(Received for publication, July 13, 1995)
From the
The technique of vanadate trapping of nucleotide was used to
study catalytic sites of P-glycoprotein (Pgp) in plasma membranes from
multidrug-resistant Chinese hamster ovary cells. Vanadate trapping of
Mg- or Co-8-azido-nucleotide (1 mol/mol of Pgp) caused complete
inhibition of Pgp ATPase activity, with reactivation rates at 37 °C
of 1.4 10
s
(t
= 8 min) or 3.3
10
s
(t
= 35 min), respectively. UV
irradiation of the inhibited Pgp yielded permanent inactivation of
ATPase activity and specific photolabeling of Pgp. Mild trypsin
digestion showed that the two nucleotide sites were labeled in equal
proportion. The results show that both nucleotide sites in Pgp are
capable of nucleotide hydrolysis, that vanadate trapping of nucleotide
at either site completely prevents hydrolysis at both sites, and that
vanadate trapping of nucleotide in the N- or C-terminal nucleotide
sites occurs non-selectively. A minimal scheme is presented to explain
inhibition by vanadate trapping of nucleotide and to describe the
normal catalytic pathway. The inhibited
Pgp
Mg-nucleotide
vanadate complex is probably an analog of
the catalytic transition state, implying that when one nucleotide site
assumes the catalytic transition state conformation the other site
cannot do so and suggesting that the two sites may alternate in
catalysis.
P-glycoprotein (Pgp), ()also called
multidrug-resistance protein, is involved in resistance of tumors to a
variety of drugs and appears to be responsible for many failures of
cancer chemotherapy (reviewed in Endicott and Ling(1989), Gottesman and
Pastan(1993), and Gros and Buschman(1993)). Pgp is a plasma membrane
protein that reduces intracellular accumulation of drugs by
transporting them out of the cell in an ATP hydrolysis-dependent
process (Ruetz and Gros, 1994) (reviewed in Shapiro and Ling(1995) and
Sharom(1995)). Coupling between catalytic sites for ATP hydrolysis on
the cytoplasmic surface and drug-binding/transport sites in the
membrane is also apparent from the drug stimulation of ATP hydrolysis
seen in plasma membrane preparations (Sarkadi et al., 1992;
Al-Shawi and Senior, 1993), partially purified and reconstituted Pgp
(Ambudkar et al., 1992; Sharom et al., 1993), and
purified, reconstituted Pgp (Shapiro and Ling, 1994; Urbatsch et
al., 1994; Sharom et al., 1995).
The amino acid
sequence of Pgp reveals a tandemly duplicated molecule, each half
containing one cytoplasmically sided nucleotide-binding site, as
diagnosed by the presence of both ``homology A'' and
``homology B'' consensus sequences (Walker et al.,
1982). A major goal of current research is to determine the mode of
operation of these two nucleotide-binding sites in the mechanism of
action of Pgp. From the data available it has not yet been possible to
determine the number of sites that are catalytically active in Pgp.
There is a single relatively high K(MgATP) around 1 mM, and ATP
hydrolysis follows simple monophasic Michaelis-Menten kinetics. MgADP
and MgAMPPNP behave as classical competitive inhibitors with K
values of 0.70 and 0.35 mM,
respectively (Al-Shawi and Senior, 1993; Urbatsch et al.,
1994). With 8-azido-ATP, which is an excellent substrate for hydrolysis
by Pgp, photoinactivation of the Pgp ATPase occurred coincidental with
covalent incorporation of approximately 2 mol of
8-azido-[
-
P]ATP/mol of Pgp, with the
incorporated analog distributed equally between N- and C-terminal
halves of the molecule. These data indicated that both
nucleotide-binding domains were capable of binding 8-azido-ATP (Georges et al., 1991; Al-Shawi et al., 1994). The inhibitor N-ethylmaleimide reacted at two sites, with the critical
sulfhydryls located equally in N- and C-terminal halves of the Pgp,
whereas NBD-Cl, another catalytic site covalent inhibitor, gave full
inactivation at
1 mol/mol of Pgp, with reaction occurring
predominantly in the C-terminal half (Al-Shawi and Senior, 1993;
Al-Shawi et al., 1994). Since MgATP afforded full protection
against inhibition by both N-ethylmaleimide and NBD-Cl, it
appeared that both nucleotide sites could bind MgATP and that
inhibition at the C-terminal site alone gave 100% inhibition of ATPase
activity.
Introduction of point mutations into either or both of the nucleotide-binding domains by site-directed mutagenesis inhibited the drug exclusion function of Pgp expressed in mammalian cells (Azzaria et al., 1989; Roninson, 1992). Loo and Clarke(1994) expressed each half of the Pgp molecule separately in Sf9 cells and found that each half-molecule displayed significant ATPase activity. This experiment showed that each nucleotide site has the potential capability for ATP hydrolysis. The state of oligomerization (homodimer? monomer?) of the expressed half-molecules of Pgp was not determined. Drug stimulation of the Pgp ATPase was apparent only in intact Pgp or when both half-molecules were expressed contemporaneously in the same cell, and Loo and Clarke concluded that coupling of ATPase activity to drug binding/transport requires interaction of both halves of the Pgp.
We have recently found that vanadate trapping of nucleotide at
catalytic sites of Pgp induces stable inhibition of ATPase activity
(Urbatsch et al., 1995). The nucleoside diphosphate was the
trapped species whether vanadate inhibition was induced with MgATP or
MgADP, and manganese or cobalt were also effective as metal cofactors.
Complete inactivation of ATPase was achieved with trapping of 1 mol of
MgADP/mol of Pgp, and full reactivation of ATPase correlated with
release of a single mol of trapped
Mg-[-
P]ADP per mol of Pgp. It has
previously been demonstrated in myosin that vanadate trapping of MgADP
at a catalytic site produces a stably inhibited complex, which
resembles the catalytic transition state (Goodno, 1979, 1982), and it
appeared that vanadate trapping induced a similar complex in Pgp,
namely Pgp
MgADP
V
.
The vanadate-trapping technique is a valuable tool for investigation of Pgp catalytic sites because it greatly increases the apparent affinity for nucleotides. Here we used vanadate trapping with the photoreactive 8-azido-ATP to investigate whether both nucleotide sites in Pgp in plasma membranes can hydrolyze nucleotide and whether vanadate trapping of nucleotide, with consequent inhibition of ATPase, occurs preferentially at one or other of the two nucleotide-binding sites. We further investigated the competition between phosphate and vanadate by measuring formation of the inhibited Pgp species. The results are interpreted in terms of mechanism of vanadate-induced inhibition of Pgp ATPase activity and the normal ATPase reaction pathway.
With 200
µM Mg-8-azido-ATP and Co-8-azido-ATP the degree of
inhibition was 85 and 95%, respectively. Similar results were seen with
Mg-8-azido-ADP and Co-8-azido-ADP. Reactivation of Pgp ATPase activity
occurred when the centrifuge column eluates were incubated at 37
°C. Fig. 1A shows the reactivation after inhibition
in the presence of 200 µM Mg-8-azido-ATP or
Mg-8-azido-ADP. The time courses for reactivation in the two cases were
identical (t= 8 min; k
= 1.4
10
s
). Fig. 1B shows the reactivation after inhibition in the
presence of the corresponding Co-nucleotides. Here, the time courses
were appreciably slower but were again the same for the two cases (t
= 35 min; k
= 3.3
10
s
).
The fact that identical reactivation rates were seen whether inhibition
was induced with 8-azido-ATP or 8-azido-ADP argues strongly that the
inhibited Pgp species contains vanadate-trapped 8-azido-ADP at
catalytic sites. This is consistent with previous work (Urbatsch et
al., 1995), which established that vanadate-trapped ADP was the
inhibitory species when Pgp was preincubated with ATP and vanadate, and
with the fact that 8-azido-ATP is known to be a good substrate for
hydrolysis by Pgp (Urbatsch et al., 1994). The reactivation of
Pgp ATPase after inhibition with 8-azido-adenine nucleotide and
vanadate was notably faster than was seen previously after inhibition
with adenine nucleotide and vanadate. Thus the fact that reactivation
was slower in the case of Co-8-azido-nucleotide proved technically
advantageous.
Figure 1: Reactivation of the ATPase activity of Pgp in plasma membranes after inducing inhibition with vanadate and 8-azido-ATP or 8-azido-ADP. Panel A, inhibition of ATPase was induced as described under ``Experimental Procedures,'' using 200 µM Mg-8-azido-ATP (squares) or 200 µM Mg-8-azido-ADP (triangles). Eluates from centrifuge columns were then incubated at 37 °C, and aliquots were taken for ATPase assay at various times. The lines are least square regression fits. Control (uninhibited) samples were preincubated with vanadate but without nucleotide. Panel B, as in A, using 200 µM Co-8-azido-ATP (diamonds) or 200 µM Co-8-azido-ADP (inverted triangles).
Permanent inactivation of Pgp ATPase was obtained in samples that had been inhibited by preincubation with vanadate and 200 µM Co-8-azido-ATP and then subjected to UV irradiation as described under ``Experimental Procedures.'' Samples that had been so treated remained 91% inhibited after 4 h of incubation at 37 °C. Thus, in contrast to the data in Fig. 1B, there was now essentially no reactivation, confirming that 8-azido-adenine nucleotide had been trapped in catalytic sites.
Figure 2:
Dependence of vanadate-induced inhibition
of Pgp ATPase activity in plasma membranes on Co-8-azido-ATP
concentration. Inhibition was induced as described under
``Experimental Procedures'' using 200 µM
vanadate, 3 mM CoSO, and 8-azido-ATP concentration
as indicated. The control (uninhibited) activity was that of a sample
preincubated without nucleotide.
We then performed experiments using a range of
concentrations of Co-8-azido-[-
P]ATP from
80 µM down to 5 µM. This concentration range
produced inhibition of Pgp ATPase from 95% down to 60% (see Fig. 2), and therefore the stoichiometry of trapped nucleotide
would range from
1 mol/mol to around 0.6 mol/mol. Fig. 3(lanes 1-4) shows the photolabeling of Pgp
in plasma membranes after vanadate trapping with
Co-8-azido-[
-
P]ATP at concentrations of 80,
20, 10, and 5 µM. Direct counting of the Pgp bands excised
from SDS gels showed that 26-54% of the trapped
P in
the centrifuge column eluates became covalently attached to Pgp on UV
irradiation and survived gel electrophoresis. For comparison,
photoincorporation of vanadate-trapped azido-nucleotide analogs in
myosin ranged from a few percent to 70% (Yount et al., 1992).
Figure 3:
Photolabeling of Pgp in plasma membranes
after vanadate trapping with
Co-8-azido-[-
P]ATP. Vanadate-induced
inhibition in the presence of varied concentrations of
8-azido-[
-
P]ATP, 200 µM vanadate, and 3 mM CoSO
and subsequent UV
irradiation of inhibited samples were performed as described under
``Experimental Procedures.'' The photolabeled samples were
run on SDS gels and subjected to autoradiography. Lanes
1-4, 80, 20, 10, and 5 µM nucleotide; lanes
5-8, same as lanes 1-4 except that the
samples were subjected to mild trypsin digestion to fragment the Pgp
into N- and C-terminal halves before being applied to SDS gels. The
position of N- and C-terminal halves of Pgp on SDS gels was confirmed
using immunoblotting with C219 anti-Pgp monoclonal
antibody.
Georges et al.(1991) showed that Pgp in Chinese hamster ovary cell plasma membranes can be fragmented into N-terminal and C-terminal ``halves'' by mild trypsin digestion. Each half contains one of the predicted nucleotide-binding domains. The N-terminal (glycosylated) fragment has an apparent molecular size of 100 kDa and the C-terminal fragment of 65 kDa on SDS gels. We previously confirmed, using the C219 monoclonal antibody, that this technique is reproducible with CR1R12 cell plasma membranes (Al-Shawi et al., 1994). Fig. 3(lanes 5-8) shows the results obtained when the photolabeled Pgp was digested with trypsin into the two halves before running on SDS gels. It is seen that even at the lowest concentration of nucleotide used, both halves of Pgp were labeled. The distribution of the radioactivity between the two halves as determined by direct counting of bands excised from gels was: N-terminal, 53%; C-terminal, 47%. This ratio was similar at all the nucleotide concentrations studied (the variation was ±9%). The fact that both nucleotide sites became labeled at all the nucleotide concentrations used showed that vanadate trapping of nucleotide in N- or C-terminal nucleotide sites occurred non-selectively.
Similar
experiments to those shown in Fig. 3were performed using
Mg-8-azido-[-
P]ATP, and they confirmed the
above conclusions. Briefly, full inhibition of Pgp ATPase was obtained
with
1 mol of trapped Mg-nucleotide/mol of Pgp, and using a range
of concentrations of Mg-nucleotide which yielded 36-74%
inhibition of ATPase activity by vanadate trapping, it was found after
subsequent UV irradiation and mild trypsin digestion of the samples
that both halves of Pgp became photolabeled (data not shown).
Figure 4:
Competition between phosphate and vanadate
during vanadate-induced inhibition of Pgp ATPase activity in plasma
membranes. Vanadate-induced inhibition of Pgp ATPase activity was
carried out as described under ``Experimental Procedures''
using varying concentrations of vanadate. Panel A, using 200
µM MgATP: diamonds, no addition; open
circles, with 200 mM sodium phosphate; closed
circles, with 133 mM NaSO
. Panel B, using 200 µM MgADP: squares, no
addition; open inverted triangles, with 200 mM sodium
phosphate; closed inverted triangles, with 133 mM
Na
SO
.
Fig. 4B shows a parallel experiment using 200
µM MgADP. Here, the concentration of vanadate producing
half-maximal inhibition was 9 µM (no P, squares) or 100 µM (plus 200 mM
NaP
, open inverted triangles). The calculated K
(P
) from these data was 22
mM, and inhibition was again competitive. The closed
inverted triangles show similar data obtained in the presence of
133 mM Na
SO
. Here the concentration of
vanadate producing half-maximal inhibition was 26 µM,
corresponding to a K
(P
) of 70
mM.
Further experiments were performed in which
V concentration was held constant at 200 µM,
and MgATP or MgADP concentration was varied from zero to 200 µM in the presence or absence of 200 mM P
. There
was no significant effect of P
on the observed inhibition
at different nucleotide concentrations.
Figure 5:
Effect of 200 mM phosphate on
ATPase activity of Pgp in plasma membranes. ATPase activity was
measured as described under ``Experimental Procedures,''
except that 133 mM NaSO
(
) or
200 mM sodium phosphate (
) was added to the assay
medium.
We used the technique of vanadate-induced inhibition by
trapped nucleotide to study catalytic properties of P-glycoprotein in
plasma membranes from multidrug-resistant CR1R12 Chinese hamster ovary
cells. It was demonstrated that in presence of vanadate plus
8-azido-[-
P]ATP and either Mg
or Co
, 8-azido-adenine nucleotide became stably
trapped in Pgp catalytic sites. Trapping of 1 mol of nucleotide per mol
of Pgp caused complete inhibition of ATPase. The evidence presented
here and previously (Urbatsch et al., 1995) indicates that
nucleoside diphosphate is the trapped inhibitory species. UV
irradiation of inhibited Pgp caused permanent inactivation and revealed
that both halves of Pgp became photolabeled. Vanadate trapping of
nucleotide in the N- or C-terminal nucleotide sites appeared to occur
non-selectively. The true distribution of vanadate-trapped nucleotide
in the two sites might be obscured by differential covalent attachment
upon UV irradiation or loss of covalent label on SDS-gel
electrophoresis. Nevertheless, the presence of radioactive label in
both halves of Pgp provides convincing evidence that both nucleotide
sites in intact Pgp are capable of hydrolyzing 8-azido-ATP to
8-azido-ADP and thus forming the stably inhibited
Pgp
Me-8-azido-ADP
V
complex. Furthermore,
vanadate trapping of nucleotide at either of the two sites must be able
to completely prevent hydrolysis at both sites in order to give
complete inhibition of ATPase activity. Therefore, both nucleotide
sites in Pgp are potentially catalytically active, but they are not
independent; indeed there is very strong cooperative interaction
between them. One likely possibility is that the two sites alternate in
catalysis.
The data presented here are consistent with previous results (Al-Shawi et al., 1994), which indicated that ATP and 8-azido-ATP bind to both of the two nucleotide-binding sites. Much higher concentrations of 8-azido-ATP were used previously than here because vanadate was not used, and this explains why labeling stoichiometries of up to 2 mol/mol of Pgp were seen in the earlier work. It was also seen previously that NBD-Cl gave full inhibition of ATPase at a labeling stoichiometry of 1 mol/mol of Pgp. This also is consistent with the results obtained here. Interestingly, NBD-Cl reacted predominantly in the C-terminal half, this preference being presumably due to a specific reactive residue in the C-terminal nucleotide site, which is either absent or less reactive in the N-terminal nucleotide site. N-Ethylmaleimide, in contrast, appeared to react in both nucleotide sites.
The data presented here are also consistent with previous mutagenic analyses of Pgp, which showed that introduction of point mutations into either or both nucleotide sites rendered the Pgp completely inactive (Azzaria et al., 1989; Roninson, 1992), and also with the data of Loo and Clarke(1994), which showed that either nucleotide site is potentially capable of ATP hydrolysis. It seems likely that ATPase activity in the experiments of Loo and Clarke(1994) resulted from formation of homodimers of the expressed ``half-molecules.''
In Fig. S1we propose a minimal reaction pathway for
vanadate-induced inhibition of Pgp. This scheme draws on work presented
here and previously (Urbatsch et al., 1995) and parallels
schemes for vanadate-induced inhibition of myosin ATPase (Goodno, 1979,
1982; Goodno and Taylor, 1982) and dynein ATPase (Shimizu and Johnson,
1983). Vanadate-induced inhibition of Pgp is seen when either MgATP or
MgADP is incubated with vanadate (V), but MgADP is the
trapped ligand in both cases. We propose that the Pgp
MgADP
species forms by either pathway k
k
k
or by pathway k
. V
can then
bind to Pgp
MgADP (step k
) to form the
inhibited Pgp
MgADP
V
complex. Reactivation of
ATPase is slow (steps k
k
) (k
= 1.4
10
s
at 37 °C) (Urbatsch et al., 1995) and
must be limited by rate k
, since overall
ATPase turnover in Pgp is
20 s
at 37 °C.
Figure S1: Scheme 1.
The finding that phosphate competes with vanadate (Fig. 4),
but not with nucleotide, during formation of the inhibited Pgp species
indicated that the stable inhibited complex cannot form when the
catalytic site of Pgp is occupied by MgADP plus P. Thus
P
appeared to compete with V
at step k
but was clearly a weakly binding ligand.
V
was shown previously to be a P
analog, which
became bound to P
sites in both myosin and dynein (Goodno
and Taylor, 1982; Werber et al., 1992; Shimizu and Johnson,
1983), and it appears that V
occupies essentially the same
position in the Pgp catalytic site as that of P
derived
from ATP during hydrolysis. The weak binding of P
by Pgp
implies that a large free energy decrease occurs on P
release (step k
) and that this step
may normally be coupled to transport of drugs.
P at a
very high concentration did inhibit ATPase activity (Fig. 5). An
explanation that is consistent with Fig. S1would be that
P
, when present at a high concentration in the medium, can
bind to the Pgp
MgADP species, which has formed by catalytic steps k
k
k
,
and that this prevents MgADP release at step k
. This would imply that P
release must precede MgADP release in catalysis and that MgADP
release (step k
) is rate-limiting.
Shimizu and Johnson(1983) analyzed presteady state kinetics of MgATP
binding and hydrolysis by dynein ATPase and found that in the presence
of vanadate the very first cycle of MgATP hydrolysis as measured by
-
P release occurred in a normal manner, but the
ATPase activity became inhibited after just a single turnover. In Pgp,
as we reported earlier, vanadate inhibition induced with MgATP was
rapid, whereas that induced with MgADP was much slower. We speculate
that the first catalytic turnover in Pgp could rapidly produce an
intermediate that favors occurrence of inhibition and that the binding
of vanadate to this intermediate is also rapid.
Overall, it appears
reasonable to assume that the inhibitory PgpMgADP
V
complex is a stable analog of the Pgp
MgADP
P
transition state complex and that the normal catalytic pathway in
Pgp follows the route (k
k
k
k
).
Understanding how the two catalytic sites interact such that presence
of the Pgp
MgADP
V
complex at either site
completely blocks ATP hydrolysis at both sites and whether this
interaction is a reflection of normal catalytic behavior remains to be
clarified. The present data suggest that once one site assumes the
catalytic transition state conformation, the other site cannot do so.