J. Biol. Macromol., 3(3) 117-125 (2003)

Ultracentrifugal Behaviors of the Tubulin-Colchicine Complex in the State of
GTP in Different Buffers
Hiroshi DOI†, Makiko KAWAGUCHI*, and Serge N. TIMASHEFF**
Department of Food Science and Nutrition, Mukogawa Women’s University,
Nishinomiya, Hyogo 663-8558, Japan*Hyogo Nutrition Vocational College,Nishinomiya, Hyogo662-0833, Japan**Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254 USA
Received August 20, 2003, Accepted September 16, 2003
The behavior of the tubulin-colchicine complex was investigated by using an analyticalultracentrifugation technique. The partial specific volumes of the tubulin-colchicine complex in
the state of GTP were 0.739 and 0.744 ml/g in imidazole and BES buffers, respectively. The
values S020.w were 5.82 and 6.06 S in an imidazole buffer in the presence of 0 and 2 mM MgCl2,
respectively. In the BES buffer, however, very interesting results were obtained. The
sedimentation coefficient S020.w increased from 5.38 S with a calcium concentration of 0 μM to5.75, 6.08 S with calcium concentration of 100 and 500 μM, respectively, in the absence ofmagnesium. This means that the protein molecule has a very flexible shape in the BES buffer andthat the shape shrinks by the addition of calcium. The increase of either the calcium or themagnesium ion concentration induced the increase of the sedimentation coefficient. This result
was in good agreement with the inducing effect of calcium on the polymerization of thetubulin-colchicine complex in the state of GDP. The sedimentation coefficient of the GDP state
was higher than that of the GTP state.
Many studies on the conformation of
tubulin have been done by using an analyticalultracentrifugation technique1-4).
Tubulinexists as a 5.8 S dimer or a 42 S double ringdepending on the magnesium concentration insolution2, 3, 5). Some researchers have found18, 30, and 36 S oligomers during
ultracentrifugation experiments of tubulin in
the presence of microtubule-associatedproteins6-8); on the other hand, results ofultrafiltration experiments and others have†recently raised the question of the state ofoligomers in solution at room temperature 9).
Many properties of tubulin, for example,
microtubule
assembly,
sedimentation
coefficient, and bindings of nucleotide andcalcium are easily affected by the magnesium
ion concentration in solution2, 10, 11).
Currently the effect of calcium on tubulin havebeen reported (12,13). Also we have indicatedtheTo whom correspondence should be addressed. TEL and FAX: +81-798-45-9883; e-mail:hiroshid@mwu.mukogawa-u.ac.jp

Abbreviations: GTP, guanosine 5’-triphosphate; GDP, guanosine

5’-diphosphate; TC-GTP, tubulin-colchicine complex in the state of GTP; TC-GDP, tubulin-colchicine
complex in the state of GDP; Tu-GTP, tubulin in the state of GTP; Tu-GDP, tubulin in the state of GDP.

118

Sedimentation of Tubulin-Colchicine

calcium binding to the tubulin-colchicine
complex in the state of GDP (14) and the effectof calcium ion on the complex in the GTP statein the BES buffer (15,16). During a calcium
binding study of the tubulin-colchicine
complex, we found that the behavior of theprotein was interesting in the experiment ofanalytical ultracentrifugation.
In this paper, the behaviors of thetubulin-colchicine complex in the state of GTP
in different buffers are described. Also, thesimilarity of the tublin-colchicine complex totubulin is indicated on the basis of ananalytical ultracentrifugation experiment.
Materials and Methods
Preparation of Calf Brain Tubulin. Tubulin
was prepared from the brain of freshlyslaughtered calves according to the modified
procedure described previously1, 3, 5). The
tubulin preparation was stored in a 10 mM
phosphate buffer, pH 7.0, containing 0.1 mM
GTP, 0.5 mM MgCl2, and 1 M sucrose underliquid nitrogen until used.
Proteinconcentration
wasdetermined
spectrophotometrically as described by Andreu
and Timasheff13). Equilibrium to the desired
buffer was carried out using a batch and acolumn of Sephadex G-25 (fine) gel
equilibrated with the desired buffer.
Preparation of the Tubulin-Colchicine
Complex. The complex formation between
tubulin and colchicine was performed by the
incubation of tubulin (40-53 mg/ml) and 1 mM
colchicine in a 10 mM phosphate buffer, pH
7.0, containing 0.1 mM GTP, 0.5 mM MgCl2,
and 1 M sucrose at 25 ℃ for 10 min as
described by Andreu and Timasheff (17).
Equilibrium to the desired buffer was
accomplished as described above.
To
prepare the GTP state, 0.1 mM GTP was
included in every step of preparation.

Density Measurement.
To determine thepartial specific volume of protein, the densityof the protein solution was measured at various
concentrations using an Anton Paar Precision
Density Meter DMA 02C. All measurements
were carried out at 20 ℃. The details of themeasurements are described in Lee andTimasheff 18).
Analytical Ultracentrifugation. Analyticalultracentrifugation was performed with a
Beckman Model E ultracentrifuge equippedwith electronic speed control and RTIC.
Protein solutions were centrifuged in a double
sector cell with a sapphire window at 20 ℃.
Schlieren patterns were traced on a Kodak
metallographic plate. The radial positions of
Schlieren patterns were recorded as the peak
position.
Analysis of the sedimentation
velocity was performed with a Nikon Model
6C microcomparator equipped with a Mitutoyo
Digimatic Micrometer 164-152.
The
measured sedimentation velocities werecorrected to water at 20 ℃ using the values ofthe partial specific volume obtained in thisstudy. The following equation was used tocorrect the sedimentation velocity, Sobs, to thesedimentation coefficient (S020.w) of protein inwater at 20 ℃19):
S0 20.w = Sobs ・η／η20.w ・(1－vρ) 20.w／（１－vρ）,
where η is the viscosity of the buffer used,η20.w is the viscosity of water at 20 ℃, v isthe partial specific volume of the protein, andρ is the density. Subscripts 20 and w
indicate 20 ℃ and water, respectively.
The viscosity of the buffers was measured
using an Ostwald viscometer at 20 ℃.
Results

119

H. Doi, M. Kawaguchi, and S.N. Timasheff

Determination of Partial Specific Volume
The partial specific volume is recognized
as an important parameter of theprotein-solvent interaction and is essential forthe determination of the sedimentation
coefficient.
Though the partial specificvolume of tubulin was determined by Lee andTimasheff 18), it is possible that the valuechanges due to the buffer because the partial
specific volume of protein is dependent on thesolvent conditions. Basically, the values oftubulin and the tubulin-colchicine complex canbe very similar. However, we have no exact
data of the partial specific volume of thetubulin-colchicine complex.
Fig. 1 shows the relationship between theapparent partial specific volume and protein
concentrations of the tublin-colchicine
complex in the state of GTP in a 10 mM
imidazole buffer, pH 7.0, containing 0.1 mM
GTP at 20 ℃. Analysis was performed by aleast squares analysis because an extrapolationis necessary to obtain the true value of a partial
specific volume14). The extrapolation of theplots gives the value of 0.739 ml/g. This
extrapolated value was the same as the meanvalue of apparent partial specific volumes.
The partial specific volume of thetubulin-colchicine complex in the state of GDP
in a BES buffer was 0.744 ml/g (Fig. 2). The
values obtained here are very close in bothbuffers.
Analytical
UltracentrifugationoftheTubulin-Colchicine Complex
There are many reports on thesedimentation of tubulin1,2,5,7,8,24). Previous
studies using ultracentrifugation indicate thattubulin consists of an α-β dimer with 5.8 S.
Also, it is indicated that colchicine binding totubulin has no apparent effects on thesedimentation of tubulin1, 9, 24). It is wellknown that the behavior of tubulin isdependent on the buffer10,21,22). In this paper,

0.80
0.78
0.76
0.74
0.72
0.70
0

3

6
9
12
Protein Concn. (mg/ml)

15

Fig. 1. Relationship between apparent partial
specific volume and protein concentration of
the tubulin-colchicine complex in the state of
GTP in 10 mM imidazole buffer, pH7.0,
containing 0.1 mM GTP at 20 ℃.

0.80
0.78
0.76
0.74
0.72
0.70
0

2

4
6
8
Protein Concn. (mg/m

10Fig. 2. Relationship between apparent partial
specific volume and protein concentration of
the tubulin-colchicine complex in the state of
GTP in 10 mM BES buffer, pH7.0, containing
0.1 mM GTP at 20 ℃.
the buffer effect and the difference between
GTP and GDP states are examined by
sedimentation analysis. Schlieren peaks are
single and symmetrical in the absence andpresence of magnesium and/or calcium. Fig.
3 shows the concentration dependence of thesedimentation velocity of TC-GTP at various
concentrations of magnesium and calcium in a
10 mM imidazole buffer, pH 7.0, containing
0.1 mM GTP at 20 ℃. In the absence ofmagnesium, the concentration dependence was120

Sedimentation of Tubulin-Colchicine

negative.
Usually, protein indicates theconcentration dependence in an analyticalultracentrifugation experiment. The results
obtained in this study indicate the typicalprofile of tubulin.
The observed
sedimentation coefficients were corrected to S0
20.w. The corrected sedimentation coefficientswere 5.82, 5.81, and 5.88 S in the presence of0, 100, and 500 μM calcium ion, respectively.
It seems that the addition of calcium induced asmall increase in the sedimentation coefficientin the absence of magnesium. However, there
was no difference in the sedimentation
coefficient of TC-GTP in the presence of 2mM MgCl2. The value of S0 20.w of TC-GTP
was 6.06 S in a 10 mM imidazole buffer, pH
7.0, containing 0.1 mM GTP and 2 mM MgCl2.
These results indicate that the addition of salt
in solution introduced a small change in the
shape of the molecule of TC-GTP in an
imidazole buffer, although it seems that theeffect of the addition of salt did not change themolecular weight because the change in thesedimentation coefficient was very small. A
further addition of magnesium led to a positive
concentration dependence of the sedimentation
coefficient, and the dependence was not astraight line, as has been observed in many
Therefore, thetubulin studies2, 23,24).
sedimentation coefficient of S0 20.w can be
determined as 6.06 S. Each pattern under
different conditions was a single symmetricalpeak, as observed in the imidazole buffer.
However, the behavior of the protein in the
BES buffer was quite different from that in theimidazole buffer.
The difference in theabsence of magnesium was particularly
remarkable. The concentration dependence
indicated a negative straight line under thecondition without magnesium, as shown in Fig.
4. The values of S020.w of TC-GTP in a 10mM BES buffer, pH 7.0, containing 0.1 mM
GTP were 5.38, 5.75, and 6.08 in the presenceof 0, 100, and 500 μ M calcium ion,

respectively. This means that the molecularshape of TC-GTP in the BES buffer is very
flexible and is easily affected by the existenceof calcium ion. In the presence of 2 mM
MgCl2, the sedimentation coefficient, S020.w, of
the protein was 6.20 S in the absence of
calcium ion.
The increase of salt
concentration led to the increase of thesedimentation coefficient in the BES buffer,too. Moreover, Figs. 3 and 4 show theinteresting result that the lines without calcium
indicate a highest sedimentation coefficient in
the presence of 4 mM MgCl2 in both buffers,
even if it appears that calcium induces theincrease of sedimentation coefficient in theFig. 3. Concentration dependence of thesedimentation
velocityofthetubulin-colchicine complex in the state of GTP
at various concentrations of magnesium andcalcium ions in 10 mM imidazole buffer,
pH7.0, containing 0.1 mM GTP.
The centrifugation was performed at 52,000rpm at 20 ℃ . Sedimentation velocity isexpressed as sobserved.
; no MgCl2,;2mM MgCl2,; 4 mM MgCl2,
2+○ ; no Ca ,● ; 100μM Ca2+,△ ;2+.
500μM Ca

H. Doi, M. Kawaguchi, and S.N. Timasheff

121

7.0, containing 0.1 mM GTP withoutmagnesium were 5.47 and 6.07 S in thepresence of 0 and

Fig. 4. Concentration dependence of thesedimentation
velocityofthetubulin-colchicine complex in the state of GTP
at various concentrations of magnesium andcalcium ions in 10 mM BES buffer, pH7.0,
containing 0.1 mM GTP.
The centrifugation was peroformed as
described in Fig. 3.
absence of magnesium. These results suggest
two roles of calcium ion in cases with andwithout magnesium.
It is interesting to compare the behaviorof the tubulin-colchicine complex to that of
tubulin in the buffer. The results of theanalytical ultracentrifugation of tubulin in theimidazole buffer are shown in Fig. 5. The
values of S020.w of Tu-GTP in a 10 mM
imidazole buffer, pH 7.0, containing 0.1 mM
GTP without magnesium were 5.84 and 5.99 S
in the absence and presence of 500 μ M
calcium ion, respectively. This indicates that
the S020.w of Tu-GTP in the imidazole buffer isthe same in the absence and presence of
calcium ion, as observed in the case of
TC-GTP in imidazole buffer.
On the other hand, in the case of the BES
buffer, the result is shown in Fig. 6. Similarly
to TC-GTP in the BES buffer, the behavior of
Tu-GTP in the BES buffer is different fromthat in an imidazole buffer.
The S020.w
values of Tu-GTP in a 10 mM BES buffer, pH

Fig. 5. Concentration dependence of thesedimentation velocity of tubulin in the state of
GTP in 10 mM imidazole buffer, pH7.0,
containing 0.1 mM GTP in the presence of 0 or
500 μM Ca2+ without magnesim ion.
The centrifugation was performed at 52,000rpm at 20 ℃. ○ ; no Ca2+, ●; 500μM
Ca2+,

Fig. 6. Concentration dependence of thesedimentation
velocityofthetubulin-colchicine complex in the state of GTP
in 10 mM BES buffer, pH7.0, containing 0.1
mM GTP in the presence of 0 or 500 μM
Ca2+ without magnesium ion.
500 μM calcium ion, respectively. These
values are quite similar to those of TC-GTP122

Sedimentation of Tubulin-Colchicine

under the same conditions. The results shownin Figs. 5 and 6 suggest that the behavior of
TC-GTP is very similar to that of Tu-GTP in
both buffers.
Discussion
The behavior of the tubulin-colchicine
complex in buffers was investigated using ananalytical ultracentrifugation technique. It is
well known that the tubulin molecule exists as
a 5.8 S dimer in solution2, 5, 7, 23). The
tubulin-colchicine complex indicates very
similar properties to those of tubulin20). The
study of tubulin is carried out using various
buffers, and it seems that the behavior of
tubulin is dependent on the buffer used21,22).
We believe that it is best to use the phosphatebuffer because tubulin is more stable in thephosphate buffer. However, because of thepeculiarities of the experiment, we will not beable to use a phosphate buffer in the study oftubulin. In this study, imidazole and BES
buffers were used, because calcium ion was
contained.
Basically, it is believed that the
behavior of tubulin in an imidazole buffer issimilar to that in a phosphate buffer. On theother hand, there have been many studies thathave used sulfonate buffers. As the behaviorof tubulin is very complicated, it is interesting
to compare the buffer effect on the behavior of
tubulin.
The partial specific volume of tubulin
has been determined as 0.736 ml/g by Lee etal.3), while the value in lysine-HCl andglutamate buffers was 0.754 ml/g21). In thecase of TC-GTP, the partial specific volumes
were 0.739 and 0.744 ml/g in 10 mM
imidazole and BES buffers, pH 7.0, containing
0.1 mM GTP, respectively. Though it appears
that there is a small difference in the partial
specific volume of TC-GTP between bothbuffers, we would like to consider that there isno difference because the value of the standard
error was 0.0031 ml/g. It is clear that thepartial specific volume obtained here is
between two values of tubulin reported before,
even if the difference between them is large.
Therefore, it is also indicated that the
tubulin-colchicine complex is similar to
tubulin from the point of view of the partial
specific volume.
The ultracentrifugal behavior of
TC-GTP was the same as that of Tu-GTP in theimidazole buffer. As expected, the behavior
of TC-GTP in the imidazole buffer isultracentrifugally the same as that in thephosphate buffer when the data of reference20)
andourpreliminary
resultsofultracentrifugation in a phosphate buffer arecompared.
A thermodynamic study has
shown that the tubulin-colchicine complex hasvery similar properties to tubulin26-28).
Tubulin still has GTPase activity after thecomplex formation with colchicine29). On theother hand, there are some differences of thegeometric structure after polymerization20,26,27).
The
criticalconcentrationforthepolymerization of TC-GTP was smaller in theimidazole buffer than in the BES
buffer(unpublished data), while the order of thecritical concentration for the microtubule
assembly of Tu-GTP was the reverse between
the imidazole and sulfonate buffers21).
Therefore,
inconclusion,thetubulin-colchicine complex is comparable withtubulin in physicochemical properties, except
for its apparent nature.
In the absence of magnesium, theconcentration dependence of the sedimentation
coefficient was observed in the imidazole and
BES buffers. There was a big difference inthe concentration dependence between theimidazole and BES buffers due to the influenceof the calcium ion concentration. While theeffect of the calcium ion concentration on thesedimentation coefficient of TC-GTP was
small in the 10 mM imidazole buffer, pH 7.0,
containing 0.1 mM GTP, the change in theH. Doi, M. Kawaguchi, and S.N. Timasheffcalcium ion concentration brought a significant
difference in the sedimentation coefficient of
TC-GTP in the 10 mM BES buffer, pH 7.0,
containing 0.1 mM GTP. This tendency wasobserved in the case of Tu-GTP as well. In
the case of the GTP state in the imidazole
buffer, the concentration dependence was very
similar between Tu-GTP and TC-GTP in theabsence of magnesium. This suggests that themolecular shape of Tu-GTP was similar to thatof TC-GTP. In the BES buffer, the behavior
of Tu-GTP was similar to that of TC-GTP in
the absence of magnesium. Therefore, it isconsidered that the behavior of thetubulin-colchicine complex in solution was
similar to that of tubulin in the imidazole and
BES buffers in the state of GTP. In otherwords, the sedimentation coefficients of
TC-GTP and Tu-GTP were 5.38 and 5.47 S,
respectively, in the BES buffer in the absenceof magnesium and calcium. This means thatthe molecular shapes of TC-GTP and Tu-GTP
are very flexible in the BES buffer without salt.
As shown in Figures 3 and 4, the concentration
dependence is smaller in the imidazole bufferthan in the BES buffer. This means that themolecular shape of TC-GTP is easily affectedby the salt concentration in the BES buffer. It
is known that the critical concentration for
microtubule formation of tubulin is lower in asulfonate buffer than it is in a phosphatebuffer21). The flexibility of the molecularshape may lead to a low critical concentration
due to the easiness of self-association of theprotein. In other words, it is believed that thedifference in the behavior of TC-GTP in thebuffers leads to the difference in microtubuleformation. The association of the protein
molecule is reflected in the value of thesedimentation coefficient.
The larger the
sedimentation coefficient, the larger the extent
oftheassociation.
Thoughthepolymerization of TC-GTP is not observed at 4mM MgCl2, it is believed that the association

123

of TC-GTP is the prestate of polymerization.
It is then reasonable to compare thesedimentation coefficient of the protein in the
different buffers at 4 mM MgCl2 without
calcium ion; the values of Sobs of TC-GTP in
the BES buffer were bigger than those in theimidazole buffer. This result is in good
agreement with the difference in the criticalconcentration
betweenbothbuffers.
Furthermore, the presence of calcium ion
reduced the value of Sobs of TC-GTP at 4 mM
MgCl2 (Figs. 3 and 4). These results are also
in good agreement with the inhibition effect of
the calcium ion on the polymerization of
TC-GTP.
In fact, the difference of Sobs
between the absence and presence of 100μM
calcium ion was larger in the BES buffer thanin the imidazole buffer in the presence of 4mM MgCl2. The inhibition ratio of thecalcium ion on polymerization was larger inthe BES buffer than in the imidazole buffer inthe presence of 6 mM MgCl2 at 7.0.
Moreover, the value of Sobs in the presence of
500μM calcium ion was between that in thepresence of 0 and 100 μM calcium ion.
The maximum inhibition of calcium
has been observed at approximately 100μM
calcium ion at 8 mM MgCl2, though thepresence of 500 μ M calcium ion still
indicates a small inhibition effect14). The fact
described above is demonstrated by the resultsof an ultracentrifugation experiment in thepresence of 4 mM MgCl2. It seems thatcalcium ion has an association effect in theabsence of magnesium ion in the state of GTP.
In fact, however, calcium ion has another effectof dissociation on TC-GTP in the presence ofmagnesium. These facts support the results
indicated previously14) that calcium ion has
two kinds of roles in microtubule assembly andthat magnesium is necessary for calcium toinhibit microtubule formation.
As shown previously, TC-GTP has
two classes of binding sites of calcium ion14).

124

Sedimentation of Tubulin-Colchicine

We believe that the inhibition effect of calcium
ion was due to the high-affinity site of calciumin the protein. Even if magnesium ion ispresent in the polymerization system, TC-GTP
has a calcium-binding activity. It is believedthat this calcium-binding activity in thepresence of magnesium is due to thehigh-affinity site because the low-affinity sites
disappeared in the presence of magnesium in
the Scatchard plot of the calcium-binding
experiment, while two classes of affinity sites
were observed in the absence of magnesium.
The results indicated in this paper support theprevious proposal. In the case of GTP,
calcium is able to bind to thetubulin-colchicine complex and indicates asmall association effect similar to that of othermetal ions in the absence of magnesium, while
calcium ion has a dissociation effect, which
induces the inhibition effect of polymerizationin the presence of magnesium.
This means that the presence ofmagnesium and the GTP state are necessary forthe inhibition effect of calcium on
polymerization. The extrapolated value ofthe sedimentation coefficient of TC-GDP
indicated the α-β dimer state of a protein
molecule14). In the absence of magnesiumand calcium, the ultracentrifugal behavior of
the tubulin-colchicine complex is the same
between the GTP and GDP states. However,
the effect of the salt concentration on thecomplex of tubulin and colchicines differs
significantly. In the absence of magnesium,
the addition of calcium made a curve in theconcentration dependence of the sedimentation
coefficient of TC-GDP, while a leastsquares-fitted straight line was obtained in theconcentration dependence of TC-GDP. In thecase of TC-GTP, the concentration dependence
was applied to a least squares fit even when 2mM MgCl2 was present in the solution. The
comparison of the results in this paper with ourprevious ones14) indicates the molecular shapesof tubulin and the tubulin-colchicine complex
changed according to the hydrolysis of GTP to
GDP and that the sizes of Tu-GDP and
TC-GDP were smaller than those of Tu-GTP
and TC-GTP, respectively.
The rates of
microtubule formation from the tubulin andpolymerization from the tubulin complex were
not in proportion to the rate of GTP
hydrolysis26,30,31) when the rate of microtubuleformation and polymerization were measured
by the increase of turbidity. We certainly do
not believe that there is only one reason.
However, one reason could be that the shape ofthe microtubule or polymer shrinks after
formation of the microtubule or polymerization
with the hydrolysis of GTP to GDP.

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Communicated by Takii Yukio