Anti‐cancer drugs and glutathione stimulate vanadate‐induced trapping of nucleotide in multidrug resistance‐associated protein (MRP)

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Abstract
Multidrug resistance‐associated protein (MRP), a member of the ABC superfamily transporters, functions as an ATP‐dependent efflux pump that extrudes cytotoxic drugs from the cells. Although glutathione has been considered to play an important role in the function of MRP, there is no convincing evidence that glutathione directly interacts with MRP. Here we demonstrate that vanadate‐induced trapping of 8‐azido‐ATP in MRP was stimulated in the presence of glutathione, oxidized glutathione and the anti‐cancer drugs VP‐16 and vincristine. MRP in membrane from a human MRP cDNA transformant was specifically photolabeled with 8‐azido‐[α‐32P]ATP by the vanadate‐trapping technique. Vanadate and Mg2+ were required for trapping of nucleotides, and vanadate trapping of nucleotides was inhibited by excess ADP as well as ATP. These results suggest that a stable inhibitory complex MRP·MgADP·Vi, an analog of the MRP·MgADP·Pi transition state complex, is formed in the presence of vanadate. Glutathione as well as anti‐cancer drugs would directly interact with MRP, and stimulate the formation of the transition state of the ATPase reaction of MRP.