Inhibition of mitochondrial adenosine triphosphatase activity by troponin component, TN-I

BIOCHEMICAL

Vol. 53, No. 2, 1973

INHIBITION

OF MITOCHONDRIAL

ACTIVITY

Yutaka

Tamaura,

Laboratory

Received

TN-I.

Shigehisa

Chemistry,

Ookayama,

TRIPHOSPHATASE

COMPONENT,

Yamazaki,

Biological

RESEARCH COMMUNICATIONS

ADENOSINE

BY TROPONIN

Shojiro

of

AND BIOPHYSICAL

Hirose

Tokyo

Meguroku,

and

Institute

Tokyo,

Yuji

of

Inada

Technology

Japan.

June 7, 1973

One of the troponin components, TN-I, strongly inhibits Summary ; the ATPase activity of AS-particles obtained from mitochondria, while troponin and the other components, TN-C and TN-T, do not. The inhibition of the ATPase activity by Component TN-I is 2+ effective only in the presence of Mg and ATP. Component TN-I digested with trypsin completely loses the inhibitory action of the ATPase activity. Adding tropomyosin does not affect the inhibitory effect of Component TN-I on the ATPase activity.

During it

the

has

tant

been role

in

by

muscle

Ca*+

and

ion.

the

The

of

actomyosin

of

Ca2+

and pling

an

the

with

troponin

Recently, into

three

inhibitory

action

TN-T(mw TN-C(

reconstitution of

the

regulatory

to

and

the

clarified

Component

the

ATPase

the

TN-I

activity

a binding

site site

components of

impor-

and

a binding

three

an

Greaser

has

system in

plays

functions(3);

mw = 18,000) of

contraction,

components

= 37,000)

tropomyosin

mitochondrial form

and

Racker(&). factor

physiological

Copyright AtI rights

that

muscle

with

leads

ATPase

presence

and

in

has

isolated

with

to

activity the

absence

ion(*).

The pure

Endo

and

Component

restoration

of

properties

Component

tropomyosin

and

troponin

shows

actomyosin,

investigations

contraction(l).

separated

= 23,000)

the

Ebashi

physico-chemical

(mw of

of

found

Gergely(2) their

course

ATPase its

properties The with

function

have

inhibitor the may

@ 1973 by Academic Press, Inc. in any form reserved.

of reproduction

inhibitor been

seems molecular be

to

been

characterized be

related 673

to

by

a component

weight

of the

in

15,000 oxidative

a

Horstman

of

the

and

its

cou-

phosphoryl-

Vol. 53, No. 2, 1973

ation

or The

to

BIOCHEMICAL

the

respiratory

present

inhibition

paper of

the

role

of

the

energy

the

the

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

control(4,5).

is

to

prove

mitochondrial ATPase

that

Component

TN-I

ATPase

activity

and

inhibitor

transformation

in

process

the of

causes to

regulation

the

clarify

mechanism

of

ATP.

Experimentals AS-particles chondria

were

by

thus

the

obtained

removal

the

tropomyosin to

the

procedure was

The

troponin

at

was

taken

~1)

To

and

subjected

sample

mM Tris-Sulfate

5 'mM phosphoenol

pyruvate

was

carried

phosphate

out

liberated

for

troponin

separated

The

concentrations

Lowry

25

--et

in

or

to

the the

assay

added

0.5

buffer(pH and

17

pg

10 min

at

30'.

was

and

15 mM

its

compo-

of ml

soluthe

of

ATPase an

assay

5 mM MgSO4,

pyruvate

measured

the

sample

7.7),

The

by

al.(s).

mM KC1

incubation,

ATP

prepara-

were

troponin

was

from

according

AS-particles

solution

15

reaction

of

with the

and

muscle

TN-C,

mM ATP,

After

the

chromatography(7).

method

preincubated

to

Troponin

The

and

0.5

due

skeletal

activity,

mM MgS04,

this

containing

4 mM ATP,

ATPase

out

probably

6 M urea(2). the

mito-

AS-particles

inhibitor.

TN-T

by

heart

Horstman(6).

DEAE-cellulose

in

30'.

beef

Gergely(2).

TN-I,

were

layer

a rabbit

and by

0.5

15 min

activity(g).

of

from

the

6.7)

for

inorganic

activity,

determined

containing

Tris-HEPES(pH

The

ATPase

Greaser

measuring

solution

and

chromatography

Before

tion(25

Racker

prepared

purified

were

heavy

ATPase

components,

proteins

nents

a high

of

further

DEAE-Sephadex

medium

of

from

mitochondrial

were

tion

of

method have

of

obtained

kinase. amount by

the

of method

Martin-Doty(l0).

Results Inhibitory

and

Discussion effect

of

troponin

and

674

its

components

on

the

ATPase

Vol. 53, No. 2, 1973

activity

of The

BIOCHEMICAL

ATPase

activity of

presence

of

of

AS-particles

AS-particles Mg

2+

and

ATP.

curves

005

tion

of

the

no

010

shows

of on

the

the

concentration

Component

TN-I activity, Component

TN-C

ATPase

activity

was

observed

0.08

acts

as probably

TN-I.

mg/ml. a

C)

and

so

small

could

ATPase

potent due

Component

the TN-I

675

component,

concentraactivity

was

concentration

in

and

its

of

at

The

a

indicate of

interaction digested

the

higher that

mitochondrial of

AS-particles with

while troponir

inhibitory

tested

results

was

case TN-C.

that be

A),

the

Component

inhibitor to

by

proteins(curve

not

These

in

inhibition

various

TN-I

mg/ml

is

the

the

The

0.16

TN-T(curve

than

Component

activity

components

of AS-particles ATPase activities of AS-particles(0.4 B) and its TN-C(O).

at

Component at

its

the

(mg/ml)

components.

lost

after

015

Concenfrdion

increasing

Component

solubility

with

by

of B),

effect

1

obtained

its

completely

inhibition

(curve

activity

and

reduced almost

ATPase

ATPase

troponin

strongly was

or

Inhibition of the ATPase activity troponin and its components. The were measured after preincubation mg protein/ml) with troponin(curve TN-I(curve A), TN-T(curve C) and

of

measured

troponin

Figure

Protein

1.

was

with

0

Fig.

RESEARCH COMMUNICATIONS

AS-particles

preincubation the

AND BIOPHYSICAL

trypsin,

Vol. 53, No. 2, 1973

and

its

inhibitory

of

AS-particles

of

the

was

BIOCHEMICAL

effect with

ATPase

the

activity

plotted

clear

from

Figure

gives

rise

to

This

indicates

the

seems

to

be

of

higher related

to

of

restoratior

with

the

trypsin

digestion.

Component

As

TN-I

the

with

ATPase

structure

the

preincubation The

digested

of

order

the TN-I.

time

restoration

the

after

TN-I

digestion

complete

RESEARCH COMMiiNICATIONS

Component

incubation

the

that

tested

Component

the 2,

also

digested by

against

inhibitor

was

AND BIOPHYSICAL

of

inhibition

trypsin

activity. the

of

is

protein

the

ATPase

activity. The

ATPase

Component

activity

TN-I

was

of

AS-particles

measured

as

in

a

the

function

presence

of

3

pH(Figure

B 0.2 B

0

Fig.

2.

5 DIgestion

Restoration the course ~~~It'~~~kg digested time the (4 w/ml). preincubation digested

Fig.

3.

IO Time (mid

O.l-

oa5

15

The

3).

0.4 -

2 -E o 0.3-

[

of

‘1-1A

Q

8.0

75

70

65

6.0

PH

of the ATPase activity of AS-particles during of the digestion of Component TN-I with Component TN-I(0.6 mg/ml) in the medium 80 mM KC1 and 6 mM Tris-HEPES(pH 6.7) was with trypsin(2 (I-g/ml) at 30' and at a given stopped by adding trypsin inhibitor digestion was The ATPase activities were measured after of AS-particles(0.4 mg protein/ml) and the Component TN-I(0.12 mg/ml).

pH-Dependence of AS-particles(0.4 (0.16 mg/ml). AS-particles presence of of Component

of

the

inhibition of the ATPase activity mg protein/ml) by Component TN-I The ATPase activities were measured after was preincubated at various pH's in the Component TN-I(curve A) and in the absence TN-I(curve B).

676

BIOCHEMICAL

Vol. 53, No. 2, 1973

pH-activity

curve

ticles

with

shape

maximal

itor(4).

The

(curve

not

of

by

curve

acidic

obtained

activity

by

the

ATPase

activity

of

AS-particles

at

the

pH

a of

pK

of

is

without pH

in

the

ATPase

between

the

value

inhibition

mitochondrial

range

forms

one-half

This

the

AS-par-

which at

side.

from

of

A,

value

ATPase

reduced

activity

7.2(pH

the

RESEARCH COMMLJNlCATl3NS

inhib-

Component

6.6

and

8.0

B).

Effects

of

ATPase

activity

It

Mg

was

,

ATP

seem

to

with

its

the

by

be

necessary

or

of

the

ATPase

carried

out

in

presence

activity

of of

and

ATPase

to

activity

of

the

presence

of

MgS04

of

without

of

the in

the

presence

an

of

AS-particles

AS-particles

by

of

the

and

without

ATP

Mg

and

677

addition

or

was

or

in

Component

both

the almost

the

that by

was

of of

in

the

ATPase

effect

indicates

ATP.

the

TN-I

with

AS-particles

in

to

Component

inhibition

Component

as

role

ATP

inhibitory

2+

ATP

examined

without

minute

of

and

TN-I

the

activity

2+

Component

and

In

This

in-

by

with

I).

mito-

ATPase

important

ATP,

the

Mg

was

and

measured. system,

ATP(Table ATPase

play

without

of

it

with

the

mitochondrial

with

reaction

EDTA

the

mitochondrial

Both of

EDTA

and was

compared

complete(93%), in

of

that

the

ATP.

AS-particles

with

the

of

Racker(4)

study,

activity

AS-particles ATP

and

them

presence

MgS04

inhibition

by

present of

of

the

the

interaction

the

both

incubation

2+

Mg the

In ATP

The

%S04

for

and

inhibited

of

2+ ,

inhibition

Horstman is

presence

on

TN-I

activity

in

Mg

tropomyosin

Component

inhibitor.

whether

TN-I.

and

by

ATPase

hibitor

the

2+

demonstrated

chondrial

caused

value

that

ATPase

shown

in

with

mitochondrial

was

pK

the

is

activity)

agreement

TN-I

TN-I

with

ATPase

good

from

Component

sigmoidal

tion

obtained

AND BIOPHYSICAL

the

obtained presence inhibiTN-I

was

Vol. 53, No. 2, 1973

Table

BIOCHEMICAL

AND BIOPHYSICAL

Effect of Mg 2+ or ATP on the inhibition activity of AS-particles(0.4 mg protein/ml) Component TN-I(O.16 mg/ml). Concentrations; and 0.5 mM ATP. EDTA, 0.5 mM MgS04

I.

ATPase

activity(pmoles

AS-particles

EDTA EDTA

+ ATP

MgSO/+ MgS04

+ ATP

Tropomyosin

plays

myosin

ATPase

effect

of

ATPase

activity

MgS04 the

and

activity

Component

Throughout Ca2+

did

this not

Component

TN-I

show on

0.306

19

0.355

0.242

32

0.364

0.037

93

important

role

Component the

in

experiment, any

difference

the

ATPase

inhibition

the

is

with

from the

Component

The

authors wish to express Mariko Ogura in preparing

the

Table

inhibitory

TN-I

alone

TN-I, and II,

and

that

tropomyosin.

the

presence in

activity

the

or

the

inhibitory of

their this

absence effect

AS-particles.

678

heartfelt manuscript.

thanks

no

effect

Acknowledgement Miss

acto-

mitochondrial

tropomyosin,

clear

between

test

Component

without

As

of To

AS-particles,

observed

together

of

and

adding

the

TN-1(2,3,11).

measured. was

in

inhibition

with

TN-I

Inhibition (%)

0.376

incubated

activity

min)

35

TN-I,

difference

Pi/IO

AS-particles + TN-I

Component

was

the ATPase by 5 mM

0.256

by

activity

ATPase

adding

were

of

0.392

on

by

ATP

ATPase

the

an

tropomyosin

significant on

RESEARCH COMMUNICATIONS

to

of by

in

Vol.

53, No.

Table

II.

BiOCHEMICAL

2. 1973

AND

BIOPHYSCAL

Effect of tropomyosin on activity of AS-particles Concentrations; AS-particles(0.4 Component TN-I(0.16 me/ml)

ATPase

the by

RESEARCH

COMMUNICA’TIC>NS

inhibition Component and

of TN-I. mg protein/ml), tropomyosin(0.6

activity(bmoles

the

mg/ml).

Pi/l0

AS-particles

ATPase

min)

Relative

activity

None

0.392

100

Tropomyosin

0.402

102

Component

TN-I

0.037

3

Component + tropomyosin

TN-I

0.050

73

References --7.

2. 3.

s. Ebashi and (1968) Greaser M. L. Muscle Regulatory Quantitative

M. and Biology.

Endo,

Progr.

Biophys.

J. Gergely, J. System in Cold 37, 1973.

p.215 4.

5. 6. 7. 8. 9. 10. 11.

Mol.

Biol. Spring Cold

Biol.,

78,

123

Chem., 246, 4226(1971) Harbour Symposia Spring Harbour Lab.,

Biol. Chem., 245, 1336(1970) Biol. Chem., =E, 3762(1963) J. Biol. Chem., a, 2547(1967) J. Biol. Chem., &, 5962(197O) A. L. Farr and R. J. Randall,

L. L. Horstman and E. Racker, M. E. Pullman and G.C. Monroy, E. Racker and L. L. Horstman, Staprans and S. Watanabe, I. 0. H. Lowry, N. J. Rosebrough, J. Biol. Chem., m,265(1951) M. E. Pullman, H. S. Penefsky, Biol. Chem., 2l.5, 3322(196o) J. B. Martin and D. M. Doty, J. M. Wilkinson, S. V. Perry, Biochem. J., 127, 215(1972)

J.

J.

A. Anal. H.

679

Datta

A.

Chem., Cole

and 21, and

E.

Racker,

I.

965(1943) P. Trayer,

J.

on