Correlation between Steady-state ATP Hydrolysis and Vanadate-induced ADP Trapping in Human P-glycoprotein

EVIDENCE FOR ADP RELEASE AS THE RATE-LIMITING STEP IN THE CATALYTIC CYCLE AND ITS MODULATION BY SUBSTRATES*

Kathleen M. KerrDagger, Zuben E. Sauna, and Suresh V. Ambudkar§

From the Laboratory of Cell Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, November 3, 2000, and in revised form, December 11, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resistance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis. The objective of this study was to understand how substrates affect the catalytic cycle of ATP hydrolysis by Pgp. The ATPase activity of purified and reconstituted recombinant human Pgp was measured using a continuous cycling assay. Pgp hydrolyzes ATP in the absence of drug at a basal rate of 0.5 µmol·min·mg-1 with a Km for ATP of 0.33 mM. This basal rate can be either increased or decreased depending on the Pgp substrate used, without an effect on the Km for ATP or 8-azidoATP and Ki for ADP, suggesting that substrates do not affect nucleotide binding to Pgp. Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal rate, they do not completely inhibit the activity. Therefore, these drugs can be classified as substrates. Vanadate (Vi)-induced trapping of [alpha -32P]8-azidoADP was used to probe the effect of substrates on the transition state of the ATP hydrolysis reaction. The Km for [alpha -32P]8-azidoATP (20 µM) is decreased in the presence of Vi; however, it is not changed by drugs such as verapamil or cyclosporin A. Strikingly, the extent of Vi-induced [alpha -32P]8-azidoADP trapping correlates directly with the fold stimulation of ATPase activity at steady state. Furthermore, Pi exhibits very low affinity for Pgp (Ki~30 mM for Vi-induced 8-azidoADP trapping). In aggregate, these data demonstrate that the release of Vi trapped [alpha -32P]8-azidoADP from Pgp is the rate-limiting step in the steady-state reaction. We suggest that substrates modulate the rate of ATPase activity of Pgp by controlling the rate of dissociation of ADP following ATP hydrolysis and that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Multidrug resistance, the reduced sensitivity to a variety of structurally unrelated, hydrophobic, chemotherapeutic agents, is a major problem in cancer treatment. This phenomenon is often associated with the overexpression of the human multidrug resistance gene (MDR1) (1, 2). MDR1 encodes a 170-kDa plasma membrane protein, P-glycoprotein (Pgp),1 that uses the energy from ATP hydrolysis to expel a variety of anticancer drugs from cells, thus making them ineffective during chemotherapy. The secondary structure of Pgp is predicted to consist of two homologous halves each containing six putative transmembrane helices and a nucleotide-binding domain. These structural elements are common to a large family of membrane transporters called the ATP-binding cassette superfamily (3, 4).

The widely accepted hypothesis of Pgp function is that substrate transport is coupled to ATP hydrolysis. However, the mechanism for this reaction is not well understood. In the absence of added substrate, Pgp catalyzes basal ATP hydrolysis (ATPase activity). This basal activity has been suggested to occur because of an endogenous lipid substrate(s) or an uncoupling of ATP hydrolysis and drug extrusion (5, 6). The basal rate is stimulated by adding any one of a variety of hydrophobic drug substrates; these drugs bind with unique apparent affinities and affect the ATPase activity of Pgp to varying degrees (7-9). However, a detailed assessment of the kinetic parameters of ATP hydrolysis in the presence of amphipathic drugs for the identification of the rate-limiting step in the catalytic cycle has not been carried out.

Expression, purification, and reconstitution procedures of endogenous and six histidine-tagged (His6) human Pgp in a heterologous expression system are well described (10-12). Studies on the ATPase activity of Pgp in crude membrane preparations have indicated various drug-stimulated effects on the ATPase activity of Pgp (5); such experiments with crude protein can be difficult to interpret. Clearly, when purifying Pgp, the lipid environment of this membrane protein affects the ATPase activity and should remain constant for comparison of drug-stimulated activities (13-16).

Kinetic schemes for the catalytic cycle of Pgp have been proposed based on binding studies and Vi-induced ADP trapping experiments (17-21). Vi is a proposed transition state analog that replaces Pi immediately after its release upon ATP hydrolysis. No phospho-enzyme intermediate of Pgp has been identified in the catalytic cycle of Pgp, indicating that all intermediates in the Pgp reaction are noncovalent (18). Senior and his colleagues (18, 19) have extensively characterized the Vi-induced ADP trapping reaction and implied that Pi release precedes ADP release and ADP release is the most likely rate-limiting step in catalysis (22). Our objective in this study is to understand how Pgp drug substrates might affect the overall kinetic mechanism of Pgp. Kinetic constants for various drugs that affect the steady-state ATPase activity of Pgp are quantified, and the extent of Vi-induced ADP trapping is compared in the presence and absence of various Pgp drug substrates. The correlation of these values has mechanistic implications. Our results suggest that ADP release is a rate-limiting step in the catalytic cycle of Pgp, and substrates and modulators exert their effect on ATPase activity by modulating this step.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Octyl beta -D-glucopyranoside and cyclosporin A were purchased from Calbiochem (San Diego, CA). ATP (disodium salt), ADP, progesterone, prazosin, valinomycin, verapamil, vinblastine, and staurosporin were purchased from Sigma; tetraphenylphosphonium chloride was from Aldrich; tamoxifen was from Research Biochemicals International (Natick, MA); rapamycin was obtained from the NCI Developmental Therapeutics Program (Bethesda, MD); and PSC833 was the generous gift of Novartis Corp. (East Hanover, NJ). Acetone-ether washed Escherichia coli bulk phospholipids, egg phosphatidylcholine, phosphatidylserine, and cholesterol were obtained from Avanti Polar Lipids (Alabaster, AL). Cycling components, as described below, were purchased from Roche Molecular Biochemicals. 8-AzidoATP and [alpha -32P]8-azidoATP (10-20 Ci/mmol) was purchased from ICN Biomedicals (Irvine, CA).

Expression and Purification of Pgp-- Human Pgp was expressed and purified as previously described (11) with minor changes. Briefly, His6-tagged Pgp was expressed using Trichoplusia ni (High FiveTM; Invitrogen, San Diego, CA) insect cells grown in monolayer cultures and infected with a recombinant baculovirus BV-MDR1(H6) that contains a human MDR1 that encodes Pgp. Crude Pgp-containing membranes were prepared by Dounce homogenization under hypotonic conditions (23), and membrane proteins were solubilized with octyl beta -D-glucopyranoside (1.25%) in the presence of 20% glycerol and lipid mixture (0.1%) (see "Routine Procedures"). Solubilized proteins were subjected to metal affinity chromatography (Talon resin from CLONTECH, Palo Alto, CA) in the presence of 0.95% octyl beta -D-glucopyranoside and 0.04% lipid; 80% purified Pgp was eluted with 100 mM imidazole (11). Pgp in 100 mM imidazole fraction was then concentrated (Centriprep-50, Amicon, Beverly, MA) to ~0.5 mg/ml and stored at -70 °C. Pgp was identified by immunoblot analysis (11) and quantitated by Amido Black protein estimation method as previously described (24).

Reconstitution of Pgp-- Purified Pgp was combined with a lipid mixture (acetone-ether washed E. coli bulk phospholipid, phosphatidylcholine, phosphatidylserine, and cholesterol (60:17.5:10:12.5 w/w/w/w) (8)), previously sonicated for 20 min in the ratio of 1:8 (protein:lipid w/w). This reconstitution mixture was dialyzed extensively (3 h with four buffer changes at 4 °C) with dialysis buffer (50 mM MES-Tris, pH 6.8, 1 mM EGTA, 1 mM dithiothreitol, and 0.1% aprotinin) using Slide-A-Lyzer cassettes (10,000 cut-off from Pierce). Pgp-containing proteoliposomes were collected by centrifugation at 140,000 × g for 1 h at 4 °C and resuspended in dialysis buffer. Protein concentration was estimated by the Amido Black assay as described above.

ATPase Assays-- ATPase activity of purified, reconstituted Pgp was measured by two methods; the end point Pi assay and the continuous cycling assay. In both assays, Pgp-specific activity was recorded as the Vi (0.3 mM)-sensitive ATPase activity. Various test agents were added from 100× stock solutions in Me2SO so that the Me2SO concentration was no greater than 1%; this concentration of Me2SO had no effect on the activity of Pgp or the cycling assay components. For the Pi assay, the amount of inorganic phosphate released over 20 min at 37 °C was measured. ATPase assay buffer (50 mM MES-Tris, pH 6.8, 50 mM N-methyl-D-glucamine chloride, 5 mM NaN3, 1 mM EGTA, 1 mM ouabain, and 2 mM dithiothreitol) was combined with either 5 mM MgCl2 or CoCl2, 0.5-2 µg of purified, reconstituted Pgp, and various Pgp drug substrates for a 5-min preincubation at 37 °C. The reaction was initiated by the addition of 5-7.5 mM ATP and quenched with SDS (final concentration, 2.5%); the amount of Pi released was quantitated using a colorimetric method as previously described (11). For the cycling assay, cycling components (3 mM phosphoenolpyruvate, 0.33 mM NADH, and 10 units/ml both pyruvate kinase and lactate dehydrogenase) were added to the ATPase assay buffer (described above) with 10-15 mM MgCl2 to link the hydrolysis of ATP directly with the oxidation of NADH (12). Purified, reconstituted Pgp (1-10 µg in 100 µl of assay volume) was preincubated with various Pgp drug substrates at 37 °C in a temperature-controlled, 96-well plate spectrophotometer (Spectra MAX 250; Molecular Devices, Sunnyvale, CA). The reaction was initiated by the addition of ATP and monitored at OD340 nm using SoftMax Pro 2.4 software (Molecular Devices) for 5-10 min. The rate of change in absorbance was converted to nmol NADH oxidized per minute using an NADH standard curve; this value is equivalent to nmol ATP hydrolyzed per minute.

Kinetic Analysis-- The drug-stimulated ATPase activities in the presence of saturating ATP concentrations (5-7.5 mM) and various Pgp drug substrates were fit to Equation 1.


&ugr;/E=[V · S/(K<SUB><UP>app</UP></SUB>+S)]+V<SUB><UP>b</UP></SUB> (Eq. 1)
upsilon /E is the ATPase activity at given concentrations of substrates, V is Vmax - Vb with Vmax as the maximal activity, S is the drug substrate concentration, Kapp is the apparent concentration at half-maximal activity, and Vb is the basal rate in the absence of added drug. The fold stimulation of ATPase activity by a given substrate is calculated by Vmax/Vb. Michaelis-Menten parameters were determined for ATP and 8-azidoATP in the presence of saturating Pgp drug substrates, verapamil (50 µM) and cyclosporin A (10 µM); for basal activity (in the absence of added drug) an equivalent volume of Me2SO was added. ATPase activities using various nucleotide concentrations were fit to Equation 2.
&ugr;/E=V<SUB><UP>max</UP></SUB> · [<UP>NTP</UP>]/(K<SUB>m</SUB>+[<UP>NTP</UP>]) (Eq. 2)
[NTP] is the concentration of ATP or 8-azidoATP and Km is the concentration of NTP at half-maximal activity. Inhibition constants for ADP were determined using various concentrations of ADP and ATP in the presence and absence of 50 µM verapamil. ATPase activities were fit to Equation 3 for competitive inhibition.
&ugr;/E=V<SUB><UP>max</UP></SUB> · [<UP>ATP</UP>]/(K<SUB>m</SUB>(1+[<UP>ADP</UP>]/K<SUB>i</SUB>)+[<UP>ATP</UP>]) (Eq. 3)
Ki is the concentration of ADP at half-maximal inhibition. All curve fits in kinetic analyses were performed using Prism 2.0 software for Power-Mac (GraphPad, San Diego, CA).

Vanadate-induced 8-azidoADP Trapping in Pgp-- Vi-induced 8-azidoADP trapping assays contained ATPase assay buffer, 0.3 mM Vi, either MgCl2 or CoCl2 (5 mM), 0.2-0.8 mg/ml purified Pgp (reconstituted), Pgp drug substrate, and 5-100 µM [alpha -32P]8-azidoATP (2.5-10 µCi/nmol). Reactions were preincubated in low light or semi-darkness in the absence of [alpha -32P]8-azidoATP at 37 °C for 5 min, initiated by the addition of [alpha -32P]8-azidoATP, and quenched by the addition of ice-cold ATP (12.5 mM). Reactions were exposed to UV light (UV-A long wave-F15T8BLB tubes, 365-nm wavelength; PGC Scientifics, Gaithersburg, MD) on ice for 10 min and subjected to SDS-PAGE and autoradiography. The extent of 32P labeling was quantified using the Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The quantification of 32P incorporation/mg Pgp was performed using SDS-PAGE samples, previously dried for PhosphorImager detection, and then dissolved in Solvable (Packard, Meriden, CT) before scintillation counting as described previously (25).

Determination of the Km for 8-AzidoATP during Vi-induced Trapping-- Purified Pgp was reconstituted into proteoliposomes and incubated in the ATPase assay buffer containing 0.3 mM Vi and increasing concentrations (1-75 µM) of [alpha -32P]8-azidoATP (3-5 µCi/nmol) in the dark at 37 °C for 3 min. The reaction was stopped by addition of 12.5 mM ice-cold ATP and placing the sample on ice. Trapping of Pgp into the Pgp·Mg8-azidoADP·Vi conformation was carried out under basal conditions and in the presence of either 50 µM verapamil or 10 µM cyclosporin A. Following SDS-PAGE on an 8% Tris-glycine gel, the radioactivity in the Pgp bands was quantified on a STORM 860 PhosphorImager system. The Km values for 8-azidoATP under basal conditions and in the presence of saturating concentrations of verapamil and cyclosporin A were obtained by fitting the data to Equation 2 using the software Prism as described above.

Inhibition of Vi-induced 8-AzidoADP Trapping by Pi-- Proteoliposomes containing purified Pgp (5-10 µg) were incubated in the ATPase assay buffer containing 0.30 mM Vi. The proteoliposomes were then treated with Me2SO (control), 50 µM verapamil, or 10 µM cyclosporin A either in the presence of 0-150 mM KH2PO4 (pH 6.8) or KCl. Finally, 50 µM [alpha -32P]8-azidoATP (2.5-5 µCi/nmol) was added in the dark and incubated at 37 °C for 5 min. The reaction was stopped by quenching with 12.5 mM ice-cold ATP solution and placing on ice. Following SDS-PAGE on an 8% Tris-glycine gel, the extent of trapping of 8-azidoADP was determined as described above.

Binding of [alpha -32P]8-AzidoATP to Pgp-- Proteoliposomes (5-10 µg of protein) were incubated in the ATPase assay buffer in the presence of Me2SO, 50 µM verapamil, or 10 µM cyclosporin A for 5 min at 37 °C and transferred to ice. After 5 min on ice, 10 µM [alpha -32P]8-azidoATP (5-10 µCi/nmol) was added to each sample in the dark and incubated at 4 °C for 5 min. The samples were then irradiated with UV light (365 nm) on ice (4 °C) as described above. Ice-cold ATP (12.5 mM) was added to displace excess noncovalently bound [alpha -32P]8-azidoATP. Excess nucleotides were removed by centrifugation at 300,000 × g at 4 °C for 10 min by using S120-AT2 rotor in a RC-M120EX micro-ultracentrifuge (Sorvall, Newtown, CT) and the pellet resuspended in 1× SDS-PAGE sample buffer. Following SDS-PAGE on a 8% Tris-glycine gel at constant voltage, gels were dried and exposed to Bio-Max MR film (Eastman Kodak Co.) at -70 °C for 12-24 h. The radioactivity incorporated into the Pgp band was quantified using the STORM 860 PhosphorImager and the software ImageQuaNT.

Routine Procedures-- The lipid mixture was made by combining acetone/ether washed E. coli bulk phospholipids, phosphotidylcholine, phosphatidylserine, and cholesterol (Avanti Polar Lipids, Alabaster, AL) in the ratio of 60:17.5:10:12.5 by weight and evaporating to dryness under N2 gas (26). This dried stock was stored at -70 °C until resuspension at 50 mg/ml in a 2 mM beta -mercaptoethanol solution. SDS-PAGE was performed using precast 8% Tris-glycine gels (Novex, San Diego, CA). Immunoblot analyses were performed as previously described (27) using the monoclonal antibody, C219 (a gift from Centocor). The Colloidal Blue Staining Kit (Novex, San Diego, CA) was used for total protein staining of SDS-PAGE samples. Sodium orthovanadate (Sigma) was prepared by boiling a 50 mM solution in water for 3 min, and concentration was determined by using molar absorbance (lambda 268 nm = 3600 M-1).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Production of Pure, Catalytically Active Human Pgp-- The previously described method was adopted for the purification of large amounts of pure Pgp (11). From ~4 × 109 cells of baculovirus-infected High Five insect cells in suspension culture, 500-600 mg of crude membrane protein was routinely obtained. 60-70% of this crude membrane protein was recovered after solubilization with octylglucoside, and 4-6 mg of purified Pgp was collected following metal affinity chromatography. The reconstitution of purified Pgp into an artificial lipid bilayer, which is required to obtain a Pgp preparation totally free of the detergent, typically yielded 65% recovery, a value common among dialysis reconstitution procedures for Pgp (12, 26, 28).

Measuring the ATPase Activity of Pgp-- The ATPase activity of purified, reconstituted Pgp was routinely measured using two techniques: 1) an end point, Pi release assay measuring the amount of Pi released by the hydrolysis of ATP in a given amount of time and 2) a continuous cycling assay linking the hydrolysis of ATP to the oxidation of NADH using pyruvate kinase, lactate dehydrogenase, and their substrates. The cycling assay was preferred to the end point Pi assay because of its ability to monitor the ATPase reaction continuously in real time; in this way, the rates of ATPase activity were easily identified as steady-state, linear rates. Although the cycling assay was used to quantitate the kinetics of ATP and all Pgp drug substrates, some drawbacks were inherent in the method. As the cycling assay measures decrease in the absorbance at 340 nm resulting from the oxidation of NADH, activity measurements using 8-azidoATP, a light-sensitive substrate, were impossible and, therefore, quantitated using the Pi assay. Furthermore, the ATPase activity could not be monitored in the presence of ADP because of its stimulatory effect of cycling in the absence of ATP hydrolysis.

A typical ATPase cycling experiment is shown in Fig. 1, where the rate (mOD340/sec) of ATPase activity specific to Pgp is determined as the Vi-sensitive activity in the presence or absence of verapamil. The rate of change of absorbance per second is converted to nmol NADH oxidized·min-1 using an NADH standard curve. This value is directly comparable with the ATPase activity of Pgp recorded as nmol ATP hydrolyzed·min-1·mg-1. Typically, verapamil-stimulated ATPase activity of purified and reconstituted Pgp was 0.6-1.2 µmol ATP hydrolyzed·min-1·mg-1, which is consistent with the values obtained using the Pi assay. This specific activity is slightly lower than previously reported for the human Pgp reconstituted by using the rapid dilution procedure (11).


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Fig. 1.   ATPase activity of purified, reconstituted human P-glycoprotein using the cycling assay. Each assay contained 5-10 µg of Pgp in ATPase assay buffer containing 15 mM MgCl2 and cycling components with (open circle ) or without (diamond ) 50 µM verapamil. Closed symbols represent non-Pgp ATPase activity in the presence of 0.3 mM Vi. The ATPase activity was measured by monitoring the oxidation of NADH at OD340 nm as described under "Experimental Procedures."

Steady-state Kinetics with Various Pgp Drug Substrates-- To characterize how human Pgp uses different drugs to stimulate ATPase activity in a concentration-dependent manner, several Pgp substrates were assayed over large concentration ranges. A true Km value cannot be obtained because of the ATPase activity in the absence of added drug substrates (the basal rate, fold stimulation = 1.0). A similar parameter, Kapp, is used to describe the concentration of progesterone, prazosin, tetraphenylphosphonium chloride ion, valinomycin, verapamil, cyclosporin A, PSC833 (a cyclosporin A analog), and rapamycin at half-maximal ATPase activity (Table I). These drugs either increased (fold stimulation > 1.0) or decreased (fold stimulation < 1.0) the ATPase activity of Pgp. The values for Kapp (often referred to as Km or K0.5) and fold stimulation vary greatly throughout the literature. Much of the published data using either human or Chinese hamster Pgp is consistent with the values obtained in this study (6, 11, 20, 28-30). However, significant differences in progesterone stimulation (30-34) and varied data using verapamil stimulation (6, 7, 11, 28, 30-32, 34-39) should be noted. In this study, many values for Kapp are given as upper or lower limits because of concentration limitations. For these experiments, the drug concentrations were at least in 5-fold excess of the amount of Pgp (nmol:nmol) and were considered to be saturated if a 10-fold increase in drug concentration did not affect the ATPase activity.

                              
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Table I
Effect of selected drug substrates on the ATPase activity of Pgp
Pgp hydrolyzes ATP in the absence of drug substrates at a constant basal rate (fold stimulation = 1.0). Kapp represents the drug concentration at half-maximal stimulation of ATPase activity; fold stimulation represents the ratio of the drug-stimulated Vmax to the basal Vmax. All assays were performed at saturating Mg·ATP concentrations (5-7.5 mM) using the cycling assay at 37 °C to monitor the ATPase activity as described under "Experimental Procedures." NA, not applicable; TPP+, tetraphenylphosphonium chloride.

Fig. 2A demonstrates both increased and decreased ATPase activity in the presence of selected agents. In the presence of saturating Mg·ATP and increasing concentrations of either prazosin or verapamil, the ATPase activity increases to a saturable Vmax value. The value of Kapp for verapamil (4.7 µM) is less than for prazosin (16 µM), indicating a higher affinity for verapamil, whereas the fold stimulation is greater for prazosin (4.5-fold) with respect to verapamil (2.0-fold); similar values have been previously reported (6, 11, 28-32, 34-36, 39). Interestingly, drugs typically considered Pgp inhibitors, like cyclosporin A, its analog PSC833, and rapamycin, are clearly depicted as substrates (Table I and data with cyclosporin A is given in Fig. 2A, inset). Upon saturation, these drugs support ATPase activity by Pgp; however, the rate is less than the basal rate in contrast to a previous report (11). Unfortunately, values of Kapp for these drugs are not able to be quantitated because of the high concentrations (0.4 µM) of Pgp required to assay for activity in either the cycling or Pi assay. However, data describing cyclosporin A as an inhibitor report low concentrations (74-400 nM) for maximal effect (6, 33, 40, 41) so the saturation of these compounds is likely.


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Fig. 2.   Effect of selected substrates on the ATPase activity of P-glycoprotein. A, ATPase activity of purified, reconstituted Pgp (5-10 µg protein) was assayed with 7 mM ATP, 14 mM MgCl2, and various concentrations of verapamil () or prazosin (black-triangle). The inset in A demonstrates the saturated ATPase activity in the presence of indicated concentrations (µM) of cyclosporin A (, CSA). B, the ATPase activity of Pgp was measured with 14 mM MgCl2 and various concentrations of ATP ranging from 0.05 to 7.5 mM in the presence of 50 µM verapamil (), 10 µM cyclosporin A (black-triangle), or in the absence of added drug (basal, black-square). Data in A and B were fit as described under "Experimental Procedures"; the dashed line in the inset in A is not a curve fit.

Effect of Selected Drug Substrates on the Affinity of Pgp for Nucleotides-- Pgp substrates, like verapamil and cyclosporin A, affect the rate at which Pgp hydrolyzes ATP; this effect is often described as fold stimulation with respect to the basal rate of ATPase activity. However, these drugs do not change the ability of Pgp to utilize nucleotides. Fig. 2B depicts the differences in maximal ATPase activity in the absence (basal) and in the presence of verapamil or cyclosporin A and suggests the similarities in values of Km, the concentration required for half-maximal stimulation. Table II reports the values of Vmax and Km for ATP (0.22, 0.33, and 0.26 mM) and 8-azidoATP (0.4, 0.6, and 0.5 mM) in the presence of verapamil, no drug, and cyclosporin A, respectively. The Km values for ATP are similar to those reported previously by us (8, 11). Clearly, the values of Km for either nucleotide are unchanged in the absence (basal) or presence of verapamil or cyclosporin A, whereas the values of Vmax remain constant in relation to each other (fold stimulation for verapamil:basal:cyclosporin A = 2:1:0.5). Additionally, the values of Ki for ADP are similar in the presence (Ki = 0.5 mM) and absence of verapamil (Ki = 0.3 mM); these data display competitive inhibition patterns with respect to ATP by intersecting on the y axis of a Lineweaver-Burke plot (data not shown), indicating that ATP and ADP bind to the same site. Other workers also have reported similar inhibition by ADP (11, 28, 39, 42).

                              
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Table II
Michaelis-Menten parameters for nucleotide hydrolysis by P-glycoprotein in the presence of selected agents
Values of Km for ATP and 8-azidoATP and Ki for ADP were determined in the presence and absence of saturating Pgp substrates representing ATPase activities both greater and less than basal activity (verapamil, 50 µM; cyclosporin A, 10 µM; all assays contained 1% Me2SO). Kinetic analyses were carried out as described under "Experimental Procedures." NA, not applicable.

Vanadate-induced [alpha -32P]8-AzidoADP Trapping in the Presence of Various Pgp Drug Substrates-- Under hydrolysis conditions (assay buffer, pH 6.8 at 37 °C), Pgp is incubated with [alpha -32P]8-azidoATP, MgCl2 or CoCl2, and Vi (0.3 mM) in the presence or absence of Pgp drug substrates. The reaction proceeds through ATP hydrolysis to products ([alpha -32P]8-azidoADP + Pi) before Vi substitutes for Pi and traps [alpha -32P]8-azidoADP on the enzyme (18). The reaction is quenched by adding excess ATP (12.5 mM) at 4 °C to inhibit any nonspecific [alpha -32P]8-azidoATP binding and to measure only the extent of trapped or occluded diphosphate nucleotide. Following UV cross-linking and gel electrophoresis, the [alpha -32P]8-azidoADP incorporated into Pgp is determined by autoradiography and PhosphorImager analysis. As shown in Fig. 3A; the extent of Vi-induced [alpha -32P]8-azidoADP trapping clearly varies in the presence of Pgp substrates. This drug effect is unlikely to be the result of inefficient UV cross-linking or populations of inactive Pgp because of the observed correlation between the extent of Vi-induced 8-azidoADP trapping and the fold stimulation of the ATPase activity in the presence of a given substrate as discussed below. In addition, the observed variation in the extent of Vi-induced [alpha -32P]8-azidoADP trapping in the presence of various drugs in Fig. 3A is not due to unequal loading of protein per lane in the gel (see the immunoblot in Fig. 3B; samples in lanes 2-8 in Fig. 3A were used for the immunodetection of Pgp with the monoclonal antibody, C219). Moreover, previous reports (43, 44) have also demonstrated drug substrate-dependent variation in the extent of [alpha -32P]8-azido ADP trapping into Pgp. [alpha -32P]8-azidoADP is incorporated into 2-5% of the Pgp in the reaction depending on the Pgp drug substrates present; these values are consistent with those previously reported for Chinese hamster Pgp in the presence of verapamil (45). It should be noted that cross-linking is highly specific using purified, reconstituted Pgp; the total protein profile of the proteoliposomes used in these studies is also shown in Fig. 3 (lane 1), having been overloaded to identify impurities. Furthermore, no [alpha -32P]8-azidoADP trapping occurs in the absence of Vi or if the reaction is performed at 4 °C a temperature at which, 8-azidoATP (or ATP) is not detectably hydrolyzed by Pgp (data not shown).


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Fig. 3.   Vanadate-induced 8-azidoADP trapping in Pgp in the presence of selected agents. A, purified, reconstituted Pgp (17 µg) was subjected to SDS-PAGE and stained as described under "Experimental Procedures" (lane 1). The protein is overloaded to emphasize impurities (the molecular masses in kilodaltons are indicated on the left). Lanes 2-8 show the effect of selected agents on the extent of Vi-induced trapping of [alpha -32P]8-azidoADP. Reconstituted Pgp (0.5 mg/ml) was incubated with 50 µM [alpha -32P]8-azidoATP (5-7.5 µCi/nmol) in ATPase assay buffer containing 5 mM CoCl2, and 0.3 mM Vi for 15 min at 37 °C in the presence of indicated drug substrate. The reaction was quenched by addition of excess ATP (12.5 mM) at 4 °C, UV cross-linked at 365 nm, run on SDS-PAGE, and subjected to autoradiography and PhosphorImager analysis as described under "Experimental Procedures." In lanes 2-8, same amount of protein (10 µg) was loaded in each lane. Lane 2, the reaction was quenched 10 s after the addition of [alpha -32P]8-azidoATP (control); lane 3, rapamycin (20 µM); lane 4, cyclosporin A (1 µM); lane 5, basal (only Me2SO); lane 6, verapamil (50 µM); lane 7, valinomycin (5 µM); lane 8, prazosin (100 µM). B, an immunoblot of protein samples in lanes 2-8 in A. The immunodetection of Pgp by using the monoclonal antibody, C219 was carried out on samples in lanes 2-8 in A (0.5 µg protein/lane) as described under "Experimental Procedures."

The Affinity (Km) for 8-AzidoATP during Vanadate-induced 8-AzidoADP Trapping Is Not Affected by Verapamil and Cyclosporin A-- We show above that Pgp substrates stimulate trapping of 8-azidoADP in the Pgp·ADP·Vi transition state complex (Fig. 3A). To quantitatively compare the effect of substrates on the ATPase activity and the extent of trapping, it is necessary to obtain the kinetic parameters for 8-azidoADP trapping into Pgp. Generating the Pgp·[alpha -32P]8-azidoADP·Vi complex in the presence of increasing concentrations of [alpha -32P]8-azidoATP and Vi and quantifying the radioactivity in the Pgp allows one to determine the Km and Vmax (extent of trapped 8-azidoADP) of [alpha -32P]8-azidoATP during trapping. We selected verapamil and cyclosporin A to assess the effect of agents, which either increase or decrease the ATPase activity of Pgp, respectively. It is clear from the data in Fig. 4 that the Vmax for [alpha -32P]8-azidoATP increases in the presence of verapamil and is reduced in the presence of cyclosporin A. The Km (25 ± 7, 23 ± 6, and 19 ± 5 µM under basal conditions, in the presence of 50 µM verapamil and 10 µM cyclosporin A, respectively) is, however, not altered by the presence of verapamil or cyclosporin A. Because the Km for [alpha -32P]8-azidoATP was ~20 µM in the presence of Vi, subsequent experiments to determine the extent of trapping in the presence of drugs were performed in the presence of 50 µM [alpha -32P]8-azidoATP. It is worth noting that the affinity of 8-azidoATP (or ATP) for Pgp is significantly increased in the presence of Vi (Km = 20 µM) when compared with the control (Km = 0.5 mM; Table II). How Vi alters the affinity of nucleotides for Pgp is not clear at present.


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Fig. 4.   Determination of Km for [alpha -32P]8-azidoATP during vanadate-induced 8-azidoADP trapping in Pgp in the presence of verapamil and cyclosporin A. Purified Pgp, reconstituted into proteoliposomes (20 µg), was incubated in the ATPase reaction mixture in the absence of drugs (black-square) and in the presence of 50 µM verapamil () or 10 µM cyclosporin A (black-triangle) at 37 °C for 3 min. Indicated concentrations of [alpha -32P]8-azidoATP (1.0-75 µM; 5-10 µCi/nmol) were then added in the dark, and the samples were incubated for an additional 10 min at 37 °C. The reaction was stopped by the addition of 12.5 mM ice-cold ATP and placing the samples on ice. The samples were photocross-linked by UV irradiation at 365 nm at 4 °C. Following SDS-PAGE on an 8% gel (10 µg of protein/lane), radioactivity in the Pgp band was estimated using a STORM 860 PhosphorImager system.

As previously reported (19, 25), the Pgp·Mg·8-azidoADP·Vi complex, trapped using Mg·[alpha -32P]8-azidoATP, dissociates rather quickly (t1/2 = 8 min at 37 °C). However, replacing MgCl2 with CoCl2 displays similar reaction kinetics within the first 2 min, whereas no dissociation of the Pgp·Co·8-azidoADP·Vi complex is seen up to 20 min (data not shown). Although Co2+ supports steady-state Pgp ATPase activity at rates 10-20-fold slower than Mg2+ (19),2 for the purpose of Vi trapping experiments, the divalent cations appear to be equivalent.

The extent of Vi trapping with 50 µM [alpha -32P]8-azidoATP and 5 mM CoCl2 in the presence of various Pgp drug substrates strongly correlates with the fold stimulation of ATPase activity by those drugs in the steady-state reaction with saturating Mg2+·ATP concentrations (Fig. 5; the data used for this analysis are given in Fig. 3A and Table I, respectively). This correlation is also seen using higher [alpha -32P]8-azidoATP concentrations (up to 500 µM) for Vi-induced trapping. In addition when CoCl2 was replaced with 5 mM MgCl2, a similar correlation between Vi-induced [alpha -32P]8-azidoADP trapping, and the fold stimulation of ATPase activity was also observed (data not shown).


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Fig. 5.   Correlation between the extent of vanadate-induced 8-azidoADP trapping and the fold stimulation of ATPase activity in the presence of Pgp substrates. The extent of Vi-induced trapping of [alpha -32P]8-azidoADP (values from PhosphorImager analysis as in Fig. 3) after a 15-min incubation at 37 °C in the presence of 50 µM [alpha -32P]8-azidoATP and 5 mM CoCl2 was correlated (r2 = 0.941) to the fold stimulation of ATPase activity under saturating MgATP conditions as shown in Fig. 2B and Tables I and II, respectively. The numbers represent the data obtained in presence of indicated agent: 1, rapamycin (20 µM); 2, cyclosporin A (1 µM); 3, Me2SO; 4, verapamil (50 µM); 5, valinomycin (5 µM); 6, prazosin (100 µM). A similar correlation between Vi-induced [alpha -32P]8-azidoADP trapping and the fold stimulation of ATPase activity was also observed when CoCl2 was replaced with 5 mM MgCl2 (data not shown).

The experiments described above clearly demonstrate that drugs, which are substrates of Pgp affect both ATP hydrolysis and the Vi-induced trapping of [alpha -32P]8-azidoATP. This raises the question as to whether the substrates influence nucleotide binding or a subsequent step during hydrolysis. We addressed this issue directly by allowing [alpha -32P]8-azidoATP to bind Pgp under nonhydrolysis conditions (at 4 °C) both in the absence and presence of verapamil or cyclosporin A. We observed that even saturating concentrations of verapamil and cyclosporin A do not affect nucleotide binding per se, suggesting that the substrate must act at a step or steps that follow binding of nucleotide (56).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

For kinetic studies of a membrane transport protein, such as Pgp, large quantities of purified protein that retains complete biological activity are necessary. The baculovirus expression system used in this study produces human Pgp at a high level in the absence of cytotoxic drug selection, and the absence of such selection pressure facilitates the assessment of the intrinsic properties of human Pgp. Because Pgp is an integral membrane protein, lipids play a key role in protein conformation and activity (13-16). Solubilization and purification in the presence of octyl glucoside and reconstitution with lipid mixture containing bulk E. coli phospholipids, phosphatidylcholine, phosphatidylserine, and cholesterol provides a system for the complete recovery of the ATPase activity of purified Pgp (8, 11, 26).

The ATPase activity of Pgp in the presence of various Pgp drug substrates can be accurately measured using purified, reconstituted Pgp in the cycling assay. In contrast, crude membrane preparations produce high background activities both in the absence of ATP because of NADH oxidases and in the presence of ATP and Vi because of the presence of Vi-insensitive ATPases. Because the cycling assay relies on the catalysis of other enzymes, we demonstrated that compounds in the assay buffer, such as Vi or Pgp drug substrates, do not affect the activity of these enzymes. Furthermore, the slow catalysis of Pgp (~2-10 s-1) with respect to cycling enzymes, pyruvate kinase, and lactate dehydrogenase is crucial to retain the tight coupling from ATP hydrolysis to NADH oxidation.

ATPase activity of Pgp is measured as the Vi-sensitive activity in the presence and absence of various Pgp substrates. The observed variability in the specific activity of Pgp upon reconstitution is likely due to preparation impurities and/or efficiencies of reconstitution; because of these variations, the ATPase activity of Pgp in the presence of drug substrates is typically considered using fold stimulation values that remain constant. The actual concentrations at which Pgp drug substrates stimulate ATP activity are unclear because all Pgp drug substrates are hydrophobic and partition into the membrane lipid surrounding Pgp at unknown local concentrations. However, characterizing drug-stimulated activities in this purified, reconstituted system is useful for comparisons among various drugs.

The determination of Michaelis-Menten parameters for various Pgp drug substrates is challenging. High amounts of Pgp are required to quantify the enzyme activity in the presence of the drug, and this amount of Pgp sets a lower limit on the amount of drug to be used for stimulation. Hence, many values for Kapp, as shown in Table I, are upper limits; however, the substrate-saturated ATPase activities in the presence of these drugs are well defined. Conversely, Pgp drug substrates are highly hydrophobic with low solubility limits making saturation at high drug concentrations problematic. For these drugs, both Kapp and drug-saturated ATPase activities are harder to define. Of particular interest is the ability of cyclosporin A and other Pgp "inhibitors" to support ATPase activity. It is clear from the data presented here that cyclosporin A, its analog PSC833, and rapamycin act as Pgp substrates, albeit at a slower ATPase rate than basal levels (Fig. 2A and Table II). The idea that these drugs act as substrates promoting ATPase activity is supported by several cyclosporin A transport studies (47-50). Additionally, human Pgp-specific monoclonal antibody, UIC2 shift assays suggest similar conformational changes with vinblastine, a common Pgp substrate, as well as cyclosporin A (51). These studies also demonstrate that the cycling assay provides a useful tool to assess whether a given modulator is a "true" inhibitor or a substrate of Pgp.

It is clear that the ATPase activity of Pgp is stimulated by a myriad of drug substrates. The number and interactions of the drug binding site(s) of Pgp has been discussed extensively in the literature (29, 31-34, 52, 53). Although these drugs have profound effects on the overall stimulation of the ATPase activity of Pgp, they have no effect on the values of Km for nucleotide substrates, ATP and 8-azidoATP (Fig. 4 and Table II). Furthermore, the value of Ki for ADP, a competitive inhibitor of ATP, remains constant in the presence of verapamil (Table II). Taken together, these data suggest that drug substrates, like verapamil and cyclosporin A, have no effect on ATP binding to Pgp; these conclusions are comparable with those suggested using mouse Pgp (mdr1b) (20).

The proposed scheme for the catalytic cycle of Pgp and the rate-limiting step is given in Fig. 6 (see the figure legend for the detailed description). To investigate other steps of the Pgp catalytic cycle where drug might have an effect, we used Vi-induced ADP trapping experiments, a method commonly used to investigate the transition state(s) of ATP-hydrolyzing proteins without covalent phosphoenzyme intermediates. Ortho-Vi can mimic Pi and bind in its place immediately after ATP hydrolysis and Pi release to trap ADP and inhibit the steady-state turnover of the enzyme (Fig. 6, step 5) (18). Therefore, the amount of ADP trapped on Pgp is indicative of the Vi-inhibited conformation, which is comparable with the transition state conformation Pgp·ADP·Pi of the ATP hydrolysis reaction. In this study, the substitution of CoCl2 for MgCl2 avoided complications with dissociation of the Pgp·8-azidoADP·Vi trapped complex while supporting apparently the same rate of reaction for the first 2 min of trapping (data not shown).


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Fig. 6.   Proposed scheme for the catalytic cycle of Pgp. The proposed scheme is similar to those previously described (20, 22, 29) and shows the rate-limiting step (step 4) in the catalytic cycle based on the work reported here. Step 1, drug substrate and ATP bind to Pgp. There is no evidence that the binding of one is either a prerequisite or inhibitory to the binding of the other. Step 2, binding of drug and ATP is followed by ATP hydrolysis, and this is accompanied by a conformational change resulting in the translocation of the drug from a high affinity (on) to a low affinity (off) site. Step 3, following hydrolysis of ATP, both Pi and drug are released, although the exact order of release is not known at present. Step 4, the ADP release, which appears to be the slowest step in the cycle (see below), is essential for regenerating Pgp for the next hydrolysis event (the box is shaded to indicate the rate-limiting nature of this step). Step 5, if Vi is provided to the system, Vi mimics Pi to trap ADP in a stable ternary conformation (Pgp·ADP·Vi). Moreover, given the chemical analogy between Pi and Vi, the general consensus is that the Pgp·ADP·Pi and the Pgp·ADP·Vi complexes are equivalent and that this transition state represents an intermediate state during the normal reaction pathway (46). Step 6, eventually, Vi and ADP dissociate from Pgp (t1/2 = 80-90 min at 37 °C (18)) to initiate the next cycle. The observed correlation between the extent of trapped 8-azidoADP in the presence of Vi and fold stimulation of ATP hydrolysis by various substrates (see Fig. 5), and the fact that Pi exhibits very low affinity for Pgp (Ki~30 mM for Vi-induced 8-azidoADP trapping in human Pgp2 and Ki~200 mM for ATP hydrolysis by Chinese hamster Pgp (19) strongly suggest that 8-azidoADP (ADP) release (step 4) is the rate-limiting step in the catalytic cycle and substrates modulate the Pgp activity by exerting effect on this step. Although not shown, both ATP and ADP are complexed with Mg2+, which has been omitted for clarity.

Although Vi inhibits all Pgp activity in steady-state experiments independent of the presence of drug substrates, different amounts of 8-azidoADP are trapped on the protein when the extent of Vi-induced trapping is complete. A striking correlation exists between the extent of Vi trapping and the steady-state fold stimulation of ATPase activity in the presence of various drug substrates. Drugs that support a higher fold stimulation of steady-state ATPase activity also demonstrate a higher extent of 8-azidoADP (or ADP) trapping; the opposite holds true for less active Pgp drug substrates (Fig. 5). A previous report (44) also suggested a close relationship between the extent of Vi-induced nucleotide trapping and the drug substrate-stimulated ATPase activity of Pgp. Directly correlating effects on the Vi-induced ADP trapped conformation and the steady-state reaction rates in the presence of Pgp drug substrates indicate that these experiments are measuring the same step in the Pgp catalytic cycle. The Vi-induced ADP trapping experiment measures the amount of ADP released (the inverse of the amount of ADP trapped) from the transition state conformation, which, by its correlation, suggests that the rate-limiting step measured in the steady-state reaction is the release of ADP (step 4 or 6 in Fig. 6).

These experiments further indicate that the release of Pi is not likely to be the rate-limiting step of the overall catalytic cycle. The low affinity of Pi for Pgp makes this step an unlikely candidate to act as the rate-limiting step of the Pgp reaction cycle (Ki ~30 mM for Vi-induced 8-azidoADP trapping in Pgp,2 and Ki ~200 mM for ATP hydrolysis (19)). Additionally, the correlation between the steady-state reaction (fold stimulation) and the amount of ADP trapping in the presence of drug substrates like verapamil and cyclosporin A indicates that the rate-limiting step is a step after Vi binds and traps Pgp, and Pi release is a prerequisite for Vi binding (Fig. 6, steps 3 and 5).

The ADP release being the rate-limiting step in the catalytic cycle is further supported by our recent observation that there is an inverse relationship between ADP release from the Pgp·MgADP·Vi complex and the recovery of the substrate binding to the transporter following the transition state step (25). In addition, the rate of the release of 8-azidoADP (or ADP) from the Vi-trapped Pgp is not affected by the addition of excess nucleotides such as ATP, ADP, or AMPPNP (56). It is now clear that most ATP-binding cassette transporters catalyze Vi-sensitive ATP hydrolysis, which is stimulated by substrates. Vi-induced 8-azidoADP trapping has been demonstrated in other transporters such as MRP1 and ATP-binding cassette R (54, 55). It is perhaps most likely that the ADP release is a rate-limiting step in the catalytic cycle of other members of the super family of ATP-binding cassette transporters.

    ACKNOWLEDGEMENTS

We thank Drs. Michael Gottesman and Ira Pastan for helpful discussions and encouragement and John Gribar and Melissa Smith for comments on the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: United States Patent and Trademark Office, Arlington, VA 22202.

§ To whom correspondence should be addressed: Lab. of Cell Biology, Bldg. 37, Rm. 1B-22, NCI, NIH, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255. Tel.: 301-402-4178; Fax: 301-435-8188; E-mail: ambudkar@helix.nih.gov.

Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M010044200

2 K. M. Kerr and S. V. Ambudkar, unpublished data.

    ABBREVIATIONS

The abbreviations used are: Pgp, P-glycoprotein; PAGE, polyacrylamide gel electrophoresis; Vi, vanadate; MES, 2[N-morpholino]ethane sulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Ambudkar, S. V., Dey, S., Hrycyna, C. A., Ramachandra, M., Pastan, I., and Gottesman, M. M. (1999) Annu. Rev. Pharmacol. Toxicol. 39, 361-398[CrossRef][Medline] [Order article via Infotrieve]
2. Gottesman, M. M., Hrycyna, C. A., Schoenlein, P. V., Germann, U. A., and Pastan, I. (1995) Annu. Rev. Genet. 29, 607-649[CrossRef][Medline] [Order article via Infotrieve]
3. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427[CrossRef][Medline] [Order article via Infotrieve]
4. Croop, J. M. (1998) Methods Enzymol. 292, 101-116[CrossRef][Medline] [Order article via Infotrieve]
5. Gottesman, M. M., Pastan, I., and Ambudkar, S. V. (1996) Curr. Opin. Genet. Dev. 6, 610-617[CrossRef][Medline] [Order article via Infotrieve]
6. Ramachandra, M., Ambudkar, S. V., Gottesman, M. M., Pastan, I., and Hrycyna, C. A. (1996) Mol. Biol. Cell 7, 1485-1498[Abstract]
7. Sarkadi, B., Price, E. M., Boucher, R. C., Germann, U. A., and Scarborough, G. A. (1992) J. Biol. Chem. 267, 4854-4858[Abstract/Free Full Text]
8. Ambudkar, S. V., Lelong, I. H., Zhang, J., Cardarelli, C. O., Gottesman, M. M., and Pastan, I. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8472-8476[Abstract]
9. Rao, U. S., and Scarborough, G. A. (1994) Mol. Pharmacol. 45, 773-776[Abstract]
10. Ambudkar, S. V. (1998) Methods Enzymol. 292, 504-514[Medline] [Order article via Infotrieve]
11. Ramachandra, M., Ambudkar, S. V., Chen, D., Hrycyna, C. A., Dey, S., Gottesman, M. M., and Pastan, I. (1998) Biochemistry 37, 5010-5019[CrossRef][Medline] [Order article via Infotrieve]
12. Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1998) Methods Enzymol. 292, 514-523[Medline] [Order article via Infotrieve]
13. Urbatsch, I. L., and Senior, A. E. (1995) Arch. Biochem. Biophys. 316, 135-140[CrossRef][Medline] [Order article via Infotrieve]
14. Doige, C. A., Yu, X., and Sharom, F. J. (1993) Biochim. Biophys. Acta 1146, 65-72[Medline] [Order article via Infotrieve]
15. Sharom, F. J. (1997) Biochem. Soc. Trans. 25, 1088-1096[Medline] [Order article via Infotrieve]
16. Romsicki, Y., and Sharom, F. J. (1999) Biochemistry 38, 6887-6896[CrossRef][Medline] [Order article via Infotrieve]
17. Liu, R., and Sharom, F. J. (1996) Biochemistry 35, 11865-11873[CrossRef][Medline] [Order article via Infotrieve]
18. Urbatsch, I. L., Sankaran, B., Weber, J., and Senior, A. E. (1995) J. Biol. Chem. 270, 19383-19390[Abstract/Free Full Text]
19. Urbatsch, I. L., Sankaran, B., Bhagat, S., and Senior, A. E. (1995) J. Biol. Chem. 270, 26956-26961[Abstract/Free Full Text]
20. Urbatsch, I. L., Beaudet, L., Carrier, I., and Gros, P. (1998) Biochemistry 37, 4592-4602[CrossRef][Medline] [Order article via Infotrieve]
21. Hrycyna, C. A., Ramachandra, M., Ambudkar, S. V., Ko, Y. H., Pedersen, P. L., Pastan, I., and Gottesman, M. M. (1998) J. Biol. Chem. 273, 16631-16634[Abstract/Free Full Text]
22. Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289[CrossRef][Medline] [Order article via Infotrieve]
23. Germann, U. A. (1998) Methods Enzymol. 292, 427-441[Medline] [Order article via Infotrieve]
24. Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 56, 502-514[Medline] [Order article via Infotrieve]
25. Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520[Abstract/Free Full Text]
26. Ambudkar, S. V., Lelong, I. H., Zhang, J., and Cardarelli, C. (1998) Methods Enzymol. 292, 492-504[Medline] [Order article via Infotrieve]
27. Germann, U. A., Chambers, T. C., Ambudkar, S. V., Licht, T., Cardarelli, C. O., Pastan, I., and Gottesman, M. M. (1996) J. Biol. Chem. 271, 1708-1716[Abstract/Free Full Text]
28. Urbatsch, I. L., al-Shawi, M. K., and Senior, A. E. (1994) Biochemistry 33, 7069-7076[Medline] [Order article via Infotrieve]
29. Dey, S., Ramachandra, M., Pastan, I., Gottesman, M. M., and Ambudkar, S. V. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10594-10599[Abstract/Free Full Text]
30. Litman, T., Nielsen, D., Skovsgaard, T., Zeuthen, T., and Stein, W. D. (1997) Biochim. Biophys. Acta 1361, 147-158[Medline] [Order article via Infotrieve]
31. Rebbeor, J. F., and Senior, A. E. (1998) Biochim. Biophys. Acta 1369, 85-93[Medline] [Order article via Infotrieve]
32. Garrigos, M., Mir, L. M., and Orlowski, S. (1997) Eur. J. Biochem. 244, 664-673[Abstract]
33. Litman, T., Zeuthen, T., Skovsgaard, T., and Stein, W. D. (1997) Biochim. Biophys. Acta 1361, 169-176[Medline] [Order article via Infotrieve]
34. Litman, T., Zeuthen, T., Skovsgaard, T., and Stein, W. D. (1997) Biochim. Biophys. Acta 1361, 159-168[Medline] [Order article via Infotrieve]
35. Lee, C. G., Gottesman, M. M., Cardarelli, C. O., Ramachandra, M., Jeang, K. T., Ambudkar, S. V., Pastan, I., and Dey, S. (1998) Biochemistry 37, 3594-3601[CrossRef][Medline] [Order article via Infotrieve]
36. Rao, U. S. (1995) J. Biol. Chem. 270, 6686-6690[Abstract/Free Full Text]
37. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 21449-21452[Abstract/Free Full Text]
38. al-Shawi, M. K., and Senior, A. E. (1993) J. Biol. Chem. 268, 4197-4206[Abstract/Free Full Text]
39. al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994) J. Biol. Chem. 269, 8986-8992[Abstract/Free Full Text]
40. Dey, S., Hafkemeyer, P., Pastan, I., and Gottesman, M. M. (1999) Biochemistry 38, 6630-6639[CrossRef][Medline] [Order article via Infotrieve]
41. Demeule, M., Laplante, A., Murphy, G. F., Wenger, R. M., and Beliveau, R. (1998) Biochemistry 37, 18110-18118[CrossRef][Medline] [Order article via Infotrieve]
42. Sharom, F. J., Yu, X., Chu, J. W., and Doige, C. A. (1995) Biochem. J. 308, 381-390[Medline] [Order article via Infotrieve]
43. Shepard, R. L., Winter, M. A., Hsaio, S. C., Pearce, H. L., Beck, W. T., and Dantzig, A. H. (1998) Biochem. Pharmacol. 56, 719-727[CrossRef][Medline] [Order article via Infotrieve]
44. Szabo, K., Welker, E., Bakos, Muller, M., Roninson, I., Varadi, A., and Sarkadi, B. (1998) J. Biol. Chem. 273, 10132-10138[Abstract/Free Full Text]
45. Senior, A. E. (1998) Acta Physiol. Scand. Suppl. 643, 213-218[CrossRef][Medline] [Order article via Infotrieve]
46. Senior, A. E., and Gadsby, D. C. (1997) Semin. Cancer. Biol. 8, 143-150[CrossRef][Medline] [Order article via Infotrieve]
47. Saeki, T., Ueda, K., Tanigawara, Y., Hori, R., and Komano, T. (1993) J. Biol. Chem. 268, 6077-6080[Abstract/Free Full Text]
48. Schramm, U., Fricker, G., Wenger, R., and Miller, D. S. (1995) Am. J. Physiol. 268, F46-F52[Abstract/Free Full Text]
49. Schinkel, A. H., Wagenaar, E., van Deemter, L., Mol, C. A., and Borst, P. (1995) J. Clin. Invest. 96, 1698-1705[Medline] [Order article via Infotrieve]
50. Didier, A., Wenger, J., and Loor, F. (1995) Anticancer Drugs 6, 669-680[Medline] [Order article via Infotrieve]
51. Mechetner, E. B., Schott, B., Morse, B. S., Stein, W. D., Druley, T., Davis, K. A., Tsuruo, T., and Roninson, I. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12908-12913[Abstract/Free Full Text]
52. Pascaud, C., Garrigos, M., and Orlowski, S. (1998) Biochem. J. 333, 351-358[Medline] [Order article via Infotrieve]
53. Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948[Abstract/Free Full Text]
54. Nagata, K., Nishitani, M., Matsuo, M., Kioka, N., Amachi, T., and Ueda, K. (2000) J. Biol. Chem. 275, 17626-17630[Abstract/Free Full Text]
55. Sun, H., Molday, R. S., and Nathans, J. (1999) J. Biol. Chem. 274, 8269-8281[Abstract/Free Full Text]
56. Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem., in press


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