛋白酶对IAARh123光标记的LmrA进行凝胶内消化，除了包含LmrA最后两个跨膜结构域的6.8 kDa多肽外，还揭示了几种IAARh123标记的多肽。
