Abstract

P-glycoprotein / MDR1 was the first member of the ATP-binding cassette (ABC) transporter superfamily to be identified in a eukaryote. In eukaryotes, ABC proteins can be classified into three major groups based on function: transporters, regulators, and channels. MDR1 / P-glycoprotein is a prominent member of eukaryotic export-type ABC proteins. MDR1 / P-glycoprotein extrudes a very wide array of structurally dissimilar compounds, all lipophilic and ranging in mass from approximately 300 to 2000 Da, including cytotoxic drugs that act on different intracellular targets, steroid hormones, peptide antibiotics, immunosuppressive agents, calcium channel blockers, and others. Nucleotide binding and hydrolysis by MDR1 / P-glycorptrotein is tightly coupled with its function, substrate transport. ATP binding and hydrolysis were extensively analyzed with the purified MDR1 / P-glycoprotein. The vanadate-induced nucleotide trapping method was also applied to study the hydrolysis of ATP by MDR1 / P-glycoprotein. When MDR1 hydrolyzes ATP in the presence of excess orthovanadate, an analog of inorganic phosphate, it forms a metastable complex after hydrolysis. Using this method, MDR1 / P-glycoprotein can be specifically photoaffinity-labeled in the membrane, if 8-azido-[α32P]ATP is used as ATP. Visualization of the structure, as well as the biochemical data, is needed to fully understand how MDR1 / Pglycoprotein recognizes such a variety of compounds and how it carries its substrates across the membrane using the energy from ATP hydrolysis. To do so, large amounts of pure and stable proteins are required. Heterologous expression systems, which have been used to express P-glycoprotein, are also described.

Keywords: P-glycoprotein, Heterologous, ATP hydrolysis