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
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ABSTRACT |
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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 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.
Materials--
Octyl 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
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.
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 [ 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
[ 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 [ Binding of [ 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 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 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.
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.
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).
Vanadate-induced [ 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·[
As previously reported (19, 25), the Pgp·Mg·8-azidoADP·Vi
complex, trapped using Mg·[
The extent of Vi trapping with 50 µM
[
The experiments described above clearly demonstrate that drugs, which
are substrates of Pgp affect both ATP hydrolysis and the Vi-induced
trapping of [ 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 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).
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
[
-32P]8-azidoADP was used to probe the effect of
substrates on the transition state of the ATP hydrolysis reaction. The
Km for [
-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 [
-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 [
-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
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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
[
-32P]8-azidoATP (10-20 Ci/mmol) was purchased from
ICN Biomedicals (Irvine, CA).
-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
-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).
(Eq. 1)
/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.
[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.
(Eq. 2)
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).
(Eq. 3)
-32P]8-azidoATP (2.5-10 µCi/nmol). Reactions were
preincubated in low light or semi-darkness in the absence of
[
-32P]8-azidoATP at 37 °C for 5 min, initiated by
the addition of [
-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).
-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.
-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.
-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 [
-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 [
-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.
70 °C until resuspension at 50 mg/ml in a 2 mM
-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 (
268 nm = 3600 M
1).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 ( ) or without (
) 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."
Effect of selected drug substrates on the ATPase activity of Pgp
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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 (
). 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 (
), or in the absence of added
drug (basal,
). 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.
Michaelis-Menten parameters for nucleotide hydrolysis by P-glycoprotein
in the presence of selected agents
-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
[
-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 ([
-32P]8-azidoADP + Pi) before Vi substitutes for Pi and traps
[
-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 [
-32P]8-azidoATP binding and
to measure only the extent of trapped or occluded diphosphate
nucleotide. Following UV cross-linking and gel electrophoresis, the
[
-32P]8-azidoADP incorporated into Pgp is determined
by autoradiography and PhosphorImager analysis. As shown in Fig.
3A; the extent of Vi-induced
[
-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
[
-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
[
-32P]8-azido ADP trapping into Pgp.
[
-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 [
-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 [ -32P]8-azidoADP. Reconstituted
Pgp (0.5 mg/ml) was incubated with 50 µM
[
-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 [
-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."
-32P]8-azidoADP·Vi complex in
the presence of increasing concentrations of
[
-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 [
-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 [
-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
[
-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 [
-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 [ -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 (
) and in the presence of 50 µM verapamil (
) or 10 µM cyclosporin A
(
) at 37 °C for 3 min. Indicated concentrations of
[
-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.
-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.
-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 [
-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
[
-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 [ -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
[
-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 [
-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).
-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 [
-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
1) with respect to cycling enzymes, pyruvate kinase, and
lactate dehydrogenase is crucial to retain the tight coupling from ATP hydrolysis to NADH oxidation.
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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.
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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.
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.
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ABBREVIATIONS |
---|
The abbreviations used are: Pgp, P-glycoprotein; PAGE, polyacrylamide gel electrophoresis; Vi, vanadate; MES, 2[N-morpholino]ethane sulfonic acid.
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