Tabas Linda B, Dantzig Anne H
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285-0424, USA.
Anal Biochem. 2002 Nov 1;310(1):61-6. doi: 10.1016/s0003-2697(02)00282-8.
This study investigated a high-throughput assay to measure multidrug resistance-associated protein (MRP1)-mediated uptake into membrane vesicles. Typically, a rapid filtration technique using a 12-filter vacuum manifold is used. We report here the development of a 96-well microtiter dish assay. MRP1-transfected HeLa cells (HeLa-T5) were used for the membrane vesicle preparations. The uptake of 50nM [3H]leukotriene C(4) (LTC(4)) was measured in a 96-well microtiter dish with rapid filtration onto a Perkin Elmer unifilter GF/B plate using a Perkin Elmer Filtermate 196. Counting of the isotype was conducted with a Perkin Elmer Top Count NXT. Uptake was adenosine 5'-triphosphate-dependent and linear over a 120-s time course. Uptake was inhibited by the leukotriene D(4) antagonist, MK 571, with a k(i) of 0.67 microM, and by the anti-MRP1 monoclonal antibody QCRL-3 but not by QCRL-1. Inhibition by estradiol-17-beta-glucuronide was 35-fold greater than inhibition by estradiol-3-beta-glucuronide. The kinetic parameters for LTC(4) uptake were determined to be a K(m) of 157nM with a V(max) of 344pmol/min/mg protein. The properties of MRP1-mediated transport of LTC(4) are consistent with those previously reported. The microtiter dish assay is a more expedient method for measuring transport into membrane vesicles and will have applications to other transporters.
本研究调查了一种高通量检测方法，用于测量多药耐药相关蛋白（MRP1）介导的膜囊泡摄取。通常，使用12孔滤膜真空歧管的快速过滤技术。我们在此报告一种96孔微量滴定板检测方法的开发。转染了MRP1的HeLa细胞（HeLa-T5）用于制备膜囊泡。在96孔微量滴定板中测量50nM [3H]白三烯C4（LTC4）的摄取，使用Perkin Elmer Filtermate 196快速过滤到Perkin Elmer unifilter GF/B板上。使用Perkin Elmer Top Count NXT进行同位素计数。摄取是依赖于腺苷5'-三磷酸的，并且在120秒的时间进程中呈线性。摄取受到白三烯D4拮抗剂MK 571的抑制，其抑制常数（Ki）为0.67 microM，也受到抗MRP1单克隆抗体QCRL-3的抑制，但不受QCRL-1的抑制。雌二醇-17-β-葡萄糖醛酸苷的抑制作用比雌二醇-3-β-葡萄糖醛酸苷的抑制作用大35倍。LTC4摄取的动力学参数确定为米氏常数（Km）为157nM，最大反应速度（Vmax）为344pmol/分钟/毫克蛋白。MRP1介导的LTC4转运特性与先前报道的一致。微量滴定板检测方法是一种更便捷的测量膜囊泡摄取的方法，并且将应用于其他转运体。
