The characteristic feature of both of these transporters is that they can recognize and transport a wide variety of hydrophobic agents including anticancer drugs, reversing agents, and hydrophobic peptides. Our major goals include elucidation of the mechanism of substrate interaction with the multidrug transporters and how the energy from ATP hydrolysis is coupled to drug transport. Such studies will provide an insight into the role of these transporters in the development of multidrug resistance in cancers. The CD spectrum of soluble or reconstituted protein in the presence or absence of verapamil is identical indicating that the substrate binding does not result in gross changes in the structure of P-glycoprotein. The properties, such as drug-stimulated ATP hydrolysis and photoaffinity labeling with azidopine or iodoarylazidoprazosin, of the pure protein are similar to those observed in native membranes.
ADP.Mg complex decreases the affinity of P-glycoprotein for substrates.
We can generate about 5 to 7 mg of pure protein from 500 mg of crude membrane protein. We plan to use pure protein for the resolution of three-dimensional structure. We collaborate with Dr. Di. Xia, LCB, for three-dimensional structure analysis. Additional work utilizing other dopamine receptor antagonists indicates that the modulation of the interaction of substrates with Pgp appears to be complex.