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C100369200v1
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Papers In Press, published online ahead of print July 12, 2001
J. Biol. Chem, 10.1074/jbc.C100369200
Submitted on July 2, 2001
Revised on July 11, 2001
Accepted on July 11, 2001

Evidence for the vectorial nature of drug (substrate)-stimulated ATP hydrolysis by human P-glycoprotein

Zuben E. Sauna, Melissa M. Smith, Marianna Muller, and Suresh V. Ambudkar

Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-4255

Corresponding Author: ambudkar{at}helix.nih.gov

P-glycoprotein (Pgp), the ATP-binding cassette (ABC) multidrug transporter, exhibits a drug (substrate)-stimulatable ATPase activity and vanadate (Vi) inhibits this activity by stably trapping the nucleoside diphosphate in the Pgp·ADP·Vi conformation. We recently demonstrated (Sauna, Z.E., Smith, M.M., Muller, M., and Ambudkar, S.V. (2001) J. Biol. Chem. 276, 21199-21208) that Vi-induced [a-32P]8-azidoADP trapping into Pgp in the absence of substrate occurs both in the presence of [a-32P]8-azidoATP (following 8azido-ATP hydrolysis) or [a-32P]8-azidoADP (without hydrolysis) and furthermore, the transition-state intermediates generated under either condition are functionally undistinguishable. In this study, we compare the effect of substrates on Vi-induced [a-32P]8-azidoADP trapping into Pgp under both non-hydrolysis and hydrolysis conditions. We demonstrate that whereas substrates stimulate the Vi-induced trapping of [a-32P]8-azidoADP under hydrolysis conditions, they strongly inhibit Vi-induced trapping under non-hydrolysis conditions. This inhibition is concentration-dependent, follows first order kinetics, and it is effected by drastically decreasing the affinity of nucleoside diphosphate for Pgp during trapping. However, substrates do not affect the binding of nucleoside diphosphate in the absence of Vi indicating that the substrate-induced conformation exerts its effect at a step distinct from nucleoside diphosphate-binding. Our results demonstrate that during the catalytic cycle of Pgp, although the transition-state, Pgp·ADP·Pi (Vi) can be generated both via the hydrolysis of ATP or by directly providing ADP to the system, in the presence of substrate the reaction is driven in the forward direction, i.e. hydrolysis of ATP. These data suggest that the substrate-stimulated ATP hydrolysis by Pgp is a vectorial process, and that Pgp, primarily functions as an ATP hydrolyzing pump.


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