Szeri Flóra, Iliás Attila, Pomozi Viola, Robinow Steven, Bakos Eva, Váradi András
Institute of Enzymology, Hungarian Academy of Sciences, Budapest, P.O. Box 7, H-1518, Hungary.
Biochim Biophys Acta. 2009 Feb;1788(2):402-9. doi: 10.1016/j.bbamem.2008.11.007. Epub 2008 Nov 20.
DMRP, an ABC transporter encoded by the dMRP/CG6214 gene, is the Drosophila melanogaster orthologue of the "long" human multidrug resistance-associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP6/ABCC6, and MRP7/ABCC10). In order to provide a detailed biochemical characterisation we expressed DMRP in Sf9 insect cell membranes. We demonstrated DMRP as a functional orthologue of its human counterparts capable of transporting several human MRP substrates like beta-estradiol 17-beta-D-glucuronide, leukotriene C4, calcein, fluo3 and carboxydichlorofluorescein. Unexpectedly, we found DMRP to exhibit an extremely high turnover rate for the substrate transport as compared to its human orthologues. Furthermore, DMRP showed remarkably high basal ATPase activity (68-75 nmol Pi/mg membrane protein/min), which could be further stimulated by probenecid and the glutathione conjugate of N-ethylmaleimide. Surprisingly, this high level basal ATPase activity was inhibited by the transported substrates. We discussed this phenomenon in the light of a potential endogenous substrate (or activator) present in the Sf9 membrane.
DMRP是一种由dMRP/CG6214基因编码的ABC转运蛋白，是人类“长型”多药耐药相关蛋白（MRP1/ABCC1、MRP2/ABCC2、MRP3/ABCC3、MRP6/ABCC6和MRP7/ABCC10）在黑腹果蝇中的同源物。为了进行详细的生化特性分析，我们在Sf9昆虫细胞膜中表达了DMRP。我们证明DMRP是其人类对应物的功能同源物，能够转运几种人类MRP底物，如β-雌二醇17-β-D-葡萄糖醛酸、白三烯C4、钙黄绿素、Fluo3和羧基二氯荧光素。出乎意料的是，我们发现与人类同源物相比，DMRP在底物转运方面表现出极高的周转率。此外，DMRP显示出非常高的基础ATP酶活性（68-75 nmol Pi/mg膜蛋白/分钟），丙磺舒和N-乙基马来酰亚胺的谷胱甘肽共轭物可进一步刺激该活性。令人惊讶的是，这种高水平的基础ATP酶活性受到转运底物的抑制。我们根据Sf9膜中存在的潜在内源性底物（或激活剂）讨论了这一现象。
