Simple purification of highly active biotinylated P-glycoprotein: Enantiomer-specific modulation of drug-stimulated ATPase activity

Citation
M. Julien et al., Simple purification of highly active biotinylated P-glycoprotein: Enantiomer-specific modulation of drug-stimulated ATPase activity, BIOCHEM, 39(1), 2000, pp. 75-85
Citations number
51
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
39
Issue
1
Year of publication
2000
Pages
75 - 85
Database
ISI
SICI code
0006-2960(20000111)39:1<75:SPOHAB>2.0.ZU;2-R
Abstract
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 po lyhistidine tail and a 100-amino acid residue biotin acceptor domain of oxa loacetate decarboxylase from Klebsiella pneumoniae at the carboxyl terminus end of Pgp (Pgp-H6BD). The expression/purification protocol for Pgp-H6BD i nvolves high level expression of the fusion protein in the yeast Pichia pas toris, biotinylation in vitro with biotin ligase, solubilization of crude m embrane fractions in detergent, and affinity purification by a combination of nickel and avidin chromatography. Biotinylated Pgp binds to immobilized monomeric avidin and can be eluted with free biotin in a high state of puri ty. This protocol is rapid and efficient and yields purified Pgp which show s robust ATPase activity, as determined by vanadate-induced trapping of pho toactive nucleotides and by direct measurement of ATP hydrolysis by Pgp-H6B D, This method should be useful for structural studies of the protein by sp ectroscopic or crystallographic approaches. This purified Pgp-H6BD preparat ion has been used to study the enantiomer-specific effects of inhibitors of Pgp-mediated drug transport on the drug-stimulated ATPase activity of the protein. A series of 1,4-disubstituted piperazine derivatives with a centra l chiral carbon and modified at the head and tail groups are shown to stimu late Pgp ATPase activity in a dose-dependent fashion. Some of these compoun ds are also capable of inhibiting either vinblastine or verapamil stimulati on of ATPase activity of Pgp in an enantiomer-specific fashion. The enantio meric specific inhibitory activity of these compounds suggests complex inte ractions at a single substrate binding site(s) on Pgp.