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 AlF4 inhibited the ATPase activity of both nucleotide binding domains, (C) 2000 Academic Press.