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

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dc.contributor.author Bebawy, M
dc.contributor.author Morris, MB
dc.contributor.author Roufogalis, BD
dc.date.accessioned 2012-02-02T05:35:35Z
dc.date.issued 1999-03-15
dc.identifier.citation Analytical Biochemistry, 1999, 268 (2), pp. 270 - 277
dc.identifier.issn 0003-2697
dc.identifier.other C1UNSUBMIT en_US
dc.identifier.uri http://hdl.handle.net/10453/14662
dc.description.abstract 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.
dc.language eng
dc.relation.isbasedon 10.1006/abio.1998.3087
dc.title A continuous fluorescence assay for the study of p-glycoprotein-mediated drug efflux using inside-out membrane vesicles
dc.type Journal Article
dc.description.version Published
dc.parent Analytical Biochemistry
dc.journal.volume 2
dc.journal.volume 268
dc.journal.number 2 en_US
dc.publocation San Diego en_US
dc.identifier.startpage 270 en_US
dc.identifier.endpage 277 en_US
dc.cauo.name GSH.Pharmacy en_US
dc.conference Verified OK en_US
dc.for 0301 Analytical Chemistry
dc.personcode 112474
dc.percentage 100 en_US
dc.classification.name Analytical Chemistry en_US
dc.classification.type FOR-08 en_US
dc.edition en_US
dc.custom en_US
dc.date.activity en_US
dc.location.activity WOS:000079508100014 en_US
dc.description.keywords Fluorescein-colchicine
dc.description.keywords Inside-out membrane vesicle
dc.description.keywords Methylene blue
dc.description.keywords Multidrug resistance
dc.description.keywords P-glycoprotein substrates
pubs.embargo.period Not known
pubs.organisational-group /University of Technology Sydney
pubs.organisational-group /University of Technology Sydney/Graduate School of Health
pubs.organisational-group /University of Technology Sydney/Strength - Health Technologies
utslib.copyright.status Closed Access
utslib.copyright.date 2015-04-15 12:17:09.805752+10
pubs.consider-herdc false
utslib.collection.history Closed (ID: 3)

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