A continuous fluorescence assay for the study of P-glycoprotein-mediated drug efflux using inside-out membrane vesicles
A continuous fluorescence assay for the study of P-glycoprotein-mediated drug efflux using inside-out membrane vesicles
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DOI：
10.1006/abio.1998.3087
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发表时间：
1999-03-15
影响因子：
2.9
通讯作者：
Roufogalis, BD
中科院分区：
文献类型：
--
作者：
Bebawy, M;Morris, MB;Roufogalis, BD
建立了一种利用膜囊泡模型测定P-糖蛋白转运活性的荧光分析方法。在本试验中，将亚甲蓝掺入由对100 ng.ml（-1）长春碱（VBL 100）耐药的人急性淋巴细胞白血病细胞及其敏感对照制备的由内而外的囊泡中。当探针穿过囊泡膜时，秋水仙碱的荧光衍生物（荧光素-秋水仙碱）的荧光被猝灭。荧光素-秋水仙碱转运依赖于P-糖蛋白的存在，需要ATP，并被钒酸盐和逆转剂维拉帕米以剂量依赖性方式抑制。此外，转运与P-gp底物长春碱和甲氨蝶呤竞争。荧光素-秋水仙碱的转运被发现是合作的P-gp（n = 1.23）。该测定快速，需要少量样品，并且消除了过去使用的放射性程序的需要。该测定应可用于表征P-gp的转运动力学，用于检查和优化化疗药物的组合，以及用于检查逆转剂和底物（其可能与荧光底物探针竞争转运）的作用。其他可能的应用包括检查纯化的P-gp在重构系统中的P-gp介导的转运特性;（C）1999 Academic Press.
A fluorimetric procedure for assaying the transport activity of P-glycoprotein (P-gp) using a membrane vesicle model has been developed. In this assay methylene blue is incorporated into inside-out vesicles prepared from human acute lymphoblastic leukemic cells resistant to 100 ng.ml(-1) vinblastine (VBL100) and their sensitive controls. The fluorescence of a fluorescent derivative of colchicine (fluorescein-colchicine) is quenched as the probe is transported across the vesicle membrane. The fluorescein-colchicine transport was found to be dependent on the presence of P-glycoprotein, required ATP, and was inhibited by vanadate and the reversal agent, verapamil, in a dose-dependent manner. Furthermore, the transport was competed against by the P-gp substrates, vinblastine and methotrexate. The transport of fluorescein-colchicine by P-gp was found to be cooperative (n = 1.23). The assay is rapid, requires small amounts of sample, and removes the need for the radioactive procedures used in the past. The assay should find use in characterizing the transport kinetics of P-gp, for examining and optimizing combinations of chemotherapeutics, and for examining the effects of reversal agents and substrates which potentially compete for transport with the fluorescent substrate probe. Other possible applications include examining P-gp-mediated transport properties of purified P-gp in reconstituted systems; (C) 1999 Academic Press.