Studies on the immunochemical and functional similarities among iron-sulfur proteins involved in mitochondrial steroid hydroxylations

Vol. 54, No. 2, 1973

STUDIES

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ON THE IMMUNOCHEMICAL AND FUNCTIONAL PROTEINS INVOLVED IN MITOCHONDRIAL Jeffrey The The

Received

Toxicology University

August

Center, of

SIMILARITIES AMONG STEROID HYDROXYLATIONS

IRON-SULFUR

Baron

The Department Iowa, Iowa City,

of Pharmacology, Iowa 52242

7, 1973

SUMMARY : The effects of a goat anti-adrenodoxin immunoglobulin fraction on the NADPHand NADH-dependent electron transport sequences in the mitochondria of steroidogenic tissues have been examined. The NADPH-cytochrome c reductase activities of sonicated mitochondrial preparations derived from bovTne adrenal cortex and rat adrenals and ovaries were inhibited in a similar manner in the presence of this antibody, while the inhibition of this activity in rat testicular mitochondrial preparations was less pronounced. The NADH-dependent reduction of cytochrome c catalyzed by these mitochondrial preparations was not affected by the antibody. These results indicate that, while they may not be identical, the iron-sulfur proteins involved in mammalian mitochondrial steroid hydroxylations exhibit immunochemical and functional similarities. The lone

oxidative

is

the

initial

adrenal,

testis,

reaction

are

require

NADPH

chain

cleavage

ovary

and

:

IlB-hydroxylase been

resolved

into

cholesterol been sulfur of

In Omura

the

intermediate cytochrome

which

(5)

between P-450.

demonstrated

the

both

cytochrome

of

deoxycorticosterone

0 1973 by Academic

by

of

system that

P-450

of

studies

and

cytochrome

for

catalyzed

Press, Inc.

bovine

of

be

human

com-

the

adrenals, to

which

(5).

has

Although

do the

and

similar

has

contain

the also

the

mitochondria

an

lib-hydroxylase

adrenocortical functions

flavoprotein

recent

requirement

(3)

not ironsystem

(6).

adrenodoxin

reduced

More

in

side-

protein

case to

mitochondria

adrenodoxin

and

NADPH-flavoprotein the

al.

tissues

ovarian

mitochondria et

the this

these

three

appear

testicular

these

replace

the

in

thus

Omura

system

mitochondria

proposed

which,

adrenocortical

components, can

an

in

cholesterol

rat into

oxidase;

systems

components

The

(2),

pregneno-

catalyzing in

(1).

resolved

protein

bovine

IIB-hydroxylase al .

of

Copyright

its

been

terminal

cleavage

adrenocortical

et

the

of

systems

activity

form

hormones

fraction

adrenocortical each

These

three

into

protein bovine

bovine

iron-sulfur

side-chain

resolved

for

to

steroid

enzyme

mitochondrial

have

system

of

The

the

adrenodoxin.

side-chain

biosynthesis

oxygen

of

an

cholesterol

placenta.

P-450,

and named

the

molecular

cytochrome

been

the

with

mitochondria

dehydrogenase; has

in and

systems

(4)

ponents

step

associated

cleavage

placental

of

an

dehydrogenase employing

adrenodoxin

by

mitochondria, as

in c

bovine 764

an

as

well

the

electron and

antibody

to

transfer oxidized adrenodoxin

NADPH-dependent as

adrenocortical

in

the

reductions

llB-hydroxylation mitochondria

(7).

BIOCHEMICAL

Vol. 54, No. 2, 1973

I

, mg

Figure

1:

Titration adrenocortical

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

/ IO

05 protem/mg

Ig

I 15

/ 20

mitochondrv3

NADPH-cytochrome mitochondria

2

protein

reductase activity goat anti-adrenodoxin

by

of

bovine immunoglubulin.

Bovine adrenocortical mitochondria were sonicated for 4 minutes in 0.5minute intervals. Each 2.2-ml reaction mixture contained 36.4 uM cytochrome c, 0.91 mM KCN, 0.1 mg of mitochondrial protein and 50 mM Tris-chloride buffer, pH 7.4. The reactions were initiated by the addition of 50 PM NADPH. Anti-adrenodoxin and preimmune immunoglobulin (lg) were substituted for buffer in the indicated concentrations. The solid line represents the activities in the presence of anti-adrenodoxin immunoglobulin, and the dashed line represents activities in the presence of preimmune immunoglobul in. In the absence of imnunoglobulin, NADPH-cytochrome 2 reductase activity was 60.7 nmoles cytochrome protein. -c reduced/min/mg

It

is

generally

tissues

assumed

function

cholesterol

in

that

the

examine

the

proteins

of

iron-sulfur

a comparable

catalyzed

summarizes

the

by

initial

manner

the

immunochemica

an

functional

an and

the of

employing

1 and ovar

in

mitochondria

results

adrenal,

proteins

in

side-chain

steroidogenic

cleavage

these

tissues.

antibody

to

similarities

testicular

other

of

This

communication

adrenodoxin among

to

the

iron-sulfur

mi tochondria.

METHODS Bovine of 160 For of were

and

rats,

studies human

mitochondria

(8).

Boyd

g Holtzman

and

employing

hours

immunoglobulin

serum,

respectively,

determined

by

cytochrome

5

the

the

ovaries

biuret

reductases

were

mitochondria,

after

the

fractions

were

injection.

were

prepared

(9).

determined

testes

were by rats

sulfate The as

765

The

previously

from

same

48

method 140-

method.

500

units

hours

and

anti-adrenodoxin immune

fractionation. activities

the

removed the

every

from

to

received

subcutaneously

last

ammonium

reaction

according

prepared male

(Squibb)

by

prepared and

mitochondria

gonadotropin 24

were

Adrenals,

testicular

chorionic

sacrificed

preimmune goat

adrenocortica

Simpson

and

and preimnune

Protein of

NADPH-

described

and (7)

was NADHafter

Vol. 54, No. 2, 1973

BIOCHEMICAL

I

I

0.5 mg

Figure

Ig

2:

The

I

1

I

15

IO

protem/mg

mlfochondriol

Titration mitochondria

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20

615’ mg

protean

NADPH-cytochrome 4 by goat anti-adrenodoxin

absence nmoles

conditions were the same as those of immunoglobulin, NADPH-cytochrome cytochrome 4. reduced/min/mg protein.

Figure

3: The

absence nmoles

the

~185

RESULTS

had

AND

The

from

of

of

bovine to the

-c

activity

was

Unlike

its

data

in

previous

y-globulin

a goat

with

for

a total

of (20

of

for

adrenal

1. In the was 90.0

of

rat n.

Figure activity

4 minutes

protein

rat

Figure activity

activity irnnunoglobuli

described 4 reductase

55 watts

1

In

using

the

64.1

was

a Branson

kc).

inhibitory

4 not

fraction

these

presence on

the

reductase

require studies

the

effect that

in

derived

Baron

be

inhibition

(7)

4 without

immune

in rabbit

766

production Figure to

of

preimmune

These

and

the

of

I,

the

sonicated NADPH-

No diminution

reductase effect

the

serum

are an

in

this

fraction.

activity, on

mitochondria, results

which

of

irnnunoglobulin

adrenocortical (5).

from

the

was

al.

in

fraction

NADPH-cytochrome

bovine

et

an

the

seen

mitochondria.

of

antibody

adrenodoxin of

can

produced of

isolated

elicits

As

immunoglobulin

activity in

adrenodoxin

mitochondria protein.

mitochondria

reductase

I show

homogeneous

anti-adrenodoxin

observed

Table

does

of

iron-sulfur

goat

NADH-cytochrome which

for

output

adrenocortical

the

adrenocortical

cytochrome

sonicated

an

m~tochandr~ol

DISCUSSION

antibody

addition

the

been at

immunization

purified

bovine

of NADPH-cytochrome 2 reductase mitochondria by anti-adrenodoxin

Sonifier

proteln/mg

activity immunoglobulin.

described c reductase -

conditions were the same as those of immunoglobulin, NADPH-cytochrome cytochrome protein. -c reduced/min/mg

mitochondria

model

an

Titration ovarian

reductase

Ig

the activity an

of activity

agreement

with

anti-adrenodoxin was

employed.

It

was

BIOCHEMICAL

Vol. 54, No. 2, 1973

Table

1.

adrenal

0 2.0

(immune)

2.0 adrenal

Rat

a The conditions except that NADH.

however,

inhibitory

that

to

protein

inhibit

of can

that

the

electron

seen

58.6

100

2.0

immune)

58.9

101

2.0

preimmune)

58.6

100

95.5

100

2.5

( immune)

95.8

100

2.5

(preimmune)

95.4

100

imnunoglobulin

goat

the

Table in

the

data

in presented

and in

results bovine Figure

was

presence

of

inhibition

strikingly

with

in

mitochondrial two

of

equal

found rat

to adrenal

bovine

the

and

antibody

‘767

that,

analogous

as

the demonstrate

NADPH-dependent exhibit the

immunochemical

functional

similarity

adrenals. 3 show

adrenoactivity

differs,

species

indicate

more

reductase

similar

these further

an

also

c

l-4, 90 pM

times

by

obtained

the

extent

involved of

was

catalyzed

results

2 are

5-10

than

NADH-cytochrome in

the

adrenals

these rat

the

observed

1 and

was

activity

activity

of

Although

proteins

Moreover,

“adrenodoxin”

was

1.

in

to

inhibition

for Figures addition of

fraction

reductase

Similar

Figures

iron-sulfur

fraction

-c reductase rabbit y-globulin.

c

mitochondria in

described by the

immunoglobulin

no

transport

The

96

2).

curves

similarity. of

56.6

NADPH-cytochrome

adrenal

be

preinnnune)

of

(Figure

titration

2.0

the

mitochondria,

rat

100

anti-adrenodoxin

mitochondria cortical

100

58.8

NADPH-cytochrome

goat the

59.0

107

immune)

same as those were initiated

concentration The

101

2.0

were the reactions

the

Percent of control activity

369.4 348.7

0

(lg)

c

100

0

testis

found,

(preimmune)

innnunoglobulin of the mito-

346.8

0

ovary

Rat

nmoles cyt reduced/minT mg protein

lg protein/mg mitochondrial protein

Tissue

Rat

RESEARCH COMMUNICATIONS

Lack of effect of goat anti-adrenodoxin on NADH-cytochrome c reductase activities chondria of steroidggenic tissues.a

mg

Bovine cortex

AND BIOPHYSICAL

to

the

results

Vol. 54, No. 2, 1973

BIOCHEMICAL

Figure

NADPH-cytochrome mitochondria

4:

Titration of rat testicular

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

c reductase by-anti-adrenodoxin

activity

of HCG-treated immunoglobul in.

The conditions were the same as those described for Figure that 0.5 mg of mitochondrial protein was present in each reaction In the absence of immunoglobulin, NADPH-cytochrome c reductase 5.0 nmoles cytochrome 4 reduced/min/mg protein.

obtained

with

rat

immunoglobulin

almost 2).

cytochrome

4

rat

the -et

untreated

-*al

(IO) contain

rats

with

of

increased

synthesis

observations, from

consistently However, were

studied, of

curve

an

testes

of the

mitochondria

of

protein.

from

In

reductase

activity

was

reductase

activity

in

NADH-dependent (Table

with

rat

reduction I).

testicular

It

is

observed of

readily

mitochondria

768

cytochrome apparent (Figure

the

with

these

and

was

not

fraction.

rats

-c

in

mitochondria low

of

protein,

results

extremely

testes

was

functionally

mitochondria

of

-c

fraction

protein

immunoglobulin the

NADH-

agreement

activity

anti-adrenodoxin

alter

iron-sulfur

NADPH-cytochrome

the

not

(HCG)

was

titration

testicular

gonadotropin

rats

this

and

although

reductase

NADPH-

mitochondria

no adrenodoxin-like

-c

of

iron-sulfur

mitochondria.

iron-sulfur

derived

the

NADPH-cytochrome

conditions

obtained

did

adrenal

untreated

immunoglobulin

inhibition same

such

antibody Thus,

that,

or

adrenal

immunochemically

chorionic

the

anti-adrenodoxin

the

in

rat

be

reported

NADPH-cytochrome

by

I). to

little

the

inhibited when

inhibition

(Table

human

of

the

appears

have

activity Indeed,

with

except mixture. activity was

anti-adrenodoxin

the

mitochondria. obtained

protein

the

inhibiting

ovarian that

activity

rats

of

mitochondria,

comparable

treatment

prepared

rat to

adrenal

reductase

mitochondria,

capable

mitochondria

to Mason

of

in

ovarian

similar

of

identical As

adrenal

was

-c reductase is

(Figure

of

bovine

fraction

cytochrome curve

and

I,

treated

with

elevated the

and

presence

(Figure

HCG

of

4).

-c was that

the

4)

differs

the

No

found

under

titration greatly

BIOCHEMICAL

Vol. 54, No. 2, 1973

from

those

for

observations

adrenal may

testicular

iron-sulfur

similarity

with

be more of may be

the

the

large

in

protein This

inability

of

reductase

activity

inhibit

of

to

these

mi tochondria

greater

detail

iron-sulfur

the

(7). the

of

may

inhibit

involved mammalian

(b)

a portion

(c)

the

iron-sulfur

protein

ovary, with

embedded most

the

relatively

of

the

the

NADPH-cytochrome

will

testis

mitochondrial

the

for

the 5

mitochondria system

while

solubilized

undoubtedly

in

among occurring

it from

define

similarities

hydroxylations

steroidogenic

comparison

the

thus

explanation

functional

steroid

in

and all in

may

membrane

likely

reductase

studies and

in

antibody;

adrenocortical of

thus

mitochondria

react

be the

bovine

Additional

might

testicular

completely

activity

and

rat

and

to

is

immunochemical

proteins

mitochondria

be able

sonicated

proteins

of

adrenal

which

the

the

immunochemical

mitochondrial

the

possibility

completely

or

testicular

not

molecules

antibody

of

activity

of

may

latter

the

effects

the

These (a)

minimal

iron-sulfur

protein;

l-3).

possibilities: only

ovarian

RESEARCH COMMUNICATIONS

(Figures

following

exhibit

reductase

proteins molecule

iron-sulfur membrane.

may

iron-sulfur

situated

the

inhibitory -c

iron-sulfur

antibody

mitochondria of

and

the

the

differently

with

one

adrenal to

involve

ovarian

to

NADPH-cytochrome

not

can

due

protein

resistent

the

and

be

AND BIOPHYSICAL

the

in

the

tissues.

ACKNOWLEDGEMENTS The

author

throughout Public

these Health

thanks

Mrs.

studies. Service

Cathy This

Grant

Hayford research

for was

her

expert

supported

technical in

part

assistence by

U.S.

GM 12675.

REFERENCES

I. 2.

1:

5. 6. 7. a. 9. 0.

Sulimovici, S.I. and Boyd, G.S., Vitam. Horm. (New York), g, 199 (1969). Simpson, E.R. and Boyd, G.S., Biochem.Biophys. Res. Commun., 8, 945 (1967). Sulimovici, S. and Boyd, G.S., Steroids, 12, 127 (1968). Mason, J.I. and Boyd, G.S., Eur. J. Biochem., 21 308 (1971). Omura, T., Sanders, E., Estabrook, R.W., Cooper, D.Y. and Rosenthal, O., Arch. Biochem. Biophys., 117, 660 (1966). Kimura, T. and Ohno, H., J. Biochem. 63, 716 (1968). Baron, J., Taylor, W.E. and Masters, B.S.S., Arch. Biochem. Biophys., 150, 105 (1972). Simpson, E.R. and Boyd, G.S., Eur. J. Biochem., 2, 489 (1971). Gornall, A.G., Bardawill, C.J. and David, M.M., J. Biol. Chem., 177, 751 Mason, J.I., Estabrook, R.W. and Purvis, J.L., Ann. N.Y. Acad. ST.,

-212 (1973).

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