Substrate-induced efflux of anions from bovine adrenal cortex mitochondria and its relationship to steroidogenesis

Vol. 35, No. 5, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

"SUBSTRATE-INDUCED EFFLUX OF ANIONS FROM BOVINE ADRENAL CORTEX MITOCHONDRIA AND ITS RELATIONSHIP TO STEROIDOGENESIS" Evan R. Simpson

Department of Biochemistry of Texas Southwestern Medical Dallas, Texas, 75235

University

Received

In recent measuring

years,

the reactions

Walter,

Mehlman,

Carefully permeability

towards

Ca++ ions

( McCarthy

other Scholz

mechanisms

pyridine

exist

the mitochondrial tissues

(Estabrook

and Thurman,

1968).

of adrenal

cortex

chondria the normal

respiratory

and Devlin,

cortex

nucleotides

mitochondria

Consequently

membrane,

as has been proposed

chain,

transfer

it

the net

are

1958;

problem

Paetkau

Boxer

because electron

display in

and

would

for

transport

1961;

interest contain, chain

that

equivalents

mitochondria

Abbreviations: aGP - a-glycerophosphate; DHAP - dihydroxyacetone a-glycerophosphate dehydrogenase; LDH - lactate dehydrogenase; DOC - deoxycorticosterone. 765

of

be expected

and Devlin,

they

an im-

the absence

of reducing

is of particular

concerned, a second

equiva-

and to a desire

(Lardy,

to effect

This

due primarily

of reducing 1961),

when incubated

1968).

and Sacktor,

is

to

1967).

adrenal

and Peron,

This

in gluconeogenesis

and Lardy,

bovine

has been devoted

in the transfer

(Boxer

involved

Walter

prepared

involved

membranes

to understand

of effort

from mitochondria.

mechanisms

mitochondrial

1965;

amount

of substances

in shuttle

across

across

at Dallas,

pyruvate from bovine adrenal cortex mitomalate respectively, has been studied. With the DHAP formed leaves the mitochondria. With pyruvate formed leaves the mitochondria. These that the "a-glycerophosphate shuttle" and the in bovine adrenal cortex.

an increasing

the efflux

to interest

shuttle

School

May 9, 1969

SUMMARY: The efflux of DHAP and chondria, incubated with aGP and aGP as substrate, virtually all 75% of the malate as substrate, observations support the concept "malate shuttle" can be operative

lents

and Rene Frenkel

of

Williamson, when mito-

in addition

to

responsible

for

phosphate; GPDH TRA - triethanolamine;

Vol. 35, No. 5, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

steroid

mixed-function

Sanders,

Estabrook,

second

electron

comb,

1955),

source

Cooper

and it

within

the mitochondrial

into

(Simpson

transfer

the mitochondria

1966;

Suzuki

in bovine

(Simpson

malic

adrenal

1968)

of cytoplasmic

generated

This

and Lipsmost

is malic

likely

enzyme

Further,

in bovine

c.onstitute

1967).

cortex,the

1969).

that,

Omura,

NADH (Sweat

oxidases

and Estabrook,

enzymes

1966;

and Kimura, than

mixed-function

and Estabrook,

(fig.

and Nelson,

NADPH rather

shown that

and mitochondrial

the net

(Harding

requires

the mitochondria

cytosol

effect

chain

has been

has been postulated the

reactions

and Rosenthal,

transport

of NADPH for

located

oxidase

a 'malate

NADPH reducing

it

adrenal

cortex,

shuttle'

to

equivalents

1).

/I

CYTOSOL

MITOCHONDRION

'malate shuttle' of bovine adrenal cortex, showing how Figure 1. Proposed the cytosol and mitochondrial malic enzymes could function together to effect net transfer of NADPH reducing equivalents into the mitochondria.

The purpose induced

efflux

of this of anions

report

of DHAP when aGP is

malate

is

adrenal METHODS: cribed with

shuttle'

two examples

cortex and (b)

observations

and the

'malate

mitochondria: the efflux

support shuttle'

of substrate- (a) of pyruvate

the concept are

the

operative

that

when the

'a-

in bovine

mitochondria.

Bovine

adrenal

cortex

(Cammer and Estabrook, 0.25M

adrenal

the substrate, These

the substrate.

cortex

to demonstrate

from bovine

efflux

glycerophosphate

is

sucrose

containing

mitochondria 1967)

except

1% bovine

were that

prepared

the mitochondria

serum albumen, 766

as previously

pH 6.8.

were

deswashed

The mito-

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 35, No. 5, 1969

chondria

were

incubated

15 mM TRA-HC1 buffer

at 24'C pH 7.4,

5mM MgC12.

Efflux

by coupling

the enzymic

chondria which

was measured

measurement measured mixture

about were

filter

total

as it

leaves

Luft

2OmM KCl,

pH 7.4,

was measured

Hedman,

of DHAP efflux,

efflux,

filters

and directly

the mito-

and Ernster,

of aliquots (Millipore

Reactions

were

on neutralized

GPDH and NADH were

LDH and NADH were

on top of a 0.65

1963)

micron

aliquots

Efflux

Bedford,

filter. with

using

The filtration

by the enzymatic

the

incubation

Massa.),

7% perchloric

for

was also

of the mitochondrial

Corp.,

stopped

used.

used;

time

acid

a was

and assays

methods

of Estabrook

(1962).

RESULTS AND DISCUSSION: adrenaL

sucrose,

buffer

the mitochondria

of the substance

by filtration

5 seconds.

and Maitra

from

of NADH (Suranyi,

of pyruvate

performed

phosphate

0.25M

fluorometrically.

on Millipore

0.8 micron

1OmM potassium

reduction

the measurement

directly

containing

of substances

to the oxidation

Thus for

in a buffer

cortex

mitochondria

DHAP in aliquots

Mw GPQH III

Fig.

UADH

2 shows DHAP formation supplied

of a reaction

with mixture

&P I

aGP.

and efflux

The solid

incubated

from

triangles

in the absence

bovine show of GPDH

NADH 1 __

40

IADH

I

Figure 2. Formation and efflux of DHAP from bovine adrenal cortex mitochondria incubated with aGP. Final concentrations: Mitochondria-2.1 mgfml; aGP - 800 FM; NADH - 57 pM; GDPH - 6.7 pg/ml. The NADH trace was taken directly from the fluorimeter recording. ‘76’7

Vol. 35, No. 5, 1969

and NADH. through

BIOCHEMICAL

The open

triangles

a Millipore

suspension

The rate

efflux.

the

NADH was added.

oxidation

This

aliquot

was initially

down to the previous

indicates

that

DHAP accumulated

system

was not

chondria.

serving

These

the

incubation

maintained

a low

mixture

filtrate

in the experiment

When the

NADH was oxidized,

NADH.

It

then

the rapid

dropped

phase

The rate

within

the mitochondria shuttle

confirmed

with

of rate

of NADH oxidation this

time

period

enzyme

of DHAP out of the mito-

by measurement

in the absence

case,

between

of the

until

level

of DHAP in aliquots

In this

the difference

steady-state

of DHAP

aliquot

coupled

GPDH and NADH.

from both

the

over

the DHAP

total

reducing

and system.

second

addition

the same time

the direct

(Simpson

measurement

min. -1 mg. protein-l,

was about

2 mpmoles

may be important

in adrenal

steroidogenesis

of

interval

as

cortex

mg. protein

as an energy

mitochondria

and Estabrook,

and the

1969;

and the steady-state

-1 .

source

The a-

in adrenal

may be inhibited Stoppani,

rate

cortex,

during

De Brignone

and

1968).

Cammer, that

pyruvate

Cooper

and Estabrook

malate-supported

formation

NADPH for bovine

diffusion

the DHAP accumulated

was 4 mkmoles

as NADH oxidation

showed

the

rate

the oxidation

during

the extra-mitochondrial

conducted

of DHAP formation

glycerophosphate

Brignone,

the mitochondria

containing

to the

burst

to the

the

and then

rapid

cortex

(1967)

and Simpson

118-hydroxylation

due to the action

llg-hydroxylation.

adrenal

-

of NADH oxidation.

of NADH oxidation level

a second

initial

passed

fluorometri-

of NADH was oxidized,

outsjde

were

added

NADH followed

rapidly

were

the DHAP was extra

with

in agreement

level,

all

was in good agreement

to facilitate

observations

of the

This

and that

almost

which

GPDH and NADH were

oxidized

rate.

when no NADH was present,

from

experiment,

initial

aliquots

can be seen that

and the oxidation

of this

After

slowed

It

In a parallel

mitochondrial tally.

show DHAP in parallel

filter.

mitochondrial.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. mitochondria

and Estabrook

of DOC was accompanied

of mitochondrial 3 shows pyruvate supplied 768

(1969)

with

malic formation malate

by

enzyme in supplying and efflux

and DOC.

from

The solid

Vol. 35, No. 5, 1969

LDH MW NADN 111

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mrlata I

NADH I

DDC 1

l

total

a

filtrate

in

presence

Of

NADY+LDH

mlnr.

Figure 3. Formation and efflux of pyruvate from bovine adrenal cortex mitochondria incubated with malate and DOC. Final concentrations: mitochondria 1.1 mg. protein/ml; malate - 3.1 mM; DOC - 140 p.M; NADH 78.5 FM; LDH - 5kg/ml. The NADH trace is the fluorimeter recording from which the contribution of endogenous pyridine nucleotide has been subtracted.

triangles which

show total was incubated

DOC resulted oxygen

pyruvate

in the absence

in a stimulation The open

uptake.

were

triangles

which

passed

pyruvate

was extra-mitochondrial.

of LDH and NADH, there

sulted

by a rate that

rapid

pyruvate

was accumulating

facilitating

ment of pyruvate to the difference in

the absence

of oxidation

of the nucleotide

when no NADH was present, was not

burst

of a second

the diffusion in the presence between

total

of LDH and NADH.

of the

the mitochondria

out

When all

reduced

769

to the

nucleotide

rate, during

again

levels

NADH was oxidized,

re-

followed showing

the interval

coupled

in the reaction the

well

enzyme system Measure

of the mitochondria.

of LDH and NADH gave and filtrate

75% of the

in the presence quite

the extra-mitochondrial of pyruvate

increased

of NADH once again

at the previous

outside

and that

conducted

aliquot

of

in parallel

Thus about

of NADH corresponding

Addition

with

concentrations

filters.

mixture

addition

concomitant

In an experiment

was oxidation

of oxidation

formation

Millipore

of a reaction As expected,

of LDH and NADH.

show pyruvate

through

in the filtrate. in an initial

in aliquots

of pyruvate

aliquots

pyruvate

concentration

that

corresponded

mixture

incubated

pyruvate

Vol. 35, No. 5, 1969

accumulated

until

state

over

level

BIOCHEMICAL

the the

second

AND BlOfliYSlCAL

addition

same time

of NADH.

interval

It

as the

RESEARCH COMMUNICATIONS

then

initial

dropped

to the steady-

rapid

burst

of NADH

1968;

Simpson

oxidation.

It has previously Estabrook,

1968)

that

118-hydroxylation, enzyme. cortex

shuttle' to effect

and thus

would

the net

and Caldwell,

+ CO2 + NADPH are of bovine

reported

are consistent involves

together

Supported

(Peron

in the presence

mitochondria

cortex

shown

pyruvate

The observations

'malate

adrenal

been

adrenal pyruvate

on

with

the scheme shown

of NADPH across

the mitochondrial

under

the control

efflux

and mitochondrial

transfer

bring

cortex

here

the cytosol

by U. S. P. H. S. Grant

a good source

steroid

NO.

from in Fig.

for

malic

bovine 1.

adrenal This

enzymes working

the mitochondrial

hydroxylase

of extra-mitochondrial

of malate

cytosol

malic

and

systems

generation

membrane, of bovine of NADPH.

GM 16488.

REFERENCES Boxer, G, E. and Devlin, T. M., Science, 134, 1495 (1961). R. W. in "Functions of the Adrenal Cammer, W., Cooper, D. Y. and Estabrook, Cortex" ed. McKerns, Appleton-Century-Crofts, 943 (1968). Cammer, W. and Estabrook, R. W., Arch. Biochem. Biophys., 122, 721, (1967). Estabrook, R. W. and Maitra, P. K., Anal. Biochem, 3, 369 (1962). Estabrook, R. W. and Sacktor, B., J. Biol. Chem., 3, 1014 (1958). Harding, B. W. and Nelson, D. H., J. Biol. Chem., 241, 2212 (1966). Lardy, H. A., Paetkau, V. and Walter, P., Proc. Natl. Acad. Sciences, 53, 1410 (1965). Mehlman, M. A,, Walter, P. and Lardy, H. A., J. Biol. Chem., 242, 4594 (1967). of the Adrenal Cortex", ed. McCarthy, J. L. and Peron, F. G., in "Functions McKerns, Appleton-Century-Crofts, 261 (1968). Omura, T., Sanders, E., Estabrook, R. W., Cooper, D. Y. and Rosenthal, O., Arch. Biochem. Biophys., m, 660 (1966). Peron, F. C. and Caldwell, B. V., Biochim. Biophys. Acta, 164, 396 (1968). Simpson, E. R. and Estabrook, R. W., Arch. Biochem. Biophys., 126, 977 (1968). Simpson, E. R. and Estabrook, R. W.,Arch. Biochem. Biophys., 129, 384 (1969). Stoppani, A. 0. M., De Brignone, C. M. C. and Brignone, J. A., Arch. Biochem. Biophys. 127, 463 (1968). Suranyi, E. M., Hedman, R., Luft, R. and Ernster, L., Acta Chem. Stand., 11, 877 (1963). Suzuki, K. and Kimura, T., J. Biol. Chem., 242, 485 (1967). Sweat, M. L. and Lipscomb, M. D., J. Am. Chem. Sot., z, 5185 (1955). R. and Thurman, R. G., Bari Symposium (1968) in Williamson, J. R., Scholtz, press.

770