From the Laboratoire de Chimie Physique des
Macromolécules aux Interfaces, Université Libre de
Bruxelles, B-1050 Brussels, Belgium and the
British Columbia
Cancer Research Centre, Vancouver,
British Columbia V5Z 1L3, Canada
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ABSTRACT |
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Ligand-dependent changes in
accessibility of purified P-glycoprotein, functionally reconstituted in
liposomes, were investigated by fluorescence measurements. Trp
quenching experiments provided evidence that P-glycoprotein adopts
different tertiary structures upon binding of drug substrates in the
absence and presence of MgATP and its nonhydrolyzable analog,
MgATP P-glycoprotein is a 170-kDa plasma membrane protein involved in
the multidrug resistance phenomenon responsible for failure of many
human cancer chemotherapies (1). A structural prediction based on its
sequence predicts two homologous halves, each containing six putative
membrane-spanning A large body of evidence suggests that the transmembrane domains of the
P-glycoprotein participate in the recognition of substrates, whereas
the ATP hydrolysis necessary for transport is carried out by both NBD
regions with a similar efficiency in an alternating fashion (11-16).
The mechanism of coupling ATP hydrolysis at the two cytoplasmic
nucleotide-binding sites to drug transport by the intramembrane
drug-binding site(s) is likely to involve substantial conformational
changes in the P-glycoprotein structure (17). Different tertiary
conformational changes have previously been shown to take place upon
addition of MgATP and MgATP-verapamil, an actively transported
chemosensitizer (18). The comprehension of the mechanism of interaction
between the drug-binding site(s) and the NBD domains is essential for
understanding how P-glycoprotein transports its substrates. The aim of
this study was to further investigate the different conformations
adopted by the protein in the presence of nucleotide ligands and drugs.
Purified P-glycoprotein from CHO cells was reconstituted into lipid
vesicles with an "inside-out" orientation, exposing its cytoplasmic
region, which contains the NBD domains, to the external medium. The
resulting proteoliposomes have previously been shown to exhibit both
ATP-dependent drug transport and drug-stimulated ATPase
activity (18).
Acrylamide quenching of Trp fluorescence was used to monitor
ligand-dependent changes in the accessibility of
reconstituted P-glycoprotein in the presence of MgATP, MgATP Materials
Daunorubicin, ATP, ATP Methods
P-glycoprotein Reconstitution--
8 µg of P-glycoprotein (0.3 mg/ml in 25 mM Tris-HCl (pH 7.4), 150 mM NaCl,
1 mM EDTA, 2 mM Chaps), purified as described
previously (20), was added to a dried film of asolectin (protein:lipid ratio of 1:20 w/w) and octylglucoside (lipid:octylglucoside ratio of
1:3 w/w). The mixture was centrifuged at 800 × g for
30 s on a 0.7 × 2-cm column of Sephadex G-50 (fine)
equilibrated with 125 mM NaCl and 50 mM
Tricine-NaOH (pH 7.4). The proteoliposomes were collected in about 60 µl. 50% of the protein was recovered.
ATP Hydrolysis--
ATP hydrolysis was measured as described by
Shapiro and Ling (20) in the absence and presence of increasing
concentrations (2, 10, and 50 µM) of each anthracycline
derivative. Protein determination was performed according to Peterson
(21).
Fluorescence Quenching Experiments--
Acrylamide quenching
experiments were carried out on a SLM Aminco 8000 fluorimeter at an
excitation wavelength of 295 nm instead of 280 nm to reduce the
absorbance by acrylamide. Control experiments established that no
emission of the anthracycline derivatives occurred at 340 nm if excited
at 295 nm (see Ref. 22 for excitation spectrum, ~390-540 nm, of
daunomycin). Acrylamide aliquots were added from a 3 M
solution to the proteoliposome suspension (1 ml in water) containing 8 µg of reconstituted P-glycoprotein and the various ligands. The final
concentration was 3 mM for MgATP and MgATP Azidopine Photolabeling--
Plasma membrane vesicles were
prepared from CHRB30 cells as described previously (23).
The plasma membrane vesicles are typically about 50% inside-out,
exposing the cytoplasmic ATP-binding sites, and are sealed (24). The
vesicles were incubated in darkness at room temperature for 1 h
with 0.3 µM [3H] azidopine and the
indicated concentrations of anthracycline derivatives. After
incubation, the samples were exposed to a UV lamp for 10 min while
being kept on ice. Laemmli's buffer was added, and the samples were
analyzed by SDS-polyacrylamide gel electrophoresis and by
autoradiography of the dried gel.
Fluorescence Experiments--
Fluorescence experiments were
performed to investigate changes in P-glycoprotein structure that occur
upon binding of drug and nucleotide substrates. Five anthracycline
derivatives, cytotoxic (iododoxorubicin, 4-demethoxy-daunorubicin, and
FCE) and noncytotoxic (daunorubicin and 4'-epi-doxorubicin) to
resistant cells, were used (Fig. 1).
These were added to a final concentration of 10 µM to
P-glycoprotein-containing proteoliposomes in the presence and absence
of MgATP (3 mM) or MgATP
Fig. 2 shows Stern-Volmer plots of
P-glycoprotein fluorescence quenching by acrylamide in the absence and
presence of MgATP and MgATP
In the presence of drugs noncytotoxic to resistant cells (daunorubicin
and 4'-epi-doxorubicin) known to be transported by P-glycoprotein,
addition of MgATP resulted in the stabilization of the enzyme in a
conformational state intermediate between that observed in the presence
of MgATP alone and that observed upon binding of the five drugs (Fig.
4A). Replacement of MgATP by its nonhydrolyzable analog,
MgATP
Upon addition of drugs cytotoxic to resistant cells (iododoxorubicin,
4-demethoxy-daunorubicin, and FCE) and MgATP or MgATP ATPase Activity Measurements in the Presence of Anthracycline
Derivatives--
Upon binding of anthracycline derivatives, except for
the FCE derivative, P-glycoprotein adopted the same conformations when MgATP was replaced by its nonhydrolyzable analog MgATP Inhibition of [3H]Azidopine Photolabeling of
P-glycoprotein by Anthracycline Derivatives--
Azidopine, an analog
of verapamil, is able to specifically label P-glycoprotein (25). The
ability to inhibit photoaffinity labeling has frequently been used as
an indicator of whether a particular drug interacts with P-glycoprotein
(26, 27). Therefore, to demonstrate the binding of the anthracycline
derivatives to P-glycoprotein and to determine whether differences in
azidopine competition could be identified between cytotoxic and
noncytotoxic anthracycline derivatives, photolabeling experiments were
performed on CHRB30 plasma membrane vesicles in which about
15% of the protein is P-glycoprotein. Fig.
6 shows the densitometric analysis of the
inhibition of [3H]azidopine labeling in the presence of
increasing concentrations (10, 30, and 100 µM) of each
anthracycline derivative. Although all the compounds were able to
compete with [3H]azidopine labeling, the observed rate of
inhibition varied as a function of the drug tested. In fact, at final
concentrations of 30 and 100 µM, the three cytotoxic
compounds (iododoxorubicin, 4-demethoxy-daunorubicin, and FCE)
exhibited a stronger inhibition of [3H] azidopine
labeling than noncytotoxic derivatives (daunorubicin and
4'-epi-doxorubicin). This suggests a higher affinity of these cytotoxic
agents for P-glycoprotein or the binding of drugs to several
P-glycoprotein sites.
Previous experiments, including infrared spectroscopy (18),
enzymatic proteolysis (28, 29), fluorescence labeling in the NBD
domains (17), and immunoreactivity experiments (30), have suggested
that P-glycoprotein may exist in different conformational states during
its catalytic cycle. Our data provide strong evidence that the protein
undergoes tertiary conformational changes depending on the nature of
the ligands.
Our experiments demonstrated that upon addition of MgATP, the enzyme
adopts a different tertiary structure, resulting in a significantly
increased solvent accessibility, according to previous data (18).
MgATP In this study, we also demonstrated the influence of the binding of
five anthracycline derivatives to P-glycoprotein. Binding of the five
anthracycline derivatives to P-glycoprotein reduces accessibility of
the protein to solvent. The enzyme therefore undergoes a first
conformational change. However, in the presence of MgATP or MgATP The existence of such different conformational states could be due to
the involvement of distinct binding sites for the anthracycline derivatives tested. Azidopine photolabeling experiments also suggest the possibility for the drugs to bind to different P-glycoprotein sites. These experiments, performed in the presence of varying amounts
of each anthracycline derivative, revealed a higher ability of the
three cytotoxic derivatives to compete with azidopine, suggesting a
stronger affinity of the cytotoxic drugs for P-glycoprotein or the
presence of several different drug-binding sites. Independent experimental data also support the hypothesis that P-glycoprotein contains several substrate sites (33). Shapiro and Ling (34) previously
showed the existence of two different and positively cooperative sites
for Hoechst 33342 and rhodamine 123 binding and transport. Furthermore,
unpublished data2 suggest the
possibility of binding of cytotoxic and noncytotoxic anthracycline
derivatives to distinct but overlapping P-glycoprotein domains. The
initial rates of rhodamine 123 and Hoechst 33342 transport in
P-glycoprotein-containing plasma membrane vesicles isolated from
CHRB30 cells were followed in the presence of varying
concentrations of each anthracycline derivative by fluorescence
monitoring. According to these data, noncytotoxic daunorubicin and
4'-epi-doxorubicin inhibit rhodamine 123 transport but stimulate
Hoechst 33342 transport. Cytotoxic iododoxorubicin and FCE seem to bind
to the Hoechst 33342 site as well as the rhodamine 123 site, whereas
4-demethoxy-daunorubicin binds more weakly to the Hoechst site. All
together, these data suggest distinct binding sites for the cytotoxic
and noncytotoxic drugs.
In conclusion, this study describes distinct P-glycoprotein
conformations corresponding to different drug and ATP binding and
hydrolysis states. Separate or simultaneous addition of MgATP and drug
substrates give rise to distinct conformational changes in the
P-glycoprotein molecule, confirming that coupling between the
drug-binding site(s) and the catalytic sites for ATP hydrolysis occurs.
Interestingly, a difference in the protein comportment during these
stages has been identified in the presence of cytotoxic and
noncytotoxic derivatives. It is currently assumed that P-glycoprotein binds and transports substrates with a low specificity via a relatively nonspecific hydrophobic binding pocket (35). However, our results demonstrate that minor changes in the anthracycline structure cause
major modifications in the conformational states adopted by the enzyme,
suggesting that if P-glycoprotein effectively binds its substrates with
a low specificity, the following steps in its catalytic cycle leading
to transport are much more dependent on the drug structure.
S. Five anthracycline derivatives were tested as drug
substrates: daunorubicin, 4'-epi-doxorubicin, iododoxorubicin,
4-demethoxy-daunorubicin, and methoxy-morpholino-doxorubicin. Among
them, daunorubicin and 4'-epi-doxorubicin have been shown to be
rejected outside the multidrug-resistant cells, whereas the three
others have been shown to accumulate in multidrug-resistant cells
overexpressing P-glycoprotein and therefore retain their cytotoxic
activity. A small conformational change was associated with nucleotide
binding and amplified after nucleotide hydrolysis. Different
conformational states were adopted by P-glycoprotein upon the addition
of the anthracycline derivatives in the absence and presence of MgATP
or MgATP
S. These conformational changes are shown to be related to
the nature of the antitumor agents and more precisely to their capacity
to accumulate in resistant cells. These data also suggest that the
cytotoxicity of iododoxorubicin and 4-demethoxy-daunorubicin is related
to the fact they are not transported by P-glycoprotein. On the
contrary, methoxy-morpholino-doxorubicin cytotoxicity may be explained
in terms of its rapid reincorporation into the plasma membrane after
being transported by P-glycoprotein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helices and a cytoplasmic nucleotide-binding domain (NBD)1 with
characteristic Walker motifs A and B. However, experimental studies
concerning this proposed topology (2-5) remain controversial. According to its sequence, P-glycoprotein is classified as a member of
a large family of membrane transporters known as the ATP-binding cassette superfamily that includes yeast, bacteria, and mammalian transporters (6, 7). P-glycoprotein is proposed to function as an
ATP-driven efflux pump, transporting through the plasma membrane an
unusually broad but well defined spectrum of structurally unrelated
cytotoxic drugs, including the Vinca alkaloids, anthracyclines, epipodophyllotoxins, and taxanes (8-10).
S, a
nonhydrolyzable analog of MgATP, and of five anthracycline antitumor
agents (daunorubicin, 4'-epi-doxorubicin, iododoxorubicin,
4-demethoxy-daunorubicin, and FCE) previously used in pharmacokinetics
studies on multidrug-resistant and sensitive K562 cells (19). Three of
these agents (iododoxorubicin, 4-demethoxy-daunorubicin, and FCE) have
been shown to accumulate within multidrug-resistant cells
overexpressing P-glycoprotein and to retain their cytotoxic activity.
On the contrary, daunorubicin and 4'-epi-doxorubicin were rejected
outside the multidrug-resistant cells. We analyze here how the
P-glycoprotein conformational changes are related to the nature of the
antitumor agents and, more precisely, to their capacity to accumulate
into resistant cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S, octylglucoside, and asolectin were
from Sigma. [3H]Azidopin (specific activity of 40 Ci/nmol) was purchased from Amersham Pharmacia Biotech.
4'-epi-doxorubicin, iododoxorubicin, 4-demethoxy-daunorubicin, and FCE
were obtained from Pharmacia-Farmitalia (Milan, Italy). Sephadex G-50
was from Amersham Pharmacia Biotech.
S and 10 µM for the various anthracycline derivatives. Fluorescence intensities were measured at 340 nm after each addition of
quencher. All measurements were carried out at 25 °C. Acrylamide quenching data were subjected to a linear fit up to 80 mM
acrylamide. Above this concentration, the static quenching by
acrylamide is responsible for the deviation from linearity in
Stern-Volmer plots.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S (3 mM), a
nonhydrolyzable MgATP analog. This analog allows discrimination between
the influence of nucleotide binding and nucleotide hydrolysis on
P-glycoprotein structure. The exposure of P-glycoprotein Trp residues
to the external solvent was subsequently determined by continuous
monitoring of P-glycoprotein fluorescence intensity in the presence of
increasing concentrations of acrylamide (0-0.1 M), a
neutral aqueous quencher.
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Fig. 1.
Anthracycline structures.
S. Addition of MgATP led to the highest
level of fluorescence quenching and therefore to the highest Trp
exposure to aqueous solvent. MgATP
S caused a smaller increase in the
Trp exposure when compared with the situation with MgATP. As shown in
Fig. 3, no significant quenching was
observed upon addition of the five anthracycline derivatives in the
absence of MgATP or MgATP
S, suggesting that drug binding to
P-glycoprotein resulted in a decreased accessibility of the Trp to the
aqueous solvent. Upon co-addition of MgATP or MgATP
S and drug
substrates, the quenching efficiency was observed to depend on the
nature of the drug added (Figs. 4 and
5).
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Fig. 2.
Stern-Volmer plots of P-glycoprotein Trp
quenching by acrylamide in the absence and presence of
nucleotides. F is the measured fluorescence intensity,
and F0 is the initial fluorescence intensity in the absence
of acrylamide. , no ligand added;
, 3 mM MgATP;
,
3 mM MgATP
S. The results are the means of three
experiments. The error bars represent the standard
deviation.
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Fig. 3.
Stern-Volmer plots of P-glycoprotein Trp
quenching by acrylamide upon addition of daunorubicin. Addition of
4'-epi-doxorubicin, iododoxorubicin, 4-demethoxy-daunorubicin, or FCE
to P-glycoprotein give identical Stern-Volmer plots (data not shown).
F is the measured fluorescence intensity, and F0
is the initial fluorescence intensity in the absence of acrylamide.
, no ligand added;
, 10 µM anthracycline
derivative. The results are the means of three experiments. The
error bars represent the standard deviation.
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Fig. 4.
Stern-Volmer plots of P-glycoprotein Trp
quenching by acrylamide upon co-addition of noncytotoxic anthracycline
derivatives and MgATP (A) or
MgATP S (B). F
is the measured fluorescence intensity, and F0 is the
initial fluorescence intensity in the absence of acrylamide. A,
, 3 mM MgATP;
, 3 mM MgATP and 10 µM daunorubicin;
, 3 mM MgATP and 10 µM 4'-epi-doxorubicin;
, 10 µM
anthracycline derivative. B,
3 mM Mg ATP;
, 3 mM Mg ATP
S and 10 µM daunorubicin;
, 3 mM Mg ATP
S and 10 µM
4'-epi-doxorubicin;
, 10 µM anthracycline derivative.
The results are the means of three experiments. The error
bars represent the standard deviation.
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Fig. 5.
Stern-Volmer plots of P-glycoprotein Trp
quenching by acrylamide upon co-addition of cytotoxic anthracycline
derivatives and MgATP (A) or
MgATP S (B). F
is the measured fluorescence intensity, and F0 is the
initial fluorescence intensity in the absence of acrylamide.
A,
, 3 mM MgATP;
, 3 mM MgATP
and 10 µM iododoxorubicin;
, 3 mM MgATP
and 10 µM 4-demethoxy-daunorubicin;
, 3 mM
MgATP and 10 µM FCE. B,
, 3 mM
MgATP;
, 3 mM MgATP
S and 10 µM
iododoxorubicin;
, 3 mM MgATP
S and 10 µM 4-demethoxy-daunorubicin;
, 3 mM
MgATP
S and 10 µM FCE. The results are the means of
three experiments. The error bars represent the standard
deviation.
S, had no effect on the levels of quenching observed (Fig.
4B). This indicates that nucleotide binding was necessary
and sufficient to modify the accessibility of the protein to the water phase.
S, two distinct
situations were observed (Fig. 5): 1) in the presence of
iododoxorubicin and 4-demethoxy-daunorubicin, no fluorescence quenching
was detected even after addition of MgATP or MgATP
S and 2) in the
presence of the morpholino derivative (FCE), no fluorescence quenching
was detected after addition of MgATP
S, but addition of MgATP led to
substantial quenching of the protein fluorescence. In the presence of
MgATP, the accessibility of the protein was identical to that observed
with transported noncytotoxic molecules. Stern-Volmer constants were
calculated in each case (Table I).
Influence of ligand binding on the Stern-Volmer constant (Ksv)
S.
Furthermore, MgATP had no influence on P-glycoprotein conformation when
iododoxorubicin and 4-demethoxy-daunorubicin were bound to the protein,
suggesting that the nucleotide could not access the ATP-binding sites
in this conformation. P-glycoprotein ATPase activity was therefore measured in the absence and presence of each of the anthracycline derivatives to determine whether ATP binding and hydrolysis still occurred. In the absence of anthracycline derivatives, an ATPase activity of 65 nmol/min/mg of protein was measured for the
reconstituted P-glycoprotein. According to Table
II, P-glycoprotein exhibited ATPase
activity in the presence of all the derivatives tested, demonstrating
that significant inhibition of ATP binding and hydrolysis did not occur
when the drugs were bound to the protein.
ATPase activity measurements of reconstituted P-glycoprotein in the
presence of 3 mM MgATP and 10 µM
anthracycline derivatives
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Fig. 6.
Photoaffinity labeling of
P-glycoprotein-containing plasma membrane vesicles with 0.3 µM 3H-azidopine in the
presence of varying concentrations of anthracycline derivatives.
The samples were run on an 8% Laemmli SDS-polyacrylamide gel. The gel
was dried, amplified, and exposed to x-ray film (Kodak) for 1 day at
70 °C. Approximately half of the sample was lost.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S, a nonhydrolyzable analog of MgATP, was used to investigate
the structural changes associated with nucleotide binding. Our
fluorescence measurements provide evidence that MgATP
S binding
increases, slightly but significantly, the accessibility of some
P-glycoprotein domains. The structural change observed upon ATP binding
(P-glycoprotein + MgATP
S) was much more pronounced when ATP
hydrolysis occurred (P-glycoprotein + MgATP).
S,
addition of cytotoxic and noncytotoxic derivatives resulted in various
degrees of Trp fluorescence quenching. In the presence of noncytotoxic
derivatives (daunorubicin and 4'-epi-doxorubicin), binding of MgATP
S
was necessary and sufficient for the protein to undergo a
conformational change. The protein was stabilized in a conformation
state intermediate between the "opened" structure observed in the
presence of MgATP alone and the "closed" structure observed upon
binding of the drug. In the presence of the cytotoxic iododoxorubicin
and 4-demethoxy-daunorubicin, neither ATP binding nor ATP hydrolysis
were capable of modifying the tertiary structure of P-glycoprotein. The
protein was maintained in the closed conformational state induced by
the binding of drug derivative. However, the ATPase activity
measurements described in this paper clearly indicated that ATP is
still able to bind to P-glycoprotein and is hydrolyzed in the presence
of the five anthracycline derivatives tested. In the presence of
noncytotoxic agents, the protein probably undergoes a conformational
change after ATP binding and hydrolysis, which might be an important
step in the catalytic cycle of drug transport. In contrast, the
cytotoxic derivatives iododoxorubicin and 4-demethoxy-daunorubicin probably inhibit one or more steps in the catalytic cycle of
P-glycoprotein. This impairs their transport by the protein and results
in an improved accumulation of these compounds within
multidrug-resistant cells. In the presence of the cytotoxic FCE
derivative, a third situation was observed: only hydrolysis of ATP
increased accessibility of the protein. Binding of MgATP
S was not
sufficient to induce a conformational change. After hydrolysis of ATP,
FCE behaves like transported noncytotoxic molecules, and this argues in
favor of transport of FCE by P-glycoprotein. It follows that
cytotoxicity of this derivative may be due to its large hydrophobicity,
which would favor rapid reincorporation into the plasma membrane after transport. Such a property has already been proposed for verapamil, a
well known chemosensitizer that is thought to trap P-glycoprotein in a
futile cycle by rapid rediffusion through the membrane (31, 32).
Briefly, these data suggest that the step of the catalytic cycle
stabilizing the P-glycoprotein depends on the nature of the drug.
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FOOTNOTES |
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* This work was supported by Caisse Générale d'Epargne et de Retraite, Action de Recherches Concertées, and Fonds National de la Recherche Scientifique.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.
§ Scientific collaborator of the National Fund for Scientific Research (Belgium).
¶ Recipient of financial support from Fonds pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture.
** To whom correspondence should be addressed: Laboratoire de Chimie Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, CP 206/2, Bd. du Triomphe, B-1050 Brussels, Belgium. Fax: 32-2-650-53-82; E-mail: jmruyss{at}ulb.ac.be.
2 A. B. Shapiro, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
NBD, nucleotide-binding domain;
FCE, methoxy-morpholino-doxorubicin;
Hoechst
33342, 2-[2-(4-ethoxyphenyl)-6-benzimidazolyl]-6-(1-methyl)-4-piperazil)-benzimidazole;
ATPS, adenosine 5'-O-(thiotriphosphate);
Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
Tricine, N[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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