2,4 dinitrophenol and the permeability of mitochondria

Vol. 28, No. 5, 1967

2,4

Dinitrophenol Henry

and the

Tedeschi

Permeability

and Helen

of Mitochondria

Wilson

Horn

Department of Biological Sciences, State University New York at Albany, Albany, New York 12203

Received

July

26, 1967

Bielawski, cal

Thomas

resistance

(DNP).

little

as 0.4% of

effect

was found effect

attributed

to

aperture

and the

the

and that

value.

required

to uncouple

the

by the the

The concentration

The difference

of

about reduces

mM.

difference

experiment

Mitchell

coupling

(e.

hypothesis

DNP by the

in the

in the

electri-

addition

of to as

for

maximal

a half

concentration

solution

between

the

was considered

g. Mitchell, predicts

study

on this

question.

was used.

required was

surrounding

the

mitochondrial

and the

out

of

to malonamicle

concentrations

of

DNP (0.1

concentrations,

for

However,

in this

it

one of the

was recognized

permeability

laboratory

in mitochondrial

our

of

elements

of oxidative-phosphorylation

in proton

The photometric

The results

permeability

>.

an increase

of variations

to be a test

hypothesis 1966

carried

tematic

experiments

and sodium

in the

acetate

mM).

Substantive

example,

Parker

course

of a sys-

have

of Tedeschi preclude

some bearing

and Harris

a large

change

induced

by relatively

uncoupling

takes

(1965)

the

only.

permeability

technique

that

reports

(1958) in the high

place

at

50% uncoupling

mMDNP. In the ing

2,4

resistance

oxidative-phosphorylation lipid

in composition

chemi-osmotic

Experiments

lower

uptake

effect

a decrease

membrane.

This of

original

report

brought

The DNP-induced

the

the

(1966)

bilayers

to be 0.5

this

synthetic

and Lehninger

of phospholipid

dinitrophenol

for

of

present

on whether

or not

study, the

the

changes

particles

have

752

in volume

with

been exposed

time

differ

to DNP (the

dependDNP

by .03

BIOCHEMICAL

Vol. 28, No. 5, 1967

preparations related

swell changes

a.

less).

behave

such

of the

Mitochondria they

However,

in volume,

Rationale

swell

must

changes,

it

simple

is

and Harris,

also

(1952), (b)

to calculate

Tedeschi,

1959 ).

see Tedeschi

the

equations

Tedeschi,

needed

volume

method

of following

law.

assumes

Bentzel

e_t &.,

From the

change, (see

osmotic

behavior,

permeability

involved (e.

it

Jacobs

to water; (c)

diffusion

in this

discussed

volume

directly

1966 )and

the

transferred

the volume

(a)

a high

been previously

(Tedeschi

of all

the

same.

The reciprocal

the

mitochondrial

used

to measure

and Harris,

rapid

index

g.

rationale

Jacobs

and

(1952),

changes

1958 ) presents

in mitochondrial

solutions

optical

active

osmotically a simple

volume

(calibrations

of the

osmotically

mitochondrial

and direct

as long

as the

and experimental)

density

is directly

active

is

kept

proportional

to

volume.

Procedures

b.

The isolation (Tedeschi,

1959 ).

viously

presented were

and the

photocell

molal

of mitochondria The details (Tedeschi

recorded

sucrose 0.05

with of

of 580 m . P

medium.

of

Since

1955 ),

of solute

constants

The assumptions

method

refractive

0.3

(1958),

amount

kinetics

(i.e.

solute.

1959 >.

relative

length

times

of

of water.

the

approach

all

have

The photometric

light

to un-

solute.

and Harris,

by that

permeability

at

by Fick's

g. Tedeschi

From the

This

and Harris

approximated

internal

penetration

to calculate

1958 >.

equilibrium

(e.

be followed

possible

osmotic

can be attributed

of

to the

system

of penetrant

(Tedeschi

effects

as i leakage

in response

entrance

rather

these

method

as an osmotic

is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

the

photometric

and Harris,

1958 ).

a Beckman

a Coleman

Each 0.10 and 0.01

ml containing

was carried

Jr.

linear-log

2,4

as previously method

recorder.

of mitochondrial

, pH 7.4, dinitrophenol

753

have

was diluted (DNP)

described been

The changes

spectrophotometer

ml sample M tris

out

in transmitted

The light were

pre-

used

source

at a wave-

stock

suspension

to 1.10

ml with

in 0.01

M tris

were

in the

same

added

to

Vol. 28, No. 5, 1967

bring

the

minutes

DNP concentration

at

or 0.137 were

31 + l°C.

molal

added.

maintained cooled

at

were

2 + l°C

systematic

error

order

of the

control,

ized

throughout

cedures ally

but

active

penetrant

figure.

penetrants

the

conclusion

warmer

than

the

original

absence

volume

where

of the

arbitrarily

as unity

(see

mM DN:

0 time

of

were was

the

the were

to

reported

Tedeschi,,

random-

identical

pro-

correspond

in 0.336

suspen-

To avoid

determinations

volume

0.1

solutions

of the mitochondria

The volumes

the mitochondrial

to the

determinations

subject

5

malonamide

containing

solutions.

were

for

spectrophotometer

penetrant

The controls

of DNP.

M tris

the

and calibration

experiment.

0.3 molal

and calibration

by deteriorations

experimental

incubated

corresponds

determinations

At

were

either

0.01

Penetrant

the

introduced

suspensions

sucrose,

of the

During

in the

taken

molal

RESEARCH COMMUNICATIONS

3 ml of

air.

the

is

These

incubation

addition

in the

24 + 2°C.

mM.

0.0697

of

by circulating

sions

this

Na acetate,

indicated

AND BIOPHYSKAL

to 0.1

After

The time

penetration

any

BIOCHEMICAL

to osmotic-

osmolal

of a non-

1958 ).

RIWJLTS Previous eral

work

(Tedeschi

non-electrolytes

(low

unaffected

by swelling.

permeability

or the

kinetics

of

the

V is

A plot

surface

volume

area

change

amounts

does (2)

that

weight,

The kinetics

osmotically

are

foilow

penetration lipid

consistent for

the

the

relatively

available

active

permeability

(see

Fig.

1A).

a slope Equation

concentration not P A t

involve =

volume

constant

of a non-penetrant

considerable which

molecular

of VL vs t provides

meability cant

the

P the

volume,

indicates

with

soluble)

expressed

sevremains

no change

penetration.

pattern

of

in either

Accordingly,

the

in equation

(1):

V2 = vo2 + 2PAV,t

(1) where

(1959))

of this

at

and A the (SPAV,) (1)

present

sucrose

in

assumption

surface

which

makes the

are

the

754

V, is (see

the

medium,

1952

of per-

that Since

initial

Jacobs,

an indication

assumption

final

no signifithere

is

Equation

a

(2)

be used.

- CiVo

CO2

is

area

externally.

should

(Co + cs>

(t),

any time

In

(

civo

+ c,v

CiVo + CoV

1

>

=

f (V)

).

Vol. 28, No. 5, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OK TIME

(Sec.)

TIME

Kinetics of swelling units (see text).

Fig. 1. relative

Malonamide penetration, The standard deviation time (set) 3 Exp. 0.09 Control 0.07

A.

B. Malonamide is time.

An analogous equation

penetration

equation

(2),

of a typical

experiment.

external

VL is

presented

concentration

expressed

in

time.

18 0.07 0.07

The function

has been previously

Co is the

V is

experimental and control. of each V is as follows: 6 9 -- 12 0.08 0.06 0.08 0.07 0.06 0.07 (control).

(Sec.)

of equation

(2),

by Schiodt

F (V)

(1933).

of non-penetrant,

C

In the

con-

S

centration a slope

of which

a number

penetrant corresponds

of practical

be obtained the

or

(2) provides

apparent

penetrability

this

to PA (Fig. problems

A plot

In practice

1B).

since

it

of f (V) vs time

depends

equation

entirely

provides (2)

on V, which

presents cannot

directly.

In (1)

and Ci = Co + C,.

equation

initial

phase

of

the

approximately

was used

(PA,

swelling the

(V lower same answer

see below)

nevertheless

is

because

755

somewhat of

its

than (Table

2),

either 1).

higher simplicity.

with

equation

Although equation

the (l),

From equation

Vol.28,No.

$1967

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE1 Comparison penetration. deviations.

of the use of equation (1) with equation (2) 4 independent experiments; the deviations PA is defined in the text. Equation (1) 0.16 + 0.03

PA

(1)

it

is

clear

straight

line.

illustrated for

The slope

relative

1A.

and the

purposes

of this

120 seconds).*

In

in

solution

osmolal

and PA given since have

it

are

gives

leaked

out

this

results

(e.

paper, of

To avoid

Table

a non-penetrant value.

as Y eq (Table

osmotically It

this

2).

appropriate

use

taken

reason,

The data

are

in

volume

after

of mitochondria as unity;

active

A, V.

is useful

internal

to decide

the

left

as PA (defined

volume

is difficult

is

of arbitrary were

volume

The equilibrium

procedures. For

the

expressed

has been

in a

This

parameters

active

result

Vo2.

V, and Veq (the

as to whether the

the

2) are

osmotically

to this

should

intercept

A, these

g.

the

has occurred.

arbitrarily

).

as penetrability),

information during

1959

time

by substituting

in specifying

relative

when equilibrium taken

Tedeschi,

paper

of

be ZPAV, and the

P can be obtained

in particular units

.336

of V2 as a function would

V. and A (see

assumptions,

Equation (2) 0.11 + 0.02

a plot

in Fig.

values

for

that

for malonamide are standard

volume

solutes precisely

at 120 seconds

expressed

in terms

of PA, V,

TABLE 2 Kinetic parameters in the presence and text. Mean of four independent standard deviations. Penetrant Malonamide Na acetate

Xwith

the

a. b. a. b.

assumptions

Control Exp. Control Exp.

and absence experiments.

PA 0.16+0.03 0.11+02 0.08+0.03 0.05+0.01

previously

vo 0.8w.20 0.749.16 0.74s.16 0.61+0.04

used

756

P-1:4

X lo-'

of DNP (see Equation (1)) The deviations are

veq 2.7eO.19 2.025.16 2.6w.11 1.58+0.10

cm&

for

was

malonamide

BIOCHEMICAL

Voi. 28, No. 5, 1967

and V eq in Table

2.

permeability

occurs.

chosen

their

the

since

simple

system

In all

the

swelling

is

of

from

countered

(Fig.

in the

to test

other

whether

a change

Na acetate.

to be slow

system

study

IA and Table

samples

control

penetrability

incubated report

value.

enough

are

a change

by about

These

in

were

to be followed

possibly

brought

about

K+ in the

presence

of DNP is well (1953)

but by the

with

and Berger

this

of

rate

with

the

difference result

internal

in a number

of

the

resistance en-

direction.

However,

the

the

in permeability

opposite

rather leakage

that

The results

changes

30-40X.

documented

clear

in electrical

Therefore

in permeability

is

DNP.

and in the

by 25-402.

shrinkage

and Mudge

with

a decrease

smaller,

decreased

2) it

The apparent

is decreased

is also

Stanbury

the

was known

experiments

the

to represent

chosen

used.

by this

ume (V,,)

were

One is malonamide,

phospholipid

99.6%

parent

Two penetrants

penetration

lower

bimolecular

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The apfinal

vol-

is not

likely

of a concomitant ions.

The loss

of experiments

(e.

of g.

(1957)). CONCLUSIONS

The results sistent

with

dria

in the

This

work

(P-183)

obtained the

concept

presence

was supported

with

both

malonamide

of a general

of 2,4

increase

and sodium in

acetate

the permeability

are not

con-

of mitochon-

dinitrophenol.

by grants

from

the American

Cancer

Society,

Inc.

and USPHS (GM 13610-02). REFERENCES

C. J. Bentzel, R. Sha'afi and A, K. Solomon, Membrane and Transport Phenomena, 10th annual meeting, Biophysical Society (1966) M. Berger, Biochim. Biophys. Acta 23:504 (1957) J. Bielawski, T. E. Thompson and A. L. Lehninger, Biochem. Biophys. Res. Comm. 24:948 (1966) M.H. Jacobs, in Modern Trends in Physiology and Biochemistry, E.S.G. Barron New York (1952) p. 149 ed., Academic Press, P. Mitchell, Biol. Rev. 41:445 (1966) V. H. Parker, Biochem. J. 97:658 (1965) E. Schiodt, J. Gen. Physiol. 16:977 (1933) S. W. Stanbury and G. H. Mudge, Proc. Sot. Exp. Biol. Med. 82:675 (1953) H. Tedeschi, J. Biophys. Biochem. Cytol. 6:241 (1959) H. Tedeschi and D.L. Harris, Arch. Biochem. Biophys. 58:52 (1955) H. Tedeschi and D.L. Harris, Biochim. Biophys. Acta 28:392 (1958)

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