THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 46, Issue of November 15, pp.

28875–28883, 1996
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Efficient Purification and Reconstitution of P-glycoprotein for

Functional and Structural Studies*
(Received for publication, February 27, 1996, and in revised form, June 17, 1996)

Maoqing Dong‡, François Penin, and Loris G. Baggetto§

From the Insitut de Biologie et Chimie des Protéines, UPR 412 CNRS, 7 Passage du Vercors,
F-69367 Lyon Cedex 07, France

Plasma membrane P-glycoprotein is known as an ATP- mdr2), but only mdr1 genes encode Pgp that is involved in the
dependent drug efflux pump that confers multidrug re- MDR phenotype (1, 2). Pgp has been described as an ATP-de-
sistance to tumor cells. None of the reported purification pendent drug efflux pump that extrudes drugs out of cells and
procedures worked properly for our P-glycoprotein-over- thus confers cell resistance by lowering intracellular drug con-
producing cell lines, i.e. murine lymphoid leukemia P388/ centration (1). However, the mechanism by which this phenom-
ADR25, rat hepatoma AS30-D/COL10, and human lympho- enon occurs remains unclear and is of particular interest be-
blastic leukemia CEM/VLB5 cells. We have thus developed a cause of its broad specificity for structurally dissimilar
general procedure for efficient purification of P-glycopro-
substrates. Two models have been proposed (3). The first one
tein by combining solubilization with sodium dodecyl sul-
states that Pgp would be organized as a hydrophilic pore
fate and chromatography on ceramic hydroxyapatite. This
through which the drug is effluxed. In the second model, Pgp

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procedure was successful for the three cell lines and
yielded 70% of the P-glycoprotein present in the starting would act as a flippase to export the drug, which interacts first
plasma membranes with more than 99% purity. After ex- with the membrane lipid bilayer before it binds to Pgp. Pgp has
changing sodium dodecyl sulfate into dodecyl maltoside also been supposed to function as a volume-regulated chloride
and reconstitution into liposomes, purified P-glycoprotein channel (4). It is clear today that Pgp exhibits an ATPase
exhibited a specific ATPase activity of about 200 nmol/min/ activity, but little is known about how this activity is coupled to
mg, which was very similar to that obtained for P-glycopro- the drug transport process.
tein solubilized and purified with 3-[(3-cholamidopropyl)- One of the most important factors limiting our understand-
dimethylammonio]-1-propanesulfonic acid. This ATPase ing of Pgp function is the lack of structural information. A
activity was sensitive to orthovanadate inhibition and stim- prerequisite for Pgp structural and functional studies is the
ulated by verapamil and other drugs. More importantly, availability of a large amount of highly purified and active Pgp.
drug transport properties of the reconstituted P-glycopro- Although several efforts have been made to purify Pgp from
tein were comparable with those of P-glycoprotein embed- multidrug-resistant cells (5–9), none of the described proce-
ded in plasma membranes. Since it is virtually devoid of
dures has been designed for preparative scale purification, due
lipids, this preparation is suitable for both functional and
to poor separation of contaminants from Pgp, to rather low
structural investigations.
ATPase activity of the resultant Pgp, or to low Pgp yield.
Recently, by using dye-ligand chromatography, Urbatsch et al.
Multidrug resistance (MDR)1 is a phenomenon encountered (10) have purified active Pgp in the presence of lipids from an
in cancer treatments in which the tumors become resistant to a octylglucoside extract of membranes from multidrug-resistant
variety of cytotoxic chemotherapeutic agents. The molecular Chinese hamster ovary (CHO) cells. However, this procedure
basis for one major type of MDR is the overexpression of the has not been successful for the purification of Pgp from Sf9
P-glycoprotein (Pgp), a plasma membrane glycoprotein with insect cells infected by bacculovirus carrying the mdr1 gene,
molecular masses ranging from 130 to 170 kDa, depending on due either to the difference of membrane composition or to the
its glycosylation state in a specific drug-resistant cell line. alteration in posttranslational modification of recombinant Pgp
There are two classes of genes in human (mdr1 and mdr2) and (11). A combination of DEAE-cellulose anion exchange with
three in rodents (mdr1a (or mdr3), mdr1b (or mdr1), and immunoaffinity chromatography has also been used for the
isolation of hamster Pgp from CHO cells after solubilization by
Zwittergent 3–12 (12). However, this procedure requires the
* This work was supported by CNRS, by Association pour la Recher- use of an expensive monoclonal antibody immunoaffinity col-
che sur le Cancer Grant 6756, and by a grant from Ligue Nationale
Contre le Cancer, Comité du Rhône (to L. G. B.). The costs of publica- umn. During the completion of this study, Sharom et al. (13)
tion of this article were defrayed in part by the payment of page reported the purification of Pgp from multidrug-resistant CHO
charges. This article must therefore be hereby marked “advertisement” cells by combining selective Chaps extraction and lentil lectin
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
affinity chromatography. The resultant purified hamster Pgp
‡ Recipient of postdoctoral fellowships from CNRS and Fondation
pour la Recherche Medicale. displayed a high basal ATPase activity.
§ To whom correspondence should be addressed. Tel.: 33 04 72 72 26 Nevertheless, all of these procedures that have been de-
29; Fax: 33 04 72 72 26 26; E-mail: lgb@ibcp.fr. signed for the multidrug-resistant CHO cell line do not work
1
The abbreviations used are: MDR, multidrug resistance; ADR, ad-
properly on our Pgp-overproducing lines, i.e. P388 murine
riamycin; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-
sulfonic acid; dodecyl maltoside, 1-O-n-dodecyl-b-D-glucopyrano- lymphoid leukemia (P388/ADR25), AS30-D rat hepatoma
syl(134)a-D-glucopyranoside; CHO, Chinese hamster ovary; COL, (AS30-D/COL10), and CEM human lymphoblastic leukemia
colchicin; DTT, dithiothreitol; HPLC, high performance liquid chroma- (CEM/VLB5) cells. This is mainly due to the poor efficiency of
tography; HAP-HPLC, ceramic hydroxyapatite HPLC; octylglucoside,
Pgp solubilization obtained with the reported mild detergents
1-O-n-octyl-b-D-glucopyranoside; PA, L-a-phosphatidic acid; PAGE,
polyacrylamide gel electrophoresis; PC, L-a-phosphatidylcholine; Pgp, (such as octylglucoside, Zwittergent 3–12, Chaps, etc.), which
P-glycoprotein; VLB, vinblastine. varies from cell line to cell line. Also, in order to preserve its

This paper is available on line at http://www-jbc.stanford.edu/jbc/ 28875

28876 Purification and Reconstitution of P-glycoprotein
ATPase activity, no attempt has been made to delipidate Pgp in performed with a Branson model 250 sonifier equipped with a micro-
the reported purification procedures. These preparations are probe. HPLC was run on a Waters apparatus equipped with a model
991 photodiode array detector. Bio-Gel hydroxyapatite, prepacked ce-
thus difficult to use for structural studies and crystallization,
ramic hydroxyapatite HPLC column (0.7 3 5.2 cm), and Bio-Beads SM2
which require a monodisperse detergent-protein complex. were from Bio-Rad. Ultima GoldTM XR scintillation medium was from
Therefore, the main purpose of the present study is to find a Packard. Tritium radioactivity counting was performed on a liquid
way for efficient solubilization of Pgp and to set up a reliable scintillation analyzer (Packard) using protocol 15 designed for 3H.
protocol for its purification that can be applied to every resist- Selection of Highly Multidrug-resistant, Pgp-overproducing Cell
ant cell line and that will yield a Pgp preparation suitable for Lines—Murine lymphoid leukemia P388 and rat hepatoma AS30-D cell
lines were maintained in DMEM, and human lymphoblastic leukemia
structural and functional investigations.
CEM cell line was maintained in RPMI 1640 medium. In all cases, the
Mild nonionic and some Zwittergent detergents are known to culture media were supplemented with heat-inactivated fetal calf se-
be able to preserve the biological activities of membrane pro- rum (10%), penicillin (100 units/ml), streptomycin (100 mg/ml), and
teins and thus are generally used for their solubilization and amphotericine B (0.25 mg/ml). Cells grew at 37 °C in a humidified
purification. However, the solubilization efficiency of these de- atmosphere under 5% CO2 in air. AS30-D cells were cultured in the
tergents is sometimes low, and it depends on membrane species presence of 5.2 g/liter glucose as described previously (26). The highly
drug-resistant cell lines P388/ADR25, AS30-D/COL10, and CEM/VLB5
and varies from protein to protein, although it can be increased
were established by growing the corresponding sensitive parental cell
by the addition of high concentrations of salt (14, 15). Further- line in the medium containing, respectively, stepwise increased concen-
more, the ability of mild detergents to disrupt aggregates of trations of adriamycin, colchicin, and vinblastine. Finally, the cells
membrane proteins is limited (16). On the contrary, SDS is a selected in this way were able to grow in the presence of the highest
powerful solubilizing and dissociating detergent that is also possible concentration of drug (25 mg/ml adriamycin for P388/ADR25,
very effective at preventing a protein of interest from aggre- 10 mg/ml colchicin for AS30-D/COL10, and 5 mg/ml vinblastine for
CEM/VLB5, respectively), and the phenotype of each cell line was stable
gating and nonspecifically associating with other proteins into
for at least 8 months. Cells used for the preparation of plasma mem-
mixed micelles, especially during chromatographic purification branes were grown for 2 days in the absence of drug, washed with cold

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steps. The drawback of using SDS is the loss of biological phosphate-buffered saline (1.8 mM KH2PO4, 10 mM Na2HPO4, 138 mM
activity of solubilized proteins. However, successful reactiva- NaCl, 2.7 mM KCl, pH 7.4) and stored in liquid nitrogen.
tion of SDS-solubilized proteins has been repeatedly reported Preparation of Plasma Membranes—Plasma membranes were pre-
(17–21) even after SDS-PAGE (22). In fact, the main point for pared by sonication and discontinuous sucrose density gradient centrif-
ugation essentially according to previous reports (5, 12). Briefly, 5 3 108
reactivation is the removal of SDS that can be achieved by
cells were quickly thawed in 5 ml of buffer containing 50 mM Tris-HCl,
exchanging it with a mild detergent (21). pH 7.4, 50 mM NaCl, 2 mM EDTA, 2 mM phenylmethanesulfonyl fluo-
Because of the poor efficiency of mild detergents, as stated ride, 2 mM leupeptin, and 4 mM pepstatin A and were sonicated for three
above, we have chosen SDS for solubilization of Pgp from 20-s bursts with 30-s cooling periods between. After removal of the
plasma membranes and for its subsequent purification. By nuclei by centrifugation (1,000 3 g, 10 min, 4 °C), the membrane ex-
comparison, a combination of Chaps with NaCl has also been tract was loaded onto a discontinuous sucrose gradient consisting of 4
ml of 45% (w/v), 3 ml of 31%, and 2 ml of 16% sucrose in 10 mM Tris-HCl
evaluated for Pgp purification. To purify Pgp in the presence of
(pH 7.4) and ultracentrifuged at 4 °C in a swinging bucket SW41Ti
detergent, we have used ceramic hydroxyapatite chromatogra- rotor (Beckman) for 18 h at 100,000 3 g. The membrane fractions
phy (HAP-HPLC), which has been successfully used for purifi- collected at the 16/31% sucrose interface (i.e. purified plasma mem-
cation of both soluble and membrane proteins (15, 23–25). branes) were diluted to 12 ml with 10 mM Tris-HCl, pH 7.4, and pelleted
Hydroxyapatite often resolves components that other chro- by ultracentrifugation (100,000 3 g, 30 min, 4 °C). The pellets were
matographic media fail to separate and is especially very useful suspended in 1 ml of 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4) and stored
in liquid nitrogen.
for separating protein-detergent complexes (15). By combining
Reconstitution of Purified Pgp into Liposomes—For the drug trans-
SDS solubilization and HAP-HPLC, we have designed a gen- port assay, reconstitution of purified Pgp into liposomes was carried out
eral protocol for effective purification of Pgp. We report here by the method developed by Rigaud et al. (27, 28) as follows: lipids (PC,
this protocol that has been successfully and reproducibly ap- 25 mg; PA, 2.5 mg) were dissolved in chloroform and dried under a
plied to purify Pgp from our three cell lines and yields large stream of nitrogen. To the lipid film was added 1.375 ml of buffer
amounts of highly purified Pgp. After SDS exchange into do- containing 20 mM Tris-HCl, pH 7.4, 75 mM NaCl, 1 mM DTT, and 0.5 mM
EDTA. The mixture was sonicated on ice until the milky mixture
decyl maltoside and reconstitution into liposomes, Pgp dis- became transparent (about 15 min). To a 100-ml aliquot of this prepa-
played a similar drug-sensitive ATPase activity to that ob- ration were sequentially added 610 ml of buffer and 40 ml of 10% dodecyl
tained for Pgp solubilized and purified with Chaps. Moreover, maltoside under constant stirring at room temperature. After 15 min,
the reconstituted SDS-purified Pgp exhibited drug transport 250 ml of 0.2 mg/ml purified Pgp were slowly added, and the mixture
properties comparable with that of Pgp embedded in plasma was incubated under constant agitation for another 15 min. To remove
membranes prepared from the same multidrug-resistant can- the detergent, Bio-Beads (previously washed sequentially in methanol
and MilliQ water) were added stepwise: 40 mg for 45 min, 40 mg for 45
cer cells. min, 40 mg for 30 min, and finally 40 mg for 30 min. Aliquots of the
supernatant containing reconstituted proteoliposomes were used for
EXPERIMENTAL PROCEDURES the drug transport assay. Reconstituted Pgp could be kept at 4 °C for 2
Materials—Cell culture media (DMEM and RPMI 1640) were from days without losing any transport activity. A similar procedure was
BioWhittaker, and fetal calf serum was from Sigma. Adriamycin (ADR), followed when egg yolk and sheep brain lipids were used.
colchicin (COL), sodium orthovanadate, verapamil, antibiotic and anti- ATPase Activity Assay—The hydrolysis of ATP by Pgp was measured
mycotic solution (containing 10,000 units/ml penicillin, 10 mg/ml strep- by an NADH fluorometric assay improved by Gonzalo et al. (29). Briefly,
tomycin, and 25 mg/ml amphotericine B), Chaps, sodium deoxycholate, the decrease of NADH fluorescence emission at 460 nm was quantita-
egg yolk L-a-phosphatidic acid (PA), egg yolk L-a-phosphatidylcholine tively recorded at 30 °C by using an SLM Aminco 8000C spectroflu-
(PC), crude egg yolk lipids, crude sheep brain phospholipids, and rou- orometer (SLM Instruments Co.) in 1 ml of reaction buffer containing
tine chemicals were from Sigma. Vinblastine (VLB) was a gift from 20 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 0.25
Roger Bellon Laboratories (France). [3H]Vinblastine (13.5 Ci/mmol) mM phosphoenolpyruvate, 26 mM NADH, 1.5 mM ATP, 30 mg/ml pyru-
was purchased from Amersham Corp. ATP (disodium salt), phos- vate kinase, 15 mg/ml lactate dehydrogenase, and 1 mg of proteolipo-
phoenolpyruvate, NADH, pyruvate kinase, and lactate dehydrogenase somes or 0.2–1 mg of purified plasma membranes. In the case of plasma
were bought from Boehringer Mannheim France. Dodecyl maltoside, membranes, ATPase activity of Pgp was assayed after the addition of
octylglucoside, and Zwittergent 3–12 were from Calbiochem, and SDS the specific ATPase inhibitor mixture (10 mM NaN3, 0.25 mM EGTA, 2
was from Serva. 5-bromo-4-chloro-3-indolyl phosphate and nitro blue mM ouabain) in the assay medium. The drug of interest was directly
tetrazolium were from Promega. Anti-Pgp monoclonal antibody C219 added to this reaction. ATPase activity was calculated from the slope
was from CIS Bio International (Centocor Diagnostics). Sonication was during the first 5 min, which was linear in all cases. Protein was
 Purification and Reconstitution of P-glycoprotein 28877
TABLE I
Pgp solubilization efficiency by different detergents
The solubilization of P388/ADR25 plasma membrane was followed by
recording the decrease of turbidity at 500 nm with a spectrophotometer
after aliquot additions of the chosen detergent from a concentrated
stock solution. When the optical density reached a plateau, the amount
of total detergent in the solution was calculated (numbers between
parentheses), and the mixture was submitted to ultracentrifugation
(100,000 3 g for 30 min). Pgp distribution in the supernatant and in the
pellet was analyzed by SDS-PAGE followed by laser scan densitometry.
Solubilization efficiency is the ratio of solubilized Pgp to overall Pgp in
plasma membranes.
Solubilization efficiency
Detergent
No NaCl With NaCl (1M)

%
Octylglucoside (1.4%) 11 39
Dodecyl maltoside (0.4%) 16 47
Chaps (1%) 24 61
Zwittergent 3–12 (0.4%) 14 51
Sodium deoxycholate (2%) 30 —a
SDS (2%) 93 NDb
a
Not soluble in the presence of 1 M NaCl.
b
Not determined.

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COL10, and human lymphoblastic leukemia CEM/VLB5),
which were, respectively, 7,830, 103, and 18,990 times more
resistant to the respective drug than their corresponding sen-
sitive parental cell lines (these data will be reported else-
where). Electrophoresis presented in Fig. 1A shows that
plasma membranes of all the resistant cell lines contain a large
FIG. 1. Overexpression of Pgp in different MDR cell lines.
Plasma membranes prepared as described under “Experimental Proce- amount of Pgp when compared with their parental cells. Pgp
dures” from different cell lines were solubilized in sample loading buffer was revealed by Western blotting using anti-Pgp monoclonal
containing 7% SDS and then resolved on 8% SDS-PAGE. A, SDS-PAGE antibody C219 (Fig. 1B), while no Pgp could be detected in
silver-stained gel. B, Western blot revealed with anti-Pgp monoclonal sensitive lines. The comparison of the electrophoretic patterns
antibody C219. Lane 1, P388-sensitive cells; lane 2, P388/ADR25; lane
3, AS30-D-sensitive cells; lane 4, AS30-D/COL10; lane 5, CEM-sensitive among the resistant and their sensitive parental lines shows
cells; lane 6, CEM/VLB5. Each lane contains 10-mg membrane proteins that Pgp was the only overproduced protein in plasma mem-
in both A and B. MW, molecular weight marker, 1 mg of protein for each branes (Fig. 1A). Pgp overproduction in each of these resistant
band. lines represents an average of 30, 27, and 25% (w/w) of total
determined by the method of Peterson (30) using bovine serum albumin detected membrane proteins for P388/ADR25, AS30-D/COL10,
as a standard. and CEM/VLB5, respectively, as estimated by laser densitom-
[3H]Vinblastine Uptake by Plasma Membranes and Reconstituted etry of electrophoresis gels. The smears of high molecular
Purified Pgp—[3H]Vinblastine uptake by either reconstituted purified weight revealed by anti-Pgp monoclonal antibody C219 are
Pgp or plasma membranes was measured according to the protocol observed even when the sample loading buffer contained up to
described by Horio et al. (Ref. 31; see legends of Figs. 4 and 5).
7% SDS, or 8 M urea. This is probably due to the existence of
SDS-PAGE and Western Blotting—Protein samples were analyzed
by SDS-PAGE in either 8% or 5–10% gradient polyacrylamide gels Pgp oligomers and/or to different levels of Pgp glycosylation.
under reducing conditions as described by Laemmli (32) and detailed
previously (33) except that samples were not heated before loading. Solubilization of Pgp from Plasma Membranes
Diluted protein samples were concentrated by the addition of 1⁄10 vol- Various detergents were evaluated for their efficiency to
ume of 0.15% sodium deoxycholate and 1⁄10 volume of 72% trichloroace-
tic acid according to the method of Peterson (30) except when SDS was
solubilize Pgp. Prior to detergent addition, peripheral mem-
present. Gels stained either with Coomassie Brilliant Blue G-250 or brane proteins and adsorbed soluble ones were removed as
with silver staining (34) were scanned in a computerized laser densi- follows. 1 ml of thawed plasma membranes were diluted with 1
tometer (Personal Densitometer SI, Molecular Dynamics) and quanti- ml of 10 mM sodium phosphate buffer (pH 7.0) containing 1 M
tatively analyzed with the ImageQuant program. For Western blots NaCl and a protease inhibitor mixture (2 mM phenylmethane-
(35), proteins were transferred onto 0.45-mm pore nitrocellulose filters sulfonyl fluoride, 1 mM leupeptin, and 4 mM pepstatin A). After
(Schleicher & Schüll) in a Mini-TransBlot electrophoretic transfer cell
(Bio-Rad) at 100 V for 1 h. Blots were blocked by a 2-h incubation at
homogenization by brief sonication and incubation at 4 °C for
room temperature in Tris-buffered saline (10 mM Tris-HCl, 0.15 M NaCl, 15 min, the membranes were ultracentrifuged (100,000 3 g, 30
pH 7.4) supplemented with 10% defatted milk and 0.1% Tween 20. min, 4 °C). The pellet was homogenized by brief sonication in 1
After washing with Tris-buffered saline containing 1% defatted milk ml of 10 mM sodium phosphate buffer (pH 7.0) containing 1 mM
and 0.1% Tween 20, the filters were incubated with anti-Pgp chicken DTT and the selected detergent at the concentration indicated
monoclonal antibody C219 (0.2 mg/ml) for 1 h at room temperature in Table I. Detergent concentration for maximal Pgp solubili-
before washing three times with the same solution. Blots were subse-
quently incubated for 45 min with the secondary antibody alkaline
zation was determined as described in the legend of Table I.
phosphatase anti-IgG conjugate (1:2,000; Sigma), washed three times This table shows that nearly all of the mild detergents tested
with the above washing solution, and developed by the 5-bromo-4- poorly solubilized Pgp from P388/ADR25 plasma membranes.
chloro-3-indolyl phosphate/nitro blue tetrazolium substrates. However, in the presence of 1 M NaCl, Pgp solubilization was
RESULTS
3– 4 times more efficient depending on the detergent used.
Among mild detergents, the most efficient Pgp solubilization
Pgp Overproduction by Different Drug-resistant Cell Lines
was achieved with Chaps in the presence of 1 M NaCl, but it
We have established several resistant cell lines (i.e. murine never exceeded 60%. In contrast, the strong dissociating agent
lymphoid leukemia P388/ADR25, rat hepatoma AS30-D/ SDS could almost completely solubilize Pgp and did not require
28878 Purification and Reconstitution of P-glycoprotein
salt addition. Similar results were obtained with both AS30-D/
COL10 and CEM/VLB5 plasma membranes (data not shown).
In conclusion, the best detergents for Pgp solubilization were
SDS and also Chaps with high salts. These two detergents were
thus chosen to set up Pgp purification procedures.

Pgp Purification from Overproducing Cells

A number of chromatographic methods were tested for the
purification of P388/ADR25 Pgp. Ion exchange on DEAE-cellu-
lose used by others (7, 12) could not be applied for the purifi-
cation of Chaps- or SDS-solubilized Pgp because of the strong
binding of these detergents to the column. Gel filtration could
poorly separate Pgp from the contaminants due to the existence
of mixed detergent-protein complexes. Among several dyes af-
finity chromatography matrix screened, only Cibacron Blue
F3-GA could be used to remove limited contaminant proteins
from Chaps-solubilized preparation without binding Pgp. Ten-
tative experiments using Bio-Gel hydroxyapatite revealed that
Pgp could be separated from most of the membrane proteins in
the presence of high concentrations of detergent, in particular
SDS or Chaps in the presence of salts. However, the chromato-

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graphic resolution of Bio-Gel hydroxyapatite was poor. Further
experiments with ceramic hydroxyapatite HPLC showed that
efficient separation of Pgp from all other membrane proteins
has been obtained. Thus, a completely new procedure for puri-
fication of Pgp has been set up and is described here.
Purification of SDS-solubilized Pgp—After removal of pe-
ripheral membrane proteins and adsorbed soluble ones, the
membranes were dissolved with 2% SDS and ultracentrifuged
for 30 min at 100,000 3 g at 20 °C. The supernatant, i.e. Pgp
crude extract, was diluted to 1% SDS with an equal volume of
10 mM phosphate buffer (pH 7.0) containing 1 mM DTT and
then applied onto a prepacked HAP-HPLC column equilibrated
with 50 mM phosphate buffer (pH 7.0), 1% SDS, and 1 mM DTT.
After washing with the equilibration buffer, the chromatogra-
phy was developed with a linear gradient of sodium phosphate
in the same buffer. The buffers and the column were main-
tained at 28 °C to prevent SDS precipitation. A typical separa-
tion profile for P388/ADR25 Pgp is shown in Fig. 2A, and the
electrophoretic analysis of eluted fractions is shown in Fig. 2B.
Pgp interacts with hydroxyapatite more strongly than all other
proteins and was eluted as a single peak at about 0.45 M
FIG. 2. Purification of Pgp by HAP-HPLC. A, elution profile of
phosphate. Since Pgp was the last protein eluted under the SDS-solubilized P388/ADR25 plasma membrane proteins with the in-
conditions used (Fig. 2A), it was thus obtained with a very high dicated phosphate gradient. The chromatography was performed at a
degree of purity as shown in Fig. 2A (lane 6). The eluted flow rate of 1 ml/min and monitored continuously at both 280 (data not
fractions containing pure Pgp were pooled and concentrated by shown) and 220 nm. Pgp was eluted as a broad single peak at about 0.45
M phosphate. B, electrophoretic analysis of eluted fractions from HAP-
HAP-HPLC (see below) followed by ultrafiltration with Centri- HPLC in A. Samples were resolved on an 8% SDS-PAGE gel followed by
con (Amicon, 30-kDa cut-off) or Centriprep devices (Amicon, silver staining. Lane 1, SDS-solubilized membrane protein crude ex-
100-kDa cut-off). In the case of large scale separation, contam- tract, 8 mg; lanes 2– 6, aliquot fractions collected at different elution
inants in the very first eluted Pgp fractions could be easily times during the phosphate gradient; lane 2, 15.5–16 min (18 ml, 0.2
mg); lane 3, 17.2–17.7 min (18 ml, 1 mg); lane 4, 18.7–19.2 min (18 ml, 3
removed by a second passage on the HAP-HPLC with almost no mg); lane 5, 22.5–23 min (18 ml, 2 mg); lane 6, 26 –26.5 min (24 ml, 2 mg).
loss of Pgp. Most of the compounds eluted between 10 and 15 min were not proteins.
Similar chromatographic and electrophoretic profiles were C, silver-stained 5–10% gradient SDS-PAGE of purified Pgp from three
obtained for AS30-D/COL10 and CEM/VLB5 Pgp. For each of cell lines. Lanes 1–3, purified Pgp from P388/ADR25, AS30-D/COL10,
and CEM/VLB5 cells, respectively (2 mg of protein for each lane). Note
these cell lines, the only modification was a slight adjustment that silver-staining was overdeveloped to reveal as many proteins as
of phosphate gradient in order to elute Pgp as a well resolved possible. A larger number of bands were thus revealed in Fig. 2B, lane
single peak. This method allowed the preparation of very pure 1, when compared with lane 2 in Fig. 1A. D, Western blotting of purified
Pgp from the three cell lines as shown in Fig. 2C (see also Fig. Pgp from the three resistant cell lines. SDS-PAGE was done as in C,
and blots were revealed with anti-Pgp monoclonal antibody C219.
2B, lane 6). In the 5–10% gradient SDS-PAGE (Fig. 2, C and D),
Lanes 1–3, purified Pgp from P388/ADR25, AS30-D/COL10, and CEM/
the higher molecular weight protein bands observed are obvi- VLB5 cells, respectively (1 mg of protein for each lane). MW, molecular
ously dimers and higher order oligomers of Pgp, since they weight marker, 1 mg of protein for each band.
were revealed by the specific anti-Pgp monoclonal antibody
C219 as shown in the immunoblots of Fig. 2D. The presence detected in P388/ADR25 and AS30-D/COL10. Failure of anti-
and amount of oligomers in purified Pgp depend on the cell line Pgp monoclonal antibody C219 to detect any band lower than
studied; while oligomers are clearly visible in the CEM/VLB5 the monomeric Pgp band indicates the absence of any proteo-
Pgp preparation (Fig. 2, C and D, lane 3), they are barely lytic Pgp fragment in the Pgp preparation. Fig. 2C also shows
 Purification and Reconstitution of P-glycoprotein 28879
TABLE II
Protein recovery during the purification of SDS-solubilized Pgp from
murine, rat, and human drug-resistant cell lines
Plasma membranes from 5 3 108 cells were prepared as described
under “Experimental Procedures.” The percentage of Pgp of total mem-
brane proteins was estimated by SDS-PAGE followed by laser scan
densitometry. The yield of Pgp at each step is calculated relative to the
overall Pgp present in plasma membranes (100%).
Cell lines and steps Protein Pgp Yield

mg % of total protein % of Pgp

P388/ADR25
Plasma 1.08 30 100
membrane
1 M NaCl wash 0.82 35 89
Pgp crude extract 0.78 35 84
HAP-HPLC 0.21 .99 65
AS30-D/COL10
Plasma 1.33 27 100
membrane
1 M NaCl wash 1.00 34 95
Pgp crude extract 0.96 35 94
HAP-HPLC 0.25 .99 70
CEM/VLB5
Plasma 1.44 25 100
membrane
1 M NaCl wash 1.08 32 96

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Pgp crude extract 1.01 33 93
HAP-HPLC 0.26 .99 72

that, apart from monomeric Pgp and some Pgp oligomers, no

other protein was detected by silver staining. Other SDS-PAGE
experiments designed to show low molecular weight proteins FIG. 3. Effects of orthovanadate and verapamil on the ATPase
(but that could not resolve Pgp oligomers) did not reveal any activity of reconstituted purified Pgp. Both SDS-purified Pgp
other bands except Pgp, as shown in Fig. 2B, lane 6 (data not (open circle) and Chaps-purified Pgp (solid circle) from the P388/ADR25
shown for AS30-D/COL10 and CEM/VLB5 cell lines). Since 2 cell line were reconstituted into egg yolk lipids (see “Experimental
Procedures”). Note that in the case of SDS-purified Pgp, SDS was
mg of Pgp were loaded for each lane of Fig. 2C and taking into exchanged into dodecyl maltoside prior to reconstitution. ATPase activ-
account that less than 0.01 mg can be detected by silver stain- ities were assayed at 30 °C in 1 ml of reaction buffer containing 1 mg of
ing, one can estimate that the purity of Pgp exceeds 99% of reconstituted Pgp in the presence of the indicated concentrations of
detected proteins. By comparison, the molecular mass of P388/ orthovanadate and verapamil. A, inhibition by orthovanadate; 50%
inhibition of the ATPase activity was calculated by the GraphPad Prism
ADR25 Pgp (140 kDa) is lower than those estimated for both
program. B, activation by verapamil. All data are presented as the
AS30-D/COL10 and CEM/VLB5 Pgp (170 kDa), likely due to means for triplicate determinations.
the lower glycosylation level in resistant P388 cells (36).
Table II summarizes the recovery of Pgp during the purifi-
HAP-HPLC. In order to reduce the phosphate concentration,
cation procedure described above for the three cell lines. The
SDS-purified Pgp was first dialyzed at room temperature
yield of purified plasma membranes (16/31% sucrose gradient
against 10 mM phosphate buffer, pH 7.0, 1% SDS, and 1 mM
interface fraction) from each of these cell lines is about the
DTT. The resultant dialysate was then injected onto the ce-
same, about 2.6 mg/109 cells. The yield of purified Pgp from
ramic HAP-HPLC column, which was equilibrated with 50 mM
plasma membranes is very high for the three cell lines, about
phosphate (pH 7.0), 1 mM DTT, and 0.1% dodecyl maltoside.
70% of overall Pgp present in starting plasma membranes (i.e.
The column was then extensively washed with at least 15
0.2 mg of Pgp/mg of membrane proteins). This results from the
column volumes of the equilibration buffer to remove SDS. Pgp
high efficiency of SDS solubilization and the excellent recovery
was eluted with 0.5 M phosphate containing 1 mM DTT and
of purified Pgp from HAP-HPLC (75% of solubilized Pgp).
0.1% dodecyl maltoside, and the phosphate concentration was
Purification of Chaps-solubilized Pgp—The conditions of Pgp
immediately reduced by dialysis. Pgp can easily be concen-
solubilization with Chaps were the same as with SDS, except
trated by this way to 0.2 mg/ml without significant loss of Pgp
that SDS was replaced by 1% Chaps and 1 M NaCl according to
and without concentrating the detergent used.
Table I. The subsequent HAP-HPLC was performed in the
presence of 1% Chaps and 0.5 M NaCl instead of SDS in all Characterization of the ATPase Activity of
buffers used. Highly purified Pgp was also obtained with only Reconstituted Purified Pgp
one-step HAP-HPLC. However, the yield of purified Pgp was 10
times lower (about 20 mg/mg of starting plasma membrane No ATPase activity could be detected for both SDS- and
proteins) than that obtained with SDS. Apart from the rela- Chaps-purified Pgp, even after exchanging SDS and Chaps into
tively lower solubilization efficiency of Chaps, the important dodecyl maltoside (not shown). However, the reconstitution
loss of Pgp during HAP-HPLC step is attributed to aggregation into lipids allowed the restoration of Pgp ATPase activity.
of Chaps-solubilized Pgp on the column matrix. However, con- Comparison of the ATPase Activities of SDS- and Chaps-
trary to the SDS-Pgp preparation, this Chaps-Pgp preparation purified Pgp—After exchanging SDS into dodecyl maltoside,
does not need any exchange of detergent prior to reconstitution Pgp samples were extensively dialyzed against 20 mM Tris-
into liposomes in order to restore Pgp ATPase activity. HCl, pH 7.4, 6 mM MgCl2, 0.5 mM EDTA, 75 mM NaCl, 1 mM
DTT, and 0.1% dodecyl maltoside and reconstituted into lipo-
Detergent Exchange and Pgp Concentration
somes as described under “Experimental Procedures.” For
Since Pgp strongly interacts with hydroxyapatite, Pgp con- Chaps-purified Pgp, the detergent exchange step into dodecyl
centration and detergent exchange can easily be done with maltoside was not necessary. The ATPase activity was meas-
28880 Purification and Reconstitution of P-glycoprotein
TABLE III TABLE IV
Comparison of the ATPase activities of reconstituted purified Pgp with Stimulatory effects of selected drugs on the Pgp ATPase activity
Pgp embedded in plasma membranes from murine, rat, and human The measurement of the specific ATPase activity of reconstituted and
drug-resistant cell lines membrane Pgp is described under “Experimental Procedures” and in
Pgp from P388/ADR25, AS30-D/COL10, and CEM/VLB5 cell lines the legend of Table III. The drug of interest was directly added to the
was purified in the presence of SDS and reconstituted into liposomes assay medium. Drug concentrations yielding to maximal stimulation
after exchanging SDS into dodecyl maltoside. Plasma membranes were are given in parentheses, in mM. Data are presented as the means of
prepared as described under “Experimental Procedures” from P388/ triplicate determinations (mean 6 S.D.). Abbreviations are defined in
ADR25, AS30-D/COL10, and CEM/VLB5 cell lines in which Pgp repre- the legend of Table III.
sents 30, 27, and 25% (w/w) of total plasma membrane proteins, respec-
Drug maximal stimulation
tively, as estimated by laser densitometry of SDS-PAGE. The Cell lines and samples
hydrolysis of ATP by Pgp was measured by NADH fluorimetric assay as Verapamil Colchicin Vinblastine
detailed under “Experimental Procedures.” The specific ATPase activity
-fold
of membrane Pgp (italic values) was calculated by taking into account
the Pgp percentage of total membrane proteins after subtraction of P388/ADR25
residual ATPase activity insensitive to NaN3, ouabain, and EGTA in PM 1.3 (10.0) 1.2 (1.5) 1.2 (1.5)
the corresponding parental sensitive cell lines (P388, AS30-D, and Pgp-EYL 2.1 (5.0) 1.6 (1.5) 1.5 (1.0)
CEM). Data are presented as the means of triplicate determinations Pgp-SBL 2.0 (5.0) 1.4 (1.5) 1.4 (1.0)
(mean 6 S.D.). Pgp-EYL, Pgp reconstituted in egg yolk lipids; Pgp-SBL, AS30-D/COL10
Pgp reconstituted in sheep brain lipids; Pgp-PC:PA, Pgp reconstituted PM 1.4 (10.0) 1.1 (1.5) 1.2 (1.5)
in PC:PA (10:1, w/w) liposomes; PM, plasma membranes; ND, not Pgp-EYL 2.1 (5.0) 1.4 (1.5) 1.5 (1.0)
determined. Pgp-SBL 2.0 (5.0) 1.4 (1.5) 1.5 (1.0)
CEM/VLB5
Specific ATPase activity PM 1.5 (10.0) 1.2 (1.5) 1.3 (1.5)
Cell line Vanadate Pgp-EYL 2.2 (5.0) 1.5 (1.5) 1.6 (1.0)
Sample Basal (50 mM) Pgp-SBL 1.9 (5.0) 1.5 (1.5) 1.5 (1.0)
nmol/min/mg Pgp

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P388/ADR25 Pgp-EYL 109 6 6 21 6 1 that obtained for Pgp reconstituted with sheep brain lipids. By
Pgp-SBL 171 6 9 53 6 3
Pgp-PC:PA 158 6 20 ND comparison, one can estimate that the above reconstituted Pgp
PM 1450 350 specific ATPase activities are 8 –12 times lower than that of
AS30-D/COL10 Pgp-EYL 121 6 6 24 6 1 Pgp embedded in plasma membranes (taking into account the
Pgp-SBL 181 6 9 56 6 3 Pgp percentage of total plasma membrane proteins; see legend
Pgp-PC:PA 209 6 22 ND
PM 2430 580
of Table III). This is mainly due to the better lipidic environ-
CEM/VLB5 Pgp-EYL 132 6 7 26 6 1 ment of Pgp in the plasma membrane (see “Discussion”). Table
Pgp-SBL 199 6 10 61 6 3 III also shows that in all cases up to 70 – 80% of the ATPase
Pgp-PC:PA 219 6 11 ND activity of reconstituted Pgp was inhibited by 50 mM orthovana-
PM 2450 560
date. This inhibition level is in the range of that observed for
Pgp in plasma membranes (about 76%). It should be noted that
ured under optimized conditions, i.e. 1.5 mM ATP and 6 mM the total inhibition of reconstituted Pgp activity by 200 mM
Mg21 at pH 7.4. When reconstituted into egg yolk lipids, SDS- orthovanadate was always obtained (see Fig. 3A), while for
purified Pgp from P388/ADR25 cells exhibited a specific plasma membranes some residual ATPase activity was ob-
ATPase activity of 110 nmol/min/mg Pgp, which was very close served that was not sensitive to both orthovanadate and known
to that obtained for Chaps-purified Pgp (130 nmol/min/mg plasma membrane ATPase inhibitors used in the ATPase assay
Pgp). Experiments with AS30-D/COL10 and CEM/VLB5 Pgp samples.
also showed that SDS- and Chaps-purified Pgp displayed al- Activators of Pgp ATPase Activity—Table IV shows that
most identical ATPase activity once they were reconstituted verapamil was the most effective activator among the drugs
into liposomes. Fig. 3A shows that the activities of SDS- and tested. Verapamil could stimulate the ATPase activity of re-
Chaps-purified Pgp from P388/ADR25 cells were sensitive to constituted Pgp (either Pgp-egg yolk lipids or Pgp-sheep brain
orthovanadate with 50% inhibition at around 12.5 mM or- lipids) up to 2-fold, while colchicin and vinblastine were less
thovanadate in both cases. At 200 mM orthovanadate, Pgp effective, and both gave a 1.5-fold maximal stimulation. In the
ATPase activity was completely inhibited. In contrast, these case of Pgp in plasma membranes, all of the drugs used were
ATPase activities could be stimulated by verapamil at low less effective than in the case of reconstituted Pgp. Further-
concentrations (Fig. 3B), which gave maximally 2.1-fold stim- more, higher concentrations of verapamil (10 instead of 5 mM)
ulation at 5 mM. However, at verapamil concentrations higher and vinblastine (1.5 instead of 1 mM) were needed to give
than 10 mM, the Pgp ATPase activity became inhibited. These maximal stimulation.
verapamil effects were also observed for Pgp in plasma mem- Taken together, all of the above data indicate that either
branes, but in that case, the maximal stimulation was obtained SDS- or Chaps-purified Pgp, after reconstitution into lipo-
with a higher drug concentration (10 mM, see Table IV). somes, displayed a substantial ATPase activity that was lipid-
Effects of Lipids on the ATPase Activity of Pgp—Table III dependent and could be modulated by a number of drugs.
shows that there were only slight differences in the specific
ATP-dependent [3H]Vinblastine Transport by
ATPase activity among purified murine (P388/ADR25), rat
Reconstituted Purified Pgp
(AS30-D/COL10), and human (CEM/VLB5) Pgp when reconsti-
tuted into the same type of liposomes. However, Pgp specific Pgp has been shown to be an ATP-dependent drug efflux
ATPase activity varied with the lipids used. An average pump in plasma membrane vesicles prepared from multidrug-
ATPase activity of 184 nmol/min/mg Pgp can be calculated for resistant cancer cells (31, 37–39) as well as in proteoliposomes
Pgp reconstituted with sheep brain lipids (L-a-phosphatidyleth- reconstituted by partially or almost purified Pgp (90% pure) (9,
anolamine extract), which is higher (about 1.5-fold) than that of 40 – 42). We therefore examined the characteristics of drug
Pgp reconstituted with egg yolk lipids (60% PC extract, 120 transport of our highly SDS-purified Pgp and compared them
nmol/min/mg Pgp). Pgp reconstituted under well controlled with those of plasma membranes. As shown in Fig. 4A, in the
phospholipid composition designed for drug transport (PC:PA, presence of 1.5 mM ATP, the amount of [3H]vinblastine taken
10:1 (w/w); see below) exhibited an ATPase activity similar to up by reconstituted CEM/VLB5 Pgp increased linearly with the
 Purification and Reconstitution of P-glycoprotein 28881

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FIG. 4. [3H]Vinblastine uptake by reconstituted purified Pgp
from CEM/VLB5 cells. SDS-purified Pgp was exchanged into dodecyl FIG. 5. [3H]Vinblastine uptake by plasma membranes from
maltoside as described before reconstitution into PC:PA (10:1) lipo- CEM/VLB5 cells. Plasma membranes from CEM/VLB5 cells were
somes (see “Experimental Procedures”). Reconstituted CEM/VLB5 Pgp prepared as described under “Experimental Procedures.” ATP-depend-
(5 mg) was incubated at 30 °C for 30 s in 200 ml of reaction medium ent [3H]vinblastine uptake was measured as a function of time (A) and
containing 10 mM Tris-HCl, pH 7.4, 1.5 mM ATP, 6 mM MgCl2, 0.25 M ATP concentration (B) as described in the legend of Fig. 4 and calcu-
sucrose, 3 mM creatine phosphate, 30 mg/ml creatine kinase before lated by subtracting blank values obtained in the presence of AMP
adding [3H]vinblastine (final concentration is 25 nM). ATP-dependent instead of ATP. In each experiment, the amount of total membrane
[3H]vinblastine uptake was measured as a function of time (A) or ATP protein in which Pgp represented about 20% (w/w) was 50 mg. Data
concentration (B) and calculated by subtracting blank values obtained points represent the mean 6 S.D. of triplicate determinations.
in the presence of AMP instead of ATP. Data points represent the
mean 6 S.D. of triplicate determinations. that calculated for both [3H]vinblastine uptake by P388/ADR25
and AS30-D/COL10 plasma membrane Pgp (13.5 6 2.6 and
incubation time during 10 min and reached a steady state at 20 13.5 6 1.0 pmol/mg protein per 20 min, respectively, i.e. 68
min. The [3H]Vinblastine uptake slowly decreased after 20 pmol/mg Pgp per 20 min for both). These results suggest that
min, suggesting that [3H]vinblastine diffused out of the proteo- the ATP-dependent drug transportation catalyzed by reconsti-
liposomes and that a continuous supply of ATP is needed for tuted SDS-purified Pgp appears as efficient as that by Pgp
vinblastine transport (31). The maximal [3H]vinblastine up- embedded in plasma membranes.
take at 20 min was 111 6 36 pmol/mg of Pgp. Fig. 4B shows
that the [3H]vinblastine uptake was dependent on ATP concen- DISCUSSION
tration and that 1.5 mM was observed as the optimum ATP We have established three highly multidrug-resistant cell
concentration. Higher concentrations of ATP inhibited drug lines, i.e. murine lymphoid leukemia P388/ADR25, rat hepa-
transport, a phenomenon also observed for the ATPase activity toma AS30-D/COL10, and human lymphoblastic leukemia
of Pgp. It should be mentioned that the optimum ATP concen- CEM/VLB5 cells. These cells constitutively overproduce Pgp up
tration for Pgp ATPase activity was also 1.5 mM, indicating the to 25–30% by weight of total plasma membrane proteins. Un-
close coupling between ATP hydrolysis and ATP-dependent fortunately, the reported methods for purification of Pgp did
drug uptake. not work properly with these cell lines. Thus, by combining
These features of [3H]vinblastine uptake by reconstituted SDS solubilization with ceramic hydroxyapatite HPLC, we de-
Pgp are very similar to those observed for plasma membranes veloped a new, simple, reliable and high yielding procedure for
(Fig. 5). However, the [3H]vinblastine uptake by CEM/VLB5 efficient purification of Pgp that was effective for the three
plasma membranes linearly increased with time up to 20 min, tumor lines. The ATPase activity of highly SDS-purified Pgp
but the steady-state uptake was also obtained at 20 min as for was restored after exchange of SDS into dodecyl maltoside and
reconstituted Pgp. Moreover, the maximum [3H]vinblastine up- reconstitution into liposomes. This activity was close to that of
take was also obtained for 1.5 mM ATP (23.4 6 0.9 pmol/mg of Pgp purified with Chaps, indicating that SDS did not irrevers-
protein per 20 min). Although the nature of the vesicles is quite ibly denature the Pgp. Also, this reconstituted ATPase activity
different in terms of lipid and protein composition, the recon- could be modulated by a number of drugs, especially those used
stituted Pgp transport activity (111 pmol/mg Pgp per 20 min) is for selection of cell resistance, and was shown to be more sensi-
very close to that obtained for plasma membrane Pgp (117 tive to drug stimulation than Pgp in plasma membranes. Fur-
pmol/mg Pgp per 20 min) calculated when taking into account thermore, the reconstituted Pgp was shown to transport [3H]vin-
the percentage of Pgp in plasma membranes (about 20% of total blastine with an efficiency comparable with plasma membrane-
proteins). Similarly, the [3H]vinblastine uptake by reconsti- embedded Pgp. The drug transport features reported in this work
tuted purified P388/ADR25 and AS30-D/COL10 Pgp (119 6 10 constitute the first detailed demonstration of ATP concentration
and 97 6 25 pmol/mg Pgp per 20 min, respectively) are close to and time dependences for highly purified reconstituted Pgp.
28882 Purification and Reconstitution of P-glycoprotein
Purification of membrane proteins by chromatographic plasma membranes and that Pgp functioning may be modu-
methods unavoidably begins with solubilization, during which lated by its state of oligomerization (44, 46). Furthermore, our
proteins are dispersed into solution from the anchoring lipid previous work also indicated the tendency of the murine Pgp
bilayer. Effective solubilization of membrane proteins is accom- C-terminal nucleotide-binding domain to form oligomers (47),
panied by the appropriate choice of detergent and buffer sys- as confirmed recently (48). Besides this oligomerization state of
tems (16). Because SDS was able to solubilize Pgp almost membrane Pgp, we assume that the high level of overexpres-
completely (Table I), we used it to set up the Pgp purification sion reached would favor an aggregation state of the protein
procedure, although SDS is well known to inactivate proteins. into the membrane, perhaps yielding the formation of Pgp
In order to compare the activity of purified Pgp after removal of clusters. This may explain the difficulties that we encountered
SDS, we also purified Pgp solubilized with Chaps because (i) it in solubilizing Pgp.
was the most effective mild detergent in the presence of high Chaps-solubilized hamster Pgp was shown to retain partial
concentrations of salt (Table I) and (ii) it was reported to allow or complete ATPase activity during the reported purification
the preparation of active Pgp (13, 43). The key point of the Pgp procedures (13, 43). However, in our case, Chaps-purified Pgp
separation was the use of ceramic hydroxyapatite HPLC col- in the presence of salt, like SDS-purified Pgp, did not display
umn, which allowed the complete purification of Pgp in only any ATPase activity. This is likely a consequence of the phos-
one step in the presence of 1% SDS or 1% Chaps after pH and pholipid depletion in Pgp-detergent mixed micelles during sol-
phosphate gradient optimization. This one-step chromato- ubilization and, probably more importantly, during the HAP-
graphic purification was possible because Pgp bound strongly HPLC in the presence of a high concentration of detergent.
to hydroxyapatite and it was the last protein eluted from the Indeed, detergent delipidation was shown to inactivate Pgp
column. The presence of DTT was required to prevent protein ATPase activity, which could subsequently be restored by the
aggregation during HAP-HPLC. Although Chaps was able to addition of phospholipids (43, 49). Accordingly, restoration of
solubilize up to 60% Pgp in the presence of 1 M NaCl, the Pgp ATPase activity and its drug sensitivity, as well as drug

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recovery of purified Pgp from HAP-HPLC in the presence of transport activity, were readily achieved in the present work by
Chaps was much lower (10%) than in the presence of SDS reconstitution into liposomes. Although SDS-purified Pgp
(75%). Obviously, this is due to the poor efficiency of Chaps in needed the exchange of SDS into mild detergent prior to recon-
disrupting nonspecific protein association, resulting in Pgp stitution, there was no significant difference in reconstituted
aggregation once the Chaps-mixed micelles interact with HAP- activity between Chaps- and SDS-purified Pgp, showing that
HPLC matrix. Indeed, it has been shown by sucrose gradient SDS does not irreversibly denature Pgp although some dena-
velocity sedimentation that Chaps solubilized Pgp essentially turation could not be completely ruled out. These results
as oligomers, while SDS yielded essentially monomers (44).
clearly demonstrate that SDS can be successfully used to purify
Moreover, SDS is known to overcome the formation of aggre-
Pgp without loss of Pgp function provided that SDS is effi-
gates during chromatographic separations (24). One main ad-
ciently removed. In fact, the detergent exchange procedure we
vantage for the use of SDS solubilization and HAP-HPLC of
report here completely fulfills this requirement.
Pgp is its general applicability, whatever the cell line used,
The basal specific ATPase activity of lipid-depleted Chaps-
with only a minor adjustment of phosphate gradient to opti-
and SDS-purified Pgp obtained after reconstitution (about 200
mize the separation of Pgp on HAP-HPLC. The yield of SDS-
nmol/min/mg Pgp for each cell line) is in the range of that
purified Pgp is considerably high for all three cell lines (i.e.
reported by Shapiro and Ling (12) for 90% pure Pgp, but it
about 70% of overall Pgp in plasma membranes; in other words,
remains about 8 times lower than that for Pgp preparations for
0.2 mg of purified Pgp/mg of plasma membrane proteins). This
which no attempt was made to delipidate the protein (11, 13).
is the highest yield of purified Pgp ever reported. In contrast,
This activity is also 8 –12 times lower than that of plasma
the yield of Chaps-purified Pgp is 10 times lower. This discrep-
membrane-embedded Pgp, although it is dependent on the
ancy between Pgp yields justifies the development of the SDS
sources of lipids used (Table III). Pgp ATPase activity was
purification procedure.
Apart from the fundamental value of hydroxyapatite for Pgp indeed reported to be modulated by phospholipids (43, 49),
purification, another very interesting application is its use for some of which allowed a great stimulation of catalytic activity
detergent exchange (45) as described here for SDS exchange whereas others produced inhibition (13). Furthermore, Sharom
into dodecyl maltoside. SDS present in Pgp preparation was and co-workers (13) reported that Pgp selectively associates
easily exchanged into a mild detergent by extensively washing with only certain phospholipid species, and their Pgp prepara-
the column with almost no loss of Pgp. In addition, hydroxyap- tion was associated with an unidentified phospholipid. Specific
atite can also be used to concentrate diluted Pgp solution with- lipids are possibly required for Pgp catalytic activity, and this
out concentrating the detergent micelles, which is the major unidentified species might play an important role. Several
problem when using ultrafiltration. The detergent exchange other factors may account for the observed different level of the
procedure would be applicable to other membrane proteins, ATPase activity between reconstituted and plasma membrane
and other detergents such as Chaps, Zwittergent 3–12, octyl- Pgp: (i) the real amount of Pgp functionally reconstituted into
glucoside, deoxycholate, octaethyleneglycol mono-n-dodecyl liposomes, (ii) the orientation of Pgp in the proteoliposomes
ether, etc. could also be efficiently exchanged by this method.2 (inside-out/right side-out Pgp distribution), (iii) the size of re-
Both SDS- and Chaps-purified Pgp were obtained with pu- constituted vesicles, (iv) the oligomerization state of Pgp, (v)
rity exceeding 99% of detected protein (Fig. 2). In addition to the protein:phospholipid ratio, (vi) the presence of traces of
monomeric Pgp, some oligomerized Pgp was also detected, es- detergent in reconstituted membranes, (vii) the lack of possible
pecially in the CEM/VLB5 cell line in comparison with the modulation of Pgp activity by other plasma membrane pro-
other two lines. Although it is a common tendency for mem- teins, etc. In addition, the enzymatic activity of membrane
brane proteins to aggregate in the detergent-solubilized state, proteins is often difficult to estimate because it varies greatly
several reports have suggested that Pgp oligomers exist in with the conditions of preparation and measurement used. This
is particularly obvious for the Pgp ATPase activity, which is
activated by several compounds. For example, in the case of
2
M. Dong, L. G. Baggetto, P. Falson, M. le Maire, and F. Penin, P388/ADR25, if the comparison between reconstituted and
submitted for publication. plasma membrane Pgp is made on the basis of the ATPase
 Purification and Reconstitution of P-glycoprotein 28883
activity obtained under maximal stimulation by verapamil, Montserret for help with chromatography; J. L. Rigaud for help with
and assuming that only 50% of reconstituted Pgp was right reconstitution experiments; A. Berne, I. Guilhot, J. Vigouroux, B.
Lermite, and C. Tenin for help with cell culture; C. Van Herrewège for
side-out, one can calculate that the reconstituted Pgp activity is art work; A. Bosch for photography; and Roger Bellon Laboratories for
only about 3-fold lower than that observed for plasma mem- the gift of vinblastine. We also thank M. le Maire and P. Falson for
brane Pgp. helpful criticisms.
Nevertheless, the reconstitution conditions used here al-
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3
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