Vol.
89,
No.
August
13,
3,
1979
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS
Pages
1979
ACTIVATION
BY ACTIN OF ATPase ACTIVITY
958-964
OF CHEMICALLY
MODIFIED GIZZARD MYOSIN WITHOUT PHOSPHORYLATION John
C. Seidel
Department of Muscle Research Boston Biomedical Research Institute 20 Staniford Street, Boston Massachusetts Department of Neurology, Harvard Medical Received
June
02114 and School
15,1979
SUMMARY : The ATPase2+aci;c; . 'tly+ of myosin from chicken gizzard measured in the presence of either Mg is increased in the absence of dithiothreitol or z;o;U5$action with Cu2+, o-iodo+sobenzoate, or N-ethylmaleimide. Iodosobenzoate produce no change in K (EDTA)-ATPase while N-ethylmaleimide produces a desfease. These treatments also make the actin-activated ATPase insensitive to Ca when assayed in the presence of tropomyosin and a partially purified myosin light chain kinajf. Phosphorylation of N-ethylmaleimide modified myosin remains dependent on Ca and therefore appears not to be required for activation by actin of the ATPase activity of modified myosin.
There
is convincing
smooth
muscle
dalton
light
of
can be regulated chain
some myosin
but
does
(5,6).
(l-41,
to
ATPase
activity
myosin
alone
myosins thiol
time
from
is
raising
modification
make the actin-activa
the
the
presence
with
evidence
with
of
that
actin-bound myosin
that
oxidation
thiol
modification
skeletal
muscle
may differ
N-ethylmaleimide
increase
.ted ATPase activity
insensitive
in
oxidation the
20,000-
activation
proteins
been reported
might
addition
from
on phosphorylation
have
and there
shows that
actin
the
regulatory
of gizzard
In
myosin
of
does not depend
actin.
work
of actin
phosphorylation
also
possibility
The present with
is
properties
by
and from
(7-9).
there
interaction
influenced
gizzard
interaction
from smooth muscle
Ca 2+ in
and the
modifiers
or its
(6)
the
by Ca 2+-dependent
Some of the enzymatic with
that
however,
preparations
respond
change
tion
evidence
to
influence the
is
the
activity
of
evidence
their
that
response
of gizzard
Ca 2+ -ATPase
to
myosin
activity
and
to Ca 2+ and to phosphoryla-
of myosin. METHODS
Myosin was prepared from fresh chicken gizzards essentially Sobieszek and Small (10) with 0.1 to 0.5 mM 1,4-dithiothreitol 0006-291X/79/150958-07$01.00/0 Copyright All rights
@ 1979 by Academic Press, Inc. in my form reserved.
of reproduction
958
as described by (DTT) being
Vol.
89,
No.
BIOCHEMICAL
3, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Tropomyosin from chicken gizzards was included in all steps of the preparation. obtained by one isoelectric precipitation (at pH 4.9) of the 37-55% ammonium tropomyosin (11) described by Aksoy et al. (1). sulfate fraction from "native" F-actin was prepared from rabbit skeletal muscle as described by Spudich and Watt The partially purified fraction containing myosin light chain kinase used (12). in this work was the 37-55% ammonium sulfate fraction of "native" tropomyosin referred to as P (1). 37-55 Ca2+ -activated ATPase activity was determined by incubation of+gizzard and 5 mM AT sr K (EDTA)myosin in ;!5 mM NaCl, 40 mM Tris, pH 7.5, 10 mM CaCl p H 7.5 and ?'mM ATP and Mg -ATPase in ATPase in 0.6M KCl, 5 mM EDTA, 40 mM Tris, 80 mM NaCl, 20 mM Tris, pH 7.5, 10 mM MgC12, 2 mM ATP, and either 0.1 mM EGTA or The reaction was terminated by adding an equal volume of 10% 0.1 mM CaCl . 't. ic acid and released inorganic gysphate de trichloroace Incorporation of P from ,-[5eLrd;inAeTdpZttohZn~st?~dw1f Fiske and Subbarow (13). measured as described previously (14). Treatment with N-ethylmaleimide was carried out for 10 min at 0' in a solution containing 0.5M KCl, 40 mM morpholinopropane sulfonic acid (MOPS), pH 7.5, 1 mM N-ethylmaleimide and 10 mg of myosin per ml, the reaction being The reaction with o-iodosobenterminated by adding O.OlM 1,4-dithiothreitol. zoate was initiated by adding iodosobenzoate to a final concentration of O.OlM in a solution containing 0.3M NaCl, O.lM MOPS, pHo7.5, and 10 mg of myosin per ml. Th5+react')n was c+arried out for 10 min at 25 , the samples placed in ice, and For activities were determined within one hour. Mg -, cafe , or K (EDTA)-ATPase measurement of ATPase activity in the presence of actin and regulatory proteins excess reagent was removed by adding 14 volumes of 1 mM MOPS, pH 7.0, containing 0.1 mM EDTA. The myosin was allowed to settle, the supernatant removed and the myosin eithler washed with a solution containing 40 mM NaCl, 1 mM MOPS, pH 7.0, and in the same solution without the washing step. 0.1 mM EDTA or sgsuspended Treatment with Cu was done by adding Cu(N03)2 to a final concentration of 3 PM for activity of myosin alone and 10 nM for actin-activated ATPase in a solution ATPase activities were determined containing 0.5M NaC12+l mM MOPS, pH 7.5. without removal of Cu . DTT was removed either by dialysis against 400 volumes of a solution containing 0.5M NaCl, 1 mM MOPS, pH 7.5, and 0.1 mM EDTA or by the dilution procedure described above. RESULTS AND DISCUSSION ATPase
The Ca2+ -activated present
throughout
ionic
strength
the
preparation
This
increase
in
the
and
are
can produce
as much as a 4-fold
observed
removal
which
produce
and iodosobenzoate
(16)
Mg 2+ -ATPase
measured
the
increase is not
effect
with
it
when
959
low of I).
by any change to define "spontaneous" Both
modifiers.
increases little
at
(Table
was difficult this
DTT
completion
in activity
sulfhydryl
and
with
measured
after
observe
2- to 3-fold
25 mM NaCl
prepared
accompanied
consistently
produce in
low
Because
activity.
we could
to
myosin
of DTT by dialysis
in 0.6M KC1 and it
ATPase
under
gizzard
consistently
but
we attempted
(15)
of
(25 mM NaCl)
is not
conditions
increase,
ATPase
preparation
K+(EDTA)-activated
standard
CU2+
the
activities
in both or
no
Ca
change
2+
-
in
Vol.
89,
No.
3,
1979
BIOCHEMICAL
AND
BIOPHYSICAL
TABLE
COMMUNICATIONS
I
ATPase Activities
of Myosin Alone ATPase Activity ~moles/mg myosinlmin
AP i, Additions
ca*+
Ca2++KC1
Mg2+
K+(EDTA)
DTT (in preparation)
0.037
0.144
0.0051
0.39
None (DTT removed)
0.176
0.154
CU(NO~)~
0.097
0.114
0.0188
0.34
o-iodosobenzoate
0.270
0.182
0.0110
0.37
N-ethylmaleimide
0.140
0.182
0.0119
0.11
the
in Ca 2+ -ATPase
K+(EDTA)-ATPase accompanied muscle only
(Table
at
in
myosin
increased
a change heavy
is not
Initial myosin
and
Pj7-55,
showed
a decrease
preparation
of myosin
influence
similar
effects.
It
the (Table
by actin
is no further may be noted
that
in
fraction
of
used
for
rabbit gizzard
pmoles/mg/min
without
Ca 2+ -ATPase
of gizzard
indicating
that
of
of
"native"
tropomyosin
that
oxidation
and that
by micromolar N-ethylmaleimide
960
Ca 2+ -
increases
presence
removal
the actin-activated
either
Ca 2+ -ATPase
the
upon
of gizzard
activation
skeletal
Treatment
P;/mg/min
is
the
filaments.
activity
suggesting
to 0.29
from
affects
conditions
increased
umoles
Ca2' -dependence II),
of myosin
obtained.
under
sulfate
Indeed,treatment increases
have
of myosin
ATPase
the activation
or N-ethylmaleimide and there
of
an ammonium in
0.137
case
N-ethylmaleimide
Iodosobenzoate
to formation
measurements
at 37'
in 0.6M KC1 from 0.17
to
increase
N-ethylmaleimide
iodosobenzoate
0.033
related
but
we also
in the K+(EDTA)-ATPase.
increase
might
with
from
I)
a result
Ca2' -ATPase
meromyosin
as in the
is done at 25' (Table
0.6M KC1 (91,
0.45
N-ethylmaleimide
of K+(EDTA)-ATPase
strength
muscle
myosin
With
When the assay
low ionic
skeletal
I).
by a loss
(17).
ATPase
II).
RESEARCH
sulfhydryl myosin
with
ATPase
of
actin,
DTT used
tropo(11, in
of sulfhydryl modifiers
the
groups might
have
Cu 2+ , iodosobenzoate, in the presence
concentrations or Cu2+ produce
of EGTA
of Ca 2+ (Table some decrease
Vol.
89,
No.
BIOCHEMICAL
:3, 1979
AND
BIOPHYSICAL
TABLE ATPase
Activities
in
RESEARCH
COMMUNICATIONS
II the
Presence
of
Actin
ATPase
Activity
-
APi, Additions
Myosin
nmoles/mg
myosin/min
Alone
Complete
+ EGTA
system
+ EGTA
+ ca2+
3.2
10.2
27.5
2.9
18.5
29.3
o-iodosobenzoate
10.6
29.9
35.7
N-eIthylmaleimide
10.5
19.0
19.6
Experiment DTT
(in
None
1
preparation)
(DTT
removed)
Experiment
2
DTT
2.4
7.2
47.1
cu(No3)2
7.0
24.0
25.0
The assay system contained 1.3 mg of myosin per ml. The complete syslcem also contained 0.2 mg of tropomyosin per ml, 0.37 mg of P nd 1.0 mg of actin per ml with N-ethylmsleimide, and 1.5 mg/ml with and without DTT and with iodosobenzoate.
1.1 mg32i55
in
the
which
actin-activated also
ATPase
was observed
different
sulfhydryl
Ca2+ suggests suggests
that
that
sensitivity
in
that
the
produce
modification
protein
Ca 2+ , tropomyosin
with
iodosobenzoate.
a loss
of sulfhydryl
of
of
presence
some experiments
modifiers
oxidation
in
of
the
groups
sulfhydryls
is seen upon removal
of DTT.
produces This
of reducing
agents
and has been observed
several
weeks after
adding
0.1 mM DTT.
It may be related
activity
previously
on storage
of gizzardmyosin
ethylmaleimide reaction
reported
resemble
of myosin
with
actin-activated
ATPase,
troponin
(18).
system
those
observed
in the rabbit
N-ethylmaleimide which
in
this
case
961
is
to changes (6).
the
regulated
Ca
and
in
Ca 2+
muscle
by
sensitivity
the
in the
myosin
for
in the ATPase
The effects
2+
on
effect
readily
of gizzard
skeletal
can abolish
the
decrease
occurs
on storage
three
of activity
for
the
change
absence
That
dependence accounts
and P,,7-55,
of N-
system where of the tropomyosin-
Vol.
89,
No.
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TABLE III Effects of Tropomyosin and an Ammonium Sulfate Fraction Containing Myosin Light Chain Kinase on ActinActivated ATPase of N-Ethylmaleimide Modified Myosin ATPase Activity nmoles/mg myosinlmin
APi,
Control
NEM Myosin
Additions
+Ca2+
+EGTA
20.4
-
Actin
+ tropomyosin
12.4
14.4
-
19.0
19.6
10.2
requirement
for P3,-55
purified
gizzard
presence still
myosin
or absence
of
is required
for
and modified
does
phorylation
ported dependent
not is
but
myosins
ATPase
of
(Table
II).
not
myosin. (5)
which
required Activation
unlike
on Ca 2+ .
the
Whether
that
alters
the
myosin
These
of
for
without present
P 37-55'
modified
modifications
having
962
myosins slightly
by
activation
in
than
tropo-
(Table that
suggest with
has
and Nwith
both
IV),
the
of modified that
phos-
N-ethylmaleimide
been
those
of
native
Although Ca 2+
ATPase
phosphorylation
unless
phosphorylation
strongly
actin-activated
2+
be replaced
other
on Ca2+ but
studies
Ca
III).
requires
depends
results
a factor
Y-[32 P] ATP into
P incorporation
native
cannot
enzymatic
presence
the
activate
on actin
(Table
of 32 P from in
32
has no effect
by actin
modification
not
in P37-55
indicating
activation
abolishes
does
alone
substance
itself
Pj7-55,
myosin
N-ethylmaleimide
actin
incorporation
modified
actin-activated
modified
whether the
actin,
27.5
II.
with
The active
tropomyosin,
we measured
in Table
myosin
by
is present.
ethylmaleimide
myosin
of
activation
determine
native
as described
modification
fraction
To
11.1
17.7
Although
myosin
11.8 + P37-55
Assay conditions
the
3.2
Actin
Actin + P37-55 + tropomyosin
+Ca2+
+EGTA
10.5 Actin
Myosin
previously
preparations
different
effects
rewere than
Vol.
89,
No.
BIOCHEMICAL
:3, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TABLE IV Effect
of N-ethylmaleimide
on Phosphorylation
of Myosin
32 P Incorporation moles per mole myosin Myosin -
+EETA
+ca2+
Control
0.39
0.86
0.23
0.76
N-ethylmaleimide
treated
The incubation medium contained 0.7 mg of myosin per ml, and 0.4$+ described for measuring Mg m3 of P37--55.per ml, under conditions ATPase activity. No incorporation occurred in the absence of myosin. does
oxidation
tions
other
questions
might than
which
The effects modification other
contribute phosphorylation
remain of
being
sulfhydryl
(I-4).
phosphorylation
suggests
actin
directly
binding in actin
which
site
reagents permits
(5,6)
have
physiological
provide
a third
activation
digestion
proteolytic
phosphorylation
the
might
results
and whether
modifica-
significance
are
to be answered.
of myosin
two
to previous
That
such
diverse
a molecular but
with
in which
the
of
of ATPase activity papain
or
modifications
mechanism
example
trypsin
phosphoryl
by actin, (14,19-21)
can mimic
involving group
the
a structural does
a chemical
not
the and
effect
of
change
in
participate
binding.
Acknowledgments: Supported by grants from NIH (HL-15391) Dystrophy ,Giation. I wish to thank Mrs. Aida Carlos for excellent technical assistance, and Dr. John Gergely of the manluscript.
and from the Muscular and Mrs. Remy Nazareno for helpful discussions
References 1. 2. 3. 4. 5. 6.
Aksoy, M.O., Williams, D., Sharkey, E.M., and Hartshorne, D.J. (1976) Biochem. Biophys. Res. Commun. 69, 35-41. Sobieszek, A., and Small, J.V. n977) J. Mol. Biol. 112, 559-576. R.S. (1977) Proc. Natl. Acad. Sci. Chacko, S., Conti, M.A., and Adelstein, USA 2, 129-133. Sherry, J.M.F., Gorecka, A., Aksoy, M.A., Dabrowska, R. and Hartshorne, D.J. (1978) Biochemistry 17, 4411-4418. Ebashi, S., Mikawa, T., Hirata, M., Toyo-oka, T., and Nonomura, Y. (1977) in Excitation Contraction Coupling in Smooth Muscle, eds. R. Casteels, T. Godfraind and J.C. Ruegg, Elsevier/North Holland, Amsterdam, pp. 325-334. Ebashi, S., Mikawa, T., Hirata,M. and Nonomura, Y. (1978) Ann. N.Y. Acad. Sci. --' 307 451-461.
963
Vol.
7.
8.
89,
10.
11. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21.
3,
1979
BIOCHEMICAL
Barany, M., Barany, K., Biophys. 113, 205-221. Yamaguchi,., Miyazawa, 216,
9.
No.
AND
Gaetjens, Y.,
BIOPHYSICAL
E.,
and Sekine,
and Bailin, T. (1970)
RESEARCH
G. (1966) Biochem.
COMMUNICATIONS
Arch. Biophys.
Biochem. Acta
411-421.
Cohen, I., Kaminski, E., Lamed, R., Oplatka, A., and Muhlrad, A. (1976) Arch. Biochem. Biophys. 175, 249-255. Sobieszek, A., and Small, J.V. (1976) J. Mol. Biol. 101, 75-92. Ebashi, S., Iwakura, H., Nakajima, H., Nakamura, R., and Ooi, Y. (1966) Biochem. Z. 345, 201-211. Spudich, J.A., and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871. Fiske., C.H. and Subbarow, Y. (1925) J. Biol. Chem. 66, 375-400. Seidel, J.C. (1978) Biochem. Biophys. Res. Commun. 85, 107-113. Torhchinskii, Y.M. (1974) in Sulfhydryl and Disulfide Groups of Proteins, pp. 51-52, Consultants Bureau, Plenum Publishing Corp., New York. Allison, W.S. (1976) Accounts Chem. Res. 9, 293-299. Kielley, W.W., and Bradley, L.B. (19561, J. Biol. Chem. 218, 653-659. Tonomura, Y., and Yoshimura, J. (1960) Arch. Biochem. Biophys. 90, 73-85. Mrwa, U., and Ruegg, J.C. (1977) in Excitation-Contraction Coupling in Smooth Muscle, eds. R. Casteels, T. Godfraind and J.C. Ruegg, Elsevier/ North Holland, Amsterdam, pp. 353-357. Marston, S.B. and Taylor, E.W. (1978) FEBS Lett. 86, 167-170. Okamoto, Y., and Sekine, T. (1978) J. Biochem. 83, 1375-1379.
964
89,
No.
August
13,
3,
1979
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS
Pages
1979
ACTIVATION
BY ACTIN OF ATPase ACTIVITY
958-964
OF CHEMICALLY
MODIFIED GIZZARD MYOSIN WITHOUT PHOSPHORYLATION John
C. Seidel
Department of Muscle Research Boston Biomedical Research Institute 20 Staniford Street, Boston Massachusetts Department of Neurology, Harvard Medical Received
June
02114 and School
15,1979
SUMMARY : The ATPase2+aci;c; . 'tly+ of myosin from chicken gizzard measured in the presence of either Mg is increased in the absence of dithiothreitol or z;o;U5$action with Cu2+, o-iodo+sobenzoate, or N-ethylmaleimide. Iodosobenzoate produce no change in K (EDTA)-ATPase while N-ethylmaleimide produces a desfease. These treatments also make the actin-activated ATPase insensitive to Ca when assayed in the presence of tropomyosin and a partially purified myosin light chain kinajf. Phosphorylation of N-ethylmaleimide modified myosin remains dependent on Ca and therefore appears not to be required for activation by actin of the ATPase activity of modified myosin.
There
is convincing
smooth
muscle
dalton
light
of
can be regulated chain
some myosin
but
does
(5,6).
(l-41,
to
ATPase
activity
myosin
alone
myosins thiol
time
from
is
raising
modification
make the actin-activa
the
the
presence
with
evidence
with
of
that
actin-bound myosin
that
oxidation
thiol
modification
skeletal
muscle
may differ
N-ethylmaleimide
increase
.ted ATPase activity
insensitive
in
oxidation the
20,000-
activation
proteins
been reported
might
addition
from
on phosphorylation
have
and there
shows that
actin
the
regulatory
of gizzard
In
myosin
of
does not depend
actin.
work
of actin
phosphorylation
also
possibility
The present with
is
properties
by
and from
(7-9).
there
interaction
influenced
gizzard
interaction
from smooth muscle
Ca 2+ in
and the
modifiers
or its
(6)
the
by Ca 2+-dependent
Some of the enzymatic with
that
however,
preparations
respond
change
tion
evidence
to
influence the
is
the
activity
of
evidence
their
that
response
of gizzard
Ca 2+ -ATPase
to
myosin
activity
and
to Ca 2+ and to phosphoryla-
of myosin. METHODS
Myosin was prepared from fresh chicken gizzards essentially Sobieszek and Small (10) with 0.1 to 0.5 mM 1,4-dithiothreitol 0006-291X/79/150958-07$01.00/0 Copyright All rights
@ 1979 by Academic Press, Inc. in my form reserved.
of reproduction
958
as described by (DTT) being
Vol.
89,
No.
BIOCHEMICAL
3, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Tropomyosin from chicken gizzards was included in all steps of the preparation. obtained by one isoelectric precipitation (at pH 4.9) of the 37-55% ammonium tropomyosin (11) described by Aksoy et al. (1). sulfate fraction from "native" F-actin was prepared from rabbit skeletal muscle as described by Spudich and Watt The partially purified fraction containing myosin light chain kinase used (12). in this work was the 37-55% ammonium sulfate fraction of "native" tropomyosin referred to as P (1). 37-55 Ca2+ -activated ATPase activity was determined by incubation of+gizzard and 5 mM AT sr K (EDTA)myosin in ;!5 mM NaCl, 40 mM Tris, pH 7.5, 10 mM CaCl p H 7.5 and ?'mM ATP and Mg -ATPase in ATPase in 0.6M KCl, 5 mM EDTA, 40 mM Tris, 80 mM NaCl, 20 mM Tris, pH 7.5, 10 mM MgC12, 2 mM ATP, and either 0.1 mM EGTA or The reaction was terminated by adding an equal volume of 10% 0.1 mM CaCl . 't. ic acid and released inorganic gysphate de trichloroace Incorporation of P from ,-[5eLrd;inAeTdpZttohZn~st?~dw1f Fiske and Subbarow (13). measured as described previously (14). Treatment with N-ethylmaleimide was carried out for 10 min at 0' in a solution containing 0.5M KCl, 40 mM morpholinopropane sulfonic acid (MOPS), pH 7.5, 1 mM N-ethylmaleimide and 10 mg of myosin per ml, the reaction being The reaction with o-iodosobenterminated by adding O.OlM 1,4-dithiothreitol. zoate was initiated by adding iodosobenzoate to a final concentration of O.OlM in a solution containing 0.3M NaCl, O.lM MOPS, pHo7.5, and 10 mg of myosin per ml. Th5+react')n was c+arried out for 10 min at 25 , the samples placed in ice, and For activities were determined within one hour. Mg -, cafe , or K (EDTA)-ATPase measurement of ATPase activity in the presence of actin and regulatory proteins excess reagent was removed by adding 14 volumes of 1 mM MOPS, pH 7.0, containing 0.1 mM EDTA. The myosin was allowed to settle, the supernatant removed and the myosin eithler washed with a solution containing 40 mM NaCl, 1 mM MOPS, pH 7.0, and in the same solution without the washing step. 0.1 mM EDTA or sgsuspended Treatment with Cu was done by adding Cu(N03)2 to a final concentration of 3 PM for activity of myosin alone and 10 nM for actin-activated ATPase in a solution ATPase activities were determined containing 0.5M NaC12+l mM MOPS, pH 7.5. without removal of Cu . DTT was removed either by dialysis against 400 volumes of a solution containing 0.5M NaCl, 1 mM MOPS, pH 7.5, and 0.1 mM EDTA or by the dilution procedure described above. RESULTS AND DISCUSSION ATPase
The Ca2+ -activated present
throughout
ionic
strength
the
preparation
This
increase
in
the
and
are
can produce
as much as a 4-fold
observed
removal
which
produce
and iodosobenzoate
(16)
Mg 2+ -ATPase
measured
the
increase is not
effect
with
it
when
959
low of I).
by any change to define "spontaneous" Both
modifiers.
increases little
at
(Table
was difficult this
DTT
completion
in activity
sulfhydryl
and
with
measured
after
observe
2- to 3-fold
25 mM NaCl
prepared
accompanied
consistently
produce in
low
Because
activity.
we could
to
myosin
of DTT by dialysis
in 0.6M KC1 and it
ATPase
under
gizzard
consistently
but
we attempted
(15)
of
(25 mM NaCl)
is not
conditions
increase,
ATPase
preparation
K+(EDTA)-activated
standard
CU2+
the
activities
in both or
no
Ca
change
2+
-
in
Vol.
89,
No.
3,
1979
BIOCHEMICAL
AND
BIOPHYSICAL
TABLE
COMMUNICATIONS
I
ATPase Activities
of Myosin Alone ATPase Activity ~moles/mg myosinlmin
AP i, Additions
ca*+
Ca2++KC1
Mg2+
K+(EDTA)
DTT (in preparation)
0.037
0.144
0.0051
0.39
None (DTT removed)
0.176
0.154
CU(NO~)~
0.097
0.114
0.0188
0.34
o-iodosobenzoate
0.270
0.182
0.0110
0.37
N-ethylmaleimide
0.140
0.182
0.0119
0.11
the
in Ca 2+ -ATPase
K+(EDTA)-ATPase accompanied muscle only
(Table
at
in
myosin
increased
a change heavy
is not
Initial myosin
and
Pj7-55,
showed
a decrease
preparation
of myosin
influence
similar
effects.
It
the (Table
by actin
is no further may be noted
that
in
fraction
of
used
for
rabbit gizzard
pmoles/mg/min
without
Ca 2+ -ATPase
of gizzard
indicating
that
of
of
"native"
tropomyosin
that
oxidation
and that
by micromolar N-ethylmaleimide
960
Ca 2+ -
increases
presence
removal
the actin-activated
either
Ca 2+ -ATPase
the
upon
of gizzard
activation
skeletal
Treatment
P;/mg/min
is
the
filaments.
activity
suggesting
to 0.29
from
affects
conditions
increased
umoles
Ca2' -dependence II),
of myosin
obtained.
under
sulfate
Indeed,treatment increases
have
of myosin
ATPase
the activation
or N-ethylmaleimide and there
of
an ammonium in
0.137
case
N-ethylmaleimide
Iodosobenzoate
to formation
measurements
at 37'
in 0.6M KC1 from 0.17
to
increase
N-ethylmaleimide
iodosobenzoate
0.033
related
but
we also
in the K+(EDTA)-ATPase.
increase
might
with
from
I)
a result
Ca2' -ATPase
meromyosin
as in the
is done at 25' (Table
0.6M KC1 (91,
0.45
N-ethylmaleimide
of K+(EDTA)-ATPase
strength
muscle
myosin
With
When the assay
low ionic
skeletal
I).
by a loss
(17).
ATPase
II).
RESEARCH
sulfhydryl myosin
with
ATPase
of
actin,
DTT used
tropo(11, in
of sulfhydryl modifiers
the
groups might
have
Cu 2+ , iodosobenzoate, in the presence
concentrations or Cu2+ produce
of EGTA
of Ca 2+ (Table some decrease
Vol.
89,
No.
BIOCHEMICAL
:3, 1979
AND
BIOPHYSICAL
TABLE ATPase
Activities
in
RESEARCH
COMMUNICATIONS
II the
Presence
of
Actin
ATPase
Activity
-
APi, Additions
Myosin
nmoles/mg
myosin/min
Alone
Complete
+ EGTA
system
+ EGTA
+ ca2+
3.2
10.2
27.5
2.9
18.5
29.3
o-iodosobenzoate
10.6
29.9
35.7
N-eIthylmaleimide
10.5
19.0
19.6
Experiment DTT
(in
None
1
preparation)
(DTT
removed)
Experiment
2
DTT
2.4
7.2
47.1
cu(No3)2
7.0
24.0
25.0
The assay system contained 1.3 mg of myosin per ml. The complete syslcem also contained 0.2 mg of tropomyosin per ml, 0.37 mg of P nd 1.0 mg of actin per ml with N-ethylmsleimide, and 1.5 mg/ml with and without DTT and with iodosobenzoate.
1.1 mg32i55
in
the
which
actin-activated also
ATPase
was observed
different
sulfhydryl
Ca2+ suggests suggests
that
that
sensitivity
in
that
the
produce
modification
protein
Ca 2+ , tropomyosin
with
iodosobenzoate.
a loss
of sulfhydryl
of
of
presence
some experiments
modifiers
oxidation
in
of
the
groups
sulfhydryls
is seen upon removal
of DTT.
produces This
of reducing
agents
and has been observed
several
weeks after
adding
0.1 mM DTT.
It may be related
activity
previously
on storage
of gizzardmyosin
ethylmaleimide reaction
reported
resemble
of myosin
with
actin-activated
ATPase,
troponin
(18).
system
those
observed
in the rabbit
N-ethylmaleimide which
in
this
case
961
is
to changes (6).
the
regulated
Ca
and
in
Ca 2+
muscle
by
sensitivity
the
in the
myosin
for
in the ATPase
The effects
2+
on
effect
readily
of gizzard
skeletal
can abolish
the
decrease
occurs
on storage
three
of activity
for
the
change
absence
That
dependence accounts
and P,,7-55,
of N-
system where of the tropomyosin-
Vol.
89,
No.
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TABLE III Effects of Tropomyosin and an Ammonium Sulfate Fraction Containing Myosin Light Chain Kinase on ActinActivated ATPase of N-Ethylmaleimide Modified Myosin ATPase Activity nmoles/mg myosinlmin
APi,
Control
NEM Myosin
Additions
+Ca2+
+EGTA
20.4
-
Actin
+ tropomyosin
12.4
14.4
-
19.0
19.6
10.2
requirement
for P3,-55
purified
gizzard
presence still
myosin
or absence
of
is required
for
and modified
does
phorylation
ported dependent
not is
but
myosins
ATPase
of
(Table
II).
not
myosin. (5)
which
required Activation
unlike
on Ca 2+ .
the
Whether
that
alters
the
myosin
These
of
for
without present
P 37-55'
modified
modifications
having
962
myosins slightly
by
activation
in
than
tropo-
(Table that
suggest with
has
and Nwith
both
IV),
the
of modified that
phos-
N-ethylmaleimide
been
those
of
native
Although Ca 2+
ATPase
phosphorylation
unless
phosphorylation
strongly
actin-activated
2+
be replaced
other
on Ca2+ but
studies
Ca
III).
requires
depends
results
a factor
Y-[32 P] ATP into
P incorporation
native
cannot
enzymatic
presence
the
activate
on actin
(Table
of 32 P from in
32
has no effect
by actin
modification
not
in P37-55
indicating
activation
abolishes
does
alone
substance
itself
Pj7-55,
myosin
N-ethylmaleimide
actin
incorporation
modified
actin-activated
modified
whether the
actin,
27.5
II.
with
The active
tropomyosin,
we measured
in Table
myosin
by
is present.
ethylmaleimide
myosin
of
activation
determine
native
as described
modification
fraction
To
11.1
17.7
Although
myosin
11.8 + P37-55
Assay conditions
the
3.2
Actin
Actin + P37-55 + tropomyosin
+Ca2+
+EGTA
10.5 Actin
Myosin
previously
preparations
different
effects
rewere than
Vol.
89,
No.
BIOCHEMICAL
:3, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TABLE IV Effect
of N-ethylmaleimide
on Phosphorylation
of Myosin
32 P Incorporation moles per mole myosin Myosin -
+EETA
+ca2+
Control
0.39
0.86
0.23
0.76
N-ethylmaleimide
treated
The incubation medium contained 0.7 mg of myosin per ml, and 0.4$+ described for measuring Mg m3 of P37--55.per ml, under conditions ATPase activity. No incorporation occurred in the absence of myosin. does
oxidation
tions
other
questions
might than
which
The effects modification other
contribute phosphorylation
remain of
being
sulfhydryl
(I-4).
phosphorylation
suggests
actin
directly
binding in actin
which
site
reagents permits
(5,6)
have
physiological
provide
a third
activation
digestion
proteolytic
phosphorylation
the
might
results
and whether
modifica-
significance
are
to be answered.
of myosin
two
to previous
That
such
diverse
a molecular but
with
in which
the
of
of ATPase activity papain
or
modifications
mechanism
example
trypsin
phosphoryl
by actin, (14,19-21)
can mimic
involving group
the
a structural does
a chemical
not
the and
effect
of
change
in
participate
binding.
Acknowledgments: Supported by grants from NIH (HL-15391) Dystrophy ,Giation. I wish to thank Mrs. Aida Carlos for excellent technical assistance, and Dr. John Gergely of the manluscript.
and from the Muscular and Mrs. Remy Nazareno for helpful discussions
References 1. 2. 3. 4. 5. 6.
Aksoy, M.O., Williams, D., Sharkey, E.M., and Hartshorne, D.J. (1976) Biochem. Biophys. Res. Commun. 69, 35-41. Sobieszek, A., and Small, J.V. n977) J. Mol. Biol. 112, 559-576. R.S. (1977) Proc. Natl. Acad. Sci. Chacko, S., Conti, M.A., and Adelstein, USA 2, 129-133. Sherry, J.M.F., Gorecka, A., Aksoy, M.A., Dabrowska, R. and Hartshorne, D.J. (1978) Biochemistry 17, 4411-4418. Ebashi, S., Mikawa, T., Hirata, M., Toyo-oka, T., and Nonomura, Y. (1977) in Excitation Contraction Coupling in Smooth Muscle, eds. R. Casteels, T. Godfraind and J.C. Ruegg, Elsevier/North Holland, Amsterdam, pp. 325-334. Ebashi, S., Mikawa, T., Hirata,M. and Nonomura, Y. (1978) Ann. N.Y. Acad. Sci. --' 307 451-461.
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RESEARCH
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COMMUNICATIONS
Arch. Biophys.
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Cohen, I., Kaminski, E., Lamed, R., Oplatka, A., and Muhlrad, A. (1976) Arch. Biochem. Biophys. 175, 249-255. Sobieszek, A., and Small, J.V. (1976) J. Mol. Biol. 101, 75-92. Ebashi, S., Iwakura, H., Nakajima, H., Nakamura, R., and Ooi, Y. (1966) Biochem. Z. 345, 201-211. Spudich, J.A., and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871. Fiske., C.H. and Subbarow, Y. (1925) J. Biol. Chem. 66, 375-400. Seidel, J.C. (1978) Biochem. Biophys. Res. Commun. 85, 107-113. Torhchinskii, Y.M. (1974) in Sulfhydryl and Disulfide Groups of Proteins, pp. 51-52, Consultants Bureau, Plenum Publishing Corp., New York. Allison, W.S. (1976) Accounts Chem. Res. 9, 293-299. Kielley, W.W., and Bradley, L.B. (19561, J. Biol. Chem. 218, 653-659. Tonomura, Y., and Yoshimura, J. (1960) Arch. Biochem. Biophys. 90, 73-85. Mrwa, U., and Ruegg, J.C. (1977) in Excitation-Contraction Coupling in Smooth Muscle, eds. R. Casteels, T. Godfraind and J.C. Ruegg, Elsevier/ North Holland, Amsterdam, pp. 353-357. Marston, S.B. and Taylor, E.W. (1978) FEBS Lett. 86, 167-170. Okamoto, Y., and Sekine, T. (1978) J. Biochem. 83, 1375-1379.
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