(Received for publication, June 26, 1995; and in revised form, October 5, 1995)
From the
We have recently described an ATP-driven, valinomycin-dependent Rb
uptake into proteoliposomes
reconstituted with mammalian P-glycoprotein (Eytan, G. D., Borgnia, M.
J., Regev, R., and Assaraf, Y. G.(1994) J. Biol. Chem. 269,
26058-26065). P-glycoprotein mediated the ATP-dependent uptake of
Rb
-ionophore complex into the
proteoliposomes, where the radioactive cation was accumulated, thus,
circumventing the obstacle posed by the hydrophobicity of
P-glycoprotein substrates in transport studies. Taking advantage of
this assay and of the high levels of P-glycoprotein expression in
multidrug-resistant Chinese hamster ovary cells, we measured
simultaneously both the ATPase and transport activities of
P-glycoprotein under identical conditions and observed 0.5-0.8
ionophore molecules transported/ATP molecule hydrolyzed. The amount of
Rb
ions transported within 1 min via the
ATP- and valinomycin-dependent P-glycoprotein was equivalent to an
intravesicular cation concentration of 8 mM. Thus, this
stoichiometry and transport capacity of P-glycoprotein resemble various
ion-translocating ATPases, that handle millimolar substrate
concentrations. This constitutes the first demonstration of comparable
rates of P-glycoprotein-catalyzed substrate transport and ATP
hydrolysis.
Inherent as well as acquired resistance to antineoplastic agents
pose a major obstacle toward curative cancer
chemotherapy(1, 2) . Multidrug resistance (MDR) ()is characterized by the development of tumor cell
resistance to diverse anticancer drugs. Mammalian cells with the
typical MDR phenotype express increased levels of P-glycoprotein (Pgp),
an integral component of the plasma membrane(3) . Consequently,
these cells display resistance to multiple cytotoxic hydrophobic
agents, mostly of natural origin, including anthracyclines, Vinca alkaloids, epipodophyllotoxins, actinomycin D, taxoids, and
dolastatin 10. Pgp, which possesses an ATPase activity, functions as an
energy-dependent extrusion pump that expels these hydrophobic cytotoxic
agents out of MDR cells(4) .
Sharom et al.(5) have extracted most of the proteins from plasma membranes of MDR cells and have reconstituted the Pgp that remained in the membranes. The reconstituted proteoliposomes displayed an ATP-dependent transport of colchicine, an established substrate of Pgp. Shapiro and Ling (7) have purified Pgp from Pgp-rich cells by a combination of anion exchange and immunoaffinity chromatography. The Pgp preparation was 90% pure and after reconstitution exhibited an ATPase activity that was highly stimulatable by several MDR type drugs and chemosensitizers. Urbatsch et al.(8) have purified Pgp to apparent homogeneity from an extremely Pgp-rich Chinese hamster ovary cell line, reconstituted it, and characterized its drug-stimulatable ATPase activity.
Sharom et al.(5) have shown that colchicine was taken up actively into
proteoliposomes with Pgp from CHO cells, against a 5.6-fold
concentration gradient. However, the rate of colchicine uptake was
about 15 pmol/mg/min with an ATPase activity of 0.5
µmol/mg/min. This extremely low stoichiometry of drug transport to
ATP hydrolysis is probably due to the hydrophobicity and membrane
permeability of Pgp substrates. Recently, Shapiro and Ling (9) have described the ATP-dependent uptake of the fluorescent
Pgp substrate, Hoechst 33342, into proteoliposomes reconstituted with
purified Pgp from highly MDR CHO cells. They reported a stoichiometry
of 1 substrate molecule transported/50 ATP molecules hydrolyzed and
attributed this low efficiency to fast rebinding of the dye to the
vesicles.
The low transport efficiency reported for Pgp, either in membrane vesicles or in reconstituted proteoliposomes, led to proposals that other mechanisms were responsible for the relatively low levels of drugs observed in MDR cells(10) . Thus, demonstration of Pgp-mediated transport similar in rate to the Pgp ATPase will prove that Pgp is indeed a drug-efflux pump and could function as such also in vivo.
We have recently described an ATP-driven,
valinomycin-dependent Rb
uptake into
proteoliposomes reconstituted with mammalian
P-glycoprotein(6) . Under these conditions mammalian Pgp
transported a cation-ionophore complex and the cation,
Rb
in this case, accumulated in the
intravesicular space. The apparent advantage of this assay is that
although the actively-transported substrate, ionophore-cation complex,
is hydrophobic, the accumulated species is the hydrophilic cations. In
the present study, we took advantage of the methodology developed for
assay of ionophore- and ATP-dependent Pgp-mediated
Rb
uptake to measure simultaneously both
the ATPase and transport functions of reconstituted Pgp. The
valinomycin- and ATP-dependent
Rb
uptake
into proteoliposomes reconstituted with Pgp was close to the ATPase
rate exhibited by these proteoliposomes under identical experimental
conditions and thus constitutes the first direct demonstration that
isolated Pgp could function as an efficient drug-efflux pump.
In transport studies the effect of
Na,K
-ATPase was abolished by careful
depletion of Na
from all reagents used in the
transport assay. The triphosphate nucleotides were treated with Dowex
50WX8. The pH of the solutions was monitored and resin was added, until
no further acidification occurred. The solutions were passed through a
0.22-µm filter and titrated to pH 7.4 with solid Tris base.
Emetine-resistant variants were derived from wild type AA8 cells
using a stepwise selection protocol of increasing drug concentrations.
Drug selection was performed by first seeding 5 10
wild type cells/25-cm
tissue culture flask (Nunc) in
growth medium (5 ml) containing 0.15 µM emetine (
3
times the LD
for parental AA8 cells). Following growth to
midconfluence, emetine-selected cells were detached by trypsinization,
counted and replated as above in the presence of a 50-100%
increment in the emetine concentration. The gradual increase in emetine
concentrations was terminated at 1 µM (
20-fold
LD
).
Proteoliposomes were formed by rapid dilution of the protein and
lipid solution into 25 volumes of reconstitution medium containing PMSF
as the sole protease inhibitor. The proteoliposomes were washed twice
by centrifugation at 130,000 g for 45 min, and
suspended in 1 ml of reconstitution medium. The proteoliposomes were
fused by adding CaCl
(25 mM final concentration),
incubated for 20 min, and diluted into 7 ml of reconstitution buffer,
and EDTA was added to a final concentration of 5 mM. The
proteoliposomes were concentrated by centrifugation for 45 min at
130,000
g and suspended in 0.5 ml of reconstitution
buffer using a 27-gauge needle. The proteoliposomes were used either
directly or after an overnight incubation on ice. Due to the high lipid
content of the samples protein was determined according to
Esen(11) , using bovine serum albumin (Fraction V, Sigma) as a
standard.
Valinomycin- and ATP-dependent Rb
uptake was assayed essentially
according to the assay strategy we have recently described(6) .
The transport activity of Pgp was assessed indirectly by measuring
uptake of
Rb
ions transported as a
Rb
-valinomycin complex into reconstituted
proteoliposomes (see Fig. 1for a scheme describing the
methodology).
Rb
serves as a convenient
monitor of K
. The assay of
Rb
uptake was based on the amplification of the isotope uptake by an
outwardly oriented concentration gradient of
K
(15) . The principle of the method relies on
the trapping of high K
concentrations within the
proteoliposomes. Upon dilution of the proteoliposomes into the assay
buffer and selective permeation of the proteoliposomes to
Rb
and K
ions by the
ionophore mobile carrier-type valinomycin, a diffusion potential is
formed, which maintains the K
gradient.
Rb
is transported into the
proteoliposomes until equilibration of its specific radioactivity with
K
is reached. Thus, even in absence of ATP hydrolysis,
the K
is concentrated in the proteoliposomes
relatively to its concentration in the buffer. This accumulation of
Rb
is transient, as during the course of
time, the cation gradient is collapsed, and the accumulated isotope
will flow out of the proteoliposomes.
Figure 1:
Scheme illustrating mechanism of
ATP-driven and valinomycin-dependent Pgp-mediated Rb
uptake. Pgp actively transports
Rb
ions into proteoliposomes by the
following mechanism. Pgp catalyzes the ATP-dependent uptake of
K
-ionophorecomplex into the vesicles. The
outward-oriented membrane potential prevents net efflux of cations, and
the actively transported
Rb
ions remain
trapped in the vesicles while the hydrophobic ionophore leaks out of
the vesicle.
Valinomycin exhibits a high
affinity toward K and
Rb
ions; thus, in the presence of relatively high cation
concentrations present in the uptake medium, valinomycin is presented
to Pgp predominantly as a cation-ionophore complex. Moreover, most
known substrates of Pgp are hydrophobic and cationic in nature, and
thus it is likely that the ionophore-cation complex is a preferred
substrate for Pgp when compared with the unloaded ionophore. The ATP-
and valinomycin-dependent
Rb
uptake
required preloading of the proteoliposomes with K
ions, and was abolished by dissipation of the K
gradient. Thus, Pgp transports the cation-ionophore complex into
the intravesicular volume of the vesicles. The
Rb
ions cotransported with valinomycin
equilibrate with the cations trapped within the vesicles. Since the
amount of
Rb
ions transported is in large
excess compared to the total amount of valinomycin present in the
medium, valinomycin plays a catalytic role and is recycled. Presumably,
the electric potential formed by the K
-gradient across
the proteoliposome membrane hinders the release of
Rb
together with the accumulated
valinomycin, and valinomycin leaks out of the proteoliposomes as the
unloaded species. Thus, although the actual Pgp substrate is the
hydrophobic
Rb
-valinomycin complex, the
accumulated substrate is hydrophilic
Rb
ions.
The transport of Rb
was
measured by rapid removal of extravesicular cations with the strong
cation exchange resin, Dowex-50WX8, 100-mesh(15) , as modified
by Garty and Karlish (16) . Unless otherwise stated, the
transport buffer contained: 25 mM Hepes-Tris (pH 7.4), 0.25 M sucrose, 8 mg/ml bovine serum albumin, 4 mM MgCl
, 2 µCi/ml carrier-free
Rb
, various amounts of valinomycin, and
either 1 mM ATP or AMPPCP in a final volume of 0.125 ml. The
buffer was preincubated for 2 min at 37 °C, and the transport was
initiated by addition of 5 µl of reconstituted proteoliposomes.
Non-hydrolyzable analogs of ATP (i.e. AMPPCP) were included in
the control samples since the cation-permeability of the
proteoliposomes was very sensitive to
Mg
-concentrations and nucleotide triphosphates are
efficient Mg
-chelators capable of altering the
Mg
-concentrations. At appropriate times, the
transport reactions were stopped by withdrawing 0.1-ml samples. The
extravesicular cations were removed as described by Garty and Karlish (15) , and the amount of radioactivity associated with the
vesicles was determined. The stopping procedure was concluded within 15
s. The amount of radioactivity associated with proteoliposomes
incubated in the absence of ionophores was less than 0.05% of the total
radioactivity added and did not increase upon a 30-min incubation at 37
°C. This amount, presumably representing nonspecific adsorption,
was routinely subtracted from all samples.
We undertook this study in order to estimate the stoichiometry of drug transport to ATP hydrolysis catalyzed by Pgp. To this end, both the ATPase and transport functions of Pgp had to be measured simultaneously under identical experimental conditions.
In
this respect, we have described an assay of valinomycin uptake into
proteoliposomes reconstituted with Pgp from rat liver, the amount of
transported valinomycin was assessed as the quantity of Rb
ions cotransported with the ionophore (6) . The amount of Pgp present in canalicular vesicles from
rat liver was low, and its presence could be detected only by Western
blotting with a monoclonal antibody(6) . As a result, its
ATPase activity was relatively low and was masked by other ATPases
present in the preparation. In contrast, Pgp ATPase activity has been
demonstrated with Pgp from multidrug-resistant CHO cells where it is
highly overexpressed(5, 8, 9, 13) .
Thus, the aim of the present study was to measure simultaneously both
ATPase and
Rb
uptake functions assayed
under identical conditions as valinomycin-dependent activities of
hamster Pgp.
Toward this end, a CHO variant (Emt)
highly-expressing Pgp was established by stepwise selection with the
MDR drug, emetine. The Pgp content in the microsomal fraction of this
Emt
subline constituted 4.5% of the total protein content.
Upon reconstitution, the relative amount of Pgp was increased to 18%,
and under the assay conditions used here all the ATPase activity was
attributable to Pgp(13) . The ATPase activity was stimulated by
known substrates of Pgp, inhibited by known inhibitors of Pgp including
vanadate and oligomycin, and insensitive to ouabain and EGTA.
Reconstitution of Pgp from Emt
plasma membranes, the Pgp
content of which was 18%, yielded proteoliposomes with a Pgp content of
40% and a consistently higher ATPase activity(13) . However,
the yield of these proteoliposomes was low, and since they did not pose
a clear advantage over proteoliposomes reconstituted with Pgp from the
microsomal fraction, the latter were routinely used.
The basal (i.e. with no substrates added) ATPase activity of
proteoliposomes reconstituted with Emt microsomal fraction
was 1.1 ± 0.25 µmol of P
/min/mg of protein (Fig. 2A and (13) ). A similar basal activity
was reported for various Pgp
preparations(5, 7, 8, 17, 18) .
This basal activity was stimulated by valinomycin and emetine, the
selecting drug used to establish Emt
cells (Fig. 2). However, a major problem became evident as the minimal
valinomycin concentrations required for demonstrating stimulation of
ATPase activity were higher than 0.1 µM, whereas
appreciable ATP- and valinomycin-dependent
Rb
uptake was already evident at a concentration of 0.1 µM valinomycin. Thus, the high basal activity demonstrated by Pgp
presumably masked the ATPase activity required for
valinomycin-dependent transport. To overcome this obstacle, we looked
for a Pgp inhibitor capable of reversibly repressing the basal ATPase
activity without exerting a deleterious effect on the proteoliposomes.
In this respect we have recently found that various hydrophobic
homopolypeptides modulate Pgp ATPase activity. (
)Poly-L-tryptophan met these expectations; at
concentrations lower than 100 nM, it inhibited both the basal
ATPase and the substrate-stimulatable activities of Pgp (Fig. 2B). These concentrations of
poly-L-tryptophan had no deleterious effects on the integrity
of the proteoliposomes as revealed by retention of encapsulated
Rb
or calcein (data not shown). Most
importantly for this study, low concentrations of
poly-L-tryptophan repressed the basal ATPase activity and, at
concentrations required for mediation of ATP-dependent
Rb
uptake (see below), valinomycin
reactivated it in a competitive manner (Fig. 3). The
Michaelis-Menten type competitive inhibition exerted by
poly-L-tryptophan on the stimulatory effect of valinomycin is
presented in Fig. 3B as a Lineweaver-Burk plot.
Figure 2:
Modulation of Pgp ATPase activity by
emetine, valinomycin, and poly-L-tryptophan. Pgp was extracted
from the MDR cells, Emt, and reconstituted into
proteoliposomes. The ATPase activity of these proteoliposomes was
determined in the presence of various concentrations of either emetine (squares) or valinomycin (circles) for 1 h and is
presented in panel A. Panel B describes the ATPase
activity of Pgp proteoliposomes determined in the presence of 100
µM (squares) or absence (circles) of
valinomycin and various concentrations of poly-L-tryptophan
(mass 5.4 kDa). The ATPase rates presented were calculated by
subtracting the values obtained in the presence of 10 µM orthovanadate.
Figure 3: Activation of poly-L-tryptophan-inhibition of Pgp ATPase by valinomycin. The ATPase activity of Pgp proteoliposomes was determined in the presence of various concentrations of valinomycin or emetine, and the following concentrations (µM) of poly-L-tryptophan (mass = 5.4 kDa): 0, circles; 1, squares; 10, triangles; 100, inverted triangles. The ATPase rates presented were calculated by subtracting the values obtained in presence of 10 µM orthovanadate. The same experimental data are presented as a Lineweaver-Burk plot on panel B.
Figure 4:
Time
course of Rb
uptake into reconstituted
proteoliposomes. Pgp fraction from Emt
cells (panel
A) and a corresponding protein fraction from Pgp-poor AA8 cells (panel B) were extracted and reconstituted for
Rb
uptake as described under
``Experimental Procedures.''
Rb
uptake was measured in an assay medium containing: 0.25 M sucrose, 8 mg/ml bovine serum albumin, 25 mM Hepes-Tris
(pH 7.4), 4 mM MgCl
, 3 mM DTT, 2
µCi/ml carrier-free
Rb
, 0.5
µM valinomycin, and either 3 mM ATP (squares) or AMPPCP (circles), in a final volume of
0.125 ml. The buffer was preincubated for 2 min at 37 °C, and the
reaction was initiated by the addition of 5 µl Pgp-reconstituted
proteoliposomes. The ATP-dependent
Rb
uptake (triangles) was calculated by subtracting the
values obtained in the presence of AMPPCP. Each point represents the
mean value ± S.D., n =
8.
In
order to discern between the ATP-dependent uptake and the
ATP-independent ionophore-mediated equilibration of Rb
, reconstituted proteoliposomes were
incubated for 3 min in a transport buffer containing valinomycin but
lacking ATP. Under these conditions,
Rb
was allowed to reach apparent equilibration with the K
trapped in the proteoliposomes, and the intravesicular
K
concentration reached a transient constant
concentration held by its diffusion potential (Fig. 4). At this
point ATP or AMPPCP was added and
Rb
uptake was determined (Fig. 5). The ATP-dependent
component of
Rb
uptake was not affected
by the preincubation. Thus, the ATP-dependent uptake results from an
authentic active
Rb
uptake and not from
effects on the ionophore-mediated equilibration of
Rb
across the proteoliposome membrane.
The ATP- and valinomycin-dependent
Rb
uptake occurred only with K
-preloaded vesicles,
indicating that the
Rb
ions were
transported into the intravesicular milieu. As shown for Pgp from rat
liver and MDR cells (6) , the ATP- and valinomycin-dependent
Rb
uptake mediated by CHO Pgp was
specific to ATP and did not occur with UTP, CTP, ADP, and
non-hydrolyzable trinucleotides (data not shown).
Figure 5:
The
effect of preincubation of reconstituted proteoliposomes in the absence
of ATP on subsequent ATP-dependent uptake of Rb
. A transport buffer similar to that
described in the legend to Fig. 4was used except that ATP was
omitted here. After preincubation for 3 min at 37 °C, 5 µl of
proteoliposomes reconstituted with either a Pgp fraction from
Emt
cells (panel A) or a corresponding protein
fraction from the Pgp-poor AA8 cells (panel B) and further
incubated for 3 min. At this time point, 1 mM ATP (squares) or AMPPCP (circles) was added. The
ATP-dependent
Rb
uptake (triangles) was calculated by subtracting the values obtained
in the presence of AMPPCP. Each point represents the mean value
± S.D., n = 4.
Determination of
the stoichiometry of drug transport to ATP hydrolysis relies on a
quantitative assay of the valinomycin-dependent ATPase and transport
activities of Pgp under identical experimental conditions. As shown in Fig. 6, measurement of Pgp-mediated ATPase and Rb
uptake as a function of increasing
valinomycin concentrations, revealed valinomycin-dependent components
of both activities. However, as pointed above, Pgp exhibited high basal
activity, in absence of added substrate, which masked the increase in
ATPase activity required to mediate the ATP-driven and
valinomycin-dependent
Rb
uptake. We have
overcome this obstacle by using poly-L-tryptophan to suppress
the basal activity of Pgp. In the presence of
poly-L-tryptophan, low valinomycin concentrations mediated an
increase in both ATPase activity and
Rb
uptake (Fig. 6B). In five independent
experiments, it was determined that 0.5-0.8
Rb
ions were transported/ATP molecule
hydrolyzed. This apparent stoichiometry of drug transport to ATP
hydrolysis was determined as the ratio of the components of
Rb
uptake to ATP hydrolysis, measured
under identical conditions, which were dependent on both ATP and
valinomycin.
Figure 6:
Pgp ATPase and Rb
uptake activities as a function of
valinomycin concentration. Pgp was extracted and reconstituted for
Rb
uptake as described in Fig. 4.
Both the ATPase and
Rb
uptake were
measured simultaneously in the same assay medium containing: 0.25 M sucrose, 8 mg/ml bovine serum albumin, 25 mM Hepes-Tris
(pH 7.4), 3 mM DTT, 4 mM MgCl
, 2
µCi/ml carrier-free
Rb
, in the
absence (panel A) or presence (panel B) of 2
µM poly-L-tryptophan (5.4 kDa), and either 3
mM ATP, or AMPPCP, in a final volume of 0.125 ml. The buffer
was preincubated for 2 min at 37 °C, and the reaction was initiated
by the addition of 5 µl of reconstituted proteoliposomes. The
Rb
uptake (circles) and ATPase
activity (squares) were assayed for 0.5 and 60 min,
respectively. The ATP-dependent
Rb
uptake
was calculated by subtracting the values obtained in the presence of
AMPPCP. Each point represents the mean value ± S.D., n = 8. The ATPase rates presented were calculated by
subtracting the corresponding values obtained in absence of
valinomycin.
An alternative approach to determine the ratio of
Pgp-dependent ATP hydrolysis to drug transport was to use inhibitors
that suppress both the ATPase and Rb
uptake and thereby determine the apparent stoichiometry as the
ratio of parallel reductions in
Rb
uptake
and ATP hydrolysis. The Pgp ATPase as well as the valinomycin- and
ATP-dependent
Rb
uptake were both
eliminated by established inhibitors of Pgp such as vanadate (Fig. 7A) and oligomycin (Fig. 7B).
High concentrations of poly-L-tryptophan competitively
inhibited both Pgp ATPase activity and the valinomycin-dependent and
ATP-driven
Rb
uptake (Fig. 7C). The ratio of the drug transport component
that was eliminated by these different three inhibitors to the fraction
of ATPase activity inhibited by these compounds was again equivalent to
0.5-0.8 mol of
Rb
transported/mol
of ATP hydrolyzed. Pgp substrates such as doxorubicin inhibited
Rb
uptake, with little or no effect on
Pgp ATPase activity (Fig. 7D). Presumably, this well
known Pgp substrate competed with valinomycin on the Pgp
pharmacophore(13) .
Figure 7:
Inhibition of Rb
uptake and ATPase activities by
vanadate, oligomycin, poly-L-tryptophan, and doxorubicin. The
experimental conditions were similar to those described in the legend
to Fig. 5, except that the valinomycin concentration here was
0.5 µM and poly-L-tryptophan was omitted except
for in panel C. The ATPase rates presented were calculated by
subtracting the values obtained in presence of 10 µM orthovanadate.
Thus, the different approaches revealed an apparent near stoichiometry of Pgp-mediated ionophore molecules transported to ATP molecules hydrolyzed.
Pgp catalyzes the ATP-driven efflux of various cytotoxic
xenobiotics out of MDR cells. However, the hydrophobicity of the
various Pgp substrates hindered efforts aimed at estimating the
stoichiometry of drug transport to ATP hydrolysis. In this respect,
using a highly Pgp-rich proteoliposome system, Shapiro and Ling (9) recently reported a stoichiometry of 1 molecule of Hoechst
33342 transported/50 ATP molecules hydrolyzed; this low stoichiometry
was attributed to the rapid rebinding of this hydrophobic chromophore
to the liposome membrane, thus suggesting that the actual rate of
transport is much faster. Recently, we devised an assay that
circumvents this obstacle of the hydrophobicity of Pgp substrates; in
this assay, Pgp-reconstituted proteoliposomes displayed an
ATP-dependent uptake of Rb
-valinomycin
complex(6) . Thus, Pgp mediated an ATP-driven
Rb
accumulation, whereas the ionophore
was recycled. Taking advantage of this assay of hydrophilic cation
accumulation, we here combined a simultaneous determination of Pgp
ATPase activity and its ability to take up
Rb
-valinomycin as reflected in the
Rb
accumulation in the Pgp-reconstituted
proteoliposomes. Using this approach, a stoichiometry of 0.5-0.8
substrate molecules transported/ATP molecule hydrolyzed was estimated.
Thus, the high specific activity of Pgp ATPase (12.5 µmol of
P
/mg of protein/min) along with its near stoichiometric
drug transport to ATP hydrolysis resemble various ion-translocating
ATPases including Na
, K
-ATPase and
Ca
-ATPase which handle millimolar substrate
concentrations. Indeed, the ATP-driven, valinomycin-dependent uptake of
Rb
ions was equivalent to an
intravesicular concentration of 8 mM.
One perplexing theme
that emerges from the present study is that 1) despite the millimolar
substrate translocation capability of Pgp, even when consisting 18%
(this paper) or 32% (8) of total plasma membrane proteins, and
2) although Pgp can surprisingly consume as much as 12% of total
cellular ATP in highly MDR cells(19, 20) , Pgp can
protect highly MDR cells only against 10 µM emetine (this
paper) or 30 µM colchicine(8) . This apparent
discrepancy of 3 orders of magnitude between Pgp's translocation
ability combined with its strong ATPase activity versus its
low efficiency in protecting cells from cytotoxic agents is highly
dependent on the preferred, rapid copartition, and rapid diffusion of
these hydrophobic drugs through the membrane. This is best exemplified
in the case of the hydrophobic peptide ionophores valinomycin and
gramicidin D. Although valinomycin proved an excellent Pgp substrate (i.e. low K and high ATPase V
), Pgp was shown to confer upon highly MDR
cells only a modest protection against this ionophore (see (13) and (21) ), the transmembrane flip-flop of which
was found to be on the order of 25
10
/s(22) . In contrast, in spite of the slow
gramicidin D translocation and consequent inhibition of Pgp ATPase
activity, Pgp proved very efficient in protecting highly MDR cells
against this channel-forming ionophore(13, 21) . This
is not surprising given the relatively slow transmembrane flip-flop (i.e. minutes half-time) gramicidin D monomers must undergo
prior to dimerization and channel formation(23) . Based on the
turnover number of Pgp, which was estimated to be 900 substrate
molecules/min (K
= 15 s; see (8) and (13) ), gramicidin D monomers, but not
valinomycin, can be efficiently extracted from the plasma membrane and
extruded.