J. Biol. Macromol., 4(1) 13-22 (2004)

ArticleStimulating Effect of High Concentration of Calcium Ion on
the Polymerization of the Tubulin-Colchicine Complex.
−Relationship between Magnesium and Calcium−
Hiroshi DOI 1 , Mariko IWASAKI 1 and Makiko KAWAGUCHI 1, 2
1

Department of Food Science and Nutrition
Mukogawa Women’s University
Nishinomiya, Hyogo 663-8558, Japan
2
Hyogo Nutrition Vocational College,
Nishinomiya, Hyogo 662-0833, Japan

Received November 21, 2003; Accepted December 10, 2003
The polymerization reaction was reversible by the addition of calcium
and/or EGTA and cooling. The extent of inhibition was affected by the pH
and magnesium ion concentration. Maximum inhibition of polymerization
was observed at a concentration of around 100µM calcium ion. The presenceof calcium in a polymerization buffer caused the critical concentration toincrease. At above 250µM calcium ion, no inhibition was observed. The
inhibitory effect is due to the high-affinity sites of calcium to thetubulin-colchicine complex and the stimulating effect is due to low-affinity
sites.
Abbreviations: EGTA, Ethlene-bis (oxyehylenenitrilo) tetraacetic acid; GTP,
guanosine 5’-triphosphate.
Key words: tubulin; colchicine; calcium; binding; GTP

Microtubules,
which
are
fundamentally composed of a protein
called tubulin 1) , play many roles incellular processes. The microtubule
protein binds to metals 2-7) as well as
drugs 8-12) . The roles of magnesiumand calcium among cations are very
important in biological systems.
For
microtubule
assembly,
magnesium ion is required, but someamount of calcium ion inhibits the
microtubule assembly 13) .
These

14

Effect of calcium on the polymerization of tubulin-colchicine

facts indicate that divalent cations
play different roles in microtubule
assembly.
It is considered that the propertiesof the tubulin-colchicine complex are
similar to those of tubulin.
For
example, both proteins polymerize inthe presence of magnesium and
GTP 14, 15) ; in addition, they have thesame sedimentation coefficient 16,17) ,very
similarthermodynamic
14,15,16)and a GTPase
properties
18, 19)activity.
Calcium has important roles on
tubulin.
Currently some papers
have
been
publishedontheinteraction between tubulin andcalcium20,21) , indicating that so many
researchers are interesting in theinteraction.
We examined thepolymerizationofthetubulin-colchicine
complexandcalcium binding to the complex in thestate of GTP in a BES buffer 22, 23) .
Also we have reported theinteractions between the complex in
the states of GTP and GDP andcalcium in an imidazole buffer 24) .
The ultracentrifugal behavior of thecomplex in a BES buffer wasdifferent from that in an imidazole
buffer 25) .
In the case of tubulin, we shouldtake care of the measurement
condition due to the instability of
tubulin as well as solvent conditions.
Tubulin is a very unstable protein,
and the stability is improved byforming a complex with colchicine 26) .
In this paper, the effects of calcium

ion on the polymerization of thetubulin-colchicine
complexare
examined in an imidazole buffer inthe presence of magnesium ion.

MATERIALS AND METHODS
Chemicals
Colchicine was obtained from Aldrich(Milwaukee, WI, USA).
Calciumcarbonate, calcium chloride and EGTA
were from the Fisher Scientific Co.
(Hampton, NH, USA). GTP was from
Sigma (St. Louis, MO, USA). All otherreagents used were special or reagent
grade.
Distilled water was further
purified through the system of charcoal
and ion exchange resin columns, andanother distillation was carried out usingaglass
distilling
apparatus.
Twice-distilled water was used in every
experiment, including the purification of
tubulin from calf brain.
Preparation of the Tubulin-Colchicine
Complex
The preparation of calf brain tubulin
was performed according to theestablished method described earlier 8, 27,
28).
The tubulin-colchicine complex
was prepared by the method ofAndreu and Timasheff 26) .
The
complex formation was confirmed
spectroscopically from absorption at
353 nm. The determination of thebound nucleotide was carried out
according to the procedure of Seckler
and Timasheff using the Beckman

Doi, Iwasaki, and Kawaguchi

15

HPLC system. The result indicatedthatonemoleculeofthetubulin-colchicine complex contained
1.87 molecules of GTP and a 0.09
molecule of GDP. This means thatthe tubulin-colchicine complex usedin this experiment was in the GTP
state.
Polymerization and Microtubule
Assembly
A polymerization reaction of thetubulin-colchicine
complexwas
performed in a 10 mM imidazole-0.1
mM GTP buffer, pH 7.0 or 6.5, in thepresence of magnesium ion without
glycerol.
The measurement ofpolymerization was carried out
turbidimetrically, as described byAndreu et al 14) .

RESULTS
Polymerization of the TubulinColchicine Complex
This section describes the effects
of magnesium ion concentration. Fig.
1 shows typical polymerization
profiles of the tubulin-colchicine
complex in a 10mM imidazole, 0.1
mM GTP buffer, pH 7.0, at different
concentrations of magnesium in theabsence and presence of calcium ion.
The tubulin-colchicine complex wasable to polymerize in an imidazole
buffer. Furthermore, an increase inturbidity was reversible by cooling.
The polymerization was inhibited bysmall
amounts
ofcalcium.

Fig.1. Polymerization profiles of thetubulin-colchicine complex in 10mM
imidazole-0.1mM GTP buffer, pH7.0,
containing 6, 8, or 12 mM MgCl 2 .

100 μ M calcium ion was added after
reaching plateau of polymerization or
before heating a reaction mixture. Protein
concentration was 1.50 mg/ml. Arrow↓indicates addition of 100μM calcium ion
and arrow⇒ does cooling of 10℃.
6mM MgCl 2 :12mM MgCl 2

8mM MgCl 2 :Turbidity obtained in the presence of
100µM calcium was the same as thatof the plateau turbidity after additionof 100µM calcium to maximum
polymerization.
Effect of the Magnesium Ion
Concentration
onthe
Polymerization
The effect of the magnesium ion
concentration on the polymerization
was examined at the protein
concentrations of 1.50 and 1.20
mg/ml at pH 7.0 and 6.5, respectively,in the absence and presence of
100µM calcium ion. The results areshown in Fig. 2.
The plateau
turbidityofpolymerizationisdependent on the magnesium ion

Effect of calcium on the polymerization of tubulin-colchicine

16

2.5

Table I. Critical Concentrations for the
Tubulin-Colchicine Complex in 10 mM
Imidazole Buffer Containing 0.1 mM
GTP in the Absence and Presence of
100µM Calcium Ion.

Turbiditu(A450）

2
1.5
1
0.5

pH

0
0

2

4
6
8
10
Magnesium Ion Concn. (mM)

12

14

Fig.2. Effect of Magenesium Ion
Concentration on the Polymerization of the
Tubulin-Colchicine Complex in 10 mM
Imidazole-0.1 mM GTP Buffer, in the
Absence and Presence of 100 µM Calcium
Ion at the Protein Concentration of 1.50
mg/ml (pH7.0) or 1.20 mg/ml (pH6.5).
▲ no calcium at pH7.0; ■ presence of 100
μM calcium ion at pH7.0; ◆ no calcium atpH6.5; ■ presence of 100 μM calcium ion
at pH6.5.

concentration.
The polymerizationof the tubulin-colchicine complex
was not observed in a 10mM
imidazole, 0.1 mM GTP buffer, pH
7.0, containing 4 mM MgCl 2 . As themagnesium ion concentration became
higher, more polymerization wasobserved. Although polymerization
did not occur at 4 mM MgCl 2 at pH
7.0, a reduction of pH from 7.0 to 6.5
introduced polymerization at 4 mM
MgCl 2 . Gaskin et al. 29) observed,
using porcine tubulin, that a greater
extent of microtubule assembly
occurred at pH 6.5 than at 7.0.
The presence of 100µM calcium
ion decreased the turbidity ofpolymerization from 1.551 to 1.164 at
a protein concentration of 1.50 mg/ml
at pH 7.0 in the presence of 8 mM

76.5

MgCl2 Concn.

Ca2+ Concn.

Critical Concn.

(mM)

(μ M)

(㎎/㎖)

8

0

0.608

100

0.792

0

0.432

100

0.577

4

MgCl 2 (Fig. 2).
In addition, theinhibition by calcium ion wasobserved at pH 6.5.
At a low
concentration of magnesium, calcium
ion inhibited polymerization much
more than at a high concentration.
The extent of inhibition was affectedby the pH and magnesium ion
concentration.
For example, theturbidity of polymerization at 6 mM
MgCl 2 was reduced from 1.617 to
1.426 by 100 µM Ca 2+ at pH 6.5(11.8% of inhibition), while it was
reduced from 0.855 to 0.515 at pH
7.0 (39.8% of inhibition).
A critical concentration of thetubulin-colchicine complex in a
10mM imidazole, 0.1mM GTP, 8 mM
MgCl 2 buffer, pH 7.0, was determinedin the absence and presence of
100µM calcium ion. TableⅠ indicatesthe critical concentration for thepolymerizationofthetubulincolchicine complex at 8 and 4 mM
MgCl 2 at pH 7.0 and 6.5, respectively.
The substitution of imidazole forDoi, Iwasaki, and Kawaguchi

phosphate resulted in a lower criticalconcentrationinmicrotubule
30)
assembly.
The addition of
100µM calcium ion led to a higher
critical concentration from 0.608 to
0.792 mg/ml. A trial measurementof the critical concentration was
performed in the absence andpresence of calcium ion.
The
existence of a critical concentration
for the tubulin-colchicine complex
polymer was confirmed.
EffectofCalcium
Ion
Concentration
onthe
Polymerization
Many reports show that many
divalent cations induce microtubule
assembly. 3, 5-7) Both magnesium andcalcium are divalent cations. It isvery interesting to clarify thedifferences between magnesium andcalcium from the cell biological pointof view.

Turbidity（A450)

1.2
1.1
1
0.9
0.8
0

100

200

300

400

500

Calcium Ion Conc. (μM)

Fig. 3. Effect of Calcium Ion Concentration
onthe
PolymerizationoftheTubulin-Colchicine Complex in 10 mM
Imidazole-0.1mM GTP Buffer, pH 6.5,
Containing 4 mM MgCl2.

17

Fig.4. Time Course of Polymerization of
the Tubulin- Colchicine Complex in 10
mM Imidazole-0.1 mM GTP Buffer, pH
7.0, Containing 8 mM MgCl 2 and Efectsof the Addition of EGTA and Calcium
Ion during Polymerization.

We tried to examine the effects of
low and high concentrations ofcalcium on polymerization because
there have been different reports on
the calcium levels required forinhibition.
The effects of calcium andmagnesium on polymerization arevery complicated, and the inhibitionof polymerization by calcium islimited at a low concentration in thecase of pH 7.0 24) .
Furthermore, a similar effect was
confirmed at pH 6.5 (Fig. 3). The
experiment was carried out at pH 6.5
at a protein concentration of 1.20
mg/ml in the presence of 4 mM
MgCl 2 . When a small amount ofcalcium was used, the inhibition
effect on polymerization could beobserved; however, at above 250 µM
Ca 2+ , no inhibition was observed.
Although 100µM calcium inhibited
polymerization by 13.7%, turbidity
with 500 µM calcium increased

18

Effect of calcium on the polymerization of tubulin-colchicine

12.0% compared with that without
calcium ion.
It is very interestingthat a small amount of calcium
inhibits polymerization and that alarge amount of calcium stimulates
the reaction.
In order to confirm these findings,
experiments using EGTA were carriedout in 10mM imidazole, 0.1mM GTP
buffer, pH 7.0, containing 8 mM
MgCl 2 (Fig. 4). As shown in Fig. 4,
further addition of 400µM calcium tothe reaction system containing
100µM
calcium
introducedanincrease in turbidity. The additionof enough EGTA caused thepolymerization to recover to theextent of that without calcium, whilea small amount of EGTA did notremove the effect of calcium.
Though the addition of 1 mM calcium
ion did not affect turbidity, a further
addition of 500 µM EGTA led to adecrease in turbidity. These results
indicate that the effect of calcium onthepolymerization
reactionisreversible.

DISCUSSION
This paper discusses the stimulating
effect of calcium ion on thepolymerization of the tubulin-colchicine
complex in the presence of magnesiumion.
Tubulin exists in the form of
microtubules, which is formed by thepolymerization.
It is well-known thatmany metal ions affect microtubule
assembly.
There are many metal ionsin a living cell.
For example,
magnesium ion exists around 0.5 mM andcalcium ion does at the range between
10−7 and 10−5 M.
In this paper, theconcentration of each metal ion was highcompared with that in a living cell.
These concentrations used in this paper
are the same as those used in the in vitro
experiments by many others.
There are many papers on theinteraction
betweentubulinand
31-34)On the other hand, themagnesium.
effect of calcium has recently reported as
described above.20, 21)
Also we22-24)
reported the calcium binding.
so,
we are interesting in the relationshipbetween magnesium and calcium on thepolymerization of tubulin.
It was Weisenberg who discovered
the inhibition effect of calcium ion in
an in vitro microtubule assembly at
low concentration 13) . The inhibition
effect of calcium on the microtubuleformation from tubulin was observedat the range of micromolar order by
Weisenberg 13) . In the case of theso-called cycle tubulin prepared bythepolymerization
anddepolymerization
method,thecalcium sensitivity was lower thanthat of pure tubulin 35) , and themicrotubules formed by tubulin in thepresence of 1 mM CaCl 2 had different
properties from those formed in 1
mM EGTA 36) .
This difference isassumed to come from the protein
preparation.
In other words, theeffect of calcium on tubulin was
weakened by the presence of
microtubule-associated proteins.

Doi, Iwasaki, and Kawaguchi

The effects of magnesium andcalcium on microtubule protein
containing
microtubule-associatedproteins has been reported in some
papers 31, 35, 37-39) . Though calcium
ion is assumed to inhibit microtubule
assembly of tubulin in two ways 35, 40) ,we investigated the intrinsic effect of
calcium on the tubulin-colchicine
complex rather than on tubulin.
Microtubule assembly in vivo isassumed to be partially regulated bymagnesium and calcium. Considering
the concentration of metal in living
cells, magnesium is the major
divalent action. Since microtubule
assembly is usually done in thepresenceofmagnesium,therelationship between magnesium andcalcium was investigated in thepolymerizationofthetubulincolchicine complex.
The tubulin-colchicine complex
can polymerize in the presence ofmagnesium at pH 7.0 (Fig. 1), and thepolymerization was inhibited by
100µM
calciumion.
Thepolymerizationofthetubulincolchicine complex has been reported
independently by Saltarelli andPantaloni 15) and Andreu et al 14) .
Both reports indicate the in vivo
polymerization
undersimilarconditions of microtubule assemblyfrom pure tubulin.
Their buffers
wereMES(4-morpholineethanesulfonate) and phosphate buffers. In
addition,therequirementofmagnesium and GTP was reported,and no glycerol was required.

19

The dependence of polymerization
on magnesium ion was examined (Fig.
2).
Polymerization measured byturbidimetry was observed at pH 7.0
at above 5 mM MgCl 2 , and turbidity
wasin
proportion
totheconcentration of magnesium, while
only
4
mM
MgCl 2
inducedpolymerization at pH 6.5.
These
facts indicate that the polymerizationof the tubulin-colchicine complex isdependent on pH, as shown in thecase of microtubule assembly from
porcine tubulin by Gaskin et al 29) .
The presence of 100µM calcium ion
inhibited polymerization at both pH
7.0 and 6.5.
The extent ofinhibition by calcium at pH 7.0 waslarger than that at pH 6.5 (Fig. 2).
In order to clarify this discrepancy,
detailed experiments of the effect of
calcium ion were carried out (Fig. 4).
First, for polymerization, we used a
10mM imidazole buffer, pH 7.0,
containing 0.1 mM GTP and 8 mM
The presence of 100µM
MgCl 2 .
calcium ion induced minimum
polymerization under the condition
used, and we could not observe
perfect inhibition by calcium at any
concentration. At more than 100µM
calcium ion, the polymerization
increased in proportion to theconcentration of calcium, and no
inhibition was then observed in thepresenceof1
mM
CaCl 2
turbidimetrically.
However, theincrease of magnesium concentrationfrom 8 to 12 mM spread theconcentration range of calcium,20

Effect of calcium on the polymerization of tubulin-colchicine

which gave minimum turbidity 24) asreported previously. These resultsmean that the affinity of calcium isaffected by the buffer conditions,
including magnesium.
Some
36,41)
researchersreportedthatmagnesium, as well as calcium,
inhibited the microtubule assembly.
The
effectofcalciumion
concentration on the polymerization
was investigated under the conditionof 4 mM MgCl 2 at pH 6.5 (Fig. 3).
Higher turbidity was obtained atabove 250µM calcium ion.
As
shown above, polymerization of thetubulin-colchicine
complexwasconsiderably
affectedbythemagnesium ion concentration and pH
in the absence of calcium, and theeffectofmagnesiumonpolymerization was affected by thepH value. Those results suggestedthatthebindingaffinityofmagnesium to the tubulin-colchicine
complex at pH 6.5 is greater than thatat pH 7.0. The experimental results
concerning the effect of calcium
indicate that calcium ion inhibits andstimulates polymerization (Fig. 3).
The result of Fig. 3 is confirmed by
the result of Fig. 4.
In ourexperiment, the presence of calcium
in a polymerization buffer caused thecritical concentration to increase,
while Saltareli and Pantaloni 15) havereported
thatthecriticalconcentration decreased in thepresence of calcium ion and thatcalcium ion could replace magnesiumion. Andreu et al 14) . showed thepartialinhibitionofthepolymerization by calcium ion at theconcentration of 10 -4 M, when themagnesium ion concentration was
The maximum
1.6×10 -2 M.
inhibitory effect of calcium wasalways observed at a concentration ofaround100µM calcium ion, and theinducing effect was readily affectedby the magnesium ion concentrationand pH.
This means that the
inhibitory effect is due to thehigh-affinity sites of calcium to thetubulin-colchicine complex and theinducing effect is due to low-affinity
sites.
The calcium binding to thetubulin-colchicine complex should be
examined in an imidazole buffer
containing different concentrations ofmagnesium ion. The results will bereported in a near future.

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Communicated by Takii Yukio