Actin polymerization by direct transphosphorylation

Vol. 8, No. 1, 1962

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACTIN POLYMl3RIZATION BY DIRECT TRANSPHOSPHORYLATION1 Teru Department and the

Received

April

tile

its

elements

is

the

The work

is

whether

the

(F)* In

zymes.

actin

dialysis

the

(1961) the

original

that

the

lated

bound

by the

nucleotide

this

(1955) material

involved,

of

sarcoin

where

The question of the

the

actin

bound

the

a

the

involved

can be phos-

nucleotide

action

questioned

The work of

contrac-

has resulted

of

can be phosohorylated

interpretation

the

Strohman

actin

of

external

he has demonstrated

has been

may be

system.

to

grounds.

en-

that

the

by CP and CPK,

by Martonosi presented

herein

and presents

can be directly

et

al.

extends evidence

phosphory-

CP and CPK system.

The CPK used Lardy

inert

actin

on technical

(1960)

nucleotide

shown that

is

interpretation

surrounding

the

actin.

experiments,

ADP of depolymerized but

has

of

mechanism

enzyme

contraction of

and Siger

bound

muscle

relationship

factors

underlying

the

(1959)

polymerized

as the

chemical

by an external

Strohman

in

"compartmentalized"

or not

phorylated

form

of Carlson

of

element

coupling"

general to

specification key

Rosenbluth

of Zoology, Columbia University, New York, N.Y., Marine Biological Laboratory, Woods Hole, Mass.

"mechanochemical in

plasm.

and Raja

24, 1962

The defined

Hayashi

to

was prepared

the

by the

penultimate

was dissolved

step in

method

of Noda,

the

and

For use,

and lyophilized.

1 mM MgC12 to

Kuby,

desired

concen-

1. Supported by National Science Foundation Grant #G-7492 and Muscular Dystrophy Associations of America Grant #QJ30C8. 2. Abbreviations: F = polymerized actin, G = non-polymerized actin, ATP = adenosine triphosphate, ADP = adenosine diphosphate, CPK = creatine phosphokinase. CP = creatine phosphate, 20

BIOCHEMICAL

Vol. 8, No. 1, 1962

tration

and treated

remove

contaminating

pared

Dowex-1

to

Grubhofer

of

free

G-ADP actin of

(0.1

"salt-induced"

polymerization

ture

As with

coefficient,

since

apparently

at O'C this

type

duced

with

ATP (100

PM) added

with

second

induction

method

causes

this

diminished zation

extent is

of

Bosenbluth

agent

at

the

(1962)

accompanying termed

Polymerization

both

due to the

replacement

the

conversion bound

have

used

of

at O°C is

The conditions viscosimeters bath,

each

with

and tris-maleate

final3

concentrations

3. "Final"

of

the

similar

containing

M&l29

depicted

concentrations

the

are

ATP,

are

with

to

un-

of polymeri-

since

method

actin

by

Hayashi

all

of

in

with

and

induction

the

the

three

Figure

is

inducing

actin,. methods

of in-

1.

follow. times

are of

Three

Ostwald

prepared

in

G-ADP actin,

final

pH at

mg/ml

of

calculated 21

be in-

dephosphorylation

present

same amounts

1.025

type

included

experiment

buffer,

is

G-ADP to G-ATP

added

in

efflux

tempera-

("ATP-salt-induced");

This

the

CP is

G-ADP actin

temperature;

G-ADP can also

salt

A third

and CPK is

This

G-ADP to polymerize

ADP by the

where

(1961).

has a high

shown a quantitative

of ATP,

The behavior duction

the

of

the

to Hayashi

at room

of

polymerization.

upon

of polymerization

29OC and O'C.

"W-salt-induced" instead

readily

process

suppressed.

method

G-ADP actin

according

and Weber

completely

(1960)

quick

simply

M MgC12)

occurs

the

was preal.

the

to

use.

and Grubhofer

be shown that

the

CPK,

to polymerize 0.001

et

to

the

1960)

actin

of Ulbrecht

before

M KCl,

F-ADP

according

nucleotide

(1960)

will

method

(1961).

and Rosenbluth

it

the

can be induced

salt

and Tsuboi,

Purified

G-ADP essentially

and Weber

was cleaned

addition

of

RESEARCH COMMUNICATIONS

(Hayashi

nucleotides.

by a modification

and converted of

with

AND BIOPHYSICAL

O°C being

G-ADP, after

a O'C CPK,

7.05.

1.33 mg/ml all

agents

The of added.

Vol. 8, No. 1, 1962

BIOCHEMICAL

AND BIOPHYSICAL

I 0.;

I- -

G-ADP

I

I

poiymerizotion

RESEARCH COMMUNICATIONS

I

I

I

with xs ATP, xs GP

0°C

0.1

08 0

0

IO

20

30

000

KCI,

GP

000

KCI,

ATP

0 0 0

KCI,

H20

40

50

60

70

MINUTES

CPK,

1 mM MgC+

CPK are

and 0.01

present

in

each viscosimeter CP-KCl,

to

final

in viscosity

The first actin

which

the

third

third,

I-$0-KCl.

the

then

from

actin

in

the the

by CP in

in

It

is

possible

ated

ADP by CP-CPK

from

the a free

actin. ADP. which

that takes That

This then

rate

it

is, free

displaces

case, the place the

The M.

is

of apparent

that

has been

converted

by way of

ADP is more

whereas

then bound

the of

free

ADP of

is

G-ADP suppressed

phosphorylation

bound

of the

viscosimeter

and by ATP in

22

first,

0.1

and extent,

The third

at O°C,

first

is

show polymerizations

two viscosimeters the

viscosimeters

polymerization

behavior first

the

the

followed.

indistinguishable

"salt-induced"

of

O'C;

to ATP,

and to

two viscosimeters are

To

to

KC1 in are

concentrations. agent:

shows no polymerization.

example

actin,

the

G-ADP and

an inducing

ATP-KCl,

of

The two proteins

equimolar

now added

second,

concentration

The changes

to

essentially is

the

M buffer.

the

the

an at

G-ADP to G-ATP

second. the

actin-associ-

nucleotide actin

phosphorylated ADP to the

lost dissociates by CP-CPK

free

condition.

BiOCtlEMICAL

Vol. 8, No. 1, 1962

This

in

turn

is

and the

etc.,

converted

mechanism

l'ATP-salt-inducedR Hayashi

the

about

7 hours,

free

ADP present

it

is

the

bound than

It

polymerizing With

unlikely

for

the

that

is

this

with

present

that

ally,

this

a basis

and Siger

by external

bound

ADP,

stated

for

the

ADP loss

the

(1960),

actin,

enzymes,

but

be in

bound

maximum is

for

with

the

in

the

system.

this

to

nucleo-

the

first is

made the more

viscosimeter* nucleotide

of

Physiologic-

mechanism significant

nuc-

an amount

first

bound

the

of about

of

ATP,

the

is

this

when comparison

in

ADP is

of unbound of

free

the

amount

added

cycling

CP-CPK

to be susceptible must

Under

ATP replacement

any time

therefore

the

an amount

100 uM of

at

provides

slow,

exhibited true

by the

finding

quite

the

the

wherein

viscosimeter,

accomplished

that

of

polymerization

can be phosphorylated

that

that

agent such

actin

cation:

more

stated

half-life

can be shown that

highly

that

have

the

experiment,

may be concluded

by Carlson

from

1 mM Mg is

nucleotide.

second

200 times

displaces

different

of

Especially

ADP is

which

(1960)

when the

can account

viscosimeter. with

not

presence

and it

3% of the bound tide

is

and Rosenbluth

o'f the

leotide,

to ATP,

RESEARCH COMMUNICATIONS

polymerization.

at O°C and in conditions

AND BIOPHYSICAL

suggested modifi-

transphosphorylation

G-ADP form.

REFERENCES Carlson, F.D., and Siger, A. Jour. Gen. Physiol. 44, 33 (1960) Grubhofer, N., and Weber, H. H. Zeit. f. Naturforsch. m, 435 (1961) Hayashi, T., and Rosenbluth, R. Biol. Bull. 119, 294 (1960) Hayashi, T., and Rosenbluth, R. Conf. on Biochem. of Muscle Contraction, Dedham, Mass. (1962) in press Hayashi, T., and Tsuboi, K. K. Fed. Proc. I& 256 (1960) Martonosi, A., Gouvea, M. A., and Gergely, J. Jour. Biol. Chem. 235, 1700 (1960) Noda, L., Xuby, S., and Lardy, H. Methods in Enzymology II, 605 (1955) ed. S. P. Colowick and N. 0. Kaplan, Acade?& Press, New York Strohman, R. C. Biochim. et Biophys. Acta 32, 436 (1959) Ulbrecht, M., Grubhofer, N., Jaisle, F., as Walter, S. Biochim. et Biophys. Acta 4J, 443 (1960) 23