Measurement of Pi dissociation from actin filaments following ATP hydrolysis using a linked enzyme assay

Vol. 143, No. 3, 1987 March 30. 1987

MEASUREMENT

BIOCHEMICAL

AND BIOPHYSICAL

OF Pi DISSOCIATION HYDROLYSIS

FROM

USING

ACTIN

A LINKED

Marie-France Laboratoire

Received

February

10,

FILAMENTS

ENZYME

FOLLOWING

ATP

ASSAY

CARLIER

d’Enzymologie

91190

RESEARCH COMMUNICATIONS Pages 1069-l 075

du

Gif-sur-Yvette,

C , N. R. S.

France

1987

SUMMARY. Using glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase as a linked enzyme assay for determination of free inorganic phosphate, as described by Trentham et al. (1972, Biochem. J. -126, 635-644) we have been able to monitor th< t&e course of P. release from F-actin following ATP hydrolysis that accompanies ATP-actin $olyqerization. The rate constant for P. dissociation from Mg-F-actin is 0.006 s at 25OC and pH 7.8, both in thk presence of 1 mM Mg and 0.1 M KC1 + 1 mM Mg. This result confirms the existence of ADP-P.-F-actin as a major intermediate in the polymerization of ATP-actin (Car-her 2nd Pantaloni, 1986, Biochemistry 7789-7792). The method is potentially useful for other enzymes 25, hydrolyzing triphosphate nuc_ptides, provided that the rate of Pi release is appreciably lower than 0.1 s , o 1987 Academic Press, Inc.

The

elucidation

polymerization

of

understand well in

the

G-actin

that

ATP-F-actin

the

complex

is

released

ADP-Pi.

F-actin

in

following

G-ATP+

F-ATP

<

fiber quench

work, filter

of the

that

scheme

Because

studied

processes

different

times

of and of

the

that

F-ADP-Pi

ATP

had

separated reaction,

actin

is the

of ATPis

hydrolysis, in

and

not that

polymerization,

15s

G-

F-ADP been

from

interference delay an

involved

alternative

times of

t P. 1

provided

F-actin,

at different

possible

reaction,

that

phosphate

,->

ADP-Pi-F-actin

the

It

on F-actin so

polymerization

inorganic

intermediate

to

cytoskeleton. (l-3))

the

following

lived

>

rapidly the

in

the

necessary

is hydrolyzed

reaction

in

1 :

Hz”\

polymerizaton

process.

medium long

involved is

of the

to actin

intermediate (4)

steps

(F-actin)

component bound

shown

for

elementary

polymerization

first

the

evidence assay

tightly

major

to the

that

the

is the

major

the

have

according

In

ATP

of

microfilaments

of this

follows we

sequence into

that

Recently,

immediately

the

dynamics

established a process

actin.

of

this in

method

using following

of the

a glass a rapid

polymerization

technique each

(1)

with

measurement

allowing

direct 0006-291X/87

1069

the at and $1.50

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 143, No. 3, 1987

faster

monitoring

of

polymerization

was

developed

by in

myosin

medium

to

the

by trapping

where

GA3P and has

enzymes in

3-PG,

assay

could

medium

by

the

do

be used NADH

into

P.1 in

presence

3-PG 1,3

PG

A slightly

We have

with

successfully

that actin

to monitor

fluorescence,

1,3

modified

shown

interfere

is

during

the

2 :

diphosphoof this

substrates

polymerization the

of to

(2)

version

the

by

equilibria

in scheme

+

the

NADH

The of the

as described

controling

of

NAD .

ATP-actin previously

rate

production of

- 3 - phosphate,

not

of

assay, the

displacement

diphosphoglycerate,

(6).

course

enzyme

(GAPDH)

the

3 phosphoglycerate.

assay

time

conversion

ensures

RESEARCH COMMUNICATIONS

to determine

dehydrogenase

described

the

thus

the

couples

glyceraldehyde

been of

technique

The

1,3-PG is

the

a linked

(5)

(PGK) 1,3

in

used

coworkers

phosphate

+ P.1 .s

medium

quantitative

kinase

GASP

assay

and

rapid

glyceraldehyde-3-

glycerate,

the

We have

ATPase.

phosphoglycerate right

in

1

Trentham

process

the

P.

sought,

AND BIOPHYSICAL

liberation

and ;

the

of free

polymerization

Pi of

ATP-actin. We find from

a value

of

ADP-Pi-Mg-F-actin,

previously

developed

MATERIALS

AND

0.006 in good

glass

filter

s

-1

for

the

agreement assay

rate with

constant the

value

of

Pi

derived

dissociation from

the

(4).

METHODS

Dithiothreitol, ATP, ADP, NAD, NADH, EGTA, sodium azide and DLglyceraldehyde-3-phosphate diethylacetal were from Sigma ; NBD-Cl from Molecular Probes ; glyceraldehyde 3-phosphate dehydrogenase from rabbit muscle and phosphoglycerate kinase from from Boehringer. yeast were Monomeric G-actin was purified from rabbit muscle by the usual procedure NBD-labeled actin (1:l) was prepared as described (9). (7-8) : Polymerization of actin was monitored spectrofluorometrically at 25OC using a Spex fluorolog 2 fluorimeter equipped with a DMIB datamate and a digital plotter. Ca-G-actin solutions, containing a proportion of 20 % NBD-labeled actin, were initially in buffer G consisting of 5 mM Tris-Cl pH 7.8, 0.2 mM dithiothreitol, 0.01 % sodium azide, 0.2 mM ATP and 0.1 mM CaC12. The 1: 1 ATP-Ca-G-actin complex was isolated free of unbound ATP by Dowex-1 treatment (10). Ca-G-actin was then converted into Mg-G-actin by a 3 min incubation in the presence of 0.2 mM EGTA and 50 PM MgCl2 (11) and immediately processed for polymerization, which was started by addition. of 1 mM MgC12 and/or 0.1 M KC1 as indicated. The ammonium sulfate suspension of linked enzymes was rapidly centrifuged, the pellet was resuspended in 10 mM Tris Cl pH 7.8 buffer (protein concentration r~) 10 mg/ml) and dialyzed against the same buffer for 2 hours before the experiment. Glyceraldehyde-3-phosphoric acid prepared by hydrolysis of the diacetal was kept at -20°C and neutralized just before use, to minimize the decomposition of the compound. ADP and NAD stock solutions were also neutralized. RESULTS In order a rate

at least

to work one

satisfactorily, order

the

of magnitude 1070

enzymatic faster

than

assay

has

the

rate

to titrate at

which

Pi at it

is

BIOCHEMICAL

Vol. 143. No. 3, 1987

5.5

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0 a

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

- 1

Z

I

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20

40

60

5.0 0

-i 80

100

Time,

120

0

10

s.

30

20

(Pi),

40

PM

Figure 1. Assay of P. added to the medium monitored by NADH fluorescence in an enzyme linked ‘assay system. The fluorescence cell contained 0.25 mM DL-glyceraldehyde-3-phosphate, 0.4 mhl ADP, 2 mM NAD, 1 mM hlgCl2, 0.5 mg/mI glyceraldehyde 3 phosphate dehydrogenase and 0.1 Mg/ml phosphoglyceratc kinase in buffer GO pH 7.8 (buffer G without free nucleotides) . At times indicated by the arrow, aliyuots of 10 nlvl inorganic phosphate (from a 0.1 M phosphate solution buffered at pH 7.8) were added to the solution. NADH fluorescence was excited at 360 nm (slits 0.5 mm) and monitored at 470 nm (slits 2.5 mm). Panel a : time course of NADH production following addition of P.. A dead time of 5 s is allowed for mixing at each addition. Panel b : depdndence of the increase in fluorescence on the concentration of Pi.

liberated

in

the

concentration

of

titrate

Pi

NAD

and

10

1 mM

MgCl2

slightly

from

that

this

al

(5).

leading

to

to

its

assay

was

G-

and a did

up

F-

last

to

forms

interfere

a

high

lo-13 of

(figure

lb).

0.4

screen

the

solution

was

J.LM

NADH

effect.

by

the

presence

that

all

actin

polymerization

(>

checked,

not 30

It

also

of

actin

an The

used

by

liberating zero

GASP

was

to

MM).

those

time

of

by

intensity

decomposed, at

NADH

deviated

and

to

to

2 mhl

in

was

close

concentrations

able

fluorescence

concentration

final

ADP,

increase

effect

are

a

are

mM

The

It

the

% GA3P

screen

affected

to

and

25-30 using

subsequent not

added

of

optimized

avoided

control not

at

ATP.

at

PGK)

GA3P,

(fig.

to

Pi

presence

the

Pi Pi

due

added

we

no

enzymes, mg/ml

mhl

dependence

were

the

minimize

containing

increasing

Because

reaction,

Finally, enzyme

the

the 0.1

0.25

of

simply

substrates

fluorescence in

was of

of

G

that

CAPDH, of

concentration upon

polymerization

both

buffer

shows

presence

concentration

--et

NADH

in the

titration

mM,

mg/ml

the

NADH

concentrations

0.25

(0.5

in

linearity

incomplete

and

la

the 2),

Trentham

Figure

mg/ml s,

versus

measuring

Pi

0.6

within

fluorescence

(fig.

medium.

of

the

higher

than

checked

that

in

solution,

2).

showed with

1071

the

components

of :

the

the

same

linked level

of

BIOCHEMICAL

vol. 143, No. 3, 1987

0

AND BIOPHYSICAL

70

20

30

40

(NADH),

RESEARCH COMMUNICATIONS

50

60

VM

Figure 2. Concentration dependence of the NADH fluorescence. Aliquots of 10 ~hl NADH were added to a solution of buffer G in the absence (0) and presence (0) of 25 UM G-actin. At the end of the calibration with C-actin, 1 mhl hlg was added to the solution and NADH fluorescence was checked not to change (*) after actin had been polymerized.

NBD

fluorescence

assay

was

experiment

concentrated added

was

mixture to

1.3

ml

s

(fig.

Polymerization within

of

be

distinguished

in

the

absence

as

follows

obtained

Pi

same

rate

presence Comparison (figure

a

led

to

and

F-actin

in

the

medium.

NADH

in

solution

assumed

in

However, a

linear

presence

of

The

screen

time

zero

the

kinetic

1st

the

of

Pi

Mg2+

due taken

analysis between 1072

Pi to

the

NADH

in

NADH (a

rate

short could

constant time

was

added

rate

limiting.

course

M

KC1

the

calibration

(fig.

of

the 3b). curve

liberated

from 25-30

account

in

the

the

insets

to

fluorescence

the The

in

0.1

been

of

to

polymerized

presence

into

In

in

same

not

had

shown the

The

with

complete

ATP-F-actin A

plus

was

was

kinetics

was

a

fluorescence.

order

was

nM

and

of

cuvette.

increase

of

actin

22-23 effect

s) NBD

enzymes

enzymes

~1

fluorimeter

data.

change

was

enzymes

curve). the

1 mM

that

and

the

a

whether

fluorescence

NAD

formation

from

or

200

in

fluorescence

the

zero,

the

by

obeyed

time

(2

increase

initial

of

3a)

in

sonication

the

that

obtained

relation

nM)

and the

at

ADP,

concentration

conclusion

at

:

monitored

turnover

(fig.

the

short

derived

% larger

NADH

2)

derivation.

50

Mg2’

the

of

was

was

mM

of

a by

Pi,

onset

the

constant 1

(26

reaction

release

that

of

solution

judged of

the

when

showing

GA3P,

polymerization at

for

KCI,

by

as

a slow

with

s-l

assay,

accelerated

was

coincident

G-actin

liberation

fluorescence, lag

0.0062

the

3a)

the

conducted 2+ + Mg -

of

was 30

contrast,

we

both

mixture. The

was

reached

PM above

fig. change

3,

BIOCHEMICAL

Vol. 143, No. 3. 1987

a

6.25

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AND BIOPHYSICAL

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RESEARCH COMMUNICATIONS

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5.95 3 8 5.65

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Figure 3. Measurement of the kinetics of P. dissociation from F-actin using the enzyme linked assay. A solution of fiBD labeled ATP-G-actin in G buffer (1: 1 complex, 26 ~thl) , extemporaneously converted into Mg-G-actinl) was polymerized upon simultaneous addition of 1 mhl MgCl2 (a) or 1 mM Mg + 0.1 hl KC1 (h) and a concentrated mixture of NADP ADP and linked enzymes (final concentrations as in fig. 1). Polymerization was accelerated by a short sonication of 2 s following the 5 s mixing of all components. Two identical consecutively ; in the first one, experiments were performed NBD fluorescence was measured (A, dashed curve ; excitation 475 nm, emission 530 nm). It was checked that the same plateau was reached when the components of the linked enzyme assay were omitted. In the second experiment, KADH fluorescence was measured (0). The excitation shutter The open circles (0) was periodically on and off to minimize bleaching. correspond to the same experiment except a 50 % larger amount of enzymes was used. In a third experiment (a, top left solid curve) actin was first polymerized with 1 mbl hlgC1, for 30 min, then the enzyme assay components were addrid. Insets-?how the” semi-logarithmic plots of the data, leading to k = 0.0062 - 0.0003 s .

1073

BIOCHEMICAL

Vol. 143, No. 3, 1987

(corresponding liberated

to

P.,

The

which of

may

be

filter

due

for

to

the

the

solution,

with

the

measurement.

When to

the

been

s

fact

dissociate developed

an additional

support

close

to

the

in

was

free

10 s (fig.

to the

Pi is

validity

F-actin full

last

linked

interferes solution

all the

the

that

Pi had

change

in

experiment

enzyme

assay

eliminated

reaction

to the

This

enzyme

continually

conditions

3a).

of the

the

reverse

added which

with

of

4 independent -1 determined s

0.005

found

accordingly

F-actin,

within

concentration of

of

of the

under

30 min,

value

case,

the

(average

value

this

RESEARCH COMMUNICATIONS

and

to only 10 %. + -1 0.0003 s

mixture

from

fluorescence

range)

no contribution

assay for

NADH

15 % higher

that

so that enzyme

-1 is

The

polymerized

totally

khl

kPi

assay.

from

had

0.0062

found

the

25-55

is accurate

ialue

experiments) using

the

AND BIOPHYSICAL

time NADH

provides

assay.

DISCUSSION We

have

described

by

reaction show

and

confirm

Because could

not shown

presence the

Pi

release

that

Pi is

liberated

our

the

have

Trentham

of ADP,

also

polymerization exchange

characteristics study

other

might

now

of actin

that

is

step,

underway, are

is

rate

(13, or

an

of Pi release

because,

in

14).

the

slow

translocating a rate

will

be

necessary

to ATP

hydrolysis.

enzymes, to elucidate

hydrolysis

in

lower and

than the Pi

the with

described faster

assay

we

that

interfered

here,

than

of Pi release this

we

which note

not

rapidly,

constant

assay,

Ca-F-actin,

experiments

rate

technique.

enzymatic

has

The

intermediate,

We should

activity,

Therefore,

with

from (4).

very

The

assay the

independent

to the

Mg-F-actin PGK

ATP

ADP-Pi-F-actin

necessary

i.e.

associated

following the

to the

(12).

GADPH-PGK

spectrofluorometrically

using

polymerized

reaction

nucleotidases,

monitor

from

from

necessary

of ATPases

be a slow

work,

the

enzyme

filaments,

ions

than

complex

to

observations

of ATP-actin

ATP-actin

linked

slowly

of Mg2+ slower

(5)

actin

to measure

to be

the

al.

previous

presence

use it

nucleotide

used

--et from

of

data

the

successfully

is

the

a general

may be useful which 0.1

s

P. release -1 1 . Further

structural

dissociation

to

changes from

the

filaments. ACKNOWLEDGEMENTS I thank this

Dr. David

R.

Trentham

for

a helpful

discussion

that

stimulated

work.

REFERENCES 1. 2.

Pardee, Pollard,

J.D., T.D..

and Spudich, and Weeds,

J.A. A.G.

(1986) J. Cell Biol. 93, 648-659. (1984) FEBS Lett. 170, 94-98. 1074

Vol.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

143,

No. 3, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Carlier, M-F., Pantaloni, D., and Korn, E.D. (1984) J. Biol. Chem. 259, 9983-9986. Carlier, M-F., and Pantaloni, D. (1986) Biochemistry 25, 7789-7792. Trentham, D.R., Bardsley, R.C., Eccleston, J.F. and Weeds, A.G. (1972) Biochem. J. 126, 635-644. Scopes, R.K. (1972) Anal. Biochem. 49, 88-94. Spudich, J.A., and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871. Eisenberg, E., and Kielley, W.W. (1974) J. Biol. Chem. 249, 4742-4748. Detmers, P., Weber, A., Elzinga, M., and Stephens, R.E. (1981) J. Biol. Chem. 256, 99-105. Mockrin, S.C., and Korn, E. D. (1980) Biochemistry lY, 5359-5362. Carlier, M-F., Pantaloni, D., and Korn, E.D. (1986) J. Biol. Chem. 261, 10785-10792. Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886. Lynm, R.W., and Taylor, E.W. (1971) Biochem. 10, 4617-4622. Johnson, K.A. (1983) J. Biol. Chem. 258, 13825-13830.

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