Nervi Pierluigi, Li-Blatter Xiaochun, Aänismaa Päivi, Seelig Anna
Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, Switzerland.
Biochim Biophys Acta. 2010 Mar;1798(3):515-25. doi: 10.1016/j.bbamem.2009.11.022. Epub 2010 Jan 14.
We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux.
我们比较了外翻质膜囊泡和活的NIH-MDR1-G185细胞中的P-糖蛋白ATP酶活性，目的是检测底物转运。为此，我们使用了六种底物，它们通过质膜的被动内流差异显著。在细胞中，含有P-糖蛋白底物结合位点的胞质膜小叶必须通过脂质膜的被动扩散来接近，而在外翻质膜囊泡中，它可直接从水相获得。与P-糖蛋白的主动外排相比，被动内流快的化合物在细胞和外翻质膜囊泡中诱导出相似的ATP酶活性谱，因为它们在胞质小叶中的浓度相似。相比之下，内流与外排相似的化合物在细胞和外翻囊泡中诱导出不同的ATP酶活性谱。它们在细胞胞质小叶中的浓度明显低于外翻膜囊泡胞质小叶中的浓度，表明P-糖蛋白可以应对被动内流。因此，P-糖蛋白以与ATP水解成比例的速率转运所有化合物（即所有化合物都是底物）。然而，只有当底物跨脂质双层的被动内流与主动外排处于相似范围内时，它才会阻止底物进入胞质溶胶。
