Binding of adenine nucleotides by mitochondria during active uptake of Ca++

Vol. 16, No. 1, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

BINDING OF ADENINE NUCLEOTIDES BY MITDCHONDRIA DURING ACTIVE Ernest0

Carafol

i”

Department The

Johns

of

UPTAKE OF CA*

and

April

The

14,

Hopkins

also

mechanisms al.,

requires

Vasington

the

ATP

suggested

necessary

electron

of

been

is

of

presence 1962;

has

Ca w

However,

Murphy,

It

of

1963). the

and

1963).

for

ATP

the

explanation,

however,

does

not

the

presence

to

the

same

completely

chondria be

and for

despite

A possible

*

U.S.

Medicine

by

Health

the

system

in

fact

this

Lehninger

Service

the the

ATP-induced

(DeLuca

and

Engstrom,

swelling

agent.

that

absence

St-‘+

has

et

role al.,(l963

Postdoctol.al

66

no

ATP

a,

b)

Fellow

in that

of

that the

supporting

action a reagent

swollen

mito-

also

of

Sr’+

by

effects

al.,

This

has

Lehriinger,

of

et 1963)

ATP

damaging

and

1961;

oligomycin, of

Furthermore,

i , Wei land specific

the

of

circumstances

al., state

fact

1963;

Brierley

structural

accumulation

that

1964; et

the

Lehninger, these

contraction

1962).

by

under

a powerful for

and

Brierley

the

supported

Ca++

1963,

1963;

or

(Carafol to

of

of

be

(Rossi

Lehninger,

account

the

may

transport

respiration-supported the

clue made

Public

mitochondria

Ca+‘,

Lehninger,

structure

finding

by

and

of

suppresses

necessary

mitochondrial

the

in

(Neubert

mitochondria

Maryland

maintenance

presence

which

of

9,

(Vasington,

the

is

in

Rossi

in

ATP

School

accumulation

mitochondria

of

Chemistry

University

accumulation

energy-conserving et

Lehninger

1364

active

Brierley

L.

Physiological

Baltimore

Received

Albert

on

been rat

shown 1 iver

the

1964). Ca’+

accumulation

significant

is amounts

Vol. 16, No. 1, 1964

of

BIOCHEMICAL

easily-hydrolyzed

accumulate

phosphate

in

phosphate. it

is

shown

liver

rat

liver

by

electron

same

observation

that

large

amounts

from

remants

Ca

as

to

been

of

the

suspending

The

uptake

id-

uptake,

its

for

7 minutes)

of in

nucleotides during

Cathis

are

is

and

inorganic

communication

accumulated

uptake

nucleotides

and

100°

further;

medium of

at

accumulation

pursued

adenine

RESEARCH COMMUNICATIONS

I 1 HCI

during

has

transport.

requi

(labile

mitochondria

This

mitochondria

AND BIOPHYSICAL

of

by

Ca*

supported

Ca++-dependent,

sensitivity

to

rat

has

inhibitors

is

the similar.

EXPERIMENTAL The Rossi

general

and

Lehninger

ATP-8-C

‘4

end

the

of

twice

counted

at

the

cold 0.25

infinite of

a biuret

the

tubes

Ca*

was

carried

Protein

was

and

the

on

and

plated,

and

apparatus.

pellets

were

according

the

the

were

acid,

gas-flow

washed

0’

pellets

formic

of

at to

The

85%

those

nucleotides,

cooled

5 minutes.

processed

determined

were

mitochondria;

quickly

in

Ca”

adenine

the

background

out,

4.6,

pH

were

dissolved a low

of of

with

x g for

in

uptake

binding

incubated

M sucrose,

buffer

(1960).

measuring

20,000

thinness

E succinate

Walser

at

the

was

period,

with

determination

measuring

For

c.p.m.),

incubation

washed

0.1

00

in

for

(1963).

(1000-1~

centrifuged

in

conditions

to

mitochondrial

When

resuspended the

method

of

suspensions

by

reaction.

RESULTS Fig. to

the

1-A

mitochondria

nucleotides that

the

labeled

Cai’

-stimulated

experiments,

it

the

limited by with

by

the its

the

rate

with

are

indicates of

compares

not

the

bound

the

binding

ATP

added

rate

of

was

bound

of

experiment

in

Fig.

ATP

and

the in

the

that

appears

The

to

67

at

accumulation 20

minute

That

this of

of of

is labeled

saturation

nucleotides The

adenine

a rate which i-b About of Ca . However,

period.

most binding

reach

Cat+.

rather

the

binding

adenine

of

hydrolyzes

present.

1-B.

labeled

but

activity possible

amount

of

accumulation

accompanies

appeared

the

binding

instantaneously,

ATPase

concentration

of

the

added

labeled

4-5% since

ATP

in

such

nucleotide

probably

true

is

was shown

nucleotide

increases

only

6.0

above

mM ATP.

Vol. 16, No. 1, 1964

BIOCHEMICAL

GO

6 MINUTES

AND BIOPHYSICAL

12 I6 OF INCUBATION

RESEARCH COMMUNICATIONS

5 IO mM ATP

t

15

A - Ca++ and ATP uptake by mitochondria. Test system contained 10 Fiqure 1. 10 mM MgC’2, 4 0 mM NaK-phosphate, 10 mM NamM Tris-HCl, pH 7.0, 80 mM NaCI, ‘4 ), 4.0 mM CaC12 succinate, 3.0 mM ATP-Na (4-1500 c.p.m. ATP-8-C and rat liver mitochondria (Is mg protein) in a total volume of 10.0. Temperature. 30”. B m ATP uptake by mitochondria as a function of the added ATP concentration. Test system as in Fig. lA, with variable amounts of ATP. Time, 20 minutes, temperature, 30”.

Typical a system

data containing

in

Table

1 show

respiratory

that

the

substrate,

Mg*,

Table Requirements

for by

labeled

binding rat liver

ATP Ca*

System

and

bound Pi.

Omission

HCl pH 7.0 3.0 mM ATP-Na (15 mg protein)

80 mM NaCl, (+I500 c.p.m. in a total

88.' 47.7 45.0 52.0 16.6

Comp 1e te Ca* omitted Complete + 1 ug

91.5 ‘9.1 91.2

0.2

oligomycin

x mg”

protein

35.0

mM 2,&dinitrophenol

68

of

any

10 mM MgCl ATP-8-CeG), volume of

Nucleotide ptake mpmoles x mg 3 protein

+

in

nucleotides

Complete Mg* omitted Succinate omitted Pi omitted Ca* omitted

Complete

maximally

1 of C14-adenine mitochondria

The test system contained ‘0 mM Tris 4.0 mM NaK-phosphate, 10 mM Na succinate, 4.0 mM CaC12, and rat liver mitochondria 10.0 ml. Incubated 20 min. at 30”.

is

BIOCHEMICAL

Vol. 16, No. 1, 1964

component with

greatly

those

limits

required

for

1962) ,( BrIerley

et

in

show

Table

1 also by

oligomycin

agreement

with

the

to

ATP

some

of adenine

the

normal

with

the

much

mitochondria

by In

several

and

were

suggest

a simple

does

accumulation

that is

It

appears

most

contains

earlier

easily-hydrolyzed

of

0.05-0.10

(1964) 1 iver

one

experiments

of

have

of

or of

that

both

of

demonstrated

per

the

umole that

Ca*c both

be

liver

a significant this

saturation Fig.

1B were or

(i.e.

-1

about may

amounts

wmole

(i.e.

high

about

160

10 be

times

compared

per

reaction

of

Ca-

both

mg protein)

recently

and

and

des-

labeled

varying

60 and

90

Ca*

adenine

concentra-

wmoles

This

adenine

of

ratio

does

nucleotide

nucleotides

adenine not

binding

accompanying

Ca*

process. of

acid-labile al., Pi)

Ca*

widely

adenine

form

et

7 min.

in

accumulated.

of

the

Lehninger P (

umole

Ca*

conspicuous

likely

not in

Ca*;

This

between

binding

a relatively

at

that

of

between

the

is

in to

protein,

uptake

intervals,

stoichiometry

indicate

as

conditions

umole

data

(1964).

which

under

per

bound

ADP

Contessa

time

bound

and

and

measured,

different

ATP

atractyloside-sensitive

in

typical

nucleotides

added

mitochondria.

the

experiments

at

nucleotide

still

Luciani

were

tions

of

Murphy,

Z,&dinitrophenol,

on

mg mitochondrial

amounts

The

appears

dependent

such

isolated

and

accumulation

There

nucleotide

freshly

in

Bruni,

nucleotide

but

smaller

by

identical

uptake.

experiments per

in

not

adenine

nucleotide

content

by

in

is

(Vasington

adenine

Ca*

1962).

nucleotide

of

of

on

are

1963).

strongly

agents

Murphy,

maximum

ATP)

binding

these

Ca+*

Lehninger,

inhibited

which

amounts

concentrations

cribed

of and

the

maximum

mpmoles

is

RESEARCH COMMUNICATIONS

requirements

of

and

aerobic

but action

of

(Rossi

the

accumulation

l.S-ZO%

The

bound

that

These

binding. accumulation

1963),

(Vasington

base-line

ATP

maximum

al.,

inhibited

mitochondria

the

AND BIOPHYSICAL

the

labeled

phosphate (1963

a,

b)

accumulated

accumulated. P32 - 1 abe led

mitochondria.

69

nucleotide

with In ATP

groups,

since

showed

that

Ca*

are

addition, and

bound

ADP

the of

Bruni are

the

bound

amounts the

order

eta., to

rat

BIOCHEMICAL

Vol. 16, No. 1, 1964

It of

Ca*

calcium Ca%

possible

binding,

and

phosphate does

Rossi in

appears

not

and

oligomycin

the

the Whitehall

in

1964).

National

the

The

aids

mitochondria, et

significance

under

supported

by

Foundation,

grants The

at

or

near

sites

forming

in

such

a manner

that

1964;

Carafoli

et

the

and

the

in

al., of

further

RESEARCH COMMUNICATIONS

bound

phosphorylation, is

Science

is

(Greenawalt

oxidative

was

nucleotide nucleotide

again

atractyloside

investigation

the

bound

out

uptake,

and

This Health,

llleaktr

Ca*

that

deposits

Lehninger,

active

that

AND BIOPHYSICAL

stabilizing the al.,

nucleotide-binding

and

in

the

sites

1964; reaction

of

action

of

investigation. from Nutrition

the

National Foundation,

Institutes Inc.,

of and

Foundation.

BIBLIOGRAPHY

Brierley, Bruni, Carafoli,

G. P., Murer, E. and Green, D. E., Science, 140, 60 (1963). Luciani, S. and Contessa, A. R., Nature, 201, 1219 (1964). E., Weiland, S. and Lehninger, A. L., Biochim. Biophys. Acta, Submitted for puklication. Carafoli, E., Rossi, C. S. and Lehninger, A. L., J. Biol. Chem., In press. DeLuca, H. F. and Engstrom, G. W., Proc. Nat. Acad. Sci. U.S., 4i’, 1744 (1961). Greenawalt, J. W., Rossi, C. S. and Lehninger, A. L., J. Cell Biol., In press. Lehninger, A. L., Rossi, C. S. and Greenawalt, J. W., Biochem. Biophys. Research comll., l-$ 444 (1963a). Lehninger, A. L., Rossi, C. S. and GreenawaIt, J. W., Fed. Proc., 22, 526 A.,

(1963b). Neubert, D. and Lehninger, Rossi, C. S. and Lehninger, Rossi C. S. and Lehninger, Schneider, W. C. in Manometric J. E. Stauffer (eds.), Vasington, F. D., J. Biol. Walser, M., Anal. Chem., 21,

A. L., Biochim. Biophys. Acta, 62, 556 (1962). A. L., Biochem. Zeit., 338, 698 (1963). A. L., J. Biol. Chem., Submitted for publication. Techniques , W. W. Umbreit, R. Burris and Burgess Press, Minneapolis, 1956, p. 188. Chem., 2& 1941 (1963). 771 (1960).

70