The development of multidrug resistance (MDR) in tumor cells is probably responsible for many clinical failures of cancer chemotherapy. The molecular basis of MDR is overexpression of the MDR1 gene, which codes for a 170 kDa integral membrane glycoprotein known as the P-glycoprotein, or Pgp. The Pgp is an ATP-dependent drug pump, capable of transporting chemotherapeutic drugs out of cells against a concentration gradient and thereby minimizing their cytotoxic effects. We have recently discovered a previously undetected high capacity, drug-stimulated, membrane-bound ATPase activity associated with the human Pgp overexpressed via a recombinant baculovirus in cultured insect cells, which makes possible a variety of new experimental approaches to understanding the properties of this clinically important enzyme. Utilizing this system for manipulating the Pgp and measuring its activity, the studies proposed in this application are designed to identify effective agents for interfering with Pgp function in a two part plan. First, we shall investigate several different aspects of the Pgp-ATPase function in isolated membranes, including its drug specificity, drug transport activity, possible physiological role, and possible regulation by protein kinases and phosphatases. Second, we shall investigate a variety of aspects of the molecular structure and mechanism of the Pgp. In these studies, we shall investigate the roles of the two proposed nucleotide binding sites in the Pgp by site-directed mutagenesis, establish conditions for detergent solubilization and purification of the Pgp in active form, and develop a reliable procedure for reconstituting the Pgp into artificial phospholipid vesicles. With the reconstituted Pgp-proteoliposomes, we shall attempt to develop a liposomal drug transport assay, attempt to establish the minimum functional unit of the Pgp, and explore the transmembrane topography of the Pgp. And finally, we shall attempt to prepare large, three-dimensional crystals of the Pgp utilizing our newly developed methodology for crystallizing integral membrane proteins. It is anticipated that better drugs and drug combinations, and possibly even new therapeutic approaches, for the treatment of cancer could emerge from the information obtained.