Native tropomyosin: Effect on the interaction of actin with heavy meromyosin and subfragment-1

Vol. 40, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NATIVE TROPOMYOSIN: EFFECT ON THE INTERACTION OF ACTIN WITH HEAVY MEROMYOSIN AND SUBFRAGMENTWan Eisenberg and W. Wayne Kielley Physiology, Laboratory of Biochemistry, National Institutes of Health, Bethesda,

Section on Cellular and Lung Institute,

National Heart Md. 20014

Received May 18, 1970 native tropomyosin markedly inhibits the SUMMARY. In the absence of Cai', acto-subfragment-l ATPase as well as the acto-heavy meromyosin ATPase despite the fact that subfragment-1, in contrast to heavy meromyosin, has only a single site for ATP. Double reciprocal plots of acto-heavy meromyosin ATPase vs. actin concentration both in the presence and absence of native tropomyosin, show that the native tropomyosin has little effect on the acto-HMM-ATPase but acts mainly by affecting the binding of actin to the HMM-ATP complex. It vivo

by release

binds the

is now generally of Caf+

to native actin

occurs

which muscle

native

tropomyosin

myosin

ATPase

with

strength,

but

the

actin

absence

in the

made it

of actin

the

native

in the

of native

allows

contraction actin-myosin tropomyosin

absence

of actomyosin

absence

or perhaps

both.

because

with

meromyosin

actin

heavy

concentration

of Cai+,

native

is

one of the tropomyosin

the

the

of Ca++, and the

tropomyosin,

in the

acto-

Ca++

and the

blocks

50

very

of

reciprocal

and therefore turnover

at

actin

low ionic

binding

of the

in vivo of

acto-

plots

In a recent

that

binding

the

to

the

rate (6).

is

difficult

double

proposed the

is

rate

state

independently authors

tropomyosin

precipitated

@MM) linear

(4)

native

turnover

question

can be obtained

can be determined

contraction,

whether

This

it

HMM-ATP complex

(Vmax)

clear

to myosin-ATP,

actomyosin

to the

ATP complex of muscle

studies

complex

vs.

in vitro,

-in

Ca++

in turn

hand,

by the

superprecipitation

whereas

binding

approach

ATPase

Analogously

which

other

inhibited

triggered This

such a way that

, on the

is

is

(1,2).

filament, in

of Caf+

both

reticulum

actin

interact

of muscle

(2,5).

the

myosin-ATP

the

to

(1,2).

inhibits

None of these reducing

along

contraction

activity,

has no effect

contraction

sarcoplasmic

absence

causes

relaxes

that

the

filaments

In the

(2,3,4).

and the

from

tropomyosin

and myosin

interaction

accepted

of of

acto-HMMmodel

in the to myosin-ATP

Vol. 40, No. 1, 1970

rather

than

BIOCHEMICAL

the

turnover

reciprocal

plot

reported

preliminary

rather

than

also

with

results

present

study

investigated

demonstrated

with

HMM which, site

technique

for

of the

actomyosin-ATP

acto-HMM, which

complex

however,

suggest

that

(7).

Szentkiralyi

Using

the

and Oplatka

tropomyosin

affects

Vmax

using

HMM and

Ka(8).

In the have

rate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

whether

We find

and HMM suggesting of native

native

tropomyosin

any effect

that

the

on the

a further

tropomyosin tryptic

that

tropomyosin

the

native

binding rate

of

of the

actin

product

on HMM., has only affects

on HMM are not find

can be

digestion

sites

We further

turnover

question

of native

two ATP sites

on the

this

two ATP binding

tropomyosin. is

effect

(S-l),

to the

ATP (9,lO).

investigated

the

subfragment-

in contrast

effect

we have

that

the

major

S-l

for

effect

to HMM-ATP, with

acto-HMM-ATP

a single

both

required

of

the of

little

if

complex.

MATERIALS AND METHODS Myosin and S-l gel G-200

was prepared

were

also

filtration

prepared

to purify

column

was then

RPM and finally tropomyosin

taken

as described

previously

using

Actin

was prepared

S-l

that

used

in all

solutions.

(7,12).

by Adelstein

into

was prepared

exception

tropomyosin

of Kielley

to F-actin

the

described

method

the

as described

polymerized

as described

by the

et al.

, pelleted

solution

twice

previously

rather

ATPase was measured

(7).

was 380 a2/q

Protein

G-200

a Sephadex

The resulting

previously

than

at 25'C,

G-actin at 30,000

(12).

Native

and Mueller

(14) with

2mM dithiothietol pHz7.0

HMM

Sephadex

using

using

concentrations

were

also

coefficient

used

for

The extinction at 278~

(11).

by centrifuqation

by Hartshorne

5mM 2mercaptoethanol

(12).

(13).

as described

as described

previously

and Harrinqton

was a pa-stat

measured

as

native

(14).

RESULTS AND DISCUSSION Table tetraacetic both

with

I shows the acid

effect

(EGTA) on the

and without

native

of ea++ and ethyleneqlycol-bis(aminoethylether) ATPase activity tropomyosin

present.

51

of acto-HMM In the

and acto-S-l absence

of native

BIOCHEMICAL

Vol. 40, No. 1, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Effect

of

Native

Tropomyosin

on Acto-HMM

and Acto-S-l

ATPase Excess EGTA

Protein Acto-HMM Acto-HMM+Native

tropcmyosin

Acto-S-l Acto-S-l+Native

tropomyosin

ATPase

(~mole/mg-min) Excess Ca++

1.48

1.36

0.09

2.26

0.85

0.02

0.16

1.00

For the experiment with HMM all samples contained 1.6mM ATP, 0.8mM There was 0.24 mg/ml HMM in MgC12 I 18mM KCl, and 0.4 mg/ml actin. the sample with excess EGTA and native tropomyosin, and 0.12 mg/ml Native tropomyosin when added equaled HMM in the other samples. 0.5 mg/ml. Samples with excess EGTA contained 5.OmM EGTA and samples with excess Ca++ contained 5.OmM EGTA and 5.2mM CaC12. For the experiments with S-l all samples contained 2mM ATP, 1mM MgC12, 2OmM KC1 0.8 mg/ml actin, and 0.20 mg/ml S-l. Native tropomyosin when added equaled 1.0 mg/ml. Samples with excess EGTA contained 1mM EGTA and samples with excess Caf+ contained 0.5mM CaC12.

tropomyosin

the

acto-H?lM This

or EGTA was present. presence

of native

tropomyosin

effect

by Hartshorne

was also

tropomyosin

increased

activating

ATPase was essentially

both

with the

of native for

in the

absence

occurred

for,

as can be seen,

marked

inhibition

of both

clear

that

previously despite

native been

the We next

fact

Ca++,

that

the

tropomyosin reported that

plot

experiment

cannot

excess

acto-HMM

the the

S-l

with

of acto-HMM be performed

acto-S-l

ATPase of the

EGTA present

the

acto-HMM

and acto-S-l

not

affects

(16)

a single

effect ATPase

but

of native vs.

actin

by adding

52

the

was reported

However

native

Thus

acto-HMM

ATPase,

site

for

was

tropomyosin

ATPases.

affects

it

tropomyosin

native

also

the

acto-S-l

caused it

is as has ATPase

ATP.

tropomyosin concentration.

a constant

native

A similar

of Ca+f

(IS).

Ca++

In the

the

ATPases.

presence

excess

ATPase.

present,

and acto-S-l

effect

only

same whether

Ca++ also

in the

major

has only the

the

actomyosin

by Kominz

investigated

reciprocal

for

tropomyosin

and Mueller of

true

the

amount

on the Clearly of native

double this tropo-

Vol. 40, No. 1, 1970

myosin then

since

the

decrease

ratio

BIOCHEMICAL

fraction

as the

of native

completely with

native

due to therefore

I,

even

was essentially

in the

the

reciprocal

a range

where

the

which

with

to hold

the

of actin

free

the

also over

ATP, the

native

EGTA was a poor

in the

reciprocal

by EGTA i.e.

shown

the

actin

We then constant

Ca++-free

of actin

so

native

concentration

effect

to Mg++.

We also

native

tropomyosin

little

below

native

tropomyosin

were noted

of

almost

employed

from

which

actin

as the

low

strength,

at the

the

maximal

effect all

in Figure

of the of these

native factors

1 to perform

our

tropomyosin into

to the

plots

very

lowest

53

in which --

actin.

the

an

Both of ATP

action

of

was still

Figure

a

dissociation (17).

of

Since

is most

accurate

KC1 concentration

consideration,

experiment.

presence

at a KC1 concentration

reciprocal

the

we worked

Mg++,

strength

of Vmax and Ka from

ionic

of HMM to

ionic

determinations

in the

was noted

of excess

may be related low

by Ca++ in

at a two to one ratio

disappeared

at very

experiment.

ATP was hydrolyzed

ratios

by working absence

this

was affected

tropomyosin

by high

in the

for

Furthermore

native

completely

15mM, an effect

tropomyosin

activated

avoided

that

chosen

Ca++-buffer.

seemed to be increased

difficulties

given

as is

used

with

had to be carefully

became markedly

of these

Taking

We

where,

Ca++ concentration

range

complexed

complex.

tropomyosin.

complexed

not

concentrations

to actin

native

were

of actin

tropomyosin

90% inhibited

a constant

actin

low actin

concentration

than

throughout

Mg++ an unusual

ATPase rate

effect

at

if

the

fraction

would

plot.

conditions

excess

but

tropomyosin

actin

tropomyosin

Similarly

the

actin-native

saturated

fraction

native

employed,

to decrease

ATPase was more

Mg++ in excess

the

lowest

was constant

Other

of

at the

an EGTA-Ca ++ buffer

tropomyosin

With

tend

of native

completely

in effect,

that,

were

tropomyosin,

of the

a ratio

acto-HMM

employed

might

dissociation

employed

the

plot,

native

with increased.

to actin

with

tropomyosin

saturated

concentration

tropomyosin

partial

in Table

of actin

actin

saturated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

at

where

observed. we chose

the

1 shows

double

conditions reciprocal

Vol. 40, No. 1, 1970

BIOCHEMICAL AND BIOPHYSICAL

0

-10

IO I /ACTIN

RESEARCH COMMUNKATIONS

20

(mg /ml )-’

FIGURE 1 Double reciprocal plots of acto-HMM-ATPase vs. actin concentration with and without native tropomyosin. All samples contained 1.6mM ATP, 0.8mM MgC12, 19mM KCl, 5mM EGTA, 2mM CaC12. (0) Native tropomyosin added at a concentration equal to 1.25 times the actin concentration, HMM concentration varied from 0.14-0.27 mg/ml. (0) No native tropomyosin added. HMM concentration varied from 0.06-O-12 mg/ml. As described previously for this type of plot (6), in all cases the ATPase of the HMM in the absence of actin was subtracted from the measured ATPase rate to give the acto-HMM ATPase rate, the reciprocal of which is plotted on the ordinate.

plots

of

absence

acto-HMM of native

tropomyosin

only

are

change a

yielded may

plots

small

in the change

essentially

native

i.e.

the

This

is

ATPase because

partial linear

of

and the

native

intercept ordinate

high, tropomysoin rate

the

at the highest

under

tropomyosin

point

this

small

the

plot Repeat

change

(Ka),

observed plot

at

is,

anything,

above

its

is probably concentration

a but

experiments

reciprocal

condition

native

to cause

on the if

and

As can be

appears

(Vmax).

tropomyosin

54

presence

where

reciprocal

Even the

native

in the

acto-HMM-ATPase.

of the

concentration

observed

the

intercept

data.

since

both

Ca++ concentration

inhibition

abscissa in the

concentration

at a free

identical

be erroneously

highest

actin

tropomyosin

causes

seen both marked

ATPase vs.

too

in Vmax the true low.

employed,

value

BIOCHEMICAL

Vol. 40, No. 1, 1970

trace

amounts

produced the

enough

reaction

during to

of myokinase

the

the

percent.

major

rate

turnover

of

of native

of the

that

under

to the

HMM-ATP complex.

recently

obtained

a comparable

This

result

fibers

which

on the

unloaded

development that skeletal little

the

velocity

(18). actin

that

It

result

resistance

ACKNOWLEDGEMENTS We would like assistance.

since

with

also

relaxed

to stretch to thank

pH-stat

but with

approach

Hartshorne

has

personal

communication).

on skinned

reduce

studies during

the binding

does markedly

to activated

out,

effect

in the

has

are dissociated

even

a small

concentration

in vivo

in

by a few

be ruled

experiments Casf

as opposed

the

to be a reduction

recent

occur

been produced

although

(D. Hartshorne,

consistent

filaments

in pH to

and therefore

cannot

a similar

the

tropomyosin

NH3 may have with

complex

of contraction is

rise

conditions,

appears

decreasing

and myosin

muscle,

our

Using

may correlate

suggest

enough

acto-HMM-ATP

of actin

a slow

as measured

tropomyosin

native

ATP was hydrolyzed,

reaction

H+ production

of the

in the

ADP to cause

of the

conclude rate

effect

all

course

We thus

on the

after

initial

reduce

and AMP-deaminase

IMP and NH3 from

mixture

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

muscle little

effect

force

which

suggest

relaxation

muscle

of

shows very

(19). Mr.

Louis

Dobkin

for

his

excellent

technical

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Weber, A., Herz, R., and Reiss, I., Proc. Royal Sot. Series B., 160, 489 (1964). Ebashi, S. and Endo, M., Prog. in Biophys. and Molecular Biol. l8, 123 (1968). Endo, M., Nonomura, Y., Masaki, T., Ohtsuki, I., and Ebashi, S., J. Biochem. 60, 605 (1966). Fuchs, F. and Briggs, F.N., J. Gen. Physiol. 5l, 655 (1968). Ebashi, S. and Ebashi, F., J. Biochem. 55, 604 (1964). E. and Moos, C., Biochemistry 1, 1486 (1968). Eisenberg, Eisenberg, E., Zobel, C.R., and Moos, C., Biochemistry 1, 3186 (1968). Szentkiralyi, E.M. and Oplatka, A., J. Mol. Biol. 43, 551 (1969). Eisenberg, E., Barouch, W.W., and Moos, C., Federation Proc. B, 536 (1969). Morita, F. and Shimizu, T., Biochim. Biophys. Acta 180, 545 (1969). W.F., Biochim. Biophys. Acta 4l, 401 (1960) Kielley, W.W. and Harrington, Eisenberg, E. and Moos, C., 3. Biol. Chem. 242, 2945 (1967). Adelstein, R., Godfrey, J.E. and Kielley, W.W., Biochem. Biophys. Res. Comm. l2, 34 (1963). Hartshorne, D.J. and Mueller, H., Biochim. Biophys. Acta 175, 301 (1969).

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BIOCHEMICAL

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AND BIOPHYSICAL

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56