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ABCB1 (P-glycoprotein/P-gp) is an ATP-binding cassette transporter well known for its association with multidrug resistance in cancer cells. Powered by the hydrolysis of ATP, it effluxes structurally diverse compounds. In this chapter, we discuss current views on the molecular basis of the substrate polyspecificity of P-gp. One of the features that accounts for this property is the structural flexibility observed in P-gp. Several X-ray crystal structures of mouse P-gp have been published recently in the absence of nucleotide, with and without bound inhibitors. All the structures are in an inward-facing conformation exhibiting different degrees of domain separation, thus revealing a highly flexible protein. Biochemical and biophysical studies also demonstrate this flexibility in mouse as well as human P-gp. Site-directed mutagenesis has revealed the existence of multiple transport-active binding sites in P-gp for a single substrate. Thus, drugs can bind at either primary or secondary sites. Biochemical, molecular modeling, and structure-activity relationship studies suggest a large, common drug-binding pocket with overlapping sites for different substrates. We propose that in addition to the structural flexibility, the molecular or chemical flexibility also contributes to the binding of substrates to multiple sites forming the basis of polyspecificity.

Human Pgp from the vinblastine-resistant cell line, KB-V1, can be purified by sequential conventional chromatography on DEAE-sepharose CL-6B resin followed by a wheat germ agglutinin column. By including glycerol (osmolyte protectant) and lipid during the solubilization and chromatography procedures most of the biological activity of Pgp can be retained. The activity of Pgp in the detergent extract or in the concentrated column fractions is stable for at least 8-10 months when stored at -80 degrees. However, repeated cycles of freezing and thawing of fractions result in considerable loss of activity. We have purified Pgp from KB-C1 (a subclone of KB 3-1 that is resistant to 1 microgram/ml colchicine) by following the same protocol. When this method was used for purification of Pgp from MDR1-transfected NIH 3T3 transfectants (N3-V2400, grown in the presence of 2.4 micrograms/ml vinblastine), the protein was eluted with 0.1 M NaCl from the DEAE-Sepharose CL-6B column as usual. However, during WGA lectin chromatography, the protein was eluted with a lower concentration of sugar (0.1 M instead of 0.25 M NAG). This altered elution pattern appears to be due to a difference in the glycosylation of human Pgp in mouse NIH 3T3 cells. This is consistent with the observation that human Pgp expressed in NIH 3T3 cells migrates faster compared to the protein from KB-V1 cells on 8-10% acrylamide gel. Similarly, other workers have purified Chinese hamster Pgp either by a single-step chromatography on Reactive Red 120 agarose or by a combination of anion exchange and immunoaffinity chromatography (see the article by Senior et al. for the purification and properties of ATPase activity of Chinese hamster Pgp). The high level of drug-stimulated ATP hydrolysis by Pgp (Table I), like other ion-transporting ATPases, indicates that this is a high-capacity pump that can function as an effective multidrug transporter. This is further supported by the qualitative demonstration of ATP-dependent vinblastine transport in proteoliposomes reconstituted with pure Pgp (see Fig. 2). Thus, these experiments provide strong evidence that purified Pgp retains its activity and that it functions as an ATP-dependent drug transporter.

Publisher Summary P-Glycoprotein (Pgp), a member of the adenosine triphosphate-binding cassette (ABC) superfamily of transporters, functions as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated lipophilic compounds. This chapter discusses the Pgp ATPase activity that can be measured in the crude membranes of mouse (NIH 3T3) transfectants. The background activity of other ATPases can effectively be blocked with sodium azide. The inclusion of other inhibitors, such as ethylene glycol bis(β- aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA) and ouabain does not have any deleterious effect on Pgp activity. NIH 3T3 transfectants selected at low drug concentration are also suitable for characterization of Pgp ATPase activity. NIH 3T3 cells are suitable for characterization of drug transport function and ATPase activity, as they offer an ideal stable expression system to assess the structure–function relationship of various regions in Pgp.

Publisher Summary This chapter discusses the methods to evaluate the differential effect of adenosine triphosphate (ATP) hydrolysis and modulator interaction on substrate binding to these two sites. The results presented in the chapter deal with P-glycoprotein (Pgp) in isolated membranes from baculovirus-infected insect cells. These assays can also be successfully used for studies with Pgp expressed in human and murine cells as well as with the purified protein on its reconstitution into proteoliposomes. Pgp shows internal homology of amino acid sequence between its N- and C-terminal halves. Each half of the protein contains six putative transmembrane regions (TMs) followed by a consensus nucleotide-binding domain (NBD). Four major steps are involved in drug transport: (1) substrate recognition, (2) ATP binding, (3) ATP hydrolysis, and (4) coupling of ATP hydrolysis to drug translocation. Photoaffinity labeling of Pgp with analogs of substrates and modulators has revealed two major areas, one on each homologous half of the protein, as primary sites of drug interaction.

Publisher Summary This chapter discusses methods for measuring protonmotive force (PMF) and the motility that is the consequence of its use. The best-studied type of bacterial motility derives from the use of external flagella. The external flagellar filaments are thin helical structures that themselves perform no transduction of metabolic energy into mechanical work. They are rotated by motors at their base, and their rotation generates thrust. The motors are reversible, and it is this that permits control of behavior, as the reversal probability is controlled by the environment. It should be noted that PMF can, under some circumstances at least, play a role in controlling the “switching” of the flageilar motor. Major examples of this are aerotaxis and phototaxis, which appear to be more strictly “PMF taxis.” Another related aspect is pH taxis and certain classes of repellent-induced taxes, where perturbation of either internal or external pH is responsible for affecting motor switching.