From the
At mole ratios of lactoperoxidase to tubulin monomers of
3-4, bovine lactoperoxidase forms 1:1 adducts with both
During experiments on the iodination of tubulin, we found
previously that lactoperoxidase (LPO)
It has now been shown by direct
photolabeling of tubulin dimers with
[
Previous demonstration of adduct formation was accomplished
by gel filtration and sucrose density gradient
centrifugation(1) . Adduct formation can also be shown by
electrophoresis on native agarose gels. As shown in Fig. 1A, LPO moves tubulin cathodally from the anodal
compartment and tubulin reduces the cathodal mobility of LPO,
presumably as a result of adduct formation. At a mole ratio of 1.0
(LPO/monomer) some residual tubulin can be seen as trailing material in
the anodal compartment. At mole ratios of 2-3, this residual
tubulin is no longer apparent and the tail is shorter. Although we do
not know the reason for this trailing, it suggests an equilibrium
between LPO and tubulin. In agreement with the above suggestion, note
that, although the LPO is overloaded, tubulin is able to reduce its
cathodal migration. At higher ionic strength there is no tubulin trail,
and more residual tubulin can be demonstrated (data not shown). This
is, perhaps, not surprising since the isoelectric point for the tubulin
monomers averages
The present study was carried out under conditions in which
the
The ready substitution of LPO for either tubulin monomer
in the adduct suggested a search for homology between LPO and the
monomers. Using the GCG GAP alignment program, there is an 18.8%
sequence identity between lactoperoxidase and rat
Recently, Shearwin and Timasheff (17) have also shown, on the
basis of linkage arguments and the fluorescence of allocolchicine
promoted by binding to tubulin that had been dissociated into monomers
by dilution, that one of the monomers alone is sufficient for
colchicicne binding. The strength of allocolchicine binding depends on
the nucleotide present in the exchangeable site, with GTP > GDP.
Because this site is located on
We have previously suggested (6, 7) that colchicine binds to
We are indebted to Dr. Dale Graham of Division of
Computer Research and Technology, National Institutes of Health, for
the GCG GAP search and to Dr. Dan Sackett for an incisive critique.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-
and
-tubulin from rat brain, thereby separating the tubulin
heterodimer into its monomers. This mixture binds colchicine normally,
and we show here by direct photoaffinity labeling that the bulk of the
[
H]colchicine becomes attached to
-tubulin
under these conditions. When the
-tubulin has been displaced by
lactoperoxidase, the ratio of label in
-tubulin to
-tubulin
is increased. The amount of label in
-tubulin decreases with a
corresponding appearance of label in lactoperoxidase. The rate of labeling of
-tubulin remains slow. We conclude that
-tubulin is not necessary for colchicine binding and propose a
model wherein the A and C rings of colchicine bind to
-tubulin,
while the B ring faces
-tubulin in the dimer.
(
)associated with tubulin in a ratio of two molecules of
enzyme to one of heterodimer. This led to splitting of the dimer into
reversible and salt-sensitive 1:1 adducts of either
- or
-monomer with one LPO molecule (1, 2) without a
requirement for unfolding and refolding of tubulin. These adducts could
be readily identified on the basis of their size by sucrose density
gradient centrifugation or gel filtration or by a change in the Soret
spectrum ascribable to an interaction of the LPO with one or more
sulfhydryl groups of tubulin(1) . However, a heme-free
lactoperoxidase can also bind tubulin, hence the necessity for a heme
interaction is not established(3) . The tubulin/LPO association
has also been observed morphologically(4) . The bulk affinity
constant for the dimer + 2 LPO molecules was 2
10
M
(1) , and although low mole
ratios of LPO copolymerized with microtubules, adduct formation
reversibly prevented polymerization of microtubule protein to
microtubules at higher mole ratios. Despite the complete separation of
the
- and
-monomers into the 1:1 adducts,
[
H]colchicine binding stoichiometry was
unaffected in the presence of excess LPO when no residual tubulin
heterodimer could be demonstrated(2, 5) , i.e. the bound colchicine was displaced toward a larger species
(consistent with a 1:1 adduct of LPO + tubulin monomer in sucrose
density gradient centrifugation). This led to the suggestion that only
one of the monomers was required for colchicine binding and that the
binding site retained its native character, but did not reveal which
monomer contained the binding site.
H]colchicine that the great bulk of the
colchicine covalently bound to the dimer resides in
-tubulin(6, 7) ; the distribution of label between
- and
-tubulin (the
/
ratio) is critically
dependent on the size of the spacer (decreasing size increases the
/
ratio) and is sensitive to incubation and irradiation
times, to urea, ionic strength, and other solution variables, and to
the age of the tubulin. It was, therefore, important to employ the
direct, zero length spacer, photolabeling method to determine whether
or not one could covalently label
-tubulin under conditions when
no
-tubulin was near, i.e. in the
-tubulin/LPO
adduct. We show here that this is feasible and that the
-monomer
is not required for colchicine binding to its site, although its
presence in the dimer has major kinetic consequences for the binding
reaction(8) .
Materials
[ring
C,methoxy-H]Colchicine, 70 Ci/mmol, was obtained
from DuPont NEN; lactoperoxidase (bovine) was obtained as a lyophilized
powder from Sigma. Rat brain microtubule protein preparation, and its
purification to tubulin was carried out as described
previously(9) . The protein was drop-frozen and stored in liquid
nitrogen, where it is stable for many months. All reactions were
carried out in Mes assembly buffer (0.1 M Mes, pH 6.9, 1
mM MgCl
, 1 mM EGTA). In some experiments
this buffer was used at half-strength.
Methods
Because the interaction between tubulin
and LPO is slow(1) , preincubations were carried out at room
temperature (23 °C) for 30-40 min in a number of experiments.
After preincubation, tubulin or tubulin/LPO mixtures were incubated in
a Dubnoff shaker in the dark at 36 °C with
[H]colchicine containing the indicated carrier
concentrations. Incubation times are as indicated in the figures.
Thereafter, colchicine-containing samples were irradiated at 4 °C
under 2 cm of 20% CuSO
5H
0 (acting as a
low UV and IR filter), with a high pressure Osram mercury lamp at 90
± 4 W delivering 49 ± 3 mW/cm
at the sample
surface over a wavelength range of 320-380 nm as measured by a
Spectroline DM-365N radiometer. Irradiation times are as indicated in
the figure legends. After irradiation, samples were denatured by
boiling for 3 min in loading buffer (0.01 M Tris-HCl, pH 6.8,
1% sodium dodecyl sulfate, 10% (v/v) glycerol, 5% mercaptoethanol, and
0.05% bromphenol blue). Samples were then subjected to gel
electrophoresis in sodium dodecyl sulfate-8% polyacrylamide gels cast
on GelBond films (FMC) and separated at 20 mA for 20 min past expulsion
of the dye front. The stained gels were then photographed and either
exposed to Kodak X-Omat AR films for autography after enhancement in
1.0 M sodium salicylate, or
5-mm bands were cut from the
lanes and dissolved in 300 µl of 30% H
O
at
65 °C, cooled, mixed with 5 ml of Ultima Gold scintillation fluid
(Packard), allowed to stand for 8-12 h at 4 °C to reduce
chemiluminescence, and counted in a 2-18.6-keV window. For native
gels samples in 10% glycerol and half-strength buffer were applied to
horizontal 1% agarose gels cast on agarose-compatible GelBond films
(FMC) and electrophoresed in an ice bath at 30 mA in half-strength
buffer with frequent mixing of the compartments to maintain the
starting pH, until expulsion of the dye front had occurred. Gels were
rinsed with water, fixed in 50% methanol, 10% acetic acid for 30 min,
flattened, air-dried, stained for 5 min with Coomassie Blue, destained,
and redried.
5.5, whereas that for the LPO isoforms is
>9.0(10) . These results also confirm earlier results (1, 5) that at appropriate mole ratios of LPO to
tubulin, no native tubulin dimer remains demonstrable.
Figure 1:
Interaction of lactoperoxidase with rat
brain tubulin. A, native electrophoresis on agarose of the
lactoperoxidase-tubulin complex at different mole ratios. Lanes 1 and 8, 6 µg of tubulin alone; lanes 2, 4,
and 6, lactoperoxidase + tubulin at mole ratios of 4.1,
2.1, and 1.3, respectively; lanes 3, 5, and 7,
comparable concentrations of lactoperoxidase alone. All samples were
preincubated at room temperature for 45 min before electrophoresis. B, photocross-linking of 3 µM [H]colchicine with tubulin or
lactoperoxidase-tubulin monomer complex at a mole ratio of 8. Panel
I, radioautograph. Note weak labeling of the faster moving portion
of the lactoperoxidase band (circled). Panel II,
Coomassie stain of the same gel. C = control; LPO = lactoperoxidase; MW = molecular weight
markers. Samples were preincubated for 40 min at room temperature and
incubated at 36 °C with label for 30 min and irradiated at 43
mW/cm
and processed as described. The
/
ratios
were 10.1 for control and 15.6 for lactoperoxidase-tubulin
complexes.
Upon
photolabeling of rat brain tubulin with colchicine in the presence of
LPO under conditions where the - and
-monomers are completely
separated in the adduct, labeling of
-tubulin occurs readily and,
as will be shown below, with a generally increased selectivity for
- vis vis
-tubulin (Fig. 1B). The
labeling pattern is a function of the mole ratio of LPO to tubulin. In Fig. 2are plotted the counts of
H incorporated into
protein bands cut out and dissolved from SDS-polyacrylamide gels (solid lines) as well as the calculated ratios of
H in
-tubulin with respect to
-tubulin
(
/
ratios). Fig. 2A represents data carried
out without preincubation of LPO with tubulin, whereas Fig. 2B depicts data collected after 40 min of
preincubation at room temperature. At low mole ratios of LPO/monomer
-tubulin radioactivity is increased or stays roughly constant,
whereas at higher mole ratios labeling of
-tubulin with colchicine
is decreased. By contrast, label in
-tubulin decreases markedly
even at low mole ratios of LPO/monomer. Comparison of the two panels
reveals that the long incubation period used here is sufficient to
overcome the preincubation requirement noted previously(1) .
Absorption of the incident UV light by the excess LPO decreases the
counts incorporated. We found it difficult to compensate for this by
longer irradiation times because of the rapid photoisomerization of
colchicine to lumicolchicine. For this reason comparisons relied on the relative
H contents of the
- and
-
monomers, called here the
/
ratio. It is a useful index of
the selectivity of the labeling process for
-tubulin. This ratio
is frequently greater in the presence of LPO than with the native
heterodimer alone, and this is due, in part, to a decrease in the
amount of label in
-tubulin. The decrease in
-tubulin
labeling in the presence of LPO was generally accompanied by an
increase in label found in the LPO band, particularly its faster
portion (no attempt was made to separate the different LPO isomers).
When label in
-tubulin was compared with the sum of
H
in
-tubulin + LPO, the ratio of
-tubulin to this sum
remained nearly constant until high mole ratios of LPO were used. This
finding suggests that part of the LPO (guest monomer) must have been
near the binding site for colchicine on
-tubulin. To check whether
or not tubulin was necessary for this reaction, similar photolabeling
experiments were carried out with LPO alone. Low level, but
significant, labeling of LPO could be demonstrated but this was always
enhanced by tubulin (average,
1.4-fold).
Figure 2:
Covalent photolabeling of
tubulin/lactoperoxidase mixtures with 3 µM
[H]colchicine as a function of the mole ratio of
lactoperoxidase to tubulin monomers.
, dpm in
-tubulin;
, dpm in lactoperoxidase;
, dpm in
-tubulin;
,
/
ratios. A, no preincubation, 45-min incubation
with label at 36 °C followed by 5-min irradiation at 43
mW/cm
. B, 40-min preincubation at room temperature
followed by 30-min incubation with label at 36 °C and irradiation
at 45 mW/cm
for 5 min as described under
``Methods.''
Colchicine binding to
tubulin is a slow reaction requiring a conformational change in tubulin
and colchicine (Ref. 11 and references in Ref. 12). Part of this can be
ascribed to the bulky B ring which acts as a kinetic
barrier to binding(8) . Because the
- and
-monomers
are separated by LPO, it was expected that the rate of binding
to
-tubulin might be faster under these conditions. It turned out,
however, that the
-tubulin/LPO adduct was labeled at approximately
the same slow rate (or even more slowly in some experiments such as
shown in Fig. 3) as the native tubulin dimer. Because binding was
followed by 5 min of irradiation, these time estimates are only
approximate. Nevertheless, it is clear that colchicine binding to the
adduct is slow and that the kinetic obstacle to colchicine binding to
-tubulin presumably presented by
-tubulin (8, 11, 12) appeared to have been replaced by
the kinetic obstacle of LPO in the adduct (Fig. 3). This suggests
that part of the LPO in the adduct is near enough to the colchicine
binding site to affect the kinetics of binding. Whether or not this LPO
contributes to the binding energy remains to be determined.
Figure 3:
The rate of binding of
[H]colchicine in the absence (left
panel) and presence (right panel) of lactoperoxidase at a
mole ratio of 3. All samples were preincubated at room temperature for
40 min without label and were then added to tubes containing
[
H]colchicine (3 µM final
concentration) and cooled immediately to 4 °C and irradiated
(``zero time'') or incubated at 36 °C for the indicated
times and then irradiated at 50 mW/cm
. All curves begin at
5 min (the time for irradiation). Samples were processed as described
under ``Methods.''
- and
-monomers of rat brain tubulin (K
for dimer dissociation
0.1-0.8 µM)(13, 14, 15, 16) were completely separated from each other by adduct
formation with bovine LPO (mean K
for
adduct dissociation
0.5 µM)(1) . Under these
conditions colchicine binds normally to the mixture of
adducts(6) , and we show here that the bulk of the label is
found on
-tubulin as it is in native dimer, with
/
ratios normally
5 or greater increasing to 15-20 in some
cases when LPO is present. This suggests strongly that the
-monomer is not required for colchicine binding to
-tubulin.
The present method does not permit a reliable estimate of the affinity
constant of colchicine for
-tubulin in the adduct, and any
contribution that the guest LPO may make to the affinity is not known
at present.
-tubulin and a
16.5% identity with
-tubulin. This is, however, of doubtful
significance, because identities between LPO and actin or bovine serum
albumin are 18.8 and 16.2%, respectively. It should be pointed out that
the isoelectric points of the two partners in the adduct are
5.5
for tubulin and >9.0 for the various isoforms of
lactoperoxidase(10) . Thus, a charge-based interaction is likely
to contribute to adduct formation, and the salt sensitivity (1) confirms this. Despite such charge contributions to adduct
formation, there is a high degree of specificity for LPO, since other
basic proteins do not disrupt the adduct (Ref. 1, and as also shown in
native electrophoresis, see above). Whatever the nature of the
LPO-tubulin contacts may be, it is clear that colchicine binding to
-tubulin does not require the presence of
-tubulin.
-tubulin(18, 19, 20) , it was argued that
the G-nucleotide effect indicated allocolchicine binding to
-tubulin. Furthermore, a comparison with podophyllotoxin binding
led to the conclusion that the G-nucleotide effect is exerted primarily
on the C
ring of colchicine or allocolchicine. The
contribution of
-tubulin to the binding energy of allocolchicine
was calculated to be
10% of the total. Whether or not
-tubulin
makes an equally small contribution to the binding energy of colchicine
remains to be determined.
-tubulin near the
interface between
- and
-tubulin because: (i) during direct
photolabeling some [
H]colchicine binds to
-tubulin. This fraction can be increased by mild denaturation.
Moreover, the proportion of label in
-tubulin is a function of the
length of the spacer, with zero length (unsubstituted) colchicine
yielding the lowest proportion of
-labeled tubulin. (ii)
Colchicine induces photo-cross-linking of the tubulin
monomers(21) . (iii) Low concentrations of urea (
1.0 M) ``tighten'' the structure of the dimer as
assessed by several functional criteria (9), and the yield of
photo-cross-linked tubulin dimers is increased. By the same reasoning,
we suggest that a part of LPO must be near the colchicine site, since
the kinetic obstacle to colchicine binding presented by
-tubulin
in the native dimer appears to have been mimicked by LPO, as judged by
the rate of binding, i.e. the rate is approximately
as slow as in the native dimer. The nearness of LPO to the site is also
suggested by the fact that LPO itself becomes labeled with colchicine,
and this occurs in a roughly reciprocal relation to the amount of label
found in
-tubulin. This leads us to the following proposal. The
bulk of the binding energy for colchicine derives from the binding of
its A
and C
rings (plus a contribution from the
cratic entropy connecting these two rings)(22) , and two ring
analogues such as 2-methoxy-5-(2`,3`,4`-trimethoxyphenyl)tropone (MTPT)
bind with similar affinity constants as does
colchicine(12, 23, 24) . Clearly both of these
rings must bind to
-tubulin. However, the rate of binding
of two ring analogues is orders of magnitude (6000-fold) faster than
for colchicine. Addition of the B
ring, and especially
bulky substituents at position C7, progressively decreases the rate,
but has only a small (
20-fold) effect on the affinity
constants(8) . Thus the B ring is primarily a kinetic barrier to
binding. A possible explanation for this is that in the binding site
the B ring faces
-tubulin, whereas the A and C rings are in
contact with
-tubulin, both near the
/
contact surface.
This would account for the labeling of
-tubulin and the kinetic
effect of bulky substituents at C7. Additional factors must be involved
in the photosensitization of monomer/monomer cross-linking, because the
two ring analogue of colchicine, MTPT (see above), also promotes such
cross-linking(21) . Replacement of
-tubulin by LPO does not
alleviate the kinetic barrier as the rate of labeling remains slow, but
does lead to LPO labeling. This proposal would predict that the
isolated native
-monomer present in dilute tubulin solutions would
bind colchicine rapidly, and colchicine should bind as rapidly as the
two ring analogues.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.