The functional purification of P-glycoprotein is dependent on maintenance of a lipid-protein interface.
P-Glycoprotein (P-gp) is a 180-kDa membrane-bound transporter which can confer the multi-drug resistance phenotype on tumor cells. We have examined the factors required to preserve activity of P-gp during its purification. The starting material for purification was plasma membranes from Chinese hamster ovary (CHrB30) cells, overexpressing P-glycoprotein. These membranes displayed drug stimulated ATPase activity (Vm = 897 +/- 55 nmol min(-1) mg(-1); Km = 1.8 +/- 0.4 mM) and high affinity binding of [3H]vinblastine (Kd = 36 +/- 5 nM; Bm = 161 +/- 11 pmol/mg). Several non-ionic detergents which readily solubilized P-glycoprotein significantly inhibited ATPase activity and drug binding at concentrations well below their respective CMC values. This inactivation was prevented by excess crude lipid mixtures, with the greatest protection afforded against dodecyl-maltoside. Furthermore, the significantly reduced binding affinity and capacity of solubilized P-gp was partly reversed by the addition of lipids. A combination of anion-exchange and hydroxyapatite chromatography were used to purify P-gp with high yield to greater than 90%. The purified, reconstituted P-gp displayed high ATPase activity (Vm = 2137 +/- 309; Km = 2.9 +/- 0.9 mM) which was stimulated by verapamil (EC50 = 3.8 +/- 0.6 microM) and inhibited by orthovanadate (3.1 +/- 0.8 microM). Pure P-gp also displayed high affinity vinblastine binding (Kd = 64 +/- 9 nM) with a capacity of 2320 +/- 192 pmol/mg. This purification scheme yields the highest P-gp activity reported to date, and indicates a dependence of function on maintaining a lipid-protein interface.
Callaghan
R
R
Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, University of Oxford, UK. callagha@icrf.icnet.uk
Berridge
G
G
Ferry
D R
DR
Higgins
C F
CF
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry; D002845:Chromatography / Q000379:methods; D006224:Cricetinae; D003902:Detergents / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D005960:Glucosides / Q000494:pharmacology; D008055:Lipids / Q000494:pharmacology; D008081:Liposomes; D008823:Micelles; D010713:Phosphatidylcholines; D010743:Phospholipids; D011994:Recombinant Proteins / Q000302:isolation & purification; D014747:Vinblastine / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003902:Detergents; D005960:Glucosides; D008055:Lipids; D008081:Liposomes; D008823:Micelles; D010713:Phosphatidylcholines; D010743:Phospholipids; D011994:Recombinant Proteins; C018619:octyl-beta-D-glucoside; D014747:Vinblastine; C024990:asolectin; D000255:Adenosine Triphosphate