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.
Kern
A
A
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina ut 29, Budapest, H-1113, Hungary.
Felföldi
F
F
Sarkadi
B
B
Váradi
A
A
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D017607:Aluminum Compounds / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D017607:Aluminum Compounds; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005033:Ethylmaleimide; D005459:Fluorides; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D011993:Recombinant Fusion Proteins; C078444:MSH3 protein, human; C421109:multidrug resistance-associated protein 1; C032311:aluminum fluoride