The problem (Zerr, 2000) ATP binding/hydrolysis is believed to be connected to substrate binding and extrusion under the influence of Pgp. However, in the absence of substrate, Pgp continues to bind and hydrolyze ATP albeit at a very low basal rate (Figure 15). This rate increases upon the addition of a known Pgp substrate. In order to clarify the relationship between nucleotide and substrate binding, the kinetic parameters of ATP hydrolysis and substrate binding were examined.

Figure 15

Substrate effect on Vi trapping (Zerr, 2000). Incubation of Pgp membranes with Vi, labeled nucleotides and a potential substrate allows for visualization of the effect of the substrate on Vi-induced trapping (Figure 16A). An SDS-PGE gel demonstrates an obvious difference between the trapping abilities of Pgp samples in the environment of a range of different substrates; the bands on the gel indicate the presence of labeled nucleotide. Lanes 1 and 2 are control lanes, and lanes 3-8 represent Pgp incubation with rapamycin, cyclosporin A, DMSO, verapamil, valinomycin, and prazosin, respectively. Each of these compounds is known to be a Pgp substrate. However, the differences in intensity between the different lanes of this gel indicates that a Pgp substrate can influence nucleotide binding. As a further control for this experiment, figure 16B demonstrates a Pgp immunoblot, showing equivalent amounts of protein in each of the experimental lanes.

Figure 16 (figure taken from Zerr, 2000)

Kinetic experiments (Zerr, 2000). Analysis of the Km values for the Pgp system revealed an interesting correlation. The addition of Pgp inhibitors or substrates did affect the rate of transport, Vmax, but it did not change the Km for the system. Even though substrate affinity was reduced, the fundamental kinetic parameters of the system did not change. This indicates that while substrates do influence the Pgp catalytic cycle, their incorporation is not the rate limiting step. The rate limiting step must occur after nucleotide binding.