Figure 3.

Relationship between the disassociation of 8AzidoADP and recovery of IAAP binding after Vi trapping. (A) Disassociation of 8Azido[α-³²P]ADP from the Pgp⋅8Azido-[α-³²P]ATP⋅Vi complex of Pgp. Crude membranes (protein, 1 mg/ml) were incubated in the dark at 37°C for 10 min with 50 μM 8Azido[α-³²P]ATP (3–5 μCi/nmol) and 250 μM Vi in the ATPase assay buffer. The reaction was stopped by adding 12.5 mM ice-cold ATP and placing the tubes on ice. Untrapped nucleotides and excess Vi were removed by centrifugation at 300,000 × g for 10 min and the membranes were resuspended in the ATPase assay buffer. The resuspended membranes were placed at 37°C. Aliquots were removed at indicated intervals and placed on ice and photocrosslinked by UV irradiation at 365 nm for 5 min. (B) Recovery of IAAP binding to Pgp subsequent to formation of transition-state conformation. Crude membranes (1 mg/ml protein) were treated with 1.25 mM 8AzidoATP and 250 μM Vi for 10 min at 37°C in the ATPase assay buffer. The samples were placed on ice, and untrapped nucleotides and excess Vi were removed by centrifugation at 300,000 × g for 10 min at 4°C. The pellet was resuspended in the ATPase assay buffer containing 1.2 mM ATP and incubated at 37°C. Aliquots were transferred to ice at the indicated intervals and photolabeled with IAAP and visualized as described in Materials and Methods. Autoradiogram is shown in both A and B. (C) Relationship between the disassociation of 8AzidoADP and the recovery of IAAP binding after Vi trapping. The radioactivity associated with the Pgp bands depicted in A and B was quantified by using the Storm 860 PhosphorImager system. Disassociation of 8AzidoADP (●) and the increase in IAAP binding (▴) after Vi trapping were plotted as a function of time. (Inset) The increase in IAAP labeling, on the x-axis vs. the disassociation of 8Azido[α-³²P]ADP on the y-axis, these are inversely proportional to each other with R² = 0.959. The data are representative of three independent experiments.