Exchange of F-actin-bound nucleotide in the presence and absence of myosin

Vol.

23, No.

3, 1966

BIOCHEMICAL

EXCHANGE OF F-ACTIN-BOUND

of Biophysics,

motion

in

tide

amount

exchange

Szent-Gyb$rgyi exchange,

took

in F-actin

In view

contraction,

found

that

absence

Martonosi,

in

have

nucleotide

of myosin,

*Supported Association *Postdoctoral

Buffalo,

N. Y.

F-actin

Fellow

of National

with is

incorporation and 5Tl-GM-718, Heart

347

Institute,

free observed

to the has re-

(Szent-Gy&gyi, studies

of nucleo-

nucleotide

into

considerable

exchange

1965). in bound-nucleotide of actin

ex-

in muscle

of the incorporation

and without

indeed

by

Szent-Gyorgyi

investigation

both

of F-actin

by USPRS Grants GM-10249 of Erie County, Inc.

(Moos,

of the role

a detailed

a rapid

(1960)

nucleotide

a change

considerations

with

was released

that

Asakura

to a transient

nucleotide

but we found

which

nucleotide

lead

of labeled

significance

into

showing

might

In our preliminary

of myosin

as resulting

is suggested

however,

incorporation

the

(Oosawa,

and Gergely

the absence

for

regarded

hypothesis

nucleotide

1966).

contraction,

the actin

of actomyosin;

observed

of the great

the bound

this

Gouvea,

superprecipitation,

might

in

change

of actin-bound

we have undertaken

of radioactive

for

is

of up to 50% of the bound

we also

during

of muscle

of the actin-bound

and Prior,

actomyosin

changeability

at Buffalo,

filaments change

test

superprecipitation

found

place

the mechanism

such a conformational

the medium.

a small

medium during

1965;

of New York

and actin

in the exchangeability

only

COMMUNICATIONS

and E. Eisenbergfi

conformational

that

nucleotide

cently

for

An experimental

the possibility

that

proposal

cyclic

1961).

increase

University

of the myosin

from a repeated and Ooi,

RESEARCH

1966

In one current relative

.I. E. Estes,

State

ReceivedApril5,

BIOPHYSICAL

NUCLEOTIDE IN TEE PRESENCE AND ABSENCE OF MYOSIN*

C. Moos, Dept.

AND

myosin.

exchangeable

of a limited and a grant USpHS.

We have

in the

amount

of labeled

from

the Heart

Vol. 23, No. 3, 1966

nucleotide change

BIOCHEMICAL

immediately at a slower

found

that

initial

steady

the addition

rate.

the interaction

rapid

change

after

is

burst

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

followed

In superprecipitating

of the actin

of exchange,

considerably

of H3ATP,

but

increased

with

that

in

by further

actomyosin,

myosin

we have

does not affect

the steady

rate

the

of further

the superprecipitated

ex-

ex-

state.

Methods Myosin

and actin

were

methods

of Szent-Gy&gyi

(19601,

respectively. F-actin

kinase

(EC 2.7.3.2)and

ATP in

the actomyosin

liberated

creatine

sired

with

reaction

myosin

the free

and Ulbrecht,

suspension

time,

and then

H3ATP was first

also

to reduce change

the free

would

exchange

occur

occurs

(Szent-Gyorgyi,

of that

ion

on addition

1965;

Moos,

if

unpublished

activity

of the

free

H3ATP in the

Contrary non-exchangeable,

to the widespread we consistently

were

Dwex-1

for

precipitated

with

either

the myosin

no additional (Very

concentration By “percent

nucleotide reaction

the de-

hwever,

to the actin. ion

of

anion-exchange

was added

results).

of the bound

Results

after

samples

so that

the magnesium

turnover

and analyzed

with

concentration

Creatine

of the

in the same manner;

acid

by incu-

For measurements

reprecipitation,

of the myosin

in actomyosin

we mean the specific centage

magnesium

out

by determination

by treatment

and Walter

and the

centrifuged

or 1,2-diaminocyclohexanetetraacetic

resin,

added,

The actin

by the

of myosin.

quickly

were

and analyzed

removed

carried

1943).

nucleotide.

washed

Jaisle,

and Gough,

by repeated

the bound

Grubhofer,

or absence

were

Elsden,

washed

muscle

were

was followed

samples

skeletal

experiments

phosphocreatine

(Eggleton,

in

rabbit

H3ATP in the presence

in actomyosin,

radioactivity with

(1951)

from

The exchange

bating

of exchange

prepared

expressed

exlittle is lw

exchange”, as a per-

mixture.

and Discussion assumption observe

348

that

F-actin-bound

incorporation

nucleotide

of radioactive

is

Vol.

23, No.

3, 1966

BIOCHEMICAL

AND

BIOPHYSICAL

Time

RESEARCH

COMMUNICATIONS

(min)

Reaction mixture contained Figure 1. Bound nucleotide exchange in F-actin. 0.2 mg/ml F-actin, 0.1 M KCI., 1 mM MgC12, 10 1184imidazole buffer, pH 1.0, at indicated times, and 50 @f H3ATP; temperature, ca. 25O. Upper curve: 10 ml aliquots were removed and mixed with 3.5 ml of 0.1 M KC1 solution containing 10 mg myosin and 50 pmoles 1,2-diaminocyclohexanetetraacetic acid at pH 7.0, and the suspension was centrifuged for exchange analysis. Lower curve: same, except 0.20 m'L. 50 mM ATP added to each aliquot 1 min. before adding myosin mixture, bringing total ATP cont. to 1.0 mM.

nucleotide This

into

incorporation

increase.

initial

but after

of the

back-exchange remaining

bound

dilution

ATP removes

burst;

a fraction

incubation

bound

ATP,

nucleotide

nearly

1, lower is

identical

all

the

curve). only

indicating

the

followed

by a continuing

that

influence samples

true

for

nucleotide

exchange

excess

or more,

subject have

of isotope

only

to rapid

shown

by continued

slow

in the

an hour is

experiments removed

curve).

incorporated

has continued

gradually

1, upper

of a 20-fold

radioactivity

Other

that

the

incubation into

the firmly

has occurred. of myosin of F-actin

or absence

of myosin,

under

(Fig.

Here again

we see exchange

2).

H3ATP (Fig.

burst

radioactive

of the F-actin

To fnvestigate

rapid

the exchange

iaotope

with

of the H3ATP by the addition

incorporated

(Fig.

unlabeled

we incubated

during

shows an initial

Sudden

of unlabeled

with

F-actin

conditions

on the actin-nucleotide with

where

H3ATP either actomyosin

in F-actin

349

alone,

exchange,

in the presence

superprecipitates with

a time

course

Vol. 23, No. 3, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-

40

-

% Exchange

/ ,,I

/

o*-e L ,./*

- I/ 0

4-

Figure 2. Bound nucleotide exchange in F-actin and actomyosin. Both mixtures contained 0.1 M KCl, 4 r&4 MgC12, 10 mM imidazole buffer, pH 7.0, 5 mM phosphocreatine, 50 ug/ml creatine kinase, 50 p H3ATP, and 0.1 mg/ml actin. Ac tomybsin mixture also contained 1 mg/ml myosin. Temperature, ca. 25’. Actin samples treated with Dowex-1 (Cl), then mixed with myosin (final cont. 1 mg/ml) and centrifuged for exchange analysis. Upper figure : incorporation of H3-nucleotide. Lower figure : hydrolysis of phosphocreatine in actomyosin suspension.

+

I-

-

mM

l-+---l---l

I+-+---

-U

Creatlne

n

/

l----l----l-----+---l--+ 20

40 Time,

similar

to that

exchange

GO

minutes

shown in Fig.

of bound nucleotide

following

of the initial

the initial burst

true

even though

time

of the ffrst In conclusion,

indeed

exchangeable.

isotope

incorporation,

rapidly-exchangeable

In the actomyosin,

was markedly

greater,

increased

exchange

This

the end of the reaction. period

1.

rapid

burst

exchange

occurred

the

50% at

entirely

whereas

as in actin

had completely

nearly

hand,

in the

the magnitude alone.

superprecipitated

This

was

before

the

measurement.

we have shwn On exposure suggesting “open”

reaching

of exchange,

was the same in actomyosin

the actomyosin

on the other

bound

that

the bound

to H3ATP, the presence nucleotide

350

there

nucleotide is

an immedfate

of a certain sites

of F-actin

number

in the actin.

is

burst of In the

of

Vol.

23, No. 3, 1966

course

of time

steady

rate,

addition

sudden

rapid

complex

appearance of turnover

actomyosin, ity

which

of exchange

actomyosin

rate

thereafter,

of myosin

the initial rate

BIOCHEMICAL

BIOPHYSICAL

additional

may indicate under burst

exchange

a slow

RESEARCH

takes

turnover

of superprecipitation

of exchange

but does greatly

'Hence,

and the onset

that

appears

that

sites

"open" is

myosin

of the F-actin

greatly does

indeed

affect

"open"

increase

sites.

The

increase

the steady

the formation

of the

do not cause

actin-nucleotide increased

at a slower

does not

of superprecipitation

of any additional of these

it

COMMUNICATIONS

place

of these

conditions

thereafter.

suggesting

or conformation

AND

sites;

the but

the

in superprecipitated the structural

stabil-

polymer.

References Eggleton, P., Elsden, S. R., and Gough, N., Biochem. J., 37, 526 (1943). Martonosi, A., Gouvea, M. A., and Gergely, J., J. Biol. Chem., 235, 1707 (1960). Moos, c., Abstr. 23rd Intl. C,ong. Physiol. Sci., Tokyo (1965). Oosawa, F., Asakura, S., and Ooi, T., Prog. Theor. Physics, Suppl. 17, p. 14 (1961). Scent-Gy&gyi, A., Chemistry of Muscular Contraction, 2nd Ed., Academic Press, New York, 1951. Szent-Gynrgyi, A. G., in Muscle, edit. by Paul, W. M., Daniel, E. E., Kay, C. M., and Monckton, G., p. 141, Pergamon Press, Oxford, 1965. Szent-Gy&rgyi, A. G., and Prior, G., J. Mol. Biol., in press (1966). Ulbrecht, M., Grubhofer, N., Jaisle, F., and Walter, S., Biochim. Biophys. Acta. 45, 443 (1960).

351