by P-glycoprotein. The stimulatory effect of vinblastine was observed only if the protein was reconstituted in proteolipo- somes, suggesting that either the ...
Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 8472-8476, September 1992 Biochemistry
Partial purification and reconstitution of the human multidrug-resistance pump: Characterization of the drug-stimulatable ATP hydrolysis (octyl glucoslde/P-glycoprotein/proteoliposome/vinblastlne/verapamil)
SURESH V. AMBUDKAR*tI, ISABELLE H. LELONG§, JIAPING ZHANG*, CAROL 0. CARDARELLI1, MICHAEL M. GOTTESMAN§, AND IRA PASTAN¶ *Division of Nephrology, Department of Medicine, and tDepartment of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; §Laboratory of Cell Biology and IMolecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
and
Contributed by Ira Pastan, June 3, 1992
binding domains, P-glycoprotein belongs to the ATP-binding cassette (ABC) superfamily of transporters (4, 6, 7). This superfamily now includes >40 members (for a recent list, see refs. 6 and 7) and the substrates transported by these members include ions, solutes, peptides, proteins, and cytotoxic natural product drugs. Several previous reports suggest the association ofATPase activity with P-glycoprotein: (i) the ATP analog 8-azido[32-P]ATP binds to P-glycoprotein (8); (ii) the immunoaffinity-purified protein exhibits a low level of ATPase activity (9); and (iii) in vitro mutagenesis of the consensus sequences of either or both ATP-binding domains of the MDR] cDNA fails to confer drug resistance in transfected cells that express the altered protein (10, 11). In addition, the ATP-dependent transport of vinblastine by membrane vesicles of multidrugresistant cells has been demonstrated (12). Thus, it has been proposed that P-glycoprotein catalyzes ATP-dependent efflux of drugs from resistant cells. These previous studies, however, do not conclusively prove that the two functions (i.e., ATP hydrolysis and drug transport) are directly mediated by P-glycoprotein. To establish that P-glycoprotein is an ATP-dependent multidrug transporter, purification and functional reconstitution into phospholipid vesicles is essential. We describe here partial purification and reconstitution of P-glycoprotein. The data suggest that P-glycoprotein exhibits a high level of substrate-stimulatable ATPase activity similar to other ion-transporting pumps.
ABSTRACT Multidrug-resistant human tumor cells overexpress the MDRI gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump. In this study we demonstrate that the partially purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis. Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/Vol) ocyl 3-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B. The 0.1 M NaCi fraction, enriched in P-glycoprotein but devoid of NaK-ATPase, was reconstituted by the detergent-dilution method. P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes. ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P-glycoprotein. The stimulatory effect of vinblastine was observed only if the protein was reconstituted in proteoliposomes, suggesting that either the substrate binding site(s) was masked by detergent or that the conformation of the soluble P-glycoprotein might not be suitable for substrate-induced activation. Several other drugs that are known to be transported by P-glycoprotein enhanced the ATPase activity in a dose-dependent manner with relative potencies as follows: doxorubicin = vinblastine > daunomycin > actinomycin D > verapamil > colchicine. The basal and vinblastine-stimulated ATPase activities were inhibited by vanadate (50% inhibition observed at 7-10 saM) but were not affected by agents that inhibit other ATPases and phosphatases. These data indicate that the P-glycoprotein, similar to other ion-transporting ATPases, exhibits a high level of ATP hydrolysis (5-12 gzmol per min per mg of protein).
MATERIALS AND METHODS Preparation of Plasma Membrane Vesides. The multidrugresistant human carcinoma KB-V1 cells, a subclone of KB3-1, were grown in the presence of vinblastine (1 ,g/ml) to confluence (13). The membrane vesicles were prepared by nitrogen cavitation and sucrose density gradient centrifugation as described (12). The final pellet of vesicles was resuspended and stored in vesicle buffer (10 mM Tris HCl, pH 7.4/50 mM NaCl/250 mM sucrose/0.5 mM phenyl-
It is well established that certain tumors and cultured cell lines develop simultaneous resistance to a wide variety of hydrophobic cytotoxic natural product agents such as vinblastine, doxorubicin, actinomycin D, and colchicine. Highly multidrug-resistant cells frequently express large quantities of a 130- to 170-kDa membrane glycoprotein, referred to as the P-glycoprotein or the multidrug transporter, that is encoded by the MDR] gene (1-3). The human MDR] cDNA encodes a protein whose deduced 1280-amino acid sequence is consistent with a secondary structure model of P-glycoprotein, which contains 12 putative transmembrane segments with two ATP (nucleotide) binding domains on the cytoplasmic surface ofthe membrane (1, 4, 5). Due to the considerable similarities in the overall topology and the sequence of ATP
methylsulfonyl fluoride) at -700C. Solubilization of P-Glycoprotein. The solubilization of membrane proteins followed the protocols established earlier (14,15). Briefly, KB-V1 membranes (1.5 mg of protein) were solubilized in a final volume of 1.2 ml with 1.4% (wt/vol) octyl 3-D-glucopyranoside in the presence of 50 mM Mops'Nmethyl-D-glucamine (NMDG), pH 7.4/20% (wt/vol) glycerol [osmolyte protectant (14, 16)]/0.4% of a phospholipid mixture containing Escherichia coli bulk phospholipid, phos-
Abbreviations: NMDG, N-methyl-D-glucamine; DTT, dithiothreitol. tTo whom reprint requests should be addressed at: Division of Nephrology, Ross Research Building, Room 945, 720 Rutland Avenue, Baltimore, MD 21205.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8472
Biochemistry: Ambudkar et al. phatidylcholine, phosphatidylserine, and cholesterol, 60:17.5:10:12.5 (wt/wt), respectively/I mM dithiothreitol (DTT)/protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride/25 gM pepstatin/1 ,uM leupeptin/1 AuM aprotinin). After a 20-min incubation on ice, the solubilized protein was separated by centrifugation for 1 h at 152,000 x g in a Beckman type 50 Ti rotor. DEAE-Sepharose CL-6B Chromatography. The DEAE-
Sepharose column (6 x 1.75 cm) was equilibrated at 40C with buffer A [20 mM Mops-NMDG, pH 7.4/1.25% octyl glucoside/20% glycerol/0.1% lipid mixture] containing E. coli bulk phospholipid (65%) and phosphatidylcholine (35%)/1 mM DTT/0.5 mM EDTA/protease inhibitors (as above). The solubilized protein (1.1-1.4 mg) was applied to the column and the protein was eluted at 0.35-0.4 ml/min with 20 ml of buffer A containing 0, 50, 100, and 150 mM NaCl. A P-glycoprotein-enriched 100 mM salt fraction (20 ml) was concentrated 10-fold by using Amicon Centriprep-30 concentrators. Reconstitution of P-glycoprotein. For reconstitution, crude detergent extract (300-400 ug of protein) or concentrated DEAE-Sepharose 100 mM salt fraction (60-100,ug ofprotein) was mixed with 9-10 mg of bath-sonicated phospholipid mixture [E. coli phospholipid/phosphatidylcholine/phosphatidylserine/cholesterol, 60:17.5:10:12.5 (wt/wt)]/1.25% octyl glucoside/50 mM Mops*NMDG, pH 7.4, in a final volume of 1 ml. The mixture was incubated for 20 min on ice and proteoliposomes or liposomes (prepared without protein) were formed at 23-25°C by a 1:25 to 1:50 dilution into buffer B (50 mM Mops*NMDG, pH 7.4/150 mM NMDG chloride/i mM DTT/0.5 mM phenylmethylsulfonyl fluoride). Proteoliposomes or liposomes were concentrated by centrifugation (14), washed once, and resuspended in 300 ,u of buffer B containing protease inhibitors. Typically, using either crude extract or partially purified fraction, we found that 20-25% of the input protein was recovered in proteoliposomes. ATPase Assays. Samples (1-3 pg of protein per ml) were incubated at 37°C with 0.6 mM [y-32P]ATP (Tris salt, 20 mCi/mmol; 1 Ci = 37 GBq) in 1 ml of 150 mM mannitol/S0 mM Mops*NMDG, pH 7/2 mM DTT/1.2 mM MgCl2. For the DEAE column fraction, 1% octyl glucoside was also present in the assay medium. At specified times, 200 ,ul was mixed with 200 ,ul of ice-cold 20% (wt/vol) trichloroacetic acid and the release of 32P, was measured as described (17). Other Methods. Protein was measured using a modification of the method of Schaffner and Weissmann (18). Protein samples, delipidated by chloroform/methanol extraction (19), were electrophoresed on a SDS/7.5% polyacrylamide gel according to Laemmli (20) and gels were stained with silver. For Western blots, proteins were transferred by standard method (21). The nitrocellulose blot was blocked for 1 h with 2.5% (wt/vol) gelatin in buffer C (150 mM NaCl/ 0.2% Triton X-100/10 mM Tris*HCl, pH 7.4) and incubated with primary antibody [P-glycoprotein-specific polyclonal antiserum 4007 (22) at 1:4000 dilution or Na,K-ATPase a-subunit-specific monoclonal antibody a-5 (1 pg/ml, ref. 23)] in buffer C containing 3% (wt/vol) bovine serum albumin for 1 h at 23°C. After three washes with buffer C, the blot was incubated with secondary antibody (horseradish peroxidaseconjugated goat anti-rabbit IgG or donkey anti-mouse IgG) at 1:4000 dilution for 1 h. The blot was washed with buffer C and developed by using an enhanced chemiluminescence (ECL, Amersham) method. Chemicals. Crude E. coli lipid, phosphatidylcholine (egg), phosphatidylserine (bovine brain), and cholesterol were obtained from Avanti Polar Lipids, Alabaster, Alabama. E. coli lipid was washed with acetone/ether as described (15), and the purified bulk phospholipid is composed of phosphatidylethanolamine (70%6), phosphatidylglycerol (15%), and cardiolipin (15%) (24). Octyl glucoside was provided by Behring Diagnostics. DEAE-Sepharose CL-6B was purchased from
Proc. Natl. Acad. Sci. USA 89 (1992)
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Pharmacia. Ly-32P]ATP (3000 Ci/mmol) was from Amersham. Topotecan and anti-Na,K-ATPase a-subunit monoclonal antibody a-5 were generously provided by L. Liu and D. Fambrough (Johns Hopkins University), respectively. All other reagents were obtained from Sigma.
RESULTS AND DISCUSSION Partial Purification and Reconstitution of P-Glycoprotein. The plasma membranes of multidrug-resistant KB-V1 cells, which are enriched in P-glycoprotein (=1% of total membrane protein) were solubilized with octyl glucoside in the presence of lipid and glycerol (osmolyte protectant). In previous studies we have used this protocol for solubilizing, without loss of activity, several transport proteins including the bacterial histidine permease, which is a member of the ATP-binding cassette superfamily (14, 16, 17, 25). With these relatively mild extraction conditions, 40-50% of the plasma membrane protein is recovered in the crude detergent extract. This detergent extract contains >90%o of the total P-glycoprotein content of the plasma membranes (data not shown). The major goal of this study is to devise conditions to assess P-glycoprotein-mediated ATP hydrolysis and the drug transport. Since plasma membranes of KB-V1 cells also contain other ATPases (e.g., Na,K-ATPase and Ca-ATPase) and phosphatases, to examine the P-glycoprotein-associated ATPase activity it is essential to separate this protein from the other ATP hydrolyzing enzymes. We tested various ion-exchange and lectin columns. Only DEAE-Sepharose CL-6B was suitable for separating P-glycoprotein from Na,K-ATPase, the other major ATP-hydrolyzing enzyme. The detergent extract was chromatographed on an anionexchange resin, DEAE-Sepharose CL-6B, and the protein was eluted with 0-0.15 M NaCl in the presence of detergent, lipid, and glycerol to prevent inactivation. The distribution of these two proteins in various DEAE-Sepharose fractions, detected by immunoblot analysis, is given in Fig. 1. Most of the P-glycoprotein (85-90%) is eluted in the 0.1 M NaCl fraction (Fig. 1A, lane 4). On the other hand, Na,K-ATPase, detected with anti-a-subunit monoclonal antibody a-5, is found only in the 0.15 M NaCl fraction (Fig. 1B, lane 5). Thus, the DEAE-Sepharose chromatography yielded a fraction enriched in P-glycoprotein but devoid of Na,K-ATPase. B
A 1
205
2
3
4
V, n
5
1
2 3 4
5
o
=MO.
116
80
:..
.:
am"
FIG. 1. Partial purification and separation of P-glycoprotein from Na,K-ATPase by DEAE-Sepharose CL-6B chromatography. The detergent extract of KB-V1 membranes (1.1-1.4 mg of protein) was applied to a DEAE-Sepharose column preequilibrated with buffer A and the protein was eluted with buffer A and with buffer A containing NaCl at 50 mM, 100 mM, and 150 mM. (A) Western immunoblot analysis with P-glycoprotein-specific polyclonal antiserum 4007 (22). (B) The blot in A was stripped and probed with anti-Na,K-ATPase a subunit monoclonal antibody a-5 (23). Lanes: 1, octyl glucoside extract; 2-5, DEAE-Sepharose column fractions eluted with 0, 50, 100, and 150 mM NaCl, respectively. The 80-kDa protein (A, lane 3) is nonspecifically recognized by 4007 antibody because it was also detected in the material from parental drug-sensitive cell line KB 3-1 (data not shown). Each lane contained 1 j&g of protein. The arrow and arrowhead show the position of P-glycoprotein and Na,K-ATPase a subunit, respectively.
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Biochemistry: Ambudkar
et
al.
A
205
116
80
FIG. 2. Reconstitution of partially purified P-glycoprotein. A silver-stained SDS/PAGE gel profile of proteoliposomes is shown. The octyl glucoside extract of KB-V1 membranes and the 100 mM NaCl eluate (Fig. 1A, lane 4) from the DEAE-Sepharose column was reconstituted by the detergent dilution method. Lanes: 1, KB-V1 plasma membranes; 2, octyl glucoside extract; 3, proteoliposomes reconstituted with octyl glucoside extract; 4, 100 mM NaCl eluate of DEAE-Sepharose column; 5, proteoliposomes reconstituted with 100 mM NaCl eluate. Other details were as described for Fig. 1.
The crude extract or the P-glycoprotein-enriched column fractions were reconstituted into phospholipid vesicles by the detergent-dilution method (14-16). Fig. 2 shows the silverstained protein profile of various fractions and proteoliposomes. The P-glycoprotein migrates in the 170-kDa region as a diffuse band. The nature of the sharp band located just above the P-glycoprotein in lanes 1-4 is at present not known. The 170-kDa protein was identified as the P-glycoprotein by the following criteria: (i) reactivity with polyclonal antiserum 4007 (see Fig. 1A) and (ii) absence in fractions derived from the drug-sensitive parental cell line KB 3-1 (data not shown). P-glycoprotein is 3- to 4-fold enriched in the DEAE fraction as compared to the crude extract (Fig. 2, lanes 2 and 4). The reconstitution of the DEAE fraction into proteoliposomes results in an additional 2- to 3-fold enrichment (Fig. 2, lanes 4 and 5). The intensity of the stained material indicates that the P-glycoprotein constitutes 25-30% of total protein in proteoliposomes reconstituted with the DEAE-Sepharose fraction (Fig. 2, lane 5). ATP Hydrolysis by Soluble and Reconstituted DEAESepharose Fraction Enriched in P-Glycoprotein. To evaluate ATPase activity of reconstituted P-glycoprotein, it is essential to determine the orientation of the protein in proteoliposomes. The covalent cross-linking of P-glycoprotein with [a-32P]ATP in intact and permeabilized proteoliposomes showed that P-glycoprotein is reconstituted with predominantly (>90%) inside-out orientation (i.e., with ATP binding sites facing the extravesicular medium; unpublished data). The ATPase activity of various fractions was examined at 370C by measuring 32p; liberation from [y-32P]ATP. The ATP hydrolysis by the proteoliposomes prepared with the crude detergent extract (Fig. 2, lane 3) was measured and the vanadate-sensitive activity ranged between 0.4 and 0.6 /Lmol per min per mg of protein. Vinblastine (20 1LM) or verapamil (100 AM) increased the activity only by 25-30%o. Similar results were obtained with the crude detergent extract (data not given). The marginal stimulation observed with these drugs may be because of high background activity due to the presence of other ATPases and phosphatases. To reduce the high background activity, we used the DEAE-Sepharose fraction that contains partially purified P-glycoprotein. The ATPase activities of the DEAE-Sepharose 0.1 M NaCl eluate (soluble fraction) and proteoliposomes prepared with the same fraction are given in Table 1. The level of activity in the soluble or reconstituted fraction was the same. The activity of the soluble fraction was measured in the presence of 1%
Proc. Natl. Acad. Sci. USA 89 (1992) Table 1. ATP hydrolysis by soluble and reconstituted DEAE-Sepharose fraction enriched in P-glycoprotein ATP hydrolysis, Aumol per min per mg of protein Treatment Sample 1.08 None Soluble fraction 1.25 Vinblastine (20 tiM) 0.10 Vanadate (250 ILM) Reconstituted 1.30 proteoliposomes None 3.85 Vinblastine (20 ,uM) 0.22 Vanadate (250 AM) DEAE-Sepharose 0.1 M NaCl eluate fraction (soluble fraction) and proteoliposomes reconstituted with the soluble fraction (Fig. 2, lanes 4 and 5; 2 ,ug of protein per ml) were incubated with the indicated drug or with equal volume of dimethyl sulfoxide for 2 min and ATPase activity was measured as described. The values are the mean of two measurements.
octyl glucoside to prevent the possible formation of proteoliposomes due to dilution of detergent in the assay medium since this fraction contains 0.1% lipid. It is very likely that the level of ATPase in the soluble fraction is underestimated due to the inhibitory effect of the detergent. In earlier studies, the ATPase activity of the immunoprecipitated P-glycoprotein was shown to be inhibited by the detergent 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) at higher (>0.2%) concentrations (26). In both soluble and reconstituted preparations of P-glycoprotein, the ATPase activity was significantly (>80-90%) inhibited by vanadate. Since the DEAE-Sepharose fraction is enriched in P-glycoprotein and totally free of Na,K-ATPase, it is likely that the observed activity may be due to P-glycoprotein, in which case the ATPase activity should be stimulated by the substrates that are known to interact with this protein. Addition of vinblastine, the substrate of P-glycoprotein, resulted in 3-fold stimulation of the ATPase activity of proteoliposomes. However, the activity of the soluble fraction was not affected by this drug (10-20% increment was observed at 20-40 uM, Table 1). Similarly, the reversing agent verapamil also failed to stimulate ATPase activity in the soluble fraction (data not shown). The lack of effect of these agents may be due to an interaction of the hydrophobic detergent with the drugsubstrate binding site(s) on the P-glycoprotein. Alternatively, the conformation of the soluble P-glycoprotein may not be suitable for substrate induced activation. Recent work by Davidson et al. (27) indicates that the maltose-stimulated ATP hydrolysis by maltose permease of E. coli is observed only with the reconstituted protein. These observations are consistent with our data. For further characterization of the substrate-stimulated ATPase activity of P-glycoprotein, we used proteoliposomes reconstituted with DEAE-Sepharose fraction (Fig. 2, lane 5). Effect of Various Drugs on ATP Hydrolysis by Proteoliposomes Enriched in P-Glycoprotein. Proteoliposomal activity was greatly stimulated by the addition of vinblastine or verapamil, and such enhancement was blocked by vanadate (Fig. 3). Verapamil, the reversing agent, has also been shown to inhibit vinblastine transport competitively in plasma membrane vesicles of KB-V1 cells (12). The stimulation of the ATPase activity was not observed with another hydrophobic antitumor agent, camptothecin, that is not transported by P-glycoprotein (28). On the other hand, topotecan, a positively charged derivative of camptothecin, which is known to interact with P-glycoprotein, enhanced ATP hydrolysis 2.5fold at 100 ,uM. These data suggest that the stimulation of ATPase activity is not nonspecifically induced due to membrane perturbation resulting from the addition of the hydro-
Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistry: Ambudkar et al.
8475
4 .0
3
0
~0a-
CD
2
E
Q-
a)
0
3 VINB
+V04
+V04
+V04
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.E a) E
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FIG. 3. Effect of various drugs on ATPase activity of proteoliposomes containing partially purified P-glycoprotein. Proteoliposomes (1-3 pg of protein per ml) were incubated with the indicated drug in the presence and absence of vanadate for 2 min and ATPase activity (,umol per min per mg) was measured. CONT, control (dimethyl sulfoxide treated); VER, 100 AM verapamil; VINB, 20 uM vinblastine; CAM, 50 uM camptothecin; +V04, 250 juM sodium orthovanadate.
phobic compounds but rather is due to the specific interaction of vinblastine, verapamil, or topotecan with P-glycoprotein. Several drugs that have previously been shown to interact with P-glycoprotein were tested for their effect on ATPase activity of proteoliposomes. All these drugs enhanced the ATPase activity in a dose-dependent manner. The halfmaximal stimulation was achieved with doxorubicin, vinblastine, and colchicine at 3 1uM, 4 ,M, and 45 ,uM, respectively (Table 2). Vinblastine transport in membrane vesicles of these cells has been observed with an apparent Km of 2 AM (12). It is important to note that due to the high lipid to protein ratio (400-600:1) of proteoliposomes, a significant portion of hydrophobic drug may bind to the lipid; therefore, the concentration required for half-maximal stimulation may be lower than the observed value. The degree of maximal stimulation was different with different drugs (Table 2). It should be noted that all drugs at higher concentrations (>100 ,uM) caused measurable inhibition; however, the mechanism of inhibitory action of the drugs is not known at present. Effect of ATP Concentraionon Vinblastine-Stimulated ATPms. The effect of ATP concentration on the proteoliposomal ATPase in the presence and absence of vinblastine is shown in Fig. 4. Although vinblastine (20 ,M) stimulated ATPase activity at ATP concentrations ranging from 0.05 to 1.2 mM, it had no effect on the apparent Km for ATP (285 ,uM and 290 ALM in the absence and presence of vinblastine, respectively; Fig. 4B). Maximal ATPase activity of 2.6 and 8.2 ,umol per min per mg of protein (average of two determinations), Table 2. Effects of selected drugs on the ATPase activity of proteoliposomes reconstituted with partially purified P-glycoprotein Fold Concentration required for stimulation half-maximal activation, ,uM Drug 1.00 None 3.15 3 Doxorubicin 3.50 4 Vinblastine 2.10 6 Daunomycin 3.10 10 Actinomycin D 3.75 35 Verapamil 1.70 45 Colchicine ATPase activity of proteoliposomes (1-3.5 pg of protein per ml) was measured as described in Fig. 3. For each drug, at least five concentrations in the range of 0.1-100 ,uM were used to estimate the values presented. Only the vanadate-sensitive activities are given. The values are the mean of two or three measurements using at least two preparations. The concentration required for maximal stimulation varied between 20 and 100 jzM, and in all cases higher concentrations (>100 ,uM) were inhibitory.
0
10
40 30 20 ATP hydrolysis/[ATP] mM
50
FIG. 4. Effect of ATP concentration on the ATPase activity of proteoliposomes. (A) The ATP/Mg molar ratio was kept constant at 1:2 and the ATPase activities were measured in the absence (open circles) and presence of 20 ,uM vinblastine (solid circles). (B) Eadie-Hofstee plot of the data shown in A. The apparent Km values, 285 and 290 AM, were obtained in the absence and presence of vinblastine, respectively.
respectively, was observed in the absence and presence of vinblastine. Based on the estimation of P-glycoprotein concentration (25-30%o) in proteoliposomes (Fig. 2, lane 5), the ATPase activity of pure P-glycoprotein ("basal level") should be 5-12 ,umol per min per mg of protein, which is comparable to the specific activities of other known iontransporting ATPases (29). Vinblastine-stimulated ATPase activity is, therefore, in the range of 15-38 ,umol per min per mg of protein. While this manuscript was in preparation, a report by Sarkadi et al. (30) appeared showing that the membranes of baculovirus-MDRI-infected insect (Sf9) cells exhibit vanadate-sensitive ATPase activity that is enhanced by its substrates. The level of drug-stimulated ATPase activity associated with P-glycoprotein expressed in Sf9 cells was estimated to be in the range of 3-5 ,mol per min per mg of protein, which is somewhat lower than that observed with material from mammalian cells. Both estimates, however, are '1000-fold higher than the activity previously reported to be associated with the purified protein (9). The mammalian drug-resistant cells exhibit higher glycolytic capacity (31) and also contain higher levels of ATP (32), which may be sufficient to support the functioning of P-glycoprotein as a highly efficient drug efflux pump. Effects of Various ATPase and Phosphatase Inhibitors on Proteoliposomal ATPase Activity. The ATPase activity of proteoliposomes was characterized with respect to its ionic requirements and inhibitor sensitivity. The presence of Na+, K+, and Cl- at 150 mM had no effect on either basal or vinblastine-stimulated activity. ATPase activity, however, required the presence of magnesium and optimal activity was observed at an ATP/Mg molar ratio of 1:2. The effect of the pH of the assay medium was tested at the pH values from 6.0 to 8.5, and maximal activity was observed at pH 7.5 (data not shown). Various agents that are known to inhibit F-, P-, and V-type ATPases (29) and phosphatases [levamisole specifically inhibits intestinal and tumor alkaline phosphatase (33)]
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Table 3. Effects of various ATPase and phosphatase inhibitors on P-glycoprotein-associated ATPase in proteoliposomes ATP hydrolysis, Concentration, Test compound mM % control No addition 100 Ouabain 1.0 97.6 Sodium azide 2.0 91.0 Oligomycin 0.003 85.0 N-Ethylmaleimide 0.050 102.5 EGTA 1.00 95.0 Sodium fluoride 110.0 5.0 Levamisole 2.00 115.0 Vanadate 0.25 10.0 Proteoliposomes (1-3 pg of protein per ml) were incubated with the indicated agent for 5 min prior to the determination of the ATPase activity. The values are the mean of two measurements. The ATPase activity of control proteoliposomes ranged between 0.95 and 1.30 ,umol per min per mg of protein. With 7-10 p&M vanadate, 50%o inhibition was observed.
were tested and, except for vanadate, all other agents were found to be without any effect (Table 3; similar results were also obtained in the presence of 20 ,uM vinblastine). It is clear that common ATPases (e.g., Na,K-, Ca-, and F0-FjATPases) and phosphatases do not contribute significantly to the activity we observed in proteoliposomes. Only 7-10 ,uM vanadate was required to obtain 50%o inhibition of ATPase activity (Table 3). Vanadate has also been shown to inhibit vinblastine transport in membrane vesicles (12). The mechanism of vanadate inhibition remains unclear at present. Earlier attempts failed to detect a phosphorylated intermediate of P-glycoprotein (34). In addition, the aspartyl-phosphate phosphorylation site, conserved from bacteria to humans in the P-type ATPases (29), is not found in the P-glycoprotein sequence (4, 5). It is possible that another site for covalent enzyme-phosphate complex may be present in P-glycoprotein and other ATP-binding cassette transporters, since the histidine (35) and maltose (27) permease-mediated activities have also been shown to be inhibited by vanadate. The activities of many pumps [e.g., Na,K-ATPase, CaATPase, arsenite/antimonite ATPase (36)] are stimulated by substrates even when the protein is in soluble form. But the substrate stimulation of P-glycoprotein-mediated ATP hydrolysis is observed only with the reconstituted protein (Table 1). This is a rather unexpected finding. For histidine (25) and maltose permeases (27), the enhancement of activity is observed only when the substrate is also concurrently transported across the liposomal membrane. Thus, it is possible that the reconstituted P-glycoprotein that exhibits drug-stimulatable ATPase activity may also be capable of the drug transport. To establish this coupling between ATP hydrolysis and drug transport, it will be necessary to purify the P-glycoprotein to homogeneity. The reconstitution system described herein should prove usefil for these drug transport studies as well as for screening potential substrates, inhibitors, and reversing agents. We thank Drs. L. Liu and D. Fambrough for the gift of topotecan and anti-Na,K-ATPase a-subunit monoclonal antibody a-5, respectively. This work (S.V.A. and J.Z.) was supported in part by the Institutional American Cancer Society Grant IRG-11-31.
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