(Received for publication, April 19, 1995; and in revised form, June 16, 1995)
From the
P-glycoprotein (Pgp or multidrug-resistance protein) shows drug-stimulated ATPase activity. The catalytic sites are known to be of low affinity and specificity for nucleotides. From the sequence, two nucleotide sites are predicted per Pgp molecule. Using plasma membranes from a multidrug-resistant Chinese hamster ovary cell line, which are highly enriched in Pgp, we show that vanadate-induced trapping of nucleotide at a single catalytic site produces stably inhibited Pgp, with t for reactivation of ATPase activity of 84 min at 37 °C and >30 h at 4 °C. Reactivation of ATPase correlated with release of trapped nucleotide. Concentrations of MgATP and MgADP required to produce 50% inhibition were 9 and 15 µM, respectively, thus the apparent affinity for nucleotide is greatly increased by vanadate-trapping. The trapped nucleotide species was ADP. Divalent cation was required, with magnesium, manganese, and cobalt all effective; cobalt yielded a very stable inhibited species, t at 37 °C = 18 h. No photocleavage of Pgp was observed after vanadate trapping with MgATP, nor was UV-induced photolabeling of Pgp by trapped adenine nucleotide observed. Vanadate-trapping with 8-azido-ATP followed by UV irradiation caused permanent inactivation and specific labeling of Pgp. Vanadate-induced inhibition was also shown with pure, reconstituted Pgp, with similar characteristics to those in plasma membranes. Vanadate trapping overcomes technical difficulties posed by lack of high affinity nucleotide-binding site(s) or a covalent enzyme-phosphate catalytic intermediate in Pgp. The finding that vanadate trapping of nucleotide at just one site/Pgp is sufficient to give full inhibition of ATPase activity shows that the two predicted nucleotide sites can not function independently as catalytic sites.
P-glycoprotein (Pgp) ()is a plasma membrane protein
which endows a multidrug resistance phenotype on cells. It acts by
hydrolyzing ATP at catalytic sites, which project from the cytoplasmic
side of the membrane, and coupling the hydrolysis of ATP to transport
of drugs or other hydrophobic molecules across the membrane to the
outside of the cell (Ruetz and Gros, 1994; Sharom et al.,
1993). Pgp has attracted considerable interest because of its possible
role in the resistance of human cancers to chemotherapy (for recent
reviews, see Endicott and Ling, 1989; Gottesman and Pastan, 1993; Gros
and Buschman, 1993).
Our laboratory has been working to characterize the catalytic sites of Pgp, and in recent work we obtained a highly Pgp-enriched plasma membrane preparation from multidrug-resistant Chinese hamster ovary cells that showed substantial ATPase activity referable to Pgp (Al-Shawi and Senior, 1993). We purified the Pgp to >95% homogeneity and reconstituted it in proteoliposomes, with retention of ATPase activity (Urbatsch et al., 1994). The ATPase activity is activated and/or inhibited by a range of drugs, and it is also sensitive to the membrane lipid environment (Urbatsch and Senior, 1995) and subject to covalent inhibition by N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 8-azido-ATP, and 2-azido-ATP (Al-Shawi et al., 1994). Several other laboratories have also demonstrated and characterized ATPase activity in plasma membrane-located Pgp (Sarkardi et al., 1992), partially purified and reconstituted Pgp (Ambudkar et al., 1992; Sharom et al., 1993), or purified, reconstituted Pgp (Shapiro and Ling, 1994).
It has been established
that Pgp-ATPase activity follows simple Michaelis-Menten kinetics with
a single K(MgATP) around 1 mM.
Free ATP is not hydrolyzed. Catalysis is relatively nonspecific in that
a wide range of magnesium nucleotides are hydrolyzed, all with
relatively high K
values. MgADP and
MgAMPPNP show competitive inhibition with high K
values, around 0.4 mM (Al-Shawi et al.,
1994; Urbatsch et al., 1994). Therefore, there is no evidence
from kinetic studies for a high affinity site involved in catalysis. We
have applied other techniques to try to detect a tight nucleotide
site(s). (
)Direct analyses of bound ATP or ADP using the
luciferin-luciferase method in Pgp-enriched plasma membranes failed to
detect any bound nucleotide, either before or after incubation with 5
mM MgATP. ``Unisite''-type ATP hydrolysis assays,
using concentrations of [
-
P]ATP
stoichiometric with Pgp (conduced as described by Penefsky(1986))
showed there was little binding or hydrolysis of the ATP under these
conditions. Direct binding experiments with
[
-
P]ATP or
[
H]ADP, using centrifuge columns, gel filtration,
or vacuum-assisted filtration methods failed to detect significant
specific binding of nucleotide to Pgp at concentrations of added
nucleotide up to
100 µM. Above this concentration,
nonspecific binding of nucleotide became problematic, in both plasma
membrane and purified reconstituted Pgp, probably due to the
particulate nature of these preparations. Finally, in an exhaustive,
and we believe conclusive, series of experiments aimed at detecting a
covalent enzyme-phosphate catalytic intermediate, we obtained negative
results with both plasma membrane and purified reconstituted Pgp.
As we point out elsewhere, the lack of a high-affinity nucleotide-binding site or covalent enzyme-phosphate intermediate in Pgp has important mechanistic implications (Senior et al., 1995). From a technical standpoint, it also renders investigation of the enzyme mechanism difficult, e.g. by impeding direct determination of number of nucleotide-binding sites by equilibrium binding techniques, by reducing stoichiometry and specificity of labeling in (photo)affinity labeling studies, by limiting applicability of fluorescent nucleotides as reporter probes, and so forth. In several ATPase enzymes, of which myosin is a pre-eminent example (Goodno, 1979, 1982; Yount, et al., 1992), vanadate-induced trapping of nucleotides in catalytic sites, with resultant generation of a stably inhibited enzyme species, has proven extremely valuable. It provides a way of stoichiometrically and specifically binding nucleotides and nucleotide analogs in the sites, such that their dissociation rate is appreciably slowed (t values of days or even weeks have been reported). Both binding and (photo)labeling experiments are thereby facilitated (e.g. Cremo et al., 1989; Garabedian and Yount, 1991; Yount et al., 1992). Moreover, the vanadate-nucleotide-enzyme complex is thought to resemble the catalytic transition state.
The ATPase activity of both plasma membrane Pgp and purified reconstituted Pgp is inhibited when vanadate is included in the ATPase assay medium. Under these conditions, vanadate causes 50% inhibition of ATP hydrolysis at around 10 µM and complete inhibition at higher concentrations (Sarkadi et al., 1992; Ambudkar et al., 1992; Al-Shawi and Senior, 1993; Urbatsch et al., 1994). Here we carried out experiments to determine whether preincubation of Pgp with nucleotide and vanadate, followed by removal of unbound ligands, generated a species of Pgp showing long-lived inhibition of ATPase activity, as seen, for example, in myosin. We found that this was the case, and in this paper we have characterized the vanadate-induced inhibited Pgp species.
Figure 8:
Reactivation of Pgp-ATPase activity and
release of trapped [-
P]nucleotide. Plasma
membranes were preincubated with 130 µM
[
-
P]ATP in the presence of 200 µM vanadate and 3 mM MgSO
, with 200 mM sodium phosphate present. All other conditions were as under
``Experimental Procedures.'' Unbound ligands were removed by
passage through centrifuge columns (time 0), and the eluates were
incubated at 37 °C. At time intervals aliquots were passed through
second centrifuge columns, and ATPase activity and bound
[
-
P]nucleotide were determined. Panel
A,
, ATPase activity (relative to control without vanadate);
▾, [
-
P]nucleotide bound. The lines are non-linear least-squares regression analysis fits to
the data. Panel B, release of vanadate-trapped
[
-
P]nucleotide plotted against relative
ATPase activity (circles). The solid line is that
expected if 100% inactivation of ATPase corresponds to trapping of 1
mol nucleotide/mol Pgp.
Here we investigated whether
vanadate induced a long-lived inhibited species of Pgp, as has been
described for example in the case of myosin. We first preincubated
plasma membranes from the multidrug-resistant Chinese hamster ovary
cell line CR1R12, which are highly enriched in Pgp, with 200 µM vanadate in the presence of 1 mM MgATP, then removed
unbound ligands by passage through centrifuge columns (see
``Experimental Procedures''). Membranes in the eluates were
then added to ATPase assay medium, and the Pgp-ATPase activity was
found to be inhibited by 90%. This indicated that preincubation
with vanadate and MgATP induced a stably inhibited Pgp species.
Conditions for reactivation of the inhibited Pgp species were
examined in Fig. 1. The closed circles show inhibited
membranes added directly to the ATPase assay medium at 37 °C.
ATPase activity was followed over several hours and was seen to
increase exponentially with an apparent rate constant (k) of 1.4
10
s
(t = 84 min). Complete
recovery of ATPase activity was eventually obtained. 10 mM
MgATP, as present in the ATPase assay medium, was not required for
reactivation; incubation of the eluate from the centrifuge column in
0.1 mM EGTA, 40 mM Tris-Cl, pH 7.4, at 37 °C
followed by assay of ATPase at various times gave the same k
for reactivation of 1.4
10
s
(Fig. 1, open
circles).
Figure 1:
Reactivation of the ATPase activity
of Pgp in plasma membranes after inducing inhibition with vanadate and
MgATP. Inhibited Pgp was induced by preincubation of plasma membranes
with 1 mM MgATP and 200 µM vanadate at 37 °C
for 20 min followed by elution through centrifuge columns in 0.1
mM EGTA, 40 mM Tris-Cl at 23 °C as described
under ``Experimental Procedures.'' ▪, uninhibited
control with no vanadate in preincubation; , eluate from
centrifuge column added to ATPase assay medium and activity determined
at indicated times;
,
, and ▾, eluates from centrifuge
columns incubated at 37, 30, and 23 °C, respectively, and aliquots
assayed at indicated times;
, eluate from centrifuge column
incubated at 4 °C and aliquots assayed at indicated times (in this
case the centrifuge column elution was also at 4 °C). The lines are
nonlinear least-squares regression fits to the
data.
The rate of recovery of ATPase activity was
temperature dependent, with k of 6.6
10
s
(t = 175
min) and 3.7
10
s
(t = 311 min) at 30 and 23 °C, respectively (Fig. 1). At 4 °C the extrapolated half-life for
reactivation of the inhibited Pgp-ATPase was >30 h. This is similar
to the situation with myosin ATPase, which is known to form a stable
transition state-like complex with vanadate ions and MgADP that has a
half-life of 3 days at 4 °C (Goodno, 1982).
In the studies reported above, the inhibited Pgp species was formed by preincubation of membranes with vanadate and MgATP at 37 °C for 20 min. Similar results were obtained if room temperature was used. For the studies reported below, we used 37 °C throughout. Additionally, verapamil was routinely present during the inhibition phase of the experiments (see ``Experimental Procedures''), but omission of it did not affect the degree or time course of the inhibition.
Figure 2: Vanadate-induced inhibition of Pgp: dependence on vanadate concentration. Plasma membranes were incubated with varied concentrations of vanadate ± 1 mM MgATP. Pgp-ATPase activity was assayed after removal of unbound ligands (see ``Experimental Procedures'').
Fig. 3, A and B show
the effects of varying the concentrations of MgATP and MgADP at
saturating concentrations of vanadate (200 µM). The
concentrations required for half-maximal inhibition were 9 µM MgATP and 15 µM MgADP. The K for MgATP hydrolysis is 1.4 mM, and the K
for competitive inhibition by MgADP is
0.4 mM (Al-Shawi and Senior, 1993), thus vanadate strongly
increased the apparent affinities of Pgp for both MgATP and MgADP. With
both nucleotides the inhibition phase of the curves was steep (see Fig. 3legend). This aspect was not further investigated here but
is of interest for future studies.
Figure 3:
Vanadate-induced inhibition of Pgp:
dependence on MgATP or MgADP concentration. Plasma membranes were
incubated with 200 µM vanadate, 3 mM MgSO, and concentrations of ATP (A) or ADP (B) as indicated. Pgp-ATPase activity was assayed after
removal of unbound ligands (see ``Experimental Procedures'').
The dotted line is in each case the curve expected for simple
non-cooperative binding (Michaelis-Menten
equation).
Figure 4:
Vanadate-induced inhibition of Pgp: time
course of inhibition with MgATP and MgADP. Plasma membranes were
incubated with 200 µM vanadate, 3 mM MgSO, and 200 µM ATP or ADP at 37 °C
for varied times, then passed through centrifuge columns to remove
unbound ligands, and the Pgp-ATPase activity assayed. The control
(100%) was incubated without vanadate.
, ATP; ▾,
ADP.
The rate constants for the reactivation of ATPase activity
were compared (Fig. 5) after Pgp was inhibited by incubation
with either MgATP or MgADP in the presence of vanadate so as to achieve
maximal inhibition. They were in fact very similar (k for MgATP-inhibited Pgp = 1.4
10
s
and for MgADP-inhibited Pgp = 1.5
10
s
). This indicated
that the inhibited Pgp species likely contained bound MgADP and
vanadate, whether inhibition was induced in presence of MgATP or MgADP.
Figure 5:
Reactivation of inhibited Pgp after
inhibition in presence of MgATP or MgADP. Vanadate-induced inhibition
was effected in the presence of 200 µM MgATP or MgADP,
followed by removal of unbound ligands (see ``Experimental
Procedures''). The eluates from centrifuge columns were incubated
at 37 °C and ATPase assays performed at intervals. The 100% value
is that of control incubated with no vanadate. and solid
line, ATP; ▾ and dashed line,
ADP.
Fig. 6shows that vanadate-trapped
8-azido-[-
P]nucleotide was, on UV
irradiation, selectively (>95%) cross-linked to Pgp in the plasma
membranes. Immunoblotting experiments (not shown) demonstrated that the
minor labeled bands seen in Fig. 6, lane 2, of
molecular sizes 65 and 100 kDa, both reacted with C219 anti-Pgp
monoclonal antibody, and correspond to proteolytic fragments of Pgp as
shown by Georges et al.(1991). It is evident therefore that
vanadate trapping offers an excellent approach to achieving specific
(photo)labeling of Pgp catalytic sites.
Figure 6:
Vanadate trapping of 8-azido-ATP and
subsequent photolabeling of Pgp. Plasma membranes were preincubated
with 200 µM 8-azido-[P]ATP, 3
mM MgSO
, in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 200
µM vanadate. Unbound ligands were removed by passage
through centrifuge columns. The degree of Pgp-ATPase inhibition was 80%
(with vanadate) or zero (without vanadate). The eluates were placed on
ice and irradiated for 5 min (
= 254 nm, 5.5
milliwatts/cm
). Samples were subjected to
SDS-polyacrylamide gel electrophoresis, then to autoradiography (lanes 1 and 2) or staining with Coomassie Blue (lanes 3 and 4).
Figure 7:
Effects of manganese and cobalt on
vanadate- induced inhibition and reactivation. A,
vanadate-induced inhibition in the presence of 1 mM ATP was
carried out as described under ``Experimental Procedures''
except that MnSO or CoSO
replaced
MgSO
. The time course of inhibition was determined as in Fig. 4. B, reactivation of the inhibited Pgp samples at
37 °C was followed as in Fig. 5.
, manganese; ▴,
cobalt.
Fig. 8B emphasizes the excellent correlation between release of vanadate-trapped nucleotide and reactivation of ATPase activity. The solid line in Fig. 8B is the expected behavior if 100% inhibition of ATPase activity occurs with trapping of 1 mol nucleotide/mol Pgp. We conclude that vanadate trapping of nucleotide at one catalytic site/Pgp molecule is sufficient to give full inhibition of ATPase activity.
In this study we demonstrate long-lived inhibition of
Pgp-ATPase activity by vanadate. Preincubation of plasma membrane Pgp
with vanadate alone caused no inhibition, yet when ATP or ADP and Mg
ions were included, a stable, enzymatically inactive complex formed
which could be isolated free of unbound ligands. Inhibition of ATPase
activity was fully reversible with a half-life of 84 min at 37 °C,
but reactivation was very slow at 4 °C with a half-life of >30
h. These observations, reminiscent of previous studies of myosin and
other ATPase enzymes, indicated that ADP, magnesium, and vanadate,
together form a stable, ternary, noncovalent, catalytic site complex.
Further evidence supporting this conclusion was that vanadate-trapped
[-
P]ADP was demonstrated
directly in the inhibited Pgp, that release of trapped, labeled
nucleotide correlated well with reactivation of the ATPase activity,
and that vanadate-trapped 8-azido-nucleotide caused permanent
inhibition of Pgp-ATPase activity after UV irradiation.
These findings open up avenues of experimentation which had been precluded or rendered difficult because of the low affinity with which Pgp binds nucleotides and analogs. The main advantage is that vanadate greatly increases the apparent affinity for the nucleotide. This allows, for example, efficient trapping of photolabeling analogs at the catalytic sites with removal of unbound ligand, which should greatly improve the specificity of incorporated photoaffinity probes at catalytic sites and help identify residues in the catalytic sites by protein chemical approaches. Vanadate trapping allows stoichiometry of nucleotide binding to be analyzed with much greater accuracy, and may be valuable for application of fluorescent nucleotides, for example in fluorescence energy transfer experiments.
Vanadate-induced inhibition of Pgp-ATPase was seen in both plasma membranes from the multidrug-resistant Chinese hamster ovary cell-line CR1R12 and in purified reconstituted Pgp. In this paper we have characterized features of the vanadate-induced inhibition of Pgp using plasma membranes, which provide a good source of material for multiple experiments. We demonstrate that a variety of nucleotides which are substrates also support the inhibition, in concert with earlier findings that the catalytic sites are of low nucleotide specificity. We characterize the time course and affinity of inhibition with adenine nucleotide, the dependence of inhibition on vanadate concentration, and the effects of different divalent cations. Reactivation of the inhibited Pgp was also characterized and was seen to be temperature-dependent. At 4 °C the half-life for reactivation was >30 h. Inhibition in the presence of cobalt ions was seen to lead to formation of an inhibited Pgp species with an extremely long half-life of reactivation.
Vanadate-induced trapping of radioactive nucleotide
was demonstrated directly in Table 3. It appeared from these
experiments that full inhibition occurred with 1 mol of nucleotide
trapped per mol of Pgp. On reactivation, release of radioactive
nucleotide correlated well with recovery of Pgp-ATPase activity (Fig. 8A). Fig. 8B indicated that full
relief of inhibition of Pgp-ATPase occurred with release of 1 mol of
trapped nucleotide/mol of Pgp.
Based on the evidence that
reactivation occurred at the same rate whether inhibition was induced
with MgATP or MgADP (Fig. 5) it seemed likely that MgADP was the
trapped nucleotide species in both cases. Using thin layer
chromatography, it was confirmed that in experiments in which
[-
P]ATP was used to induce
inhibition the resultant trapped nucleotide was
[
-
P]ADP. In myosin, the
vanadate-trapped nucleotide species is also the nucleoside diphosphate
(Goodno, 1979, 1982). Considering the similarity in structure between
vanadate and phosphate, the
Pgp
Mg
MgADP
V
complex may be a
stable analog of the Pgp
Mg
MgADP
P
complex formed by ATP hydrolysis in Pgp.
Finally, we note that vanadate-induced inhibition was demonstrated to occur in purified, reconstituted Pgp, with characteristics the same as those seen in plasma membrane Pgp. Therefore, the procedure of vanadate trapping of nucleotide may also be applied with advantage to studies of pure Pgp.