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J. Biol. Chem., Vol. 275, Issue 34, 26265-26276, August 25, 2000
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From the
Received for publication, April 12, 2000, and in revised form, May 11, 2000
We have determined the kinetic scheme and the
reaction rates of binding to microtubules of two fluorescent taxoids,
7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]Taxol (Flutax-1) and
7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-L-alanyl]Taxol (Flutax-2). Flutax-1 and Flutax-2 bind to microtubules with high affinity (Ka Microtubules are polymers assembled from Taxol drives inactive GDP-tubulin into microtubules, replacing the need
of the Taxoid binding affects the microtubular structure (26, 27). In a
previous study (23), kinetic measurements of the interchange of
radioactive labeled Taxol and docetaxel showed that taxoids are able to
bind and dissociate freely from microtubules in less than 3 min. The
same work demonstrated that the addition of Taxol to preformed
microtubules made of pure tubulin alters their structure in less than 2 min. Those results indicate that the taxoid binding site is accessible
in the microtubules. This is supported by the rapid Taxol-induced
increase of flexibility of assembled microtubules (28) and by the fast
binding and dissociation of
7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]Taxol (Flutax-1) to cellular microtubules (24). All of these characteristics would be compatible with a taxoid binding site in a zone between the
protofilaments (20, 26, 27, 29). However, in the model structure of
Taxol stabilized microtubules (obtained by fitting into an electron
microscopy density map (30) the biochemically well supported
(31-33) high resolution model of the tubulin-Taxol complex (34)), the
Taxol binding site is at one side of the In order to unveil the kinetic mechanisms of molecular recognition of
ligands by the Taxol binding site of microtubules, we have employed two
taxoids labeled at position 7 of the taxane ring either with
fluorescein (Flutax-1; Refs. 24 and 35) or difluorofluorescein
(7-O-[N-(2,
7-difluoro-4'-fluoresceincarbonyl)-L-alanyl]Taxol; Flutax-2). The use of these probes, which retain the microtubule assembly activity, has allowed a detailed kinetic characterization of
the reversible interaction of microtubules and taxoids. The results
evidence an amazingly fast initial binding reaction and provide further
insight into the interaction of microtubules with Taxol.
Tubulin, Microtubules, and Taxoids--
Purified calf brain
tubulin and chemicals were as described (27). For glycerol-induced
assembly, tubulin was directly equilibrated in 10 mM
phosphate, 1 mM EDTA, 0.1 mM GTP, 3.4 M glycerol, pH 6.7, buffer. All tubulin samples were
clarified by centrifugation at 50,000 rpm, 4 °C, for 10 min using
TL100.2 or TL100.4 rotors in a Beckman Optima TLX centrifuge. After
centrifugation, 6 mM MgCl2 and up to 1 mM GTP were added to the solution, final pH 6.5 (GAB).
Microtubules were assembled by raising the temperature to 37 °C for
30 min. The length and morphology of the assembled microtubules were
checked by negative stain electron microscopy as described (27).
Axonemes from sea urchin (Strongylocentrotus purpuratus)
sperm tail were kindly provided by Dr. Philippe Huitorel,
Université Pierre et Marie Curie Villefranche-sur-Mer, France)
and diluted a minimum of 100 times in the experimental buffer.
Docetaxel was kindly provided by Rhône-Poulenc Rorer (Antony,
France). Flutax-1 was synthesized as described (35). Their concentrations were measured spectrophotometrically (10, 24). Flutax-2
was synthesized by the reaction of
7-O-(L-alanyl) Taxol with Oregon Green 488 carboxylic acid, succinimidyl ester ("5 isomer," Molecular Probes
reference no. 0-6147), following the described procedures (35), and
purified by preparative TLC on silica gel with
chloroform/methanol/acetic acid (4:1:0.15, v/v/v) as eluent; mass
spectrum and NMR data were in accordance with its structure
(58). Flutax-2 purity (high performance liquid chromatography;
Ref. 24) was 94%. Flutax-2 induced the assembly of GDP-tubulin
similarly to Flutax-1 (24) except for the critical tubulin
concentration, which was coincident with Taxol. Flutax-2 concentrations
were measured in 0.5% SDS, 50 mM sodium phosphate buffer,
pH 7.0, employing an extinction
coefficient2 of 49,100 ± 1100 M Preparation of Cross-linked Microtubules--
In order to
stabilize microtubules against disassembly by dilution and low
temperatures, 50 µM tubulin in GAB was assembled at
37 °C for 30 min, and then 20 mM glutaraldehyde
(EMscope, microscopy grade) was added to the solution, which was
incubated at 37 °C for 10 min more. The remains of the cross-linking
agent were quenched by adding 60 mM NaBH4
(Fluka) on ice and the mixture degassed. Under these conditions, 90%
of the tubulin was found to be incorporated into the microtubules. The
morphology of the cross-linked microtubules was checked by electron
microscopy and found to be normal. They were found to be stable against
dilution and low temperatures. The taxoid binding was found to be
unaffected by the treatment as judged by the stoichiometry and the
kinetics of the binding reaction, which were not altered. The number of
active sites was found to decay at a relatively slow rate (~5% decay
in 24 h at 4 °C).
Binding of Fluorescent Taxoids to Microtubules--
The binding
of Flutax-1 and Flutax-2 to the microtubules was measured using a
centrifugation assay. Samples of cross-linked microtubules were
incubated for 1 h at different temperatures and taxoid
concentrations. The samples were then centrifuged for 20 min at 50,000 rpm in a TL100 rotor employing a Beckman Optima TLX ultracentrifuge.
The supernatants were taken, and the pellets were resuspended in a 10 mM phosphate buffer, pH 7.0, containing 1% SDS. The
pellets and supernatants were diluted 1:5 in the same buffer, and their
fluorescence was measured employing a Shimadzu RF-540
spectrofluorometer (excitation wavelength 492 nm, emission wavelength
522 nm, 5-nm excitation and emission slits). The concentration of
ligand in the samples was calculated using Flutax-1 and Flutax-2 spectrophotometric concentration standards.
The percentage of inactive ligand in the stock solutions was obtained
by measuring the titration curves at two different concentrations of
sites, 1 and 0.1 µM. The binding constant has to be the
same at both concentrations of sites and can be calculated from the apparent binding constant by discounting the percentage of the inactive
ligand from the total free ligand. The value fitting both curves with
the same binding constant and the minimal error was calculated using a
program based on the Marquardt algorithm (36). The percentages found at
each experimental temperature were averaged, and the effective binding
constants were calculated using the averaged values.
The binding of Flutax-1 and Flutax-2 can also be monitored by the
change in ligand anisotropy. The anisotropy of the fluorescence of
Flutax-1 and Flutax-2, bound and free, were measured in a SLM-8000D fluorometer, using an excitation wavelength of 470 nm and an emission wavelength of 560 nm, with 2-nm excitation and emission slits. The
binding of Flutax-1 and Flutax-2 to the microtubules was measured using
a ligand anisotropy assay. Samples of taxoids were incubated for 15 min
at different temperatures and concentrations of cross-linked microtubules. The anisotropy of the samples were measured in a Polastar-Galaxy (BMG Labtechnologies) plate reader using the 485-P excitation filter and the 520-P emission filter. Since with this method
the binding constants are determined using the free concentration of
sites (total measured binding sites minus bound ligand measured), they
are not influenced by the percentage of inactive ligand.
Kinetics of Binding and Dissociation of Fluorescent Taxoids to
Microtubules--
The kinetics of Flutax-1 and Flutax-2 binding to and
dissociation from microtubules were measured by following the change of
intensity of the fluorescence of the probe, employing a High-Tech Scientific SS-51 stopped-flow device equipped with a fluorescence detection system. A wavelength of 492 nm (2-nm slit) in the excitation pathway and a filter with a cut-off of 530 nm in the emission pathway
were used. The dead time of the instrument was determined using the
reaction of N-bromosuccinimide with
N-acetyltryptophanamide (37) and was found to be ~2 ms.
With these conditions, both Flutax-1 and Flutax-2 are photostable
within the time of the measurement.
The kinetics of the binding of Flutax-1 and Flutax-2 to microtubules
were also measured by the change in the fluorescence anisotropy of the
probe in a Spex spectrofluorometer (Fluorolog 1691) (excitation
wavelength 492 nm, emission wavelength 570 nm, 16-nm slits; a cut-off
filter of 550 nm was employed in the emission pathway to eliminate any
contribution due to scattered light). The device was equipped with a
stopped-flow module designed and built at the Laboratory of
Biomolecular Dynamics (K. U. Leuven). Flutax-2 is photostable
under these conditions, while Flutax-1 shows appreciable photoquenching
(1% of the total intensity per second) during the measurement. The
dead time of the instrument was measured as described above and was
found to be ~10 ms. A minimum of 8 curves were averaged for each
measurement. The slower dissociation of Flutax-1 and Flutax-2 from
microtubules was measured by the decrease in fluorescence anisotropy in
the SLM-8000D fluorometer equipped with a home-built mixing device
consisting of two 1-ml syringes fixed to a thermostated aluminum block
in such a way that they moved simultaneously and connected to a
three-way Hamilton valve, which acted as a mixing chamber. The output
of the valve was connected to the cuvette. The dead time of the
complete system was about 2 s, comparable with the 1-s time
constant employed in the fluorometer.
The fitting of the kinetic curves was done using a nonlinear least
squares fitting program based on the Marquardt algorithm (36) when
pseudo-first order conditions were used; otherwise, the FITSIM package
(38) was employed.
X-ray Scattering by Microtubule Solutions--
Measurements were
made at station 2.1 of the Daresbury Laboratory Synchrotron Radiation
Source. Instruments employed, data acquisition and processing, and
interpretation of the microtubule x-ray scattering were as described
previously (23).
In order to determine the signal to monitor the binding of the
fluorescent taxoids, the spectroscopic properties of the bound and free
Flutax-1 and Flutax-2 were investigated. Fig.
1 shows the fluorescence emission spectra
of the bound and free forms of Flutax-1 and Flutax-2. To match the
scattering contribution of microtubules, the spectrum of the free
species was obtained by displacing the bound form with a large excess
of the nonfluorescent taxoid docetaxel. This closely related compound
binds to the Taxol binding site and has a larger solubility (10).
Centrifugation of the solution after displacement confirmed that the
fluorescent taxoids were dissociated from the microtubules. It can be
seen clearly that the fluorescence intensity of the bound form of
Flutax-1 is 50% larger than that of the free form. The spectrum shows
a small shift toward the blue. The emission spectrum of bound Flutax-2 shows a larger shift toward the blue but a smaller change in
fluorescence intensity. The intensity change is due to the shift of the
ionization equilibrium of the fluorescein group upon binding (24);
i.e. the bound group is mostly in the dianionic form, which
has a larger quantum yield than the monoanion. By choosing adequately
the pH of the buffer, it was possible to maximize the change of
intensity of fluorescence upon binding, which was found to be optimum
at pH 6.5. Since the pK value for the ionization of the
difluorofluorescein group of Flutax-2 is much lower than that of
Flutax-1, little change in fluorescence is observed upon binding to its
site.
In addition, the immobilization of the fluorescent group that results
from the binding reaction produces a large increase in the fluorescence
anisotropy of Flutax-1 and Flutax-2. The anisotropy values of these
probes under different conditions are shown in Table
I.
Molecular Recognition of Taxol by Microtubules
KINETICS AND THERMODYNAMICS OF BINDING OF FLUORESCENT TAXOL
DERIVATIVES TO AN EXPOSED SITE*
§,
**, and
Centro de Investigaciones Biológicas,
Consejo Superior de Investigaciones Científicas,
C/Velázquez, 144, 28006 Madrid, Spain, the ¶ Laboratory
of Biomolecular Dynamics, Katholieke Universiteit Leuven,
Celestijnenlaan 200D, B-3001 Heverlee, Belgium, and Instituto de
Química Orgánica, Consejo Superior de Investigaciones
Científicas, C/Juan de la Cierva 3, 28006 Madrid, Spain
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
107
M
1, 37 °C). The binding
mechanism consists of a fast bimolecular reaction followed by at least
two monomolecular rearrangements, which were characterized with
stopped-flow techniques. The kinetic constants of the bimolecular
reaction were 6.10 ± 0.22 × 105
M1 s1
and 13.8 ± 1.8 × 105
M1 s1
at 37 °C, respectively. A second slow binding step has been measured employing the change of fluorescence anisotropy of the probe. The
reversal of this reaction is the rate-limiting step of
dissociation. A third step has been detected using small angle x-ray
scattering and involves a 2-nm increase in the diameter of
microtubules. It is suggested that the first step entails the binding
of the Taxol moiety and the second a relative immobilization of the
fluorescent probe. The equilibrium and some kinetic measurements
required the use of stabilized cross-linked microtubules, which
preserved taxoid binding. The results indicate that the Taxol binding
site is directly accessible, in contrast with its location at lumen in
the current model of microtubules. An alternative structural model is
considered in which the binding site is located between protofilaments,
accessible from the microtubule surface.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin
heterodimers. Among other functions, they form the mitotic spindle,
which segregates the chromosomes during cell division. Due to this
function, they are the target of antimitotic drugs like
Vinca alkaloids and taxoids, used in cancer treatment (1,
2). Taxol1 is extensively
employed in therapy of ovarian cancer, metastatic breast cancer, head
and neck cancer, and lung cancer (3-5). Taxol arrests cell division by
blocking microtubule dynamics, which is necessary for their function
(6). These dynamics are normally controlled by the nucleotide content
of tubulin; the heterodimer binds two molecules of guanine nucleotide:
a nonexchangeable GTP molecule at the -subunit, which plays a
structural stability role (7), and a second exchangeable molecule bound
to the -subunit, which can be either GTP or GDP. This second
nucleotide molecule controls the activation state of the protein. In
the GDP-bound state, the protein is in an inactive conformation,
forming double rings (8), whereas in the GTP state it is active for
microtubule assembly. GTP hydrolysis in the polymers and GDP/GTP
exchange in the dimers control the assembled state of tubulin (9).
-phosphate of GTP to activate the protein (10). It was the
first microtubule assembly-promoting drug to be discovered (11). Taxol
stabilizes microtubules by binding preferentially to assembled purified
tubulin with an exact 1:1 stoichiometry (10). Thus, the cellular
microtubule cytoskeleton should have a very large number of
binding sites for Taxol or other endogenous ligands, whose function is
unknown. Unassembled tubulin shows insignificant affinity for Taxol
(12-14), indicating that the binding site is mostly formed by the
polymerization process. The interaction of microtubules with Taxol has
been widely studied (10, 12-25). However, since Taxol binding and
microtubule assembly are linked reactions and the binding affinity
seems to be high (12, 14), direct measurements of the binding
thermodynamics are extremely difficult. In addition, the coincidence of
the protein and Taxol absorption bands has precluded, up to now, the
use of spectroscopic techniques to study the kinetics of the reaction.
-tubulin subunit, clearly
facing the microtubular lumen.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
cm1 at 496 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
A, fluorescence emission spectra of 1 µM Flutax-1 in GAB buffer in presence of microtubules
assembled from 20 µM tubulin before 
max = 521 nm (solid line) and after max = 522 nm (dashed line) being displaced by 50 µM docetaxel. B, 1 µM Flutax-2
in GAB buffer in the presence of microtubules assembled from 20 µM tubulin before max = 520 nm
(solid line) and after max = 524 nm (dashed line) being displaced by 50 µM docetaxel. The spectra were taken in a Shimadzu RF-540
spectrofluorometer, the excitation wavelength was 480 nm, and the
bandwidth of the excitation and emission slits was 5 nm. The chemical
structure of both probes is shown in A.
Anisotropy of the fluorescence emission of the fluorescent taxoids
(1 µM) in GAB at 37 °C (ex = 470 nm, cms = 560 nm)
Equilibrium Binding of Fluorescent Taxoids to Cross-linked Microtubules-- The equilibrium constants of binding of Flutax-1 and Flutax-2 to microtubules assembled from pure tubulin in GAB were measured by sedimentation and anisotropy using diluted cross-linked microtubules. This was necessary, since in preliminary experiments using nonstabilized microtubules, the reaction was essentially displaced toward the bound ligand state due to the high concentration of binding sites. The cross-linking employed did not affect the binding stoichiometry or the kinetics of binding (described below).
The titration curves (corrected for the small percentage of inactive
ligand in the experiments in the case of the sedimentation assays) are
shown in Fig. 2. The stoichiometry was
found to be 1:1 (0.99 ± 0.07 mol of Flutax-2/mol of assembled
tubulin), and the affinity was high (Ka = 6.6 ± 1.0 × 107 M1
(centrifugation) or 6.0 ± 0.2 × 107
M1 (anisotropy) for Flutax-1, and
Ka = 6.1 ± 1.7 × 107
M1 (centrifugation) or 5.9 ± 0.3 × 107 M1
(anisotropy) for Flutax-2 at 27 °C). The affinity constants
determined at different temperatures are summarized in Table
II. The reactions were exothermic and
characterized by negative entropy changes (Table IV).
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Kinetics of Binding of Flutax-1 and Flutax-2 to Microtubules Followed by Fluorescence Intensity-- The mechanism of Taxol binding to microtubules was studied with kinetic methods. The binding reaction was found to be very fast, requiring stopped-flow techniques. The length of the microtubules before and after passing through the stopped flow system was checked using electron microscopy (average length 4.85 ± 2.15 nm and 3.70 ± 2.10 nm, respectively), and their morphology was found to be normal.
Fig. 3A (curve
a) shows the time course of binding of Flutax-1 to a 20-fold
excess of microtubule sites. Microtubules have been assembled from pure
tubulin in GAB at 37 °C. To check that the change of fluorescence is
due to the specific binding of the probe to the Taxol site on the
microtubules, the same experiment was performed with microtubules
saturated with a nonfluorescent taxoid (docetaxel); in this case, no
change of fluorescence can be observed (Fig. 3A,
curve b). The kinetics of the reaction can be
expressed as one single exponential; a fitting to a sum of two or more
exponentials does not improve the residuals and yields practically
identical rate constants, indicating that a single process is observed.
Fig. 3B shows the dependence of the observed rate constant
on the concentration of sites at different temperatures. The observed
rate constant depends linearly on the concentration of sites
(calculated for each experiment from the critical tubulin concentration, measured as described in Ref. 10), indicating a simple
mechanism for the binding of the ligand to its site.
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1 as
follows.
![k<SUB><UP>obs</UP></SUB>=k<SUB>+1</SUB>[<UP>sites</UP>]+k<SUB>−1</SUB>](/content/vol275/issue34/fulltext/26265/img003.gif)
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(Eq. 1) |
1 from the extrapolation of the
regression to concentration zero. The value of
k+1 can be obtained with sufficient precision.
But in order to obtain the value of the dissociation constant with enough precision, the value of the product
k+1*[sites] has to be low enough so that
k1 has a significant weight in kobs. In order to do that, the concentration of
tubulin has to be 1 order of magnitude lower than the one necessary to
assemble microtubules. These concentrations were achieved employing
cross-linked microtubules; in this way, the concentration of sites can
be decreased, and the value of k1 can be
properly determined. The results of these experiments are shown in an
inset of Fig. 3B. The values of
k+1 obtained with cross-linked microtubules were
very similar to those obtained with normal microtubules (Table
III).
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Measuring the rate constants of the binding of Flutax-2 to microtubules
is more difficult, since very little change in fluorescence intensity
is observed upon binding to its site. Nevertheless, it is possible to
follow the reaction due to the shift to the blue of the spectrum (Fig.
1B). By using a 530-nm cut-off filter (coincident with the
isosbestic point), the part of the spectrum at which the intensity of
the bound form is smaller than that of the free form can be selected.
It is possible then to monitor the binding reaction by the small
decrease of the observed fluorescence intensity (Fig.
4A). In this way, the values
of k+1 and k1 for the
binding of Flutax-2 to the taxoid binding site can be determined. As in
the case of Flutax-1, the binding reaction is monophasic (within the
experimental indetermination due to the much lower signal/noise ratio)
and is blocked by docetaxel. The observed kinetic rate depends linearly
on the concentration of sites (Fig. 4B). The kinetic
constants of the binding of Flutax-1 and Flutax-2 to its site are
summarized in Table III.
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Kinetic Homogeneity of the Binding Sites--
Since the change in
fluorescence occurs in the ligand and not in the protein, the reactant
in excess to achieve pseudo-first order conditions needs to be the
assembled tubulin. The use of microtubules as the ligand in excess
implies the assumption that all of the sites in the microtubule are
equal. Since the site of Taxol in microtubules has been mapped in the
lumen of the tube using electron diffraction, electron microscopy, and
docking methods (30, 34), it might be possible that the drug has to
diffuse through the ends of the tube or through openings in the tube
wall in order to gain access to the site. In this way, the sites closer to the outer solution would be more accessible than the more internal sites. When those sites are in excess over the drug (under the pseudo-first order conditions), it might be possible that only the
outer ones become filled. To evaluate this possibility, experiments were performed at equimolecular concentrations (5, 10, and 20 µM) of Flutax-1 and sites, and the resulting kinetic
curves were analyzed using the proposed kinetic model (Scheme 1) with
the kinetic analysis package FITSIM (38) (Fig.
5). The data fit the predicted
bimolecular reaction scheme with the same kinetic constants within the
experimental error as those determined under pseudo-first order
conditions, thus demonstrating that all taxoid binding sites in the
microtubule are equal from the kinetic point of view.
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On the other hand, structural differences, like changes in the number
of protofilaments or microtubule openings, might affect the kinetics of
binding. Microtubule solutions assembled from purified tubulin consist
of a mixture of microtubules with a different number of protofilaments
typically ranging from 11 to 15 (26) and have a certain percentage of
open microtubules. If microtubule-organizing centers are used to
nucleate microtubule growth, the number of protofilaments in
microtubules assembled from pure tubulin is fixed at 13, and the
fraction of open microtubules is reduced (39, 40). Microtubules were
assembled from pure tubulin in GAB in the presence of 1, 2, and 5%
(w/w) axonemes from sea urchin sperm (it was checked by electron
microscopy that most of the microtubules were attached to the
axonemes). The observed kinetics of Flutax-1 binding were not affected
by the presence or absence of axonemes (k+1 at
37 °C was 6.27 ± 0.51 M1
s1; with 1% axonemes, 6.38 ± 0.43 M1 s1;
with 2% axonemes, 6.36 ± 0.38 M1 s1;
with 5% axonemes, 6.39 ± 0.49 M1 s1).
This indicates that the kinetics of binding of the fluorescent taxoid
were not significantly modified by the heterogeneity of in
vitro assembled microtubules.
Kinetics of Binding of Flutax-1 and Flutax-2 to Microtubules
Followed by Fluorescence Anisotropy--
A large change in the
fluorescence anisotropy of the probe is observed upon binding (Table
I). The process was studied with stopped-flow anisotropy measurements
in order to obtain more information about the binding reaction. Fig.
6A shows the time course of
the binding of Flutax-1 to microtubules followed both by the change in
intensity (inset) and anisotropy of the fluorescence. It can be seen that the change in fluorescence anisotropy is much slower than
the change in intensity. The apparent rate constant of the anisotropy
change upon binding is very weakly dependent on the concentration of
sites (kobs with 5 µM sites,
0.22 ± 0.07 s1; 10 µM
sites, 0.26 ± 0.04 s1; 20 µM sites, 0.28 ± 0.05 s1;
37 °C), indicating that a monomolecular reaction is observed. The
change in anisotropy can be expressed with a single exponential, indicating that a single process is being observed (within the experimental limitations due to the technical difficulty of the measurement; see "Experimental Procedures"). The change in
anisotropy is blocked by docetaxel. Since photostability controls
showed that Flutax-1 suffers photolysis (about 17% of the total
intensity during the experiment), measurements were done mainly with
Flutax-2, which is devoid of this effect. Fig. 6B shows the
time course of Flutax-2 binding to microtubules. The observed rate
constant is much slower than that measured by the fluorescence
intensity change and shows a weak dependence on the concentration of
binding sites (kobs 5 µM sites,
1.40 ± 0.61 s1; 10 µM
sites, 1.66 ± 0.54 s1; 20 µM sites, 2.21 ± 0.78 s1;
37 °C).
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The data suggest that the change in anisotropy is due to a second
monomolecular reaction subsequent to the high affinity binding of the
taxoid to its site. Such a rearrangement cannot be observed by
fluorescence intensity unless there is a displacement of the equilibrium of the first step or a change in the intensity due to this
second step. The reaction should then follow the following mechanism.
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![k<SUB>S(<UP>obs</UP>)</SUB>=<FR><NU>K<SUB>1</SUB>k<SUB>+2</SUB>[<UP>sites</UP>]</NU><DE>1+K<SUB>1</SUB>[<UP>sites</UP>]</DE></FR>+k<SUB>−2</SUB>](/content/vol275/issue34/fulltext/26265/img006.gif)
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(Eq. 2) |
2
and K1 constants, but k+2
can be measured and the order of magnitude of K1 can be determined with a moderate accuracy if the value of
k2 obtained from the dissociation measurements
(see below) is used. K1 can be estimated to be
on the order of 106
M1 (similar to the ones measured
from the change in intensity) in the range of temperatures studied. The
kinetic data obtained by measuring the change of anisotropy upon
binding of Flutax-1 and Flutax-2 to its site are summarized in Table
III.
Kinetics of Dissociation of Flutax-1 and Flutax-2 from
Microtubules--
The dissociation rate constant can be measured by
displacing the fluorescent drug bound to microtubules with a large
excess of a nonfluorescent competitor. Assuming the product
k+1*[docetaxel] is much higher than
k1, all of the free sites are rapidly filled
with docetaxel, and the reaction (Scheme 2) is displaced toward the
left side at a kinetic rate of k1 or
k2, depending on the limiting step. Since the
condition is fulfilled and the concentration of docetaxel is 100 µM, k+1 for docetaxel has to be in
within 1 order of magnitude with k+1 of Flutax-1 and Flutax-2. The dissociation reaction followed by fluorescence intensity and anisotropy for Flutax-1 had identical rate constants. An
example of dissociation of Flutax-2 is shown in Fig.
7. The values of the rate constants of
dissociation (Table III) were much lower than the values determined for
k1, indicating that the rate-limiting step of
the reaction is the reversal of the conformational transition
(k2). Control measurements with cross-linked
microtubules indicated that the dissociation rate constant is not
affected by cross-linking (k2 = 2.51 ± 0.14 × 102
s1, Flutax-1, 37 °C).
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Thermodynamic Parameters of Binding of Fluorescent Taxoids-- Analyzing the dependence on the temperature of the kinetic constants of the individual steps of the reaction and the equilibrium constants of binding, it is possible to obtain the thermodynamic parameters of the overall and individual reactions of binding (Table IV). The kinetics of the reaction were fully characterized for Flutax-2 only due to the limited photostability of Flutax-1. The Arrhenius plots of the individual binding and dissociation reactions of Flutax-2 are shown in Fig. 8A, and the reaction scheme is shown in Fig. 8B. The enthalpy and entropy values obtained from the kinetic measurements and from the equilibrium measurements are compatible (Table IV). The binding of Flutax-1 and Flutax-2 to their site in microtubules assembled from purified tubulin is exothermic and enthalpy-driven, the overall entropy change being negative.
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Microtubule Structural Changes Induced by Flutax-1 and Flutax-2 Binding to Microtubules-- Small angle x-ray solution scattering and electron microscopy results have shown that the addition of Taxol to preformed microtubules produces a 0.8-nm reduction of the mean diameter and in the mean number of protofilaments of the microtubules from 13.1 to 12.3 (23). Similar experiments have been performed, adding Flutax-1 and Flutax-2 to preassembled microtubules in GAB buffer. Unexpectedly, a displacement in the positions of the J0 maxima of the x-ray scattering profile toward a smaller value has been measured (Table V, Fig. 9A), indicating a 9% increase in the mean diameter of the microtubules. Therefore, Flutax-1 and Flutax-2 microtubules should have an average of 14.3 protofilaments, roughly one protofilament more than control microtubules, contrary to the result with Taxol.
|
|
The time course of the change in position of the
J01 maxima induced by Flutax-2 on glycerol
microtubules was measured (Fig. 9B). The results showed a
time-dependent displacement between the expected values,
distinct from the noise of the individual kinetic measurement, with a
half-life of approximately 60 s under the conditions of these
experiments, similar to the change induced by Taxol (23). Nevertheless,
it should be pointed out that this structural rearrangement should
affect neither the binding equilibrium measurements, since it should be
blocked by cross-linking the microtubules, nor the kinetic
measurements, since it cannot be observed in the time ranges employed
in the kinetic measurements.
| |
DISCUSSION |
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|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Mechanism of Binding of Fluorescent Taxoids to Microtubules-- Two fluorescent derivatives of Taxol, Flutax-1 and Flutax-2, are used in this work as probes for the Taxol binding site. The interaction of Flutax-1 and Flutax-2 with microtubules is a fast bimolecular reaction followed by at least two monomolecular rearrangements of the system. The first step is a fast binding of the ligand with micromolar affinity. This reaction does not affect the mobility of the fluorescent group (either fluorescein or difluorofluorescein), and since it is blocked by docetaxel it seems to be contributed by the binding of the Taxol moiety itself. Subsequent to the bimolecular step, there is a monomolecular reaction, which involves a rearrangement in the system, resulting in the immobilization of the fluorescent group. This step probably implies a weak binding of the fluorescein moiety to the microtubules. The tight binding of these Taxol derivatives has been recently characterized by measurements of fluorescence lifetimes and polarization of the bound forms with picosecond time resolution, showing that the interaction with the protein shifts the fluorescein chromophore toward the dianion structure.3 The last step observed is a slower relaxation of the microtubule structure, which implies an increase in the mean radius of the microtubules from 12.1 to 13.2 nm, opposite of that observed with Taxol (23). This supports the notion of microtubule wall dynamics (23) and the existence of a certain interaction of the fluorescent moieties of the probes with microtubules, which modifies the lateral interactions between protofilaments.
There are strong reasons to assume that Flutax-1 and Flutax-2 are bona fide probes of the Taxol binding site, taking into account the differences in size and charge due to the fluorescent moiety and its effects. These include the following. 1) Flutax-1, Flutax-2, Taxol, and docetaxel are able to induce the assembly of inactive GDP-tubulin (10, 24). 2) All of them compete for the same site with affinities in the same range of magnitude (10, 23, 24). 3) Docetaxel blocks the Flutax binding reaction at its first step (this is a direct consequence of binding the same site). 4) The Flutax dissociation results imply that the kinetic rate of binding of docetaxel should be in the same order of magnitude as those of Flutax-1 and Flutax-2. 5) Polymorphism in microtubule protofilament number does not affect the binding and dissociation kinetics; mixtures of microtubules with different protofilament number behave homogeneously from the kinetic point view.
The first two steps of the reaction of Flutax-2 binding are similar from the thermodynamic point of view; both the binding of the Taxol group to its site and the conformational change are exothermic and entropically disfavored, so the reaction is enthalpy-driven (Fig. 8B). The first step of the reaction, the binding itself, has large activation energy, even with a fast kinetic constant (i.e. a large preexponential factor), indicating that the entrance and exit of the ligand from its site are relatively difficult. The second step has a lower activation energy; however, it is a slow reaction. In both steps of the reaction, the change in entropy is negative, as expected from a reaction that implies the loss of degrees of freedom.
The equilibrium constants calculated from the kinetic data for Flutax-2
are about 5 times larger than the equilibrium measured ones, while
those calculated for Flutax-1 are 3 times smaller (Table II). These
differences give offsets of 
4 kJ mol1 and 3 kJ mol1 in the global free energy changes of
binding of Flutax-2 and Flutax-1, respectively, whereas the enthalpy
and entropy change values are compatible. They cannot be attributed to
the structural change of the microtubules (see "Results"). Since
any possible fast equilibrium between an active and an inactive
conformer of Flutax-2 in the solution would decrease both the binding
constant and the kinetic rate of the bimolecular binding reaction, we
cannot offer at present an explanation of these small differences
between the kinetic and equilibrium measurements. The taxoid binding
affinities (Table II) were high (on the order of 107
M1). Apparent binding constants
(from measurements necessarily affected by the linkage of binding and
microtubule elongation (see Introduction)) previously reported for
other taxoids were as follows:
2-debenzoyl-2-(m-aminobenzoyl)Taxol, 2.0 ± 0.9 × 107 M1 (21);
3'-N-m-aminobenzamido-3'-N-debenzamido-Taxol,
1.63 ± 0.16 × 107
M1 (high affinity site) (25); and
Taxol, 1.15 × 107
M1 (12).
The free energy change of binding of Flutax-2 to its site at 37 °C
is 48 ± 2 kJ mol1, roughly 35 kJ
mol1 of them coming from the initial step
including the binding of the Taxol moiety and 13 kJ
mol1 coming from the immobilization of the
fluorescein group, which contributes significantly to the binding
constant (note that the binding of isolated fluorescein would not be
observed, since the intrinsic affinity is far too low,
K2 of ~10 for Flutax-1 and 100 for Flutax-2).
These values imply that if the presence of the fluorescent group did
not in any way disturb the binding of the taxoid group, the affinity of
Flutax-2 should be larger than that of Taxol. Nevertheless, competition
studies of the binding of Flutax-2 and Taxol with Rotax
(7-O-[N-4-tetramethylrhodaminecarbonyl)-L-alanyl]Taxol) for its site indicate that Taxol has an affinity similar to that of
Flutax-2.2
Stabilized Taxoid Binding Sites Provided by Cross-linked Microtubules-- In order to be able to precisely measure binding and kinetic constants of ligands with high affinities or fast binding reactions, low concentrations of binding sites are required. Unfortunately, due to the mechanism of microtubule assembly, a condensation nucleation polymerization (41), in which a free tubulin concentration over a certain limit (so called critical concentration) is necessary to keep the protein assembled, purified microtubules are naturally unstable against dilution (42-43). The system has an additional complication; tubulin undergoes relatively fast aging even at 4 °C (44). This means that purified tubulin manipulation to produce microtubules is a relatively complex procedure. A treatment widely used to prevent microtubule disassembly by dilution and low temperature is a mild fixation with glutaraldehyde (45-47). 0.2% glutaraldehyde has been used to fix the cytoskeleton of Ptk2 cells, maintaining their ability to specifically bind Flutax-1 (24). In this work, we have demonstrated that this treatment conserves the taxoid binding properties of the microtubules in vitro. The binding stoichiometry and the kinetic parameters of the binding are unaffected by the fixation treatment. The bound Flutax-1 or Flutax-2 is displaced from the site by the nonfluorescent docetaxel, indicating that the binding is specific. These results show that cross-linked microtubules can be a very useful tool in the study of the taxoid-microtubule interactions.
The search of drugs whose targets are microtubules is a fundamental task in anticancer research. Since fixed microtubules are not dependent on temperature or GTP supply, they can be stored at low temperatures ready for use (for a week at 4 °C and probably for larger periods at lower temperatures; nevertheless, they cannot be frozen in the buffer employed).
Ligand Access to the Taxol Binding Site-- In a previous study (23), it was shown that the Taxol binding sites in microtubules were easily accessible; bound Taxol exchanges with docetaxel and modifies the structure of previously assembled microtubules in 1 min. In that paper, we proposed several hypotheses on the possible mechanisms that may hold for Taxol to reach its site in the microtubules in the time range observed. The types of mechanisms proposed were as follows. 1) Taxol binds near the outer microtubule surface. 2) Taxol binds into the lumen due to microtubule dynamic instability, permitting endwise depolymerization and reassembly with taxoid. 3) Taxol diffuses through the open microtubule ends and binds into the lumen. 4) Taxol diffuses through the microtubule wall and binds into the lumen. With the data available, mechanisms 2 and 3 could be definitively excluded, so only mechanisms 1 and 4 were left. Now, with the insight provided by the fast kinetic data now available, a more in depth discussion on ligand access to the binding site in the microtubules can be made.
The diffusion control limit for the binding of Flutax to microtubules
can be estimated from the number of collisions of Flutax to a rod of
the size of a microtubule (48) and can be calculated as follows,
|
(Eq. 3) |
9 m
diameter) in an aqueous buffer containing 30% glycerol at 37 °C
calculated using the Stokes-Einstein equation is 1.6 × 1010 m2 s1, while the value for the microtubule can
be neglected); ds is the reciprocal linear
density of sites in the microtubule (6.15 × 1010 m/site), L is the length of
the microtubule, and r is its radius (12 × 109 m). Considering a 4 × 106-m-long microtubule, the diffusion limit
of the bimolecular rate will be 6.4 × 104
mol1 m3 s1 (6.4 × 107
M1 s1)
(note that this number is a maximal estimation, since it does not
consider the repulsive electrostatic interaction between the negative
net charges of both molecules). The measured bimolecular rate constant
of Flutax-2 binding to microtubules at 37 °C is 1.38 × 106 M1
s1; thus, 2.1% of the collisions have to be
effective. The problem of the efficiency of collisions has been
addressed for protein-protein recognition (49, 50). A typical factor
between frequency of collisions and frequency of binding is about
and lower for protein-protein interactions. This value
holds on the percentage of the surface area of the proteins that forms the binding interface (typically 10% of the exposed area) and other
factors like electrostatic or hydrophobic interactions. The value of
can be a possible ratio for an exposed site in the
microtubule. The interface area of Flutax can be roughly estimated
in 25% of its total surface by superimposing the taxane moiety
of Flutax with the docetaxel molecule bound to tubulin (34). The
percentage of interface area of tubulin is also increased (with respect
to unassembled protein), since the exposed area of -tubulin
available for collision with ligand is largely reduced in the
microtubules, thus increasing the chances of an effective collision.
In this way, mechanism 1 is the easiest possible explanation for the kinetic data. This is supported as well by preliminary results which show that the presence of microtubule-associated proteins, which bind to the outer microtubule surface, slows down the binding of Taxol.4 Mechanisms of type 1 are in contrast with the location of the Taxol site in the high resolution model microtubules (30), which shows the Taxol site clearly facing the lumen, thus reducing its accessibility.
Let us consider the alternative mechanisms of type 4; Taxol may reach the binding site by diffusing though the microtubule wall. It can be shown that in order to feed the binding reaction at the observed association rate, the effective viscosity of the microtubule wall has to be only 100 times larger than that of water (i.e. approximately the viscosity of glycerol), which is highly unreasonable for a protein. Therefore, holes in the microtubule wall would be required. Following the high resolution model of microtubules (30) and recent three-dimensional reconstruction of electron microscopy (51), there are fenestrations in the microtubule wall of roughly 1 nm diameter. Nogales et al. (30) suggested that Flutax-1 may pass through the fenestrations to explain the rapid labeling observed by Evangelio et al. (24). The Taxol molecule has ellipsoidal axes of 1.7 × 1.5 × 1.5 nm, so it would hardly fit through a 1-nm channel. Flutax-1 and Flutax-2 have a larger size, 2.8 × 1.7 × 1.4 nm in their extended configuration, and it may be impossible for them to pass through such a hole. Nevertheless, let us consider the hypothesis that the real size of the pores could be large enough (say 2.5-nm diameter) to allow ligand diffusion through them and calculate how such a process would influence the kinetics of the binding reaction. Having a pore of 2.5-nm diameter and 3-nm depth (the size of the pore in the model of Nogales et al. (30) is about 1.6 × 0.8 nm with 3-nm depth), a molecule of 2-nm diameter will have only 0.82% of the microtubule surface available to collide with it, and only 5.29% of the collisions will have the right angle to pass through the pore. This means that roughly only 1 of every 2300 collisions will transport the ligand inside the lumen, still less than necessary (1 of 50) to feed the reaction. Therefore, the flux of ligand would not be fast enough to feed the binding reaction even if once the ligand is inside the lumen the reaction is purely diffusion-controlled. This combined with the fact that the fenestrations in the MT models are smaller than the ligand makes this mechanism improbable.
A second previously suggested possible mechanism of type 4 (23, 30) would be one in which microtubules would be completely opened in certain parts. It is evident that this would not be a static situation. A kind of microtubule breathing should exist; otherwise, a part of the sites would be freely accessible (those close to the openings), and the ligand access to other sites would need to diffuse from the open parts to the closed parts giving heterogeneous kinetics (the case for the parts far away from the openings would be equivalent, then, to diffusion from the microtubule ends (24)). This possibility can be excluded, since cross-linking of microtubules should extinguish the breathing, resulting in microtubules with two types of sites, the accessible and the inaccessible, thus giving heterogeneous kinetics as discussed above. This was not the case, since cross-linking affected neither the mechanism nor the observed kinetic rates.
A third type of mechanism that may be considered is one of an allosteric type, in which microtubules would have many binding sites, only a few of them being accessible. Taxol binding would alter the geometry of the tubulin molecule, giving access to the other sites. This would be supported by the known fact that binding of taxoids alters the structure of the microtubules (Ref. 23 and this work). However, with such a type of mechanism, the observable kinetic curve cannot be expressed by a single exponential, since the concentration of accessible sites is not constant during the reaction unless each taxoid bound gives access only to the next site, making the observed rate constant dependent on the initial concentration of accessible sites. This is not the case, since cross-linking of the microtubules does not affect the kinetics of the binding reaction, which should have been modified, since cross-linking reduces the conformational freedom of the microtubule.
In summary, we found no feasible explanation of how Taxol might get through the microtubule wall in order to bind so rapidly to a site in the lumen, and retract from such previous discussions (23) in view of the present results. Therefore, we propose that the Taxol binding site has to be at the same reaction compartment as the bulk solution, i.e. accessible at the microtubule surface.
Possible Location of the Taxol Binding Site and Consequences for
Microtubule Structure--
The location of Taxol at the microtubule
lumen in the high resolution model of microtubule (30) is in
contradiction with the measured kinetic data and the above discussion.
Nevertheless, let us point out that the three-dimensional model of
tubulin structure (34) is not incompatible with the kinetic data; only
the three-dimensional model of microtubules obtained by fitting the
tubulin structure into a microtubule electron microscopy density map
(30) is apparently contradictory with the data. How would it be
possible to reconcile the experimental results of this work with the
well supported position of the binding site in a defined zone of
-tubulin (31-33, 52, 53)?
A useful hint is provided by the results with a benzophenone derivative
of Taxol at nonessential position 7 (33) (the same position as Flutax),
which labels -tubulin Arg282 at the M loop, thus
pointing from the inner zones of binding of the essential side
chain at position 13 and benzoyl group at position 2 toward the
interprotofilament zone. A model similar to that of Nogales et
al. (30) has been constructed, in which the taxane moiety of
Flutax-1 is superimposed with the docetaxel molecule (Fig.
10A). Interestingly, this
superposition places the ionizable proton of fluorescein close to
Arg282, whose positive charge should displace the
ionization equilibrium of the fluorescein chromophore toward the
dianionic form, as observed experimentally. Then the tubulin dimers
have been rotated clockwise (as seen from the plus end of the
microtubule) in 5° steps. A rotation of 30-45° is enough to
generate a model in which the taxoid binding site is in the
interprotofilament area, accessible to the drug from the outside (Fig.
10B). It has to be pointed out that the cross-correlation
coefficient used to dock the structure of tubulin into microtubules
(Fig. 1; Ref. 30) shows a broad peak around the best fitting position,
allowing in principle a certain rotational freedom for suboptimal
fitting. Second, the parts that may produce steric conflicts if rotated
are loops difficult to trace at the resolution of the structure (3.7 Å) and that do not need to have the same structure in
Zn2+-induced sheets and microtubules, which have in
addition different interprotofilament contacts. So in principle it is
possible to consider alternative microtubule models in which the
protofilament is rotated or its structure modified in a presently
unknown manner, so the taxol site is exposed.
|
We have analyzed the compatibility of both types of microtubule model
with the surface mapping of the tubulin dimer and Taxol-induced microtubules obtained with limited proteolysis (54). Contradictions include the following. 1) The proteolytic sites
(Glu290-Ile291) and
(Ala294-Cys295) are accessible in Taxol
microtubules (54). In the model of Nogales et al. (30) (Fig.
10A), these residues are in the interprotofilament space
forbidden to proteases, while in the rotated model (Fig. 10B) they lie in the outer surface. 2) The proteolysis sites
(Lys280-Ala281) and
(Tyr281-Arg282-Ala283), both in
the M loop (flanking the Taxol binding site), are accessible in tubulin
dimers and protected in microtubules (54). In the high resolution model
(30), those residues (and thus the Taxol binding site) are accessible
at the lumen of the microtubule (proteases should be able to diffuse
through the lumen in the 20 min of incubation time employed). In the
rotated models, those residues lie between the protofilaments, being inaccessible.
A microtubule structure such as in the rotated protofilament model
(Fig. 10B) would allow a fast kinetic binding and
dissociation of taxoids while keeping the mapped position of the Taxol
binding site at the -tubulin subunit (30). In fact, the location of Taxol in the interprotofilament space has been proposed on structural (26), thermodynamic (14), and assembly kinetic results (20, 23). It is
consistent with the perturbing effects of modifications of the taxoid
side chain (27) and at position 7 (this work) on the curvature of the
microtubule wall. A rotated protofilament model (Fig. 10B)
has important structural implications for the mainly electrostatic
interprotofilament interactions and the interaction with motor
proteins. The M loop (30) is displaced outwards and makes a closer
approach to helix H3 (displaced inwards) instead of the H1-S2 loop of
the neighboring subunit. In addition, the C-terminal helices H11 and
H12 are displaced and should engage in modified interactions with the
microtubule binding sites of kinesin motors (51). Experimental
distinction between the two types of models (Fig. 10, A and
B) with direct low resolution structural methods is
difficult. However, locating the fluorescein moiety of Flutax at the
outer microtubule surface would strongly favor the rotated model. This
problem may be addressed by employing macromolecular fluorescence
quenchers, antifluorescein antibodies, and perturbants of the binding kinetics.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Francisco Amat-Guerri and Dr. Ulises Acuña for continued support with the synthesis of fluorescent taxoids and useful clarifying discussions, Dr. Pilar Lillo for help with the use of the SLM-8000 fluorometer, Dr. Juan Evangelio for initial help with the fluorescent probes, Dr. Consuelo López for use of the stopped-flow apparatus, Dr. Greg Diakun and Dr. Pablo Chacón for help with small angle x-ray scattering measurements, Dr. P. Huitorel for the axonemes, and Aurelio Hurtado and Lorenzo Alonso (CIB Technical Services) for helping with the design and manufacturing of the manual mixing device.
| |
FOOTNOTES |
|---|
* This work was supported by Comunidad Autonoma de Madrid Grant 07B/0025/99, Dirección General de Enseñanza Superior e Investigación Científica (DGESIC) Grants PB-95-0116 and APC 96-0071, and Fundación Científica de la Asociación Española contra el Cancer (to J. M. A.) and DGESIC Grant PB-96-0852 (to U. Acuna).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.
§ To whom correspondence should be addressed. Tel.: 34-915611800 (ext. 4380); Fax: 34-915627518; E-mail: fer@akilonia.cib.csic.es.
** Present address: Faculdade de Quimica, Universidade Pontificia Catolica do Rio Grande do Sul, 90619-900, Porto Alegre-RS, Brasil.
Published, JBC Papers in Press, May 18, 2000, DOI 10.1074/jbc.M003120200
2 J. A. Evangelio and J. M. Andreu, unpublished observations.
3 O. Cañadas, P. Lillo, and A. U. Acuña, manuscript in preparation.
4 J. F. Díaz and J. M. Andreu, unpublished observations.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
Taxol® (Bristol-Myers Squibb) (paclitaxel), 4,10-diacetoxy-2-(benzoyloxy)-5,20- epoxy-1,7-dihydroxy-9-oxotax-11-en-13-yl(2R,
3S)-3-[(phenylcarbonyl)amino]-2-hydroxy-3-phenylpropionate;
docetaxel (Taxotere®, Rhône-Poulenc Rorer RP56976),
4-acetoxy-2-(benzoyloxy)-5,20-epoxy-1,7,10-trihydroxy-9-oxotax-11-en-13-yl(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropionate.
| |
REFERENCES |
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|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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