Multi-drug resistance is a situation encountered in cancer patients in which the tumor becomes resistant to a variety of cytotoxic anti-cancer chemotheraputic agents. It often involves overexpression of P-glycoprotein (Pgp), a plasma membrane protein of 1280 amino acids, composed of two related halves, each of which contains six predicted transmembrane helices and one nucleotide binding site. There is firm evidence that Pgp acts in an ATP-dependent manner to exclude drugs and a wide range of other hydrophobic compounds from cells. The investigator's lab and others have established that Pgp displays substantial drug-stimulated ATPase activity, and the most widely considered current model is that Pgp is an ATP-driven drug efflux pump. The investigator has generated a Chinese hamster ovary cell line that over-expresses Pgp. Two defined systems for biochemical investigation of Pgp have been developed from these cells: 1) isolated plasma membanes which are considerably enriched in Pgp (up to 32 percent w/w) and 2) purified, reconstituted Pgp. A further system will be developed to allow combined mutagenic and biochemical analyses.
The aim of this proposal is to characterize the structure and function of the Pgp catalytic sites. Approaches include covalent labeling, specific insertion of tryptophans as fluorescent probes, mutational analyses, tests of the proposed catalytic cycle and direct structural analysis by scanning force microscopy. Basic knowledge of this kind will be invaluable in devising ways to disable P-glycoprotein in cells and overcome multidrig resistanmce in patients
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