Vol. 91, No. 3, 1979 December 14, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 755-760

TUBULIN DOES NOT BIND GLYCEROL IRREVERSIBLY James R. Zabrecky Department Received

of Biochemistry,

October

2,

and R. David

University

Cole

of California,

Berkeley,

CA 94720

1979

SUFlMARY: Tubulin incorporates radioactivity when incubated with preparations of]-glycero 1, but the material that binds to tubulin is a contaminant rather than glycerol itself. The apparent binding of glycerol by tubulin was not stoichiometric, nor was it significantly affected by dilution of the radioactive glycerol preparation with unlabelled glycerol. Radioactive glycerol purified by prior exposure to protein was an order of magnitude less effective in binding to tuhulin than unpurified glycerol. Cytochrome-C and bovine plasmaalbumin, as well as tubulin , incorporated radioactivity from isotopically labelled glycerol. The amount of radioactivity incorporated into tubulin was unaffected by assembly-disassembly. Glycerol

is

stabilize

this

tubules.

Indeed,

protein

requirement

for

procedures

(3,4)

of glycerol, physic0

routinely

tubulin

isolation

chemical

Detrich

into

rings

irreversible

binding

mations

and alter

results.

This

studies

of tubulin

(8)

their call

and 3 that

which

reported

bound

occurs,

differ

to

micro-

appears

preparation

to be a Where

in the absence

in some of their

of access0 ry proteins experiments

that are

w'ith over

(5).

tubulin

the

the

and

nature

tubulin

of

that

might

molecules. distort

had been exposed

previous

conclusions

to glycerol.

Since

binds

upon poly-

when tubulin

in such a way as to give

question

dimer

exchangeable

22 glycerol

glycerol

interactions into

into

technique.

some concern

have reported

each dimer

actually

would

with

They also

of 26 dimers,

glycerol

(l),

(6,7).

2 irreversibly

merizationldepolymerization.

(2)

content

and other

protein

--et al.

of glycerol;

obtained

be viewed

this

assembly

tubulin

and in their

may therefore

Recently,

for

in purification

with

--in vitro

of tubulin

by the polymerization

proteins

properties

purification

sources

have been developed

interaction

5 moles

its

some biological

the microtubular

microtubules

in the

and to enhance

for

The use of glycerol

its

employed

aggregated If

protein

confor-

misleading drawn

from

irreversible

0006-291X/79/230755-06$01.00/0 755

Copyright @I 1979 by Academic Press, Inc. All rights of reproduction in anyform reserved.

Vol. 91, No. 3, 1979

binding

did

different

BIOCHEMICAL

not

seem likely,

explanation

irreversible

we undertook

for

glycerol

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

data

binding

experiments

that

were

that

previously

provided

a

interpreted

as

by tubulin. MATERIALS

AND METHODS

The tubulin used was isolated from bovine brains using DEAE cellulose (Whatman DE-52) chromatography according to Weisenberg --et al, (9), and stored at -20" C in phosphate-magnesium buffer (10 mM phosphate, 5 rrMMgCl*, pH 7.0) containing 1 M sucrose. Prior to use, tubulin was freed from sucrose by dialysis overnight against a 400 fold excess of buffer. Protein samples were incubated with ["'Cl-glycerol (Amersham, CFB 174 Batch 4) for 30 to 60 min at 4' C or for 30 min at 37" C. Unbound ['4C]-glycerol was then separated from the protein either by chromatography on Sephadex G-25 (1.0 x 27 cm; phosphate-magnesium buffer) or by extensive (72 hr) dialysis at 4' C against 3 changes of a 400 fold excess of phosphate-magnesium buffer. On some occasions dialysis was also performed against phosphate-magnesium buffer containing 3.4 M glycerol. For assembly into microtubules, assembly buffer (10 mM [2-N-morpholino) mM MgClp, 3.4 M glycerol, pH 7.0) and (10 uCi) were added. Polymerization 350 nm when the temperature was raised avoided polymerization used identical GTP.

tubulin was dialysed overnight against ethansulfonic acid], 1 mM EGTA, 5 then GTP (to 1 mM) and ["'Cl-glycerol was monitored by light scattering at to 37" C. Control samples that conditions except for the omission of

Horse heart cytochrome C was obtained from Sigma and crystallized bovine plasma albumin was from Armour Pharmaceutical. Radioactivity bound to protein was measured in a Searle Mark II liquid scintillation counter and protein concentration was determined by the method of Lowry --et al. (10).

RESULTS AND DISCUSSION Table with

the

['?I-glycerol

small it

1 gives

amount

and then

amounts

active

results indicate

experiment

per mole

glycerol

bound glycerol

in Table that

to remove appeared

of glycerol, a number

of ['%]-glycerol)

of unlabelled

dialysed indeed

as moles

this

to 0.9 moles

of an experiment

of radioactivity

was calculated

repeating

results

the

to tubulin according

the radioactivity

it

ought

(using

Although

irreversibly,

different

In batches

of glycerol

ranged

to be displaced

by high

such was not

of microreversibility,

the case.

by tubulin

756

These

was not

a if

stoichiometric.

covalently,

law

was incubated

glycerol.

was binding

to the

bound

tubulin

to be bound

binding

Unless

excess

was not

of times

apparent

tubulin.

I show that

it

in which

and from

0.2

any radioconcentrations but the

observations glycerol.

Vol. 91, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

Table Apparent

Stoichiometry

Protein (2-4 mg/ml) Ci/mnole and then dialysed for 72 h at 4" C.

moles glycerol moles dimer

4"

Phosphate-magnesium Glycerola

0.7

37ob

Phosphate-magnesium

1.8

indication fact

the amount

that

that

in which

unlabelled

glycerol,

this

about

the

had the effect

proportional

in Table

1 for

plus

with

to

of increasing

rather

incubation

10.5

ul

the omission

but the radioactivity

was not

was incubated

[14C]-glycerol

was repeated

10" fold

to tubulin

protein

The results

by use of 1.5 nCi experiment

bound

was 3.4 M. polymerization.

bound was approximately

glycerol.

When this

glycerol

radioactivity

glycerol

of total

obtained

the

the amount

glycerol.

a factor

x 10-2 mM of buffer

0.7

of radioactive

at 37" were

unlabelled

of the

the specific

activity

bound

increased

only

purity

of their

['"Cl-

by

of 2.5.

The suppliers glycerol

(Amersham)

as 98%, and since

protein

represented

taminants

less

If radiochemical

proved

into

than

in Table

radioactivity

C

the radiochemical

easily

decomposition tubulin,

II were obtained. with

proteins

radioactivity

protein

757

for

accounted ought

['4C]-glycerol

30 min in the cold Sephadex

the

incorporated

have accounted

When 1.0 uCi for

total

products

other

and incubated

associated

of radioactivity

0.1% of the

could

to be the case.

1% cytochrome

list

the amount

in the glycerol

of radioactivity

shown

(1.05 excess

Phosphate-magnesium

is the

the amount

This

90 mM glycerol of a 400 fold

When glycerol was used in dialysis the concentration Incubation at 37" was under conditions that avoided

glycerol

tion.

Binding

4"

A further

of the

of Glycerol

Dialysis Buffer

Temp.

than

1

was incubated with against 3 changes

Incubation

(a) (b)

RESEARCH COMMUNICATIONS

added, all for

the the

con-

incorporaincorporation

to be similarly

labelled.

was added or at 37",

G-25 chromatography peak.

into

As in the case

to 0.5 ml of the

results

showed of tubulin,

Vol.91,

No.

3, 1979

BIOCHEMICAL

AND

RIOPHYSICAL

of

COMMUNICATIONS

II

Table Retention

RESEARCH

Radioactivity

by

Protein

Protein was incubated with ["Cl-glycerol preparations as described text, and separated from excess glycerol by dialysis or chromatography Sephadex G-25 (at 4' when sam les had been incubated at that temperature, otherwise at room temperature concentrations and radioactivity ! . Protein were determined for fractions across chromatographic peaks and averaged give the value presented in CPM/mnol of 110,000 dalton dimer. Incubation

Separation

Temp.

Method

Tubulin

4O

37"

Tubulina Cytochrome

the

level

again,

than

removed

4"

Chranatography

175

0.2

37"

Chranatography

175

1.5

used in at 4'

for

to protein

(Table

The process did

not

Tubulin

affect

at the calculated

as

of

As expected, from

less

when glycerol

was

was found

the

18 h in protein

in the unretained,

isolated by nearly

had not

for

of

was incubated

fraction,

radioactivity,

be

Incubation albumin

subsequently

was used that

could

to separate

retained

and it

glycerol.

plasma

radioactivity

The tubulin

bovine

temperature,

[14C]-glycerol

chromatography

the

with

to protein.

0.5 ml of 10% bovine

by Sephadex

incubated

higher

of the preparations

had incorporated than

was first

on Sephadex G-25 (see text).

in preparations

30 min at 37".

magnitude,

experiment

when

The glycerol

chromatography

this

and separated

was greater

contaminant

glycerol.

tubulin

3.6

0.4

was followed

peak.

1.05.10-2

175

with

protein

Dialysis

Chromatography

2 pCi ['?I-glycerol

free

1.4

37O

exposure

from

1.05.10-2

8.9

by prior

the cold

Dialysis

stoichiometric

The binding

10-7)

175

of incorporation

less

(x

Chranatography

18 hr

to

CPM/mmole

Act.

(mCi/mmol)

37"

C

(a) ["Cl-glycerol plasma albumin

Specific

in on

with

by Sephadex an order

been exposed

of

previously

II). of assembly

the

and disassembly

incorporation

was put through

a cycle

of radioactivity

of microtubules from

tubulin

[x4C]-glycerol.

of polymerization/depolymerization

758

from

in the

Vol.91,

No.3,

1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMlJNlCATlONS

Table Binding

III

During Microtubule

Assembly

Tubulin (ca. 4 mg/ml) was incubated about 10 min in 10 ti [Z-(Nmorpholino) ethanesulfonic acid], 3.4 M glycerol, with and without 1 mM GTP. The final specific activity of ["'C]-glycerol was 2.9.10-3 uCi/mnol. The incubation at 37" C was sufficient for light scattering (350 n) to reach a plateau. The mixtures were then dialyzed against 3 changes of a 400 fold excess of phosphate-magnesium buffer for at least 72 hrs. CPM/mm01 Tubulin Assembly

(+GTP)

No assembly

presence

of ['4C]-glycerol

polymerization

glycerol

that

binds

than

glycerol

itself.

that

glycerol

interacts

behavior show that

as discussed, the

of tubulin

useful

(-GTP)

1.5 x 108

and compared

was prevented

In conclusion,

1.4 x 108

by the

we find

that

irreversibly

it

may be exploited

is an impurity

example,

with

(and

tubulin

other

which

the assembly

do not to affect

of permanent

bear its (11).

buffer.

of ["Cl-

proteins)

on stabilization

the worry

for

in preparations

by Lee and Timasheff

of glycerol without

sample

of GTP from

our experiments

reversibly

effects

omission

to tubulin

Of course

for

to a control

rather on the

notion

thermodynamic Our findings

and self

assembly

modification

of

the protein.

ACKNOWLEDGEMENTS: This work was supported by National Institutes Grants GMS 20338, EHS P30 ES01896, GM) 7232. The costs of the of this article must therefore be hereby marked "advertisement" with 18 U.S.C. Section 1734 solely to indicate this fact.

of Health publication in accordance

REFERENCES: 1. 2. 3. 4. 5. 6. 7.

Shelanski, M.L., Gaskin, F., and Cantor, C.R. (1973) Proc. Nat'1 Acad. Sci. c, 765. Nagle, B.W., Doenges, H.H., and Brian, J. (1977) Cell 12, 573. Borisy, G.G., Olmsted, J.B., Marcum, J.M., and Allen, C. (1974) Fed. Am. Sot. Exp. Biol. 2, 167. Doenges, K.H., Weissinger, M., Fritzsche, R., and Schroeter, D. (1979) Biochem. 18, 1699. Scheele, R.B., and Borisy, G.G. (1976) BBRC 70, 1. Kirschner, M.W. (1978) Int. Rev. of Cytology 54, 1. Olmsted, J.B., Marcum, J.M., Johnson, K.A., Allen, C., and Borisy, G.G. (1974) J. Supramol. Struct. 2, 429.

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Vol. 91, No. 3, 1979

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