Intramitochondrial release and binding of mitochondrial aspartate aminotransferase and malate dehydrogenase in the presence and absence of exogenous succinate

Intramitochondrial release and binding of mitochondrial aspartate aminotransferase and malate dehydrogenase in the presence and absence of exogenous succinate

BIOCHEMICAL Vol. 42, No. 4, 1971 INTRAMITOCHONDRIAL ASPARTATE THE AND BIOPHYSICAL RESEARCH COMMUNICATIONS RELEASE AMINOTRANSFERASE PKESENCE Cent...

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BIOCHEMICAL

Vol. 42, No. 4, 1971

INTRAMITOCHONDRIAL ASPARTATE THE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RELEASE

AMINOTRANSFERASE PKESENCE

Centre

AND MALATE

AND ABSENCE A.

lUAKSMAN*

Neurochimie

de

BINDING OF NiITOCHONDRIAL

ANu

DEHYDRUGENASE

OF EXOGENOUS and

A.

du CNRS,

67-Strasbourg

IN

SUCCINATE

RENDON-

Faculte

de Medecine,

(Francs)

Received January 12, 1971 SUMMARY

DifPerential malate shown

to occur. This extramitochondrial

of the

It

this

is

that system

membrane

mitochondrial rhe

present

mitochondrial

AA.T and

rebind

to the

environmental

Y

*

to

may

recherche de recherche

defined

reversible,

be

to the

rather

then

integrated

sx-

(AAT)

mitochondrial

belong

this

membrane.

would

mitochondrial to

mem-

per-

constitute

structural

and

functional

system.

shows unequivocally malate

dehydroqsnase

mitochondrial

conditions

Charge de Attach6

study

inner

the

temporarily, of the

under

that

aminotransferase

from Pound

some enzymes

elements

manent

was

only

(l,2)

aspartate

released

phenomenon

Suggest

may

compartment.

conditions,

(LC 2.6.1.1)

brane

of aspartate aminotransferase and between submitochondrial fractions was phenomenon is dependent upon the nature

has been shown previously

perimsntal

If

shuttling

dehydrogenase

of

the

that (MDH)

released (EC

membrane,

depending

orqanelle

(1).

au CNRS, au CNRS. 745

inira-

1.1.1.37)

upon the

Vol. 42, No. 4, 1971

BIOCHEMICAL

AND BIOPHYSICAL

EXPLRIMENTAL

liver

Rat

et al.

(3),

under be

the

mitochondria

electron

in the

for

5U mm).

euccinate,

parallel

lb

min

with

termined

suring

in

0.25

presence

or

absence

in sucrose,

sucrose

were of

the

in which

euc-

mito-

sucrose

con-

each

37OC.

5 min periods

were run

to

(5).

M sucrose

consecutively

in either

the

at

mitochon-

or in sucrose-con-

alone

by

fractionation,

digitonin,

as modified

the

Hackenbrock

experiments,

incubations

were found

by

for

Hare1

When examined

mitochondria

suspended

of

succinate.

treated

in

method

In rebinding

For submitochondrial

(4)

the

(4),

as described

sucrose,

and

were incubated

taining

these

3’/OC in the

were incubated

taining

dria

5 min at

(2 .mM to

chondria

In

state

by

et al.

Levy

of mitochondria

incubated cinate

by

prepared

microscope,

condensed

Aliquote

METHODS

were

as described

RESEARCH COMMUNICATIONS

according

Schnaitman

by

measuring

to

et

rate

the

the

exogenous

MDH (1).

the

of

oxidation

of

at

AAT

activity

in the

NADH

in a Cary

method

Lowry

type

et al. --

(7)

was

for

used

Levy

et --

assayed

340 ml.4 by

of

measurements at

al.

was de-

presence

14 epectrophotometer

were

NADH at

of

MDH was

340 mp. The spectrophotometric

performed of

of

of oxidation

of

acetate,

method

al 0 (6).

presence rate

the

mitochondria

mea-

oxalowere

27OC. The determinations.

protein

RESULTS

Localization cubation The

cubated is these

of of

AAT

of

in a 0.25 in

mitochondria

IDH

Fig.

1 clearly

M sucrose the

activities

in sucrose,

mitochondria

results

localized

and

inner

after

successive

succinate,

and

show

medium,

that

wers transferred 746

to

sucrose.

in mitochondria

86 $ of the

membrane-enriched

in-

AAT activity

fraction.

a euccfnate

in-

When

medium (50

Vol. 42, No. 4, 1971

BIOCHEMICAL

AND BIOPHYSICAL

SUCROSE

RESEARCH COMMUNICATIONS

SUCCINATE

Fiqure 1 e - Effect of consecutive incubation of mitochondria In sucrosaaauccinate-sucrosa on AAT localization. Mf I enriched internal membrane fraction ; ME : enriched external membrane fraction ; MS t soluble matrix. figures on top of the circle8 indicate the per cent recovery of AAT activity. Figures in the circles indicate the per cent AAT activity bound to MI.

mm),

about

partment the

96 and

$ of

into

passed

the

AAT

soluble

mitochondria

in the

OP 75 (k of

inner

rebinding

membrane-enriched

zation-binding

of succinate

than

solubilization

the

protein).

This

re.lease

branes

rise

may

to

torily trations the

of the react

enzymes

the

Por

the

of

succinate of

AS may

at which magnitude

a sucrose

medium

solubilized

AAT

but

critical

seen

these

747

to

the

concen-

protein)

level

which

OP certain

Fig.

enzymes that

re-

mitochondrial

compounds,

in

of

mitochondrial

the

cycles

as

lower

at

substrate

that

com-

OP solubili-

mitochondrial

towards

be

cycle

MDH,

release-binding

proteins.

membrane

Raincubation

pmolss/mg

suggests

SpeciPically

bound

order

in

pmoles/mg

in

the

matrix.

oP AAT (0.8

difperence

1ePt

A similar

observed

(0.2

difPerentia1

same

Praction.

was also

trations

of

bound

succinate-incubated

sulted

the

total

the

required

2,

the

memgive transi-

concen-

were released to

for

produce

are a

Vol. 42, No. 4, 1971

BIOCHEMICAL

MDH activity

Fiaure 2. - AAT and succinate concentration.

morphological tron

This

preliminary

of

Figure

MI and

:

MS 8s

pM NADH

mitochondria

the

demonstration mitochondrial

illustrates

AAT in the

initial

of

RESEARCH COMMUNICATIONS

a Function

of

oxidized/mine

detectable

by

elec-

(5).

of membrane-bound This

in

Ordinate

change

microscopy

AND BIOPHYSICAL

the

different

increase

is

enzymes

variation

in

specific

in

specific

Fractions.

activity

a differential

release

confirmed

the

submitochondrial

in the

membrane indicatis

of a differential

3,

activity lndeed,the

of AAT in the

solubilitation

Fig.

of

inner

non-AAT

protein. It

is

lease into

conceivable

From

urith

membranal

space.

molecules

of

an

interesting

tween

the

during

new

and

They may,

both

extra

and

possibility

membrane and soluble

period

the

some of the

membrane,

the

contact

that

solubilized

complex

in

these

environment

conditions,

intramitochondrial is

that

Fraction 748

the

play

re-

transient enzymes

come

OP the

interact origin

shuttling

might

of

of

a role

inter-

with (B).Thus

enzymes

in the

be-

Vol. 42, No. 4, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AAT

20 - Specific activity ractions 88 8 function

transport

of constituents

the

membranal

inner

of of

of the

mM

Succinate

50.

submitochonAAT in different succinate concentration,

intermembranal

space

towards

comportment.

REFERENCES 1.

2. 3. 4. 5. 6. 7.

8.

A. and Waksman, A. Biochem. Biophys. Res. Commun., 324 (1969). Estrada-0, S., Mental, M., CBlis, H. and Carabez, A. Europ. 3. t!iochem., 12, 227 (1970). Harel, L., &cob, A. 8nd Moul6, Y. Bull. Sot. Chim. Bfol., 39, 819 (1957). IAvy, RI., Toury, R. and Andrs, J. Biochim. Biophys. Acta, 135, 599 (1967), Hackenbrock, C.R., 3. Cell Biol., 30, 269 (1966). Schnaitmen, C., Erwin, V.G. and Greenawalt, JoW., 3. Cell 32, 71~ (1967). Biol,, Lowry, O.H., Rosenbrouqh, N.J., Farr, A.L. and Randall, H.J, 3. Biol. Chem., 195, 265 (1951). Viqn8is, P.V., DuBe, E.D., Colomb, M., Reboul, A., Cherny,A., Barzu, 0. 8nd Viqnais, P.M. Bull. Sot. Chim. Biol., 52, 33 (1970). Rendon, 35,

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