Vol.
178,
August
No. 15,
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1991
EVIDENCE
THAT
UNPHOSPHORYLATED SMOOTH
Istituto
di via
Received
June
Chimica Borsari
17,
MUSCLE
Grazi
Enrico
SMOOTH
MUSCLE
MYOSIN
967-973
SUPPORTS
CONTRACTION
and
Giorgio
Biologica, 46, 44100
Trombetta Universith Ferrara,
di
Ferrara,
Italy
1991
SUMMPRY : Unphosphorylated smooth muscle myosin filaments do not disassemble in MgPTP, provided that the solution is supplemented either by 25% serum albumin or by 6% polyethylene glycol 6000. These filaments are able to support actomyosin retraction but their ATPase activity is not activated by tropomyosin-decorated F-actin. 0 1991 Academic Press. Inc.
It kDa
is
generally
light
chains
muscle
(gizzard)
myosins
into
sin
assumed of
myosins
filaments
are
in
fact
having
sedimentation
The
myosin
is
(6,
myosin It
8)
monomers
thus
not
rather
appears
support
If be
stable,
a)
the
meric
they
this
myosin
salt,
and
has shape
which
is extended
at
MgATP
a folded
sufficiently
that
(1,
form 5,
6).
conforma-
6s
of (9,
10). do
in
MgATP.
forces
the
consti-
conformation.
myosin
provided
and
filaments
disassemble
because
unpho-
characteristic
myosin
in
smooth
these
a folded
sediment
they
and
while
11'S
extended
20
myo-
(l-4),
of
unphosphorylated
strength the
MgATP
coefficient 7)
the
Phosphorylated
MgATP
filament
MgATP,
of
in
because
in
in
assembly
unphosphorylated
the
true
even ionic
high
(thymus)
disassemble
(6,
disassemble
of
is
in
the
that
monomers
stable
than
contraction
Furthermore, tuent
in
the (1,2).
monomeric
of
muscle
vitro
filaments
species
tion
non
regulates in
myosin
11s
phosphorylation
vertebrate
filaments
sphorylated
that
filaments
should
: high
to
maintain
mono-
shape 0006-291X/91 967
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
178,
No.
b)
3, 1991
the
ced
association
by
lene
BIOCHEMICAL
high
We show
that
myosin
(11, the
albumin
(or
myosin
weight
6000)
12,
solutes
prediction
are
RESEARCH
into
COMMUNICATIONS
filaments
(serum
is
albumin,
for-
polyethy-
13). is
polyethylene
filaments
BIOPHYSICAL
monomeric
molecular
glycol
serum
of
AND
correct
glycol
stable
in
: in 6000)
MgPTP
and
the
presence
of
unphosphorylated
support
actomyosin
retraction. MATERIALS
AND ME'IHODS
G-actin from rabbit muscle and tropomyosin from chicken gizzard were prepared and assayed as previously described (12). Unphosphorylated myosin was prepared from chicken gizzard (14). Molar concentration of myosin was calculated on the basis of the absorption coefficient A 1% = 5.58 (14) and of the molecular 280 nm mass of 470 kDa (15). Protein was determined by the Coomassie blue method (16). Light scattering was determined at 500 nm with a 90° observation angle. To mimic the fluid composition of muscle, actomyosin retraction was studied at the following electrolytes concentrations : 148 mM K+, 13 mM Na+, 15.5 mM Mg2+, 9.5 mM sulfate, 6 mM phosphate, 15 mM creatine phosphate, 6.5 mM PIP, 66 mM propionate, 25%, w/v, serum albumin (corresponding to a proteinate concentration of 50 meq/L). When serum albumin was replaced by 6% (w/v) polyethylene glycol 6000, propionate concentration was increased from 66 mM to 116 mM. pH was 7.0.
RESULTS Unphosphorylated are
not
disassembled
As ments was of
myosin
it
was
solution
microscope
neither
by by
the
the
decrease
dissociation
Unphosphorylated decorated
myosin F-actin,
is is
trace
A)
(Fig.
2a
not
known
that
not
activated
the
and
addition of
the
of
myosin
even
glycol
6000,
myosin
fila-
ATPase by
The
the
light
directly,
phenomenon scattering
by
electron
2b).
of light
6000
was
1.66
mM ATP
present
scattering
not
activated
activity
tropomyosin-decorated
was
(Fig.
polyethylene
in
(Fig.
filaments is
in
(l-4).
of
and,
glycol
ATPase
MgA'IP
decrease
polyethylene
the
of
the 1,
mixtures,
polyethylene
unphosphorylated
presence
following (Fig.
6% (w/v)
incubation
myosin
the
observation
When
It
reported,
in by
in
MgATP
previously
monitored
nor
by
disassemble
the
filaments,
followed 1,trace
2c by
the
and
B)
2d).
tropomyosin-
glycol of
unphosphorylated F-actin.
Since
Vol.
178, No. 3, 1991
BIOCHEMICAL
ATP ADDED 4 cx-J...o .._...._
p ---.--
DILUTION 0.4.
AND BIOPHYSICAL
B
6’ 1:
:
I
E e 8
o
RESEARCH COMMUNICATIONS
ATP ADDED
11
0.2. e-e-0 e-e-0
5 i
\I
0.1 -
‘\‘\
I 0
5
10
l -h@
15
TIME
A 20
(min)
Fig.
1. Light scattering of unphosphorylated myosin filaments in the presence of polyethylene glycol 6000 and P'TP. The incubation mixtures contained 0.26 PM unphosphorylated smooth muscle myosin, 0.13 M KC1, 10 mM MgS04, 0.2 mM EGTA, 1 mM Z-mercaptoethanol and 25 mM imidazole buffer, pH was 7.0, temperature 22oc. After 7 min of incubation the mixture was dividedinto two aliquots. A) The first aliquot was diluted by the addition of an equal volume of the salt-buffer solution (lower tra0-O). B) The second aliquot was diluted by the addition ce, of an equal volume of the salt-buffer solution containing 12% (w/v) polyethylene glycol 6000, so that final concentration of polyethylene
glycol
indicated Ordinate
we
the of
shown
have
1.1
6% (upper trace, O----O). 1.66 mM ATP was added to scattering, arbitrary units.
in
6% polyethylene
maintain
tested
presence
was
arrows
that,
filaments
whether of
an
of
9.5
presence
of
To cell
conditions
but,
most
molecules.
plus
not
important, The
activity
with
experiments
is
PM
not
with
P'IPase increased
(data
actomyosin effort respect
respect were 969
to thus
the
MgA'IP,
activity by
not not
in
the
ad-
even
in
shown).
retraction was
to
in
increased
tropomyosin,
glycol
retraction only
was
1.34
supports
actomyosin
shape
F-actin.
myosin
myosin
study
ATPase
6% polyethylene
Unphosphorylated
unphosphorylated
normal
tropomyosin-decorated
PM F-actin
At the time both aliquots.
glycol,
apparently
their
PM unphosphorylated
dition the
the
: light
have
myosin we
by
made
to
electrolyte
composition
concentration
performed
simulate
in
of the
macro-
presence
Vol.
178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Fig. 2. Electron micrographs of unphosphorylated myosin filaments in the presence of polyethylene glycol 6000 and A'TP. The unphosphorylated myosin filaments suspensions were prepared with the procedure described in fig. 1, with the exception that KC1 was 0.28 M in the samples containing polyethylene glycol. Unphosphorylated myosin filaments in 0.13 M KCl, (a); Unphosphorylated myosin filaments in 0.13 M KC1 plus 1.66 mM P'TP, samples were taken 7 min after the addition of ATP, (b); Unphosphorylated myosin filaments in 0.28 M KCl, 6% polyethylene glycol 6000 and 1.66 mM ATP, (c) and (d). Bar represents 500 nm in (a), (c) and (d) and 330 nm in (b).
970
Vol.
BIOCHEMICAL
178, No. 3, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 3. Retraction of the system formed by unphosphorylated myosin and tropomyosin-decorated F-actin in the presence of polyethylene glycol plus A'IP. The mixture contained 1 PM unphosphorylated myosin, 7.9 PM ( as monomer) F-actin, 1.1 PM tropomyosin and 6% (w/v) polyethylene glycol 6000. Salt-buffer composition was as described under materials and methods. After the addition of 6.5 mM A'IP, the mixture (0.5 ml), at the temperature of 2OC, was transferred to a glass tube (inner diameter 6 mm) immersed in a water bath at 37OC. Photographs were taken 12 min (C) and 30 min (D) after the 1 min (A), 8 min (B), transfer of the solution to the water bath.
of
either
co1
25%
6000.
It
containing and
with
the in
lene
glycol
was
complete the
amount. traction fact
r" 1.1
serum found
r
fig.
solution, control
required
that
min
polyethylene to
In
was
time,
the was it
the
less
971
or
by the
of
than found
system.
myosin
experiment
replaced
amount
was
a solution
M (as monomer) r a reaction which ended
visualization, and
gly-
7.9
cloth.
albumin
complete
either
ATP
started
experiments the
when
serum
30
at
of myosin,
a better
about
6% (w/v)
addition
a retraction
where
insure
in
observed
that
or
M tropomyosin, of
3, to
In
albumin
m unphosphorylated
formation
shown
in
was 1
F-actin
(w/v)
retraction
protein 5% of
that
found the
or
ATP
original
actomyosin
No retraction actin
polyethy-
was were
rein
omitted.
Vol.
BIOCHEMICAL
178, No. 3, 1991
Retraction
did
glycol
were
not
when
be
from
37OC
was
increased,
the
addition
by
did
to
22OC,
not
occur
unless
the
over
the
either
of
polyethylene
concentration,
On the
did
contrary,
propio-
concentration
when
temperature
ionic
strength
found
in
M KC1
or
same
equivalent
value 0.1
the 6000.
an
RESEARCH COMMUNICATIONS
albumin
at
glycol
replaced
Retraction
either
Glycerol,
polyethylene
could
ride.
occur
omitted.
replace
nate
not
AND BIOPHYSICAL
or
of
the
0.1
of
chlo-
was
decreased
the
solution
skeletal
muscle,
M potassium
by
propio-
nate. DISCUSSION In
vitro,
in
ethylene
glycol,
supports
the
the
retraction
their
lation
of
tended
shape.
are at
thus
both likely
is
phosphorylated kinase scle
vice
system
into must
four
thus
the have
"fast" emerged
same
conditions,
disassemble
MgPTP,
specific
phosphory-
not
a myosin
in
and
rate
of
larger The to
by
in
requires only
the
phosphorylated trigger
tropomyosin-de-
unphosphorylated muscle acto
the
albeit
- phosphorylated
than the rate of 2+ Ca -calmodulin-light the "slow"
rate
of
myosin acto
- unchain
smooth
myosin.
A different
de-
contraction.
REFERENCES
2.
Suzuki, H., Onishi, H., Takahashi, K. and Watanabe, (1978) J. Biochem. (Tokyo) 84, 1529-1542. Scholey, J.M., Taylor, K.A. and Kendrich-Jones, J. Nature 287, 233-235. 972
mu-
unphosphorylated
Acknowledgment: This work was supported by grants 90/40/05/016 and 90/60/05/038 of the Minister0 dell'llniversita e della Ricerca Scientifica e Tecnologica.
1.
ex-
myosins
contraction,
regulate
converts
to
the
gizzard
this
ATPase.
: it
chicken
poly-
activated
smooth
appears
not
or
not
and
times
myosin
contraction
myosin
: the
Under do
chains
support
rate about
is
albumin
from
actomyosin.
phosphorylated to
a different
ATPase
light
serum
myosin
Apparently,
myosin
vivo,
either
filaments
A'IPase
F-actin.
In
of
myosin
nevertheless
of
unphosphorylated
unphosphorylated
corated
presence
S. (1980)
Vol.
178,
No.
3, 1991
BIOCHEMICAL
AND
3.
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Scholey, J.M., Taylor, K.A. and Kendrich-Jones, J. (1981) Biochimie 63, 255-271. 4. Scholey, J-M., Smith, R.C., Drenckhahn, D., GrBschel-Stewart and Kendrich-Jones, J. (1982) J. Biol. Chem. 257, 77377745. 5. Kendrich-Jones, J., Tooth, P., Taylor, K.A. and Scholey,J.M. (1982) Cold Spring Harbour Symp. Quant. Biol. 46, 929-938. 6. 'Irybus, K.M., Huiatt, 'I.W. and Lowey, S. (1982) Proc. Nat. Pcad. SC. U.S. 79, 6151-6155. 7. Suzuki, H., Kamata, I., Onishi, H. and Watanabe, S. (1982) J. Biochem. (Tokyo) 91, 1699-1705. 8. Onishi, H. and Wakabayashi, 7. (1982) J. Biochem. (Tokyo) 92 t 871-879. 9. Elliot, A. and Offer, G. (1978) J. Molec. Biol. 123, 505-519. 10. Lowey, S., Slayter, H.S., Weeds, P.G. and Baker, H. (1969) J. Molec. Biol. 42, l-29. 11. Suzuki, A., Yamazaki, M. and Ito, 'I. (1989) Biochemistry 6513-6518. 28, 12. Grazi, E., Trombetta, G. and Guidoboni, M. (1990) Biochem. Biophys. Res. Communs. 167, 1109-1114. 13.Grazi, E., 'Irombetta, G., Magri, E. and Cuneo, P. (1990) FEBS Letters 272, 149-151. 14. Frederiksen, D.W. and Rees, D.D. (1982) Methods in Enzymol. 85, 292-298. 15. Gorecka, A\-, Aksoi, M-0. and Hartshorne, D.J. (1976) Biochem. Biophys. Res. Communs. 71, 325-331. 16. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254.
913
178,
August
No. 15,
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1991
EVIDENCE
THAT
UNPHOSPHORYLATED SMOOTH
Istituto
di via
Received
June
Chimica Borsari
17,
MUSCLE
Grazi
Enrico
SMOOTH
MUSCLE
MYOSIN
967-973
SUPPORTS
CONTRACTION
and
Giorgio
Biologica, 46, 44100
Trombetta Universith Ferrara,
di
Ferrara,
Italy
1991
SUMMPRY : Unphosphorylated smooth muscle myosin filaments do not disassemble in MgPTP, provided that the solution is supplemented either by 25% serum albumin or by 6% polyethylene glycol 6000. These filaments are able to support actomyosin retraction but their ATPase activity is not activated by tropomyosin-decorated F-actin. 0 1991 Academic Press. Inc.
It kDa
is
generally
light
chains
muscle
(gizzard)
myosins
into
sin
assumed of
myosins
filaments
are
in
fact
having
sedimentation
The
myosin
is
(6,
myosin It
8)
monomers
thus
not
rather
appears
support
If be
stable,
a)
the
meric
they
this
myosin
salt,
and
has shape
which
is extended
at
MgATP
a folded
sufficiently
that
(1,
form 5,
6).
conforma-
6s
of (9,
10). do
in
MgATP.
forces
the
consti-
conformation.
myosin
provided
and
filaments
disassemble
because
unpho-
characteristic
myosin
in
smooth
these
a folded
sediment
they
and
while
11'S
extended
20
myo-
(l-4),
of
unphosphorylated
strength the
MgATP
coefficient 7)
the
Phosphorylated
MgATP
filament
MgATP,
of
in
because
in
in
assembly
unphosphorylated
the
true
even ionic
high
(thymus)
disassemble
(6,
disassemble
of
is
in
the
that
monomers
stable
than
contraction
Furthermore, tuent
in
the (1,2).
monomeric
of
muscle
vitro
filaments
species
tion
non
regulates in
myosin
11s
phosphorylation
vertebrate
filaments
sphorylated
that
filaments
should
: high
to
maintain
mono-
shape 0006-291X/91 967
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
178,
No.
b)
3, 1991
the
ced
association
by
lene
BIOCHEMICAL
high
We show
that
myosin
(11, the
albumin
(or
myosin
weight
6000)
12,
solutes
prediction
are
RESEARCH
into
COMMUNICATIONS
filaments
(serum
is
albumin,
for-
polyethy-
13). is
polyethylene
filaments
BIOPHYSICAL
monomeric
molecular
glycol
serum
of
AND
correct
glycol
stable
in
: in 6000)
MgPTP
and
the
presence
of
unphosphorylated
support
actomyosin
retraction. MATERIALS
AND ME'IHODS
G-actin from rabbit muscle and tropomyosin from chicken gizzard were prepared and assayed as previously described (12). Unphosphorylated myosin was prepared from chicken gizzard (14). Molar concentration of myosin was calculated on the basis of the absorption coefficient A 1% = 5.58 (14) and of the molecular 280 nm mass of 470 kDa (15). Protein was determined by the Coomassie blue method (16). Light scattering was determined at 500 nm with a 90° observation angle. To mimic the fluid composition of muscle, actomyosin retraction was studied at the following electrolytes concentrations : 148 mM K+, 13 mM Na+, 15.5 mM Mg2+, 9.5 mM sulfate, 6 mM phosphate, 15 mM creatine phosphate, 6.5 mM PIP, 66 mM propionate, 25%, w/v, serum albumin (corresponding to a proteinate concentration of 50 meq/L). When serum albumin was replaced by 6% (w/v) polyethylene glycol 6000, propionate concentration was increased from 66 mM to 116 mM. pH was 7.0.
RESULTS Unphosphorylated are
not
disassembled
As ments was of
myosin
it
was
solution
microscope
neither
by by
the
the
decrease
dissociation
Unphosphorylated decorated
myosin F-actin,
is is
trace
A)
(Fig.
2a
not
known
that
not
activated
the
and
addition of
the
of
myosin
even
glycol
6000,
myosin
fila-
ATPase by
The
the
light
directly,
phenomenon scattering
by
electron
2b).
of light
6000
was
1.66
mM ATP
present
scattering
not
activated
activity
tropomyosin-decorated
was
(Fig.
polyethylene
in
(Fig.
filaments is
in
(l-4).
of
and,
glycol
ATPase
MgA'IP
decrease
polyethylene
the
of
the 1,
mixtures,
polyethylene
unphosphorylated
presence
following (Fig.
6% (w/v)
incubation
myosin
the
observation
When
It
reported,
in by
in
MgATP
previously
monitored
nor
by
disassemble
the
filaments,
followed 1,trace
2c by
the
and
B)
2d).
tropomyosin-
glycol of
unphosphorylated F-actin.
Since
Vol.
178, No. 3, 1991
BIOCHEMICAL
ATP ADDED 4 cx-J...o .._...._
p ---.--
DILUTION 0.4.
AND BIOPHYSICAL
B
6’ 1:
:
I
E e 8
o
RESEARCH COMMUNICATIONS
ATP ADDED
11
0.2. e-e-0 e-e-0
5 i
\I
0.1 -
‘\‘\
I 0
5
10
l -h@
15
TIME
A 20
(min)
Fig.
1. Light scattering of unphosphorylated myosin filaments in the presence of polyethylene glycol 6000 and P'TP. The incubation mixtures contained 0.26 PM unphosphorylated smooth muscle myosin, 0.13 M KC1, 10 mM MgS04, 0.2 mM EGTA, 1 mM Z-mercaptoethanol and 25 mM imidazole buffer, pH was 7.0, temperature 22oc. After 7 min of incubation the mixture was dividedinto two aliquots. A) The first aliquot was diluted by the addition of an equal volume of the salt-buffer solution (lower tra0-O). B) The second aliquot was diluted by the addition ce, of an equal volume of the salt-buffer solution containing 12% (w/v) polyethylene glycol 6000, so that final concentration of polyethylene
glycol
indicated Ordinate
we
the of
shown
have
1.1
6% (upper trace, O----O). 1.66 mM ATP was added to scattering, arbitrary units.
in
6% polyethylene
maintain
tested
presence
was
arrows
that,
filaments
whether of
an
of
9.5
presence
of
To cell
conditions
but,
most
molecules.
plus
not
important, The
activity
with
experiments
is
PM
not
with
P'IPase increased
(data
actomyosin effort respect
respect were 969
to thus
the
MgA'IP,
activity by
not not
in
the
ad-
even
in
shown).
retraction was
to
in
increased
tropomyosin,
glycol
retraction only
was
1.34
supports
actomyosin
shape
F-actin.
myosin
myosin
study
ATPase
6% polyethylene
Unphosphorylated
unphosphorylated
normal
tropomyosin-decorated
PM F-actin
At the time both aliquots.
glycol,
apparently
their
PM unphosphorylated
dition the
the
: light
have
myosin we
by
made
to
electrolyte
composition
concentration
performed
simulate
in
of the
macro-
presence
Vol.
178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Fig. 2. Electron micrographs of unphosphorylated myosin filaments in the presence of polyethylene glycol 6000 and A'TP. The unphosphorylated myosin filaments suspensions were prepared with the procedure described in fig. 1, with the exception that KC1 was 0.28 M in the samples containing polyethylene glycol. Unphosphorylated myosin filaments in 0.13 M KCl, (a); Unphosphorylated myosin filaments in 0.13 M KC1 plus 1.66 mM P'TP, samples were taken 7 min after the addition of ATP, (b); Unphosphorylated myosin filaments in 0.28 M KCl, 6% polyethylene glycol 6000 and 1.66 mM ATP, (c) and (d). Bar represents 500 nm in (a), (c) and (d) and 330 nm in (b).
970
Vol.
BIOCHEMICAL
178, No. 3, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 3. Retraction of the system formed by unphosphorylated myosin and tropomyosin-decorated F-actin in the presence of polyethylene glycol plus A'IP. The mixture contained 1 PM unphosphorylated myosin, 7.9 PM ( as monomer) F-actin, 1.1 PM tropomyosin and 6% (w/v) polyethylene glycol 6000. Salt-buffer composition was as described under materials and methods. After the addition of 6.5 mM A'IP, the mixture (0.5 ml), at the temperature of 2OC, was transferred to a glass tube (inner diameter 6 mm) immersed in a water bath at 37OC. Photographs were taken 12 min (C) and 30 min (D) after the 1 min (A), 8 min (B), transfer of the solution to the water bath.
of
either
co1
25%
6000.
It
containing and
with
the in
lene
glycol
was
complete the
amount. traction fact
r" 1.1
serum found
r
fig.
solution, control
required
that
min
polyethylene to
In
was
time,
the was it
the
less
971
or
by the
of
than found
system.
myosin
experiment
replaced
amount
was
a solution
M (as monomer) r a reaction which ended
visualization, and
gly-
7.9
cloth.
albumin
complete
either
ATP
started
experiments the
when
serum
30
at
of myosin,
a better
about
6% (w/v)
addition
a retraction
where
insure
in
observed
that
or
M tropomyosin, of
3, to
In
albumin
m unphosphorylated
formation
shown
in
was 1
F-actin
(w/v)
retraction
protein 5% of
that
found the
or
ATP
original
actomyosin
No retraction actin
polyethy-
was were
rein
omitted.
Vol.
BIOCHEMICAL
178, No. 3, 1991
Retraction
did
glycol
were
not
when
be
from
37OC
was
increased,
the
addition
by
did
to
22OC,
not
occur
unless
the
over
the
either
of
polyethylene
concentration,
On the
did
contrary,
propio-
concentration
when
temperature
ionic
strength
found
in
M KC1
or
same
equivalent
value 0.1
the 6000.
an
RESEARCH COMMUNICATIONS
albumin
at
glycol
replaced
Retraction
either
Glycerol,
polyethylene
could
ride.
occur
omitted.
replace
nate
not
AND BIOPHYSICAL
or
of
the
0.1
of
chlo-
was
decreased
the
solution
skeletal
muscle,
M potassium
by
propio-
nate. DISCUSSION In
vitro,
in
ethylene
glycol,
supports
the
the
retraction
their
lation
of
tended
shape.
are at
thus
both likely
is
phosphorylated kinase scle
vice
system
into must
four
thus
the have
"fast" emerged
same
conditions,
disassemble
MgPTP,
specific
phosphory-
not
a myosin
in
and
rate
of
larger The to
by
in
requires only
the
phosphorylated trigger
tropomyosin-de-
unphosphorylated muscle acto
the
albeit
- phosphorylated
than the rate of 2+ Ca -calmodulin-light the "slow"
rate
of
myosin acto
- unchain
smooth
myosin.
A different
de-
contraction.
REFERENCES
2.
Suzuki, H., Onishi, H., Takahashi, K. and Watanabe, (1978) J. Biochem. (Tokyo) 84, 1529-1542. Scholey, J.M., Taylor, K.A. and Kendrich-Jones, J. Nature 287, 233-235. 972
mu-
unphosphorylated
Acknowledgment: This work was supported by grants 90/40/05/016 and 90/60/05/038 of the Minister0 dell'llniversita e della Ricerca Scientifica e Tecnologica.
1.
ex-
myosins
contraction,
regulate
converts
to
the
gizzard
this
ATPase.
: it
chicken
poly-
activated
smooth
appears
not
or
not
and
times
myosin
contraction
myosin
: the
Under do
chains
support
rate about
is
albumin
from
actomyosin.
phosphorylated to
a different
ATPase
light
serum
myosin
Apparently,
myosin
vivo,
either
filaments
A'IPase
F-actin.
In
of
myosin
nevertheless
of
unphosphorylated
unphosphorylated
corated
presence
S. (1980)
Vol.
178,
No.
3, 1991
BIOCHEMICAL
AND
3.
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Scholey, J.M., Taylor, K.A. and Kendrich-Jones, J. (1981) Biochimie 63, 255-271. 4. Scholey, J-M., Smith, R.C., Drenckhahn, D., GrBschel-Stewart and Kendrich-Jones, J. (1982) J. Biol. Chem. 257, 77377745. 5. Kendrich-Jones, J., Tooth, P., Taylor, K.A. and Scholey,J.M. (1982) Cold Spring Harbour Symp. Quant. Biol. 46, 929-938. 6. 'Irybus, K.M., Huiatt, 'I.W. and Lowey, S. (1982) Proc. Nat. Pcad. SC. U.S. 79, 6151-6155. 7. Suzuki, H., Kamata, I., Onishi, H. and Watanabe, S. (1982) J. Biochem. (Tokyo) 91, 1699-1705. 8. Onishi, H. and Wakabayashi, 7. (1982) J. Biochem. (Tokyo) 92 t 871-879. 9. Elliot, A. and Offer, G. (1978) J. Molec. Biol. 123, 505-519. 10. Lowey, S., Slayter, H.S., Weeds, P.G. and Baker, H. (1969) J. Molec. Biol. 42, l-29. 11. Suzuki, A., Yamazaki, M. and Ito, 'I. (1989) Biochemistry 6513-6518. 28, 12. Grazi, E., Trombetta, G. and Guidoboni, M. (1990) Biochem. Biophys. Res. Communs. 167, 1109-1114. 13.Grazi, E., 'Irombetta, G., Magri, E. and Cuneo, P. (1990) FEBS Letters 272, 149-151. 14. Frederiksen, D.W. and Rees, D.D. (1982) Methods in Enzymol. 85, 292-298. 15. Gorecka, A\-, Aksoi, M-0. and Hartshorne, D.J. (1976) Biochem. Biophys. Res. Communs. 71, 325-331. 16. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254.
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