A continuous fluorescence assay for the study of P-glycoprotein-mediated drug efflux using inside-out membrane vesicles

Academic Press Inc
Publication Type:
Journal Article
Analytical Biochemistry, 1999, 268 (2), pp. 270 - 277
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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 z ml21 vinblastine (VBL100) and their sensitive controls. The fluorescence of a fluorescent derivative of colchicine (fluoresceincolchicine) is quenched as the probe is transported across the vesicle membrane. The fluoresceincolchicine 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 fluoresceincolchicine by P-gp was found to be cooperative (n 5 1.23).
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