Inhibition of actin polymerization by mercurials without removal of bound nucleotide

Vol. 5, No. 5, 1961

INHIBITION

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF ACTIN POLYMERIZATION BY MERCYRIALS WITHOUT REMOVAL OF BOUND NUCLEOTIDE

W. Drabikowski*,

W. M. Kueh13

and J. Gergely

Cardiac Biochemistry Research Laboratory, Massachusetts General Hospital and Harvard Medical School, and Department of Muscle Research, Retina Foundation, Boston, Mass. Received

13, 1961

July

Kuschinsky is unable

and Turba

to polymerize,

addition

and Strohman

various

mercurials

This

second

SH groups free

actin

SH groups, ing,

are

is unable not

salyrgan double

(1961),

for

SH blocking

loss

without

by measurement

reversed

Martonosi

shown that

whether

with

can preferentially

G-actin

of polymerizability,

nucleotide

content

of the radioactivity

or whether for

ATPcertain

ATP bind-

presented

below in-

of ATP.

Cl4 -ATP was treated

determined

(DRF) (Asakura,

since

1961)

with

with

measurements

was essentially

of G-actin, remaining

of

and reversibly loss

containing

with

ATP of G-actin.

required

The results

a corresponding

and Gouvea

process,

1961), those

by subsequent

the involvement

binding

(Asakura,

G-actin

treatment

of the bound

polymerization. reagents

salyrgan-treated is

the question

identical

of flow

The bound

recently

to the nucleotide

Dowex-treated the

have

raises

necessarily

refraction

aneous.

Barany -A et al

to polymerize

polymerization When

effect

to the release

restricted

essential

show that hibit

leads

that

this

(1961)

observation is

reported

and that

of mercaptans.

(1960,

first

in

which

of

instant-

was determined

the solution

after

1)

This work was supported by grants from the National Heart Institute (H-5949), the Muscular Dystrophy Association of America, Inc., and the Life Insurance Medical Research Fund.

2)

Permanent Department

3)

Supported Research

address: Nencki of Biochemistry,

Institute Warsaw,

with grants by the National Program, Harvard University. 389

of Experimental Poland. Science

Biology,

Foundation,

Student

EIOCHEMICAL

Vol. 5, No. 5,196l treatment (Fig.

with

1).

conditions about

Dowex l-X8,

Prolonged

AND BIOPHYSICAL RESEARCH COMMJNlCATlONS

was decreased

incubation

of the experiment

led

by only

to a slow

shown in Fig. For a considerably

20% in 60 minutes?

a negligible

loss

1 this

amount

of ATP. loss

longer

Under

amounted

time

the to

of incuba6.

100’

0 x

80

X

A

0 Time Fig.

1.

of incubation

Time course of salyrgan on the bound nucleotide

in minutes

action on the polymerizability of G-actin.

and

G-actin, prepared and labelled with C14-ATP as described previously (Martonosi al 1960), was treated with Dowex l-X8 (Cl-et -*, form, 200-400 mesh, 0.1 meq/ml) in order to remove free nucleotides. This actin solution, containing 3.4 mg of protein per ml, was incubated with 8.8 moles of salyrgan per mole (MW=60,000) of actin at 23' in 2 mM Tris, pH 7.0. At each time indicated on the abscissa, three samples were taken. One was polymerized by adding 0.1 M KC1 and 1 mM MgC12 (final concentrations); the second was similarly polymerized but in the presence of 0.01 M glutathione; the third sample was treated with Dowex l-X8 (0.1 meq/ml) and an aliquot, freed from the resin by centrifugation, was dried on filter paper and counted in a Packard Tri-Carb Scintillation Counter essentially according to Loftfield and Eigner (1960). The DRF of the polymerized actin was measured 15 to 20 minutes after addition of salts. Samples treated with salyrgan:-: bound nucleotide; l e DRF after polymerization without glutathione; d: DRF after polymerization with glutathione: Control samples (without salyrgan):a rcS: bound nucleotide;e-0: DRF after polymerization without glutathione. 4)

It appears frcnn the data of Martonosi loss of ATP&ollowing treatment with when the Mg - precipitation method the bound ATP. 390

and Gouvea (1961) that the salyrgan is considerably faster is used for the determination of

BIOCHEMICAL

Vol. 5, No. $1961 tion,

20 to 24 hours,

to a 50% decrease

there

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was a complete

in untreated

actin

release

that

was also

of ATP, compared kept

at room

temperature. Figure 60 minutes,

2 shows

as a function

The polymerizability er than groups

the salyrgan

in G-actin.

of the molar

completely

4 or 5, in close

effect,

et&

ratio

disappeared

agreement

(Kominz

for

with

an incubation

time

of inhibitor when this

the reported

of

to actin. ratio

number

was highof SH

1957).

100 .

80 A x

A

x

x

x

0

m

2

6

10

Moles of salyrgadmole

loss

Fig. 2. of bound

14 of,Gactin

Comparison of inhibition of actin polymerizability ATP as a function of salyrgan to actin ratio.

G-actin-C14-ATP (Fig. was incubated with various pH 7.7, for 60 minutes. content were measured as nucleotide;a-0: )t------J(: DRF after DRF after polymerization salyrgan and subsequently

l), at a concentration of 3.1 amounts of salyrgan at 23' in The polymerizability and bound described in Fig. 1.&M: DRF after polymerization without polymerization with glutathione;a-a: (without glutathione) of actin treated with Dowex-1. 391

m

18

and

mg per ml, 2 mM Tris, nucleotide bound glutathione; incubated

with

BIOCHEMICAL

Vol. 5, No. 5,1961 On addition

of glutathione

bility,

but

to an extent

bound.

This

the

loss

tion

only

suggests

of bound

with

ly,

treated

with

per mole

of incubation; with

absorbed ly,

recovery

after

It

curials. to salyrgan

of salyrgan

per mole

these

appears except

for

somewhat

decreased

of salyrgan

with

Since

and with

this

that

longer

times

was observed

salyrgan

open the possibility

is

readily

that,

initial-

may not be due to a specific

the rate

of methyl

compound

The results

it

clearly

interac-

for

binding.

release'of

reaction

indirectly,

with

is about

the free from

At present

it

involved

produced

similarly ten times

is even more rapid, to separate

the

necessary

for

SH groups those

that

is difficult

the influence

SH groups

may be required

to decide

of salyrgan

in the binding

by the mercurial

of ATP, or,

in the structure

REFERENCES Arch.

Biochem.

Biophys.

Barany, M., Nagy, B., Finkelman, Proc. 20. 298 (1961).

92.

F.,

140 (1961)

and Chrambach,

392

whether

is due to a

actin.

A.,

mer-

on ATP binding.

distinct

are

ATP under

to changes

hydroxide

that

of various

behaves

has not yet been possible

show that

of G-actin

the effect

of ATP release

mercury

from

polymerization nucleotide

to compare

p-hydroxymercuribenzoate

on polymerizability

Asakura,

5 minutes

surprising-

2) no recovery

of actin.

inhibition

that

The action

the slow

salyrgan

of 20 moles

(Fig.

observations

are now in progress

faster.

effect

60 minutes

the incuba-

SH groups.

Studies

slow

partial

paralleling

of polymerizability. with

This

by Dowex,

with

was incubated

even at a ratio

some of the salyrgan

tion

on the loss

Dowex the polymerizability,

%.,

10 moles

in actin

of 1 mM ATP during

recovered,

of actin.

of polymeriza-

to the ATP remaining

change

The presence

of G-actin

was partially

was a recovery

corresponding

had no effect

When a sample and then

there

an irreversible

ATP.

salyrgan

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A.,

Fed.

more of

BIOCHEMICAL

Vol. 5, No. 5,196l

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Kominz, D. R., Hough, A., Symonds, Biophys. 50. 148 (1954). Kuschinsky,

G. and Turba,

F.,

Loftfield, R. B. and Eigner, 62 (1960)

Biochem. E. A.,

Martonosi, A. and Gouvea, 17QO (1960).

M.A.,

Martonosi,

M. A.,

Strobman,

A. and Gouvea, R.,

Fed. Proc.

P.,

20.

and Laki, Biophys.

Biochem.

and Gergely, J. Biol.

299 (1961).

393

K., Acta

Biophys. J.,

Arch. L

426 (1951). Res.

J. Biol.

Chem. 35?,

Biochem.

Comm. 2, Chem. 132

1345 (1961).