Screening method for substrates of multidrug resistance-associated protein Original Research Article

Multidrug resistance-associated protein (MRP) confers multidrug resistance (MDR); it transports many chemically unrelated cytotoxic reagents to the extracellular side, thereby becoming an obstacle in the chemotherapy of cancer. In this work, a simple kinetic method of analysis is presented to assay the transport selectivity of MRP for its substrates. This method is in contrast to the conventional method in which the absolute amount of transported substrates into MRP-bearing vesicles was measured. MRP is a non-selective ATP-dependent transporter, the only thing common to all processes of transport of various kinds of substrates by MRP is consumption of ATP. Thus, kinetics of ATP consumption by MRP was chosen as a measure for estimating selectivity of MRP for its substrates. NADH oxidation was monitored at 340 nm as an index for ATP consumption. The Km values obtained for LTC4, LTD4, LTE4 (LT, leukotriene), oxidized glutathione (GSSG), daunorubicin (Dau) and vincristine (Vin) were 0.15±0.04, 0.71±0.02, 1.18±0.04, 90.9±5.2, 1.47±0.35 and 2.19±0.16, respectively. This method is thus proposed for screening the substrates of MRP, which is expected to contribute to the chemotherapy of cancer.