Release and binding of proteins and enzymes with isolated inner mitochondrial membranes

Vol. 50; No. 3,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RELEASEANDBINDING OF PROTEINSANDENZYMES WITH ISOLATED INNERMITOCHONDRIAL MEMBRANES ** Alvaro RENDON*and Albert WAKSMAN Centre de Neurochimie du CNRS, 11 rue Humann, 67085 Strasbourg Cedex,France

Received

November

23,1972 SUMMARY

Extramitochondrial substances trigger specific and reversible release of proteins and enzymes from inner membranal compartment towards intermembranal space. This shuttling phenomenonhas been shown to occur with isolated inner membranes. In previous experiments we were able to show that rat liver respond to variations

of the extramitochondrial

mitochondria

environment by releasing pro-

teins and enzymes from the inner membranal compartment towards the intermembranal space. This phenomenonshows somespecificity released proteins

and is characterized

as for the nature of the

by the capacity of the inner membrane

to recognize various extramitochondrial

"releasing

effecters".

Furthermore

rebinding of released proteins is observed when the "releasing withdrawn from the extramitochondrial

effecters"

are

medium12 3 .

The present paper compares results obtained with inner membranesprepared by two different

methods3p4. They show good concordance in the effects

produced by the "releasing

effecters"

on these two preparations.

the equivalences of the inner membranesobtained by different as lity

the

release

and

for digitonin

binding

phenomena

are

concerned.

methods as far

They exclude

to synergize the action of the "releasing

They suggest

the

effector"

possibior to

weaken the membraneas to provoke the release and binding process. They also suggest independance of the release phenomenonfrom ionic strength.

*

** Attach&? de Recherche au CNRS. Maltre de Recherche au CNRS.

Copyright @ 19 73 by Academic Press. Inc. AN rights of reproduction in any form reserved.

814

BIOCHEMICAL

Vol. 50, No. 3, 1973

EXPERIMENTAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

METHODS

Rat liver

mitochsndria

were

prepared

by Levy et aZ.5 .Mitochondrial

cribed digitonin

method3y5

the release

or by the

responding

trations.

Final

After

incubation,

in a

type

were

collected

water

sucrose the

40 rotor

Aspartate the rate

concentration

at 4 'C, were

resuspended

(AAT)

methods were

by the

incubated

volume

for

For

and cor-

diverse

for

concen-

was

1 ml.

15 min at 60,000

L centrifuge.

x g

Supernatants

and homogenized

activity

dehydrogenase

in presence

tric

were

meter

both

M ; final

model

either

in

3 ml ice

cold

at 4 'C.

of NADH oxidation measurements

with

was centrifuged

in a Spinco

as des-

method4.

effectors"at

was determined

of NADH at 340 nm in

Malate

et az.6

prepared

preparation,

was 0.25

assay mixture

aminotransferase

dehydrogenase.

obtained

mitochondrial

overnight

of oxidation

were

of various"releasing

and pellets

and stored

subfractions

membrane

to 25 mg of initial

5 min at 37 "C in presence

to Hare1

swelling-condensing-sonication

inner

experiments,

according

The method

For binding

experiments

of exogenous

(MDH) was assayed

of oxalacetate

performed

at 22 'C.

the presence

by measuring

at 340 nm7.The

in a Beckman Acta

of Lowry

by measuring

III

the rate

spectrophotome-

type

et aZsO was used for

malate

spectrophoto-

protein

determi-

nations.

were

suspended

in sucrose

above.

They were

tionned

et aZ.3, and the pellet mentionned

was dialyzed containing

then

treated

was separated

against

fraction

to 100 mg of protein

50 mM succinate,

and incubated

with

digitonin

from

the supernatant

0.25

was divided

M sucrose,

12.5 mM succinate.

at 4 'C for

branes

containing

corresponding

according

as men-

to Schnaitman

under

the already

conditions.

The supernatant

dialyzed

mitochondria

18 h, against

supernatants obtained

from

were either

and the other

Dialysis five than

in two equal

changes

one of which

one against

was performed

under

of

of dialyzing

incubated

digitonin

parts,

10 volumes with

treated,

815

freshly succinate

0.25

M sucrose

constant

prepared exposed

stirring fluid.

inner

The mem-

mitochon-

Vol. 50; No. 3,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Influence of iomc strength on potein release

AAT

St sH t2 f7J .:.:.:. .:.:.:: ::::::: ” :/q: /.j/jij ::::::: ::::::: :i:::::

4

S.+ 4l

-Digitonln-

-Swelling-

St

S-

: ?I 2 .:::::: :j:j:j:;gj;:;, .:::::: y::::: ::::j:j ::::.:. ;::::: .,:...: y:/:j ::>>:::::::: ..,... i:i:/: :.::::: ;;j:j:i ;::;:;: :/::,:: ::.:.:: J;z :.. .. .

4. 5, 6. 7, 8, 91

P

MSF IM Proteins

Fig.

Fig.

1.

2.

Fig.

1. Release of proteins by isolated inner membranes at equivalent ionic strength. Results are expressed as percent of total protein.Succinate 10 mM-acetate 30 mM-citrate 5 mM. IM : inner membrane. IMSF : intermembranal soluble fluid. Asp :aspartate ; Acet : acetate ; Glut : glutamate ; SOH : g-hydroxybutyrate ; Pyr : pyruvate ; oKG : a-ketoglutarate ; Succ : succinate ; Ma1 : malate ; Fum : fumarate; Citr : citrate ; OAA : oxalacetate.

Fig.

2. Release and binding of proteins and AAT by inner membranes isolated either by the digitonin or by the swelling-condensing-sonication method. Sucrose : in presence of sucrose alone (control). S+ : in presence of succinate containing intermembranal fluid. S- : in presence of sucrose dialyzed succinate containing intermembranal fluid. IM : inner membrane ; IMSF : internmmbranal soluble fluid.

dria

(25 mg of protein),

exposed 0.25

mitochondria

M sucrose

trifuged

swelling-condensing-sonication,

(25 mg of protein).

was run

at 60,000

or from

Thereafter

simultaneously.

x g for

A control

incubation

in presence

the preparations

15 min at 4 'C in a Spinco

816

succinate

model

were

L centrifuge.

of cen-

Vol. 50; No. $1973

Table

BIOCHEMICAL

I - Release

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

of Proteins and Enzymes : Comparison the Swelling Method

of the Digitonin

Sucrose

Subfractions Digitonin

and

Succinate

Swelling

Digitonin

Swelling

Protein

IM IMSF

88.6 11.4

73 27

83 I7

65 35

AAT

IM IMSF

96.2 3.8

88.8 11.2

23.4 76.6

33.7 66.3

MDH

IM IMSF

90 IO

50.5 49.5

51.3* 48.7

28 72

Results are expressed as percent oftotalactivity for AAT and MDH, and as percent of total protein. IM : inner membrane. IMSF : inter-membranal soluble fluid. Columns compare the results obtained either by the digitonin method or by the swelling-condensing-sonication method. *IO mM succinate.

The pellets tants

were

were

nations

homogenized

allowed

were

to stay

Suspended

in 3 ml water. overnight

made on the different

pellets

and supema-

at 4 OC. AAT, MDH and protein

determi-

fractions.

RESULTS AND DISCUSSION

Release of AAT, MDHand proteins Table tained

I compares

by either

They show that and that These responsible

this

results

is

the release,

membrane

reacts

proteins

and enzymes.

are

able

of the same order

exclude

in response

release

from

inner

membranes

or the swelling-condensingsonication

preparations

release

for

AAT, MDH and protein

the digitonin both

from isolated inner membrrmes

the possibility and thus

to release

suggest

to exogenous

817

in both

digitonin that

effector

method.

AAT, MDH and proteins

of magnitude for

ob-

to be the agent

the inner succinate

preparations.

mitochondrial by releasing

Vol. 50; No. 3, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

InfZuence of ionic strength on protein Fig. 1 shows the differential

release

effect

of diverse releasing effecters

at

comparable ionic strength as far as protein release is concerned. The results,

expressed in percent of total

release, suggest that the observed phe-

nomenais not due to changes in ionic strength only, for at equivalently ionic strength protein release is not equal in each assay. Binding of released AAl', bDH and protein

on isohted

For these experiments inner mitochondrial above described methods were incubated either branal fluid

inner membranes

membranesobtained by the in sucrose, or in intermem-

from succinate treated mitochondria dialyzed

or sucrose containing

succinate.

The results

against sucrose

in Fig. 2 show that under our

experimental conditions both types of inner nmmbraneshave still properties

rebinding

and that there is good agreement between the two preparations

although the isolation are quite different

procedures of the organels and their

in both cases. Thus inner mitochondrial

tained either by digitonin characterized

membranesob-

or sonication show releasing capacity

by rebinding potentiality.

seemto be directly

subfractions

and are

The observed phenomenondoes not

dependent upon the variation

of ionic strength of the

system.

ACKNOWLEDGEMENTS Wewish to thank Mrs. E. Bogner for skilled

assistance.

REFERENCES 1 A. WAKSMAN and A. RENDON,Biochem. Biophys. Res. Cormmuz.,42, 745 (1971). 2 A. RENDON and A. WAKSMAN,Eochem. Biophys. Res. Comvun., 42,1214 (1971). 3 C. SCHNAITMAN,V.G. ERWINand J.W. GREENAWALT,J.Cezz tiol., 32, 719 (1967). 4 G.L. SOTTOCASA,B. KUYLIENSTEBNA,L. ERNSTER and A. BERGSTRAND, Methods Enzym21.J 10, 448 (1967).

818

Vol. 50; No. 3, 1973

5 6 7 8

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMJNICATIONS

M. LEVY, R. TOURYand J. ANDRE,Biochim. Biophys. Acta, 135, L. HAREL, A. JACOBand Y. MOULE,Buzz. SOC. Chim. Bioioz. FT., A. RENDON and A. WAKSMAN,Biochem. Biophys. Res. Commua., 35, O.H. LOWRY,N.J. ROSENBROUGH, A.L. FARRand R.J. RANDALL,J. 193, 265 (1951).

819

599 ( 1967) . 39, 819 (1957). 324 (1969). Biol. Chem.,