Expression and Characterization of the N- and C-Terminal ATP-Binding Domains of MRP1

The His 6 -tagged N- and C-terminal nucleotide binding (ATP Binding Cassette, ABC) domains of the human multidrug resistance associated protein, MRP1, were expressed in bacteria in fusion to the bacterial maltose binding protein and a two-step affinity purification was utilized. Binding of a fluorescent ATP-analogue occurred with micromolar dissociation constants, MgATP was able to inhibit the ATP-analogue binding with 70 and 200 micromolar apparent inhibition constants, while AMP was nearly ineffective. Both MRP1 nucleotide binding domains showed ATPase activities (V max values between 5–10 nmoles/mg protein/min), which is fifty to hundred times lower than that of parent transporter. The K M value of the ATP hydrolysis by the nucleotide binding domains were 1.5 mM and 1.8 mM, which is similar to the K M value of the native or the purified and reconstituted transporter, N -ethylmaleinimide and A1F 4 inhibited the ATPase activity of both nucleotide binding domains.