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| - Simple Purification of Highly Active Biotinylated P-Glycoprotein: Enantiomer-Specific Modulation of Drug-Stimulated ATPase Activity
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| - A simplified method for the expression and purification of P-glycoprotein (Pgp) is presented.This method is based on the in-frame fusion of both a polyhistidine tail and a 100-amino acid residuebiotin acceptor domain of oxaloacetate decarboxylase from Klebsiella pneumoniae at the carboxyl terminusend of Pgp (Pgp-H6BD). The expression/purification protocol for Pgp-H6BD involves high-level expressionof the fusion protein in the yeast Pichia pastoris, biotinylation in vitro with biotin ligase, solubilizationof crude membrane fractions in detergent, and affinity purification by a combination of nickel and avidinchromatography. Biotinylated Pgp binds to immobilized monomeric avidin and can be eluted with freebiotin in a high state of purity. This protocol is rapid and efficient and yields purified Pgp which showsrobust ATPase activity, as determined by vanadate-induced trapping of photoactive nucleotides and bydirect measurement of ATP hydrolysis by Pgp-H6BD. This method should be useful for structural studiesof the protein by spectroscopic or crystallographic approaches. This purified Pgp-H6BD preparation hasbeen used to study the enantiomer-specific effects of inhibitors of Pgp-mediated drug transport on thedrug-stimulated ATPase activity of the protein. A series of 1,4-disubstituted piperazine derivatives witha central chiral carbon and modified at the head and tail groups are shown to stimulate Pgp ATPaseactivity in a dose-dependent fashion. Some of these compounds are also capable of inhibiting eithervinblastine or verapamil stimulation of ATPase activity of Pgp in an enantiomer-specific fashion. Theenantiomeric specific inhibitory activity of these compounds suggests complex interactions at a singlesubstrate binding site(s) on Pgp.
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