Vol. 68, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CYTOCBALASIN A INHIBITS THE --IN VITRO POLYNERIZATION OF BRAIN TUBULIN AND MUSCLE ACTIN Richard
Received
H. Himes,
December
29,
Roderick N. Kersey, Mary Ruscha Department of Biochemistry University of Kansas Lawrence, Kansas 66045
and L. L. Houston
1975
SUMMARY
Cytochalasin A (CA) inhibits the self-assembly of beef brain tubulin. The concentrations necessary to cause the inhibition are only slightly higher than the tubulin concentration. Cytochalasin B (CB) at identical and higher concentrations has no noticeable effect. Cytochalasin A also inhibits colchicine binding activity suggesting that it denatures the tubulin molecule. The results indicate that the reaction of CA with the sulfhydryl groups of tubulin is responsible for its action. CA also prevents the conversion of G-actin to F-actin, probably via a similar mechanism. INTRODUCTION The cytochalasins
are
of effects
on cellular
are
cytochalasin
known,
been
studied
to stop
extrusion
inhibit
some cells
bibliography
A74 of the Aldrich
with
surface.
and there
is
There Copyright All rights
doesn't
still appear
(4), cells
of other
Chemical
affects
cell
cellular
Co.)
However,
some question
(6)
and cause
have also
the
all
as to
cells
the actual
1362
to suggest
inhibit
induce
nuclear
halt
the
"arborization" (For
been noted.
of an
see publication
(8-10) are
(l),
(5), the
evidence
structures not
(3),
phagocytosis
has
has been shown
cells
of cytochalasins
to be any evidence
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
stop
Although
microfilament
compound
range
cytochalasins
availability,
secretion
effects
of the effects
CB apparently the
muscle
This
have a wide seven
of multinucleated
movement
cardiac
greater
the others.
with
which
at least
of its
production
interfere
A number
(7).
extensive
the
cell
of embryonic
than
products
Although
because
B (CB),
with (2),
(l),
beating
(1).
more extensively
transport
metabolic
activities
cytokinesis
hexose
fungal
is not perhaps
affected target(s) that
form
conclusive, by interacting
in
the
same way
of CB (11,lZ). the cytochalasins
Vol. 68, No. 4, 1976
interact
with
BIOCHEMICAL
microtubules
effects
of cytochalasin
inhibits
the --in vitro
or tubulin. A are
to tubulin.
preventing
the polymerization is
MATERIALS
Moreover,
sparse.
of tubulin
CA is also
reports
We provide
polymerization
colchicine
effects
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
data
on the biological here
to show that
and the binding
of
shown to be more effective
of muscle
G-actin.
CA
than
A mechanism
for
CB in these
proposed. AND METHODS
Cytochalasin A and B were purchased from the Aldrich Chemical Co. Solutions of these compounds were made in dimethyl sulfoxide (DMSO). In all experiments in which CA or CB was used the final DMSO concentration was 1%. This concentration of DMSO was included in all controls and did not affect any of the regctions studied. GTP and colchicine were products of Sigma Chemical Co. H-Colchicine was obtained from New England Nuclear Co. Beef brain tubulin was purified by the polymerization method of Shelanski previously (14). The extraction and reassembly --et al. (13) as described buffer was 20 ti (2[N-morpholino] ethane sulfonic acid) (MES), 70 mM NaCl, 1 mM EGTA, 0.5 mM MgC12, pH 6.4. The protein was purified through two polymerization cycles and was stored at -80' in the buffer containing 2 M glycerol. G-actin was extracted from an acetone powder of rabbit muscle by the procedure of Reese and Young (15) and was dialyzed at 4' overnight against 0.5 mM ATP, 0.5 mM 2-mercaptoethanol, 0.2 mM CaCl pH 7.7, before use. Colchicine binding was performed as described E'y Borisy (16). The loss of sulfhydryl content of tubulin was determined by the reaction with 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) (17). RESULTS The inhibition and is
compared
involved
In other
up to 200 uM, had no effect confirmed
by electron
samples.
In the presence
the concentrations to those large
normally
as the
with
GTP it
on the
is it
with
was found
examination
--in vivo of tubulin.
of uranyl
experiments Moreover,
1363
CA is that
of 100 PM CA no microtubules
for
experiments
less
they
1
which before effective
CB, in concentrations
reaction.
the inhibition
shown in Fig.
the inhibitor
seen that
self-assembly
of CA used to cause
concentration
by CA is
In these
of tubulin
experiments
microscopic
used
of tubulin
by colchicine.
preincubation
of polymerization
colchicine.
self-assembly
to inhibition
a two minute
initiation than
of the
This acetate were
are
result stained
evident. fairly
are only the effective
was
high
Although compared
4 to 10 times concentrations
as
Vol. 68, No. 4, 1976
Fig.
1
lower
2, for
UM CB did
addition
if
the preincubation
example, not
CA did
using
inhibit
not
show any observable
stained
samples
taken
normal
microtubules. In an attempt samples containing
was shown
that
20 minutes
to determine as well
effect
such a procedure
This
is
preincubation
shown in with
on preformed
microtubules.
was complete
did
microscopic after
examination
the addition
whether
the effect
as unreacted
1 M glycerol.
longer.
100
reaction.
polymerization
and electron
is
A 20 minute
the self-assembly
to decrease
inhibited
period
50 PM CA.
of 100 uM CA after
turbidity
buffer
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Inhibition of self-assembly of tubulin by CA and colchicine. Tubulin (10 PM) was preincubated for 2 minutes at 37O in 0.5 ml of reassembly buffer containing 0.32 M glycerol and the concentrations of CA and GTP (final concentration, 0.5 mM) was then added colchicine shown. to initiate the reaction.
of CA are Fig.
BIOCHEMICAL
samples
By measuring removed
about
1364
were
not
The
cause
the
of negatively
showed
the presence
of
of CA is
reversible,
the
dialyzed
overnight
against
the absorbance 90% of the CA.
at 240 nm it After
the
BIOCHEMICAL
Vol. 68, No. 4, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1 min
5cu z x 4$ 8
3-
W‘ zo $ g2 s
I-
/
/
Y/
I/f
2
4 MINUTES
3
16min I
6
a
Effect of time of incubation of tubulin with CA on the inhibition of polymerization. Tubulin (10 uM) was incubated with 50 ~-84 CA at 37' in 0.5 ml of reassembly buffer containing 0.32 M glycerol for the times shown before the addition of GTP.
[COLCHICINE]
Fig.
10 min
21 min
2
Fig.
5 min
MM
Tubulin (1.5 nmoles) was incubated at 37' in the absence and presence of CA for 20 minutes in reassembly buffer. At this time different aliquots of a colchicine solution (1 mM, 5 mcfml) were added and The reactions were stopped incubation was continued for 60 minutes. by placing the samples on ice and the amount of colchicine bound was determined by the filter assay method (16).
1365
Vol. 68, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL
I 5
I 10
I 15
RESEARCH COMMUNICATIONS
I 20
I 25
MINUTES
Fig.
4
The effect of CA on the sulfhydryl content of tubulin. Tubulin (0.7 mg) was incubated in 0.6 ml of reassembly buffer containing A sample (0.1 ml) was removed and added 0.3 M glycerol at 37'. to 0.4 ml of triethanolamine*HCl, pH 8.0 containing SDS and DTNB. The final concentrations of the latter two were 0.1% and 1 mM, The absorbance at 412 nm was read immediately. CA respectively. (5 1.11 of a 10 mM solution in DMSO) was added to the tubulin solution and samples were removed at different times for sulfhydryl determinations as described above.
dialysis
the CA treated
The untreated
tubulin
To determine cule, Fig.
its
tubulin was stable
whether
ability
of the data CA but
cytochalasins and CB is
lactone.
philic
across
the
With content addition
this
of CA there
inhibition
Fig. is
changes
was examined.
reactive
on the
1366
loss
CA
and CA is an
CA had any effect
rapid
The
between
addition
results
nature.
experiments.
the Michael
the
in
Reciprocal
the difference
makes CA quite
i.e.,
mole-
The results
activity.
in these
18) but
change
whether
4 contains an initial
binding
y-OH-lactone
bond,
in the tubulin
was of a noncompetitive
(1,
This
to reassemble.
procedure.
as an inhibitor
double
in mind we determined of tubulin.
dialysis
binding
an a,&unsaturated
y-keto
addition
the
structures
a,B-unsaturated
any ability
of colchicine
CB was effective
CB is
recover
structural
colchicine
that
are complex that
to this
inhibition
showed
not
not
CA caused
to prevent
3 show a definite
plots
did
of one experiment. of about
10 per
cent
to nucleoreaction. sulfhydryl Upon the of the
Vol. 68, No. 4, 1976
Fig.
free
5
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The effect of 2-mercaptoethanol on inhibition by CA. 1. Polymerization of tubulin in reassembly buffer (0.84 mg/ml) without CA. (1 mM) were incubated 2. CA (100 I.IM) and 2-mercaptoethanol together at 37' in 0.42 ml of reassembly buffer for 1 minute. Tubulin (0.42 mg in 0.08 ml) was added and after 11 minutes the polymerization was initiated by the addition of GTP. 3. CA (100 PM) and tubulin (0.84 mg/ml) were incubated at 37' for 11 minutes before the addition of GTP. The glycerol concentration in all experiments was 0.32 M. The absorbance values are X 102.
sulfhydryl
minutes
content
a decrease
followed
of 40 per
theoretical
value
expected
was about
180 uM.
There
was used.
These
molecular reaction.
weight
results thiol
Such is
by a slower
loss.
occurred.
This
cent
since
the total
was no change suggest might
that
destroy
the case as is
if
content
an identical
ability
shown in Fig.
1367
was actually
sulfhydryl
a prior its
Over a period
close
to inhibit Prior
to the
of the tubulin
concentration
incubation
5.
of 25
of CB
of CA with the
a low
self-assembly
incubation
of 1 mM
Vol. 68, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
I
The Effects of Cytochalasin A and B on the Polymerization of'G-Actin Five ~1 of DMSO, 10 mM CA or 10 mM CB were added to 0.5 ml of G-acgin (1.5 mg/ml> in 0.5 ml4 ATP, 0.5 mM Z-mercaptoethanol, 0.2 mM CaC12 at 22 . After 2 minutes 10 ~1 of 4M KC1 were added. Thirty minutes later the viscosity of the solutions were determined using a Cannon semi-microviscometer. In these experiments the free sulfhydryl content of the G-actin is approximately 0.17 mM. Thus both the G-actin and 2-mercaptoethanol compete for the CA. In the experiment in which CA was first reacted w!Lth 2-mercaptoethanol, 167 pM CA was incubated with 2.17 mM 2-mercaptoethanol in 0.3 ml for 1 minute prior to the addition of the actin in a 0.2 ml volume. The experiment was then continued as described above.
Additions
KC1
"sp
DMSO
0.08
DMSO
+
1.55
CB, 100 PM
+
1.30
CA,
20 PM
+
1.06
CA,
50 PM
+
0.67
CA, 100 PM
+
0.35
CA, 100 LIM + 2-MET
+
1.19
2-mercaptoethanol
with
effect
polymerization.
on tubulin Previously
it
100 PM CA for
was shown that
when KC1 is added to a solution This
is
shown
dramatically
in Table inhibits
tubulin
polymerization
CA with
2-mercaptoethanol.
That
CA reacts
the increase of G-actin
I together the
G-actin
this
with
inhibition
sulfhydryl
1 minute
with
is
results
to F-actin
almost
in viscosity lower
1368
in
using
was also
blocks
which
CA.
of CB (9).
The latter As observed
by prior
demonstrated
its
results
the presence
conversion.
is decreased
groups
completely
incubation
compound in of
in another
Vol. 68, No. 4, 1976
BIOCHEMICAL
CA has a greater
way.
absorbance
the a,B -unsaturated was added from
ketone
in the
of 0.95
to 0.52,
concentration
sulfhydryl
230-240
structure.
of CB.
is
group
adds to the double
clear
from the
than
within
the absorbance
the result bond
CB due to
of 2-mercaptoethanol
at 240 nm decreased
approximately This
nm region
When an excess
to 100 nM CA the absorbance
a value
equal
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2 minutes
value
one would
of an
expect
if
the
of CA.
DISCUSSION It
is
self-assembly the
of brain
conversion
binding exerts
its
This
bation sulfhydryl with
is
its
reagents
inhibit
of tubulin
free
groups
to inhibit both
(19).
sulfhydryls may also
react
expected
to occur
has been
found
by methyl
are
that
acrylate
work
does not
explain
this
type
of reaction (21)
it
affect
buried
covalent
used
sulfhydryl
groups
must be kept that
inhibitory
1369
sulfhydryl activities
at the number
Although would
not
amino be
of cytochalasins
it
modified
(20).
by CA indicated
effect
of
For example
6 to 7.5
in mind when CA is the
that
be selectively
of proteins
of action
CA; 3) CA reacts
a large
experiments.
in the pH region
free
2-mercaptoethanol
structure.
could
2) the
binding
this
of the
1) preincu-
with
is known
that
CA
groups
microtubules
in these
modification
the mechanism
was noted
incubation
esters
that
effects;
of CA with
tubular
colchicine
facts:
maximal
intact
a,B unsaturated
protein
following
by assuming
in the
the
sulfhydryl
It
the
CB in preventing
suggest
and colchicine
be explained
and acrylamide
the
the
polymerization.
at the pH value
Obviously
publication
with
with
reaction
CA does not
used could
strongly
during
the polymerization
That
also
by the
decreases
than
CB inhibits
CA destroys
to achieve
and 4) prior
ability
concentrations
supported required
of tubulin
2-mercaptoethanol;
CA but not
Moreover
by interacting is
tubulin
that
The results
role
content
destroys
the
to F-actin.
suggestion
of CA with
presented
and is more effective
of tubulin.
inhibitory
proteins.
tubulin
of G-actin
activity
results
in
used in vivo.
by this general
but
In one
of CA on the uptake
Vol. 68, No. 4, 1976
BIOCHEMICAL
of 2-deoxy-D-glucose effect
embryo
of CB was reversible.
inhibitions also
by chick
its
processes
be taken
in interpreting
and variable
levels
some of the
observed
activity
would
in yeast
and its
(22).
results
Finally
effects
explain
observed our
of CB with
while
the
irreversible
of CA proposed
of experiments
of contamination
RESEARCH COMMUNICATIONS
was irreversible
The mode of action
antibiotic
cellular
fibrolasts
Our results
by CA --in vivo.
explain
AND BIOPHYSICAL
multiple
results in which
suggest
here
might
effects that
CB is used.
CA may be responsible
on caution Low for
of CB.
ACKNOWLEDGEMENT This
work
was supported
by NIH grant
NS-11360.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21. 22.
Carter, S. B. (1972) Endeavour 113, 77-82. Mizel, S. B. and Wilson, L. (1972) J. Bio. Chem. 247, 4102-4105. Thoa, N. B., Wooten, G. F., Axelrod, .I., and Kopin, I. J. (1972) Proc. Nat'1 Acad. Sci. USA 69, 520-522. Carter, S. B. (1967) Nature 213, 261-264. Allison, A. C., Davies, P., and De Petris, S. (1971) Nature 232, 153-155. Manasek, F. J., Burnside, B., and Stroman, J. (1972) Proc. Nat'1 Acad. Sci. USA%, 308-312. Holtzer, H., Sanger, J., Ishikawa, H., and Strahs, K. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 549-566. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T., Yamada, K. M. (1971) Science 171, 135-143. Spudich, J. A. and Lin, S. (1972) Proc. Nat'1 Acad. Sci. USA 69, 442-446. Spudich, J. A. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 585-593. Croop, J. and Holtzer, H. (1975) 3. Cell Biol. 65, 271-285. Forer, A., Fmmerson, I., and Behnke, 0. (1972) Science 174, 774-776. Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Nat'1 Acad. Sci. USA 70, 765-768. Lee, Y. C., Samson, F. E. Jr., Houston, L. L., and Himes, R. H. (1974) J. Neurobiol. 2, 317-330. Rees, M. K. and Young, M. (1967) J. Biol Chem. B, 4449-4458. Borisy, G. G. (1974) Anal. Biochem. 50, 373-385. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. Angew. Chem. Internat. Edit. Binder, M., and Tamm, C. (1973) l2, 370-380. J. Biochem. 76, 651-654. Kuriyama, R. and Sakai, H. (1974) M. (1968) J. Biol Chem. 243, 3357Cavins, J. F. and Friedman, 3360. (1972) J. Biol. Kleztzien, R. F., Perdue, J. F., and Springer, A. Chem. 247, 2964-2966. Ann. N. Y. Acad. Sci. 235, Kuo, S-C and Lampen, J. 0. (1974) 137-148.
1370
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CYTOCBALASIN A INHIBITS THE --IN VITRO POLYNERIZATION OF BRAIN TUBULIN AND MUSCLE ACTIN Richard
Received
H. Himes,
December
29,
Roderick N. Kersey, Mary Ruscha Department of Biochemistry University of Kansas Lawrence, Kansas 66045
and L. L. Houston
1975
SUMMARY
Cytochalasin A (CA) inhibits the self-assembly of beef brain tubulin. The concentrations necessary to cause the inhibition are only slightly higher than the tubulin concentration. Cytochalasin B (CB) at identical and higher concentrations has no noticeable effect. Cytochalasin A also inhibits colchicine binding activity suggesting that it denatures the tubulin molecule. The results indicate that the reaction of CA with the sulfhydryl groups of tubulin is responsible for its action. CA also prevents the conversion of G-actin to F-actin, probably via a similar mechanism. INTRODUCTION The cytochalasins
are
of effects
on cellular
are
cytochalasin
known,
been
studied
to stop
extrusion
inhibit
some cells
bibliography
A74 of the Aldrich
with
surface.
and there
is
There Copyright All rights
doesn't
still appear
(4), cells
of other
Chemical
affects
cell
cellular
Co.)
However,
some question
(6)
and cause
have also
the
all
as to
cells
the actual
1362
to suggest
inhibit
induce
nuclear
halt
the
"arborization" (For
been noted.
of an
see publication
(8-10) are
(l),
(5), the
evidence
structures not
(3),
phagocytosis
has
has been shown
cells
of cytochalasins
to be any evidence
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
stop
Although
microfilament
compound
range
cytochalasins
availability,
secretion
effects
of the effects
CB apparently the
muscle
This
have a wide seven
of multinucleated
movement
cardiac
greater
the others.
with
which
at least
of its
production
interfere
A number
(7).
extensive
the
cell
of embryonic
than
products
Although
because
B (CB),
with (2),
(l),
beating
(1).
more extensively
transport
metabolic
activities
cytokinesis
hexose
fungal
is not perhaps
affected target(s) that
form
conclusive, by interacting
in
the
same way
of CB (11,lZ). the cytochalasins
Vol. 68, No. 4, 1976
interact
with
BIOCHEMICAL
microtubules
effects
of cytochalasin
inhibits
the --in vitro
or tubulin. A are
to tubulin.
preventing
the polymerization is
MATERIALS
Moreover,
sparse.
of tubulin
CA is also
reports
We provide
polymerization
colchicine
effects
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
data
on the biological here
to show that
and the binding
of
shown to be more effective
of muscle
G-actin.
CA
than
A mechanism
for
CB in these
proposed. AND METHODS
Cytochalasin A and B were purchased from the Aldrich Chemical Co. Solutions of these compounds were made in dimethyl sulfoxide (DMSO). In all experiments in which CA or CB was used the final DMSO concentration was 1%. This concentration of DMSO was included in all controls and did not affect any of the regctions studied. GTP and colchicine were products of Sigma Chemical Co. H-Colchicine was obtained from New England Nuclear Co. Beef brain tubulin was purified by the polymerization method of Shelanski previously (14). The extraction and reassembly --et al. (13) as described buffer was 20 ti (2[N-morpholino] ethane sulfonic acid) (MES), 70 mM NaCl, 1 mM EGTA, 0.5 mM MgC12, pH 6.4. The protein was purified through two polymerization cycles and was stored at -80' in the buffer containing 2 M glycerol. G-actin was extracted from an acetone powder of rabbit muscle by the procedure of Reese and Young (15) and was dialyzed at 4' overnight against 0.5 mM ATP, 0.5 mM 2-mercaptoethanol, 0.2 mM CaCl pH 7.7, before use. Colchicine binding was performed as described E'y Borisy (16). The loss of sulfhydryl content of tubulin was determined by the reaction with 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) (17). RESULTS The inhibition and is
compared
involved
In other
up to 200 uM, had no effect confirmed
by electron
samples.
In the presence
the concentrations to those large
normally
as the
with
GTP it
on the
is it
with
was found
examination
--in vivo of tubulin.
of uranyl
experiments Moreover,
1363
CA is that
of 100 PM CA no microtubules
for
experiments
less
they
1
which before effective
CB, in concentrations
reaction.
the inhibition
shown in Fig.
the inhibitor
seen that
self-assembly
of CA used to cause
concentration
by CA is
In these
of tubulin
experiments
microscopic
used
of tubulin
by colchicine.
preincubation
of polymerization
colchicine.
self-assembly
to inhibition
a two minute
initiation than
of the
This acetate were
are
result stained
evident. fairly
are only the effective
was
high
Although compared
4 to 10 times concentrations
as
Vol. 68, No. 4, 1976
Fig.
1
lower
2, for
UM CB did
addition
if
the preincubation
example, not
CA did
using
inhibit
not
show any observable
stained
samples
taken
normal
microtubules. In an attempt samples containing
was shown
that
20 minutes
to determine as well
effect
such a procedure
This
is
preincubation
shown in with
on preformed
microtubules.
was complete
did
microscopic after
examination
the addition
whether
the effect
as unreacted
1 M glycerol.
longer.
100
reaction.
polymerization
and electron
is
A 20 minute
the self-assembly
to decrease
inhibited
period
50 PM CA.
of 100 uM CA after
turbidity
buffer
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Inhibition of self-assembly of tubulin by CA and colchicine. Tubulin (10 PM) was preincubated for 2 minutes at 37O in 0.5 ml of reassembly buffer containing 0.32 M glycerol and the concentrations of CA and GTP (final concentration, 0.5 mM) was then added colchicine shown. to initiate the reaction.
of CA are Fig.
BIOCHEMICAL
samples
By measuring removed
about
1364
were
not
The
cause
the
of negatively
showed
the presence
of
of CA is
reversible,
the
dialyzed
overnight
against
the absorbance 90% of the CA.
at 240 nm it After
the
BIOCHEMICAL
Vol. 68, No. 4, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1 min
5cu z x 4$ 8
3-
W‘ zo $ g2 s
I-
/
/
Y/
I/f
2
4 MINUTES
3
16min I
6
a
Effect of time of incubation of tubulin with CA on the inhibition of polymerization. Tubulin (10 uM) was incubated with 50 ~-84 CA at 37' in 0.5 ml of reassembly buffer containing 0.32 M glycerol for the times shown before the addition of GTP.
[COLCHICINE]
Fig.
10 min
21 min
2
Fig.
5 min
MM
Tubulin (1.5 nmoles) was incubated at 37' in the absence and presence of CA for 20 minutes in reassembly buffer. At this time different aliquots of a colchicine solution (1 mM, 5 mcfml) were added and The reactions were stopped incubation was continued for 60 minutes. by placing the samples on ice and the amount of colchicine bound was determined by the filter assay method (16).
1365
Vol. 68, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL
I 5
I 10
I 15
RESEARCH COMMUNICATIONS
I 20
I 25
MINUTES
Fig.
4
The effect of CA on the sulfhydryl content of tubulin. Tubulin (0.7 mg) was incubated in 0.6 ml of reassembly buffer containing A sample (0.1 ml) was removed and added 0.3 M glycerol at 37'. to 0.4 ml of triethanolamine*HCl, pH 8.0 containing SDS and DTNB. The final concentrations of the latter two were 0.1% and 1 mM, The absorbance at 412 nm was read immediately. CA respectively. (5 1.11 of a 10 mM solution in DMSO) was added to the tubulin solution and samples were removed at different times for sulfhydryl determinations as described above.
dialysis
the CA treated
The untreated
tubulin
To determine cule, Fig.
its
tubulin was stable
whether
ability
of the data CA but
cytochalasins and CB is
lactone.
philic
across
the
With content addition
this
of CA there
inhibition
Fig. is
changes
was examined.
reactive
on the
1366
loss
CA
and CA is an
CA had any effect
rapid
The
between
addition
results
nature.
experiments.
the Michael
the
in
Reciprocal
the difference
makes CA quite
i.e.,
mole-
The results
activity.
in these
18) but
change
whether
4 contains an initial
binding
y-OH-lactone
bond,
in the tubulin
was of a noncompetitive
(1,
This
to reassemble.
procedure.
as an inhibitor
double
in mind we determined of tubulin.
dialysis
binding
an a,&unsaturated
y-keto
addition
the
structures
a,B-unsaturated
any ability
of colchicine
CB was effective
CB is
recover
structural
colchicine
that
are complex that
to this
inhibition
showed
not
not
CA caused
to prevent
3 show a definite
plots
did
of one experiment. of about
10 per
cent
to nucleoreaction. sulfhydryl Upon the of the
Vol. 68, No. 4, 1976
Fig.
free
5
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The effect of 2-mercaptoethanol on inhibition by CA. 1. Polymerization of tubulin in reassembly buffer (0.84 mg/ml) without CA. (1 mM) were incubated 2. CA (100 I.IM) and 2-mercaptoethanol together at 37' in 0.42 ml of reassembly buffer for 1 minute. Tubulin (0.42 mg in 0.08 ml) was added and after 11 minutes the polymerization was initiated by the addition of GTP. 3. CA (100 PM) and tubulin (0.84 mg/ml) were incubated at 37' for 11 minutes before the addition of GTP. The glycerol concentration in all experiments was 0.32 M. The absorbance values are X 102.
sulfhydryl
minutes
content
a decrease
followed
of 40 per
theoretical
value
expected
was about
180 uM.
There
was used.
These
molecular reaction.
weight
results thiol
Such is
by a slower
loss.
occurred.
This
cent
since
the total
was no change suggest might
that
destroy
the case as is
if
content
an identical
ability
shown in Fig.
1367
was actually
sulfhydryl
a prior its
Over a period
close
to inhibit Prior
to the
of the tubulin
concentration
incubation
5.
of 25
of CB
of CA with the
a low
self-assembly
incubation
of 1 mM
Vol. 68, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
I
The Effects of Cytochalasin A and B on the Polymerization of'G-Actin Five ~1 of DMSO, 10 mM CA or 10 mM CB were added to 0.5 ml of G-acgin (1.5 mg/ml> in 0.5 ml4 ATP, 0.5 mM Z-mercaptoethanol, 0.2 mM CaC12 at 22 . After 2 minutes 10 ~1 of 4M KC1 were added. Thirty minutes later the viscosity of the solutions were determined using a Cannon semi-microviscometer. In these experiments the free sulfhydryl content of the G-actin is approximately 0.17 mM. Thus both the G-actin and 2-mercaptoethanol compete for the CA. In the experiment in which CA was first reacted w!Lth 2-mercaptoethanol, 167 pM CA was incubated with 2.17 mM 2-mercaptoethanol in 0.3 ml for 1 minute prior to the addition of the actin in a 0.2 ml volume. The experiment was then continued as described above.
Additions
KC1
"sp
DMSO
0.08
DMSO
+
1.55
CB, 100 PM
+
1.30
CA,
20 PM
+
1.06
CA,
50 PM
+
0.67
CA, 100 PM
+
0.35
CA, 100 LIM + 2-MET
+
1.19
2-mercaptoethanol
with
effect
polymerization.
on tubulin Previously
it
100 PM CA for
was shown that
when KC1 is added to a solution This
is
shown
dramatically
in Table inhibits
tubulin
polymerization
CA with
2-mercaptoethanol.
That
CA reacts
the increase of G-actin
I together the
G-actin
this
with
inhibition
sulfhydryl
1 minute
with
is
results
to F-actin
almost
in viscosity lower
1368
in
using
was also
blocks
which
CA.
of CB (9).
The latter As observed
by prior
demonstrated
its
results
the presence
conversion.
is decreased
groups
completely
incubation
compound in of
in another
Vol. 68, No. 4, 1976
BIOCHEMICAL
CA has a greater
way.
absorbance
the a,B -unsaturated was added from
ketone
in the
of 0.95
to 0.52,
concentration
sulfhydryl
230-240
structure.
of CB.
is
group
adds to the double
clear
from the
than
within
the absorbance
the result bond
CB due to
of 2-mercaptoethanol
at 240 nm decreased
approximately This
nm region
When an excess
to 100 nM CA the absorbance
a value
equal
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2 minutes
value
one would
of an
expect
if
the
of CA.
DISCUSSION It
is
self-assembly the
of brain
conversion
binding exerts
its
This
bation sulfhydryl with
is
its
reagents
inhibit
of tubulin
free
groups
to inhibit both
(19).
sulfhydryls may also
react
expected
to occur
has been
found
by methyl
are
that
acrylate
work
does not
explain
this
type
of reaction (21)
it
affect
buried
covalent
used
sulfhydryl
groups
must be kept that
inhibitory
1369
sulfhydryl activities
at the number
Although would
not
amino be
of cytochalasins
it
modified
(20).
by CA indicated
effect
of
For example
6 to 7.5
in mind when CA is the
that
be selectively
of proteins
of action
CA; 3) CA reacts
a large
experiments.
in the pH region
free
2-mercaptoethanol
structure.
could
2) the
binding
this
of the
1) preincu-
with
is known
that
CA
groups
microtubules
in these
modification
the mechanism
was noted
incubation
esters
that
effects;
of CA with
tubular
colchicine
facts:
maximal
intact
a,B unsaturated
protein
following
by assuming
in the
the
sulfhydryl
It
the
CB in preventing
suggest
and colchicine
be explained
and acrylamide
the
the
polymerization.
at the pH value
Obviously
publication
with
with
reaction
CA does not
used could
strongly
during
the polymerization
That
also
by the
decreases
than
CB inhibits
CA destroys
to achieve
and 4) prior
ability
concentrations
supported required
of tubulin
2-mercaptoethanol;
CA but not
Moreover
by interacting is
tubulin
that
The results
role
content
destroys
the
to F-actin.
suggestion
of CA with
presented
and is more effective
of tubulin.
inhibitory
proteins.
tubulin
of G-actin
activity
results
in
used in vivo.
by this general
but
In one
of CA on the uptake
Vol. 68, No. 4, 1976
BIOCHEMICAL
of 2-deoxy-D-glucose effect
embryo
of CB was reversible.
inhibitions also
by chick
its
processes
be taken
in interpreting
and variable
levels
some of the
observed
activity
would
in yeast
and its
(22).
results
Finally
effects
explain
observed our
of CB with
while
the
irreversible
of CA proposed
of experiments
of contamination
RESEARCH COMMUNICATIONS
was irreversible
The mode of action
antibiotic
cellular
fibrolasts
Our results
by CA --in vivo.
explain
AND BIOPHYSICAL
multiple
results in which
suggest
here
might
effects that
CB is used.
CA may be responsible
on caution Low for
of CB.
ACKNOWLEDGEMENT This
work
was supported
by NIH grant
NS-11360.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21. 22.
Carter, S. B. (1972) Endeavour 113, 77-82. Mizel, S. B. and Wilson, L. (1972) J. Bio. Chem. 247, 4102-4105. Thoa, N. B., Wooten, G. F., Axelrod, .I., and Kopin, I. J. (1972) Proc. Nat'1 Acad. Sci. USA 69, 520-522. Carter, S. B. (1967) Nature 213, 261-264. Allison, A. C., Davies, P., and De Petris, S. (1971) Nature 232, 153-155. Manasek, F. J., Burnside, B., and Stroman, J. (1972) Proc. Nat'1 Acad. Sci. USA%, 308-312. Holtzer, H., Sanger, J., Ishikawa, H., and Strahs, K. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 549-566. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T., Yamada, K. M. (1971) Science 171, 135-143. Spudich, J. A. and Lin, S. (1972) Proc. Nat'1 Acad. Sci. USA 69, 442-446. Spudich, J. A. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 585-593. Croop, J. and Holtzer, H. (1975) 3. Cell Biol. 65, 271-285. Forer, A., Fmmerson, I., and Behnke, 0. (1972) Science 174, 774-776. Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Nat'1 Acad. Sci. USA 70, 765-768. Lee, Y. C., Samson, F. E. Jr., Houston, L. L., and Himes, R. H. (1974) J. Neurobiol. 2, 317-330. Rees, M. K. and Young, M. (1967) J. Biol Chem. B, 4449-4458. Borisy, G. G. (1974) Anal. Biochem. 50, 373-385. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. Angew. Chem. Internat. Edit. Binder, M., and Tamm, C. (1973) l2, 370-380. J. Biochem. 76, 651-654. Kuriyama, R. and Sakai, H. (1974) M. (1968) J. Biol Chem. 243, 3357Cavins, J. F. and Friedman, 3360. (1972) J. Biol. Kleztzien, R. F., Perdue, J. F., and Springer, A. Chem. 247, 2964-2966. Ann. N. Y. Acad. Sci. 235, Kuo, S-C and Lampen, J. 0. (1974) 137-148.
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