P-Glycoprotein Kinetics Measured in Plasma Membrane Vesicles and Living Cells
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- 作者：Pä ; ivi Ä ; ä ; nismaa ; Anna Seelig
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：March 20, 2007
- 年：2007
- 卷：46
- 期：11
- 页码：3394 - 3404
- 全文大小：190K
- 年卷期：v.46,no.11(March 20, 2007)
- ISSN：1520-4995
文摘
P-glycoprotein (MDR1, ABCB1) is an ATP-dependent efflux transporter of a large variety ofcompounds. To understand P-glycoprotein in more detail, it is important to elucidate its activity in thecellular ensemble as well as in plasma membrane vesicles (under conditions where other ATP dependentproteins are blocked). We measured P-glycoprotein activity in inside-out vesicles formed from plasmamembranes of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) for comparison withprevious measurements of P-glycoprotein activity in living NIH-MDR1-G185 cells. In plasma membranevesicles activity was measured by monitoring phosphate release upon ATP hydrolysis and in living cellsby monitoring the extracellular acidification rate upon ATP synthesis via glycolysis. P-glycoprotein wasstimulated as a function of the concentration with 19 structurally different drugs, including local anesthetics,cyclic peptides, and cytotoxic drugs. The concentrations of half-maximum P-glycoprotein activation, K1,were identical in inside-out plasma membrane vesicles and in living cells and covered a broad range ofconcentrations (K1 ~ (10-8-10-3) M). The influence of the pH, drug association, and vesicle aggregationon the concentration of half-maximum P-glycoprotein activation was investigated. The turnover numbersin plasma membrane vesicles and in living cells were also approximately identical if the latter were measuredin the presence of pyruvate. However, in the absence of pyruvate they were higher in living cells. Therate of ATP hydrolysis/ATP synthesis decreased exponentially with decreasing free energy of drug bindingfrom water to the transporter, 
(or increasing binding affinity). This suggests that drug releasefrom the transmembrane domains has to occur before ATP is hydrolyzed for resetting the transporter.
(or increasing binding affinity). This suggests that drug releasefrom the transmembrane domains has to occur before ATP is hydrolyzed for resetting the transporter.