2002年11月
A novel electron paramagnetic resonance approach to determine the mechanism of drug transport by P-glycoprotein
JOURNAL OF BIOLOGICAL CHEMISTRY
- ,
- 巻
- 277
- 号
- 47
- 開始ページ
- 45688
- 終了ページ
- 45694
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1074/jbc.M206479200
- 出版者・発行元
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
ATP-driven pumping of a variety of drugs out of cells by the human P-glycoprotein poses a serious problem to medical therapy. High level heterologous expression of human P-glycoprotein, in the yeast Saccharomyces cerevisiae, has facilitated biophysical studies in purified proteoliposome preparations. Membrane permeability of transported drugs and consequent lack of an experimentally defined drug position have made resolution of the transport mechanism difficult by classical techniques. To overcome these obstacles we devised a novel EPR spin-labeled verapamil for use as a transport substrate. Spin-labeled verapamil was an excellent transport substrate with apparent turnover number, K-m and K-i values of 5.8 s(-1), 4 mum, and 210 muM, respectively, at pH 7.4 and 37 degreesC. The apparent affinities were similar to10-fold higher than for unlabeled verapamil. Spin-labeled verapamil stimulated ATPase activity similar to5-fold, was relatively hydrophilic, and had a very low flip-flop rate, making it an ideal transport substrate. The K-m for MgATP activation of transport was 0.8 mm. By measuring the mobility of spin-labeled verapamil during transport experiments, we were able to resolve the location of the drug in proteoliposome suspensions. Steady state gradients of spin-labeled verapamil within the range of K-i/K-m ratios were observed.
- リンク情報
- ID情報
-
- DOI : 10.1074/jbc.M206479200
- ISSN : 0021-9258
- PubMed ID : 12244102
- Web of Science ID : WOS:000179404800136