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. 2000 Mar 14;97(6):2515–2520. doi: 10.1073/pnas.97.6.2515

Figure 1.

Figure 1

(A) Substrate binding to Pgp in Vi-induced ADP trapped conformation. Crude membranes (20 μg protein) were labeled with 5 nM IAAP after pretreatment with ATP or 8AzidoATP in the presence or absence of Vi as described in Materials and Methods. Autoradiogram shows untreated Pgp (lane 1); Pgp pretreated at 37°C for 10 min with, 1.25 mM ATP (lane 2); 250 μM Vi (lane 3); 1.25 mM ATP and 250 μM Vi (lane 4); 1.25 mM 8AzidoATP (lane 5), or 1.25 mM 8AzidoATP and 250 μM Vi (lane 6). (B) Saturation binding of IAAP to Pgp in normal and transition-state conformation. Crude membranes (20 μg protein) were labeled with increasing concentrations (0.7–516 nM) of IAAP after pretreatment with AMPPNP or ATP in the presence of 250 μM Vi. Samples were untreated (●), treated with AMPPNP (▴), or treated with ATP (■) as described in Materials and Methods and then labeled with IAAP. The data were fitted by using the software graphpad prism 2.0 for the PowerPC MacIntosh and are representative of three independent experiments.