Soluble cytochrome P.450 from bovine adrenocortical mitochondria

BIOCHEMICAL

Vol. 43, No. 4, 1971

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SOLUBLE CYTOCHROMEP.450 FROM BOVINE ADRENOCORTICAL MITOCHONDRIA" Saburo Isaka The Russell

Received

March

and Peter

F. Hall

Grimwade School of Biochemistry, Victoria, Australia.

University

of Melbourne,

24, 1971

SUMMARY A method is given for solubilizing at least one species of cytochrome P.450 from bovine adrenocortical mitochondria. The soluble P.450 supports side-chain cleavage of cholesterol but not lip-hydroxylation, thus clearly separating these two activities for the first time. Soluble P.450 shows a substrate-induced difference spectrum with both cholesterol and ll-deoxycorticosterone; these difference spectra are opposite in type (peak at 420 nm) to spectra observed when these substrates are added to crude P.450. It is suggested that there are at least two cytochromes P.450 in bovine adrenocortical mitochondria with distinct enzymatic activities namely, side-chain cleavage and lip-hydroxylation. The biosynthesis three

mitochondrial

of adrenal enzyme systems,

of the steroid

ring

the

of cholesterol.

side-chain

require

at specific

TPNH which

system consisting

steroids

reduces

involves

each responsible

carbon

atoms,

cytochrome

P.450

combines with

steroid

substrate

about

hydroxylation.

(e.g.

how the

specificity

alternatives

there

are several

there

may be one cytochrome

an electron

It

mitochondrial

it

side-chain could

cytochromes

which

serves

several

remains

the

to

is achieved etc.).

Among that

or alternatively protein

* These investigations were supported by the National and Medical Research Council grant No. 69/1011. 747

transport

and with

be suggested P.450

and

(Omura et al.,

oxygen

of each hydroxylation

Cl1 as opposed to the cholesterol

a number of plausible

'18 reactions

and non-heme iron

Reduced cytochrome

hydroxylation

namely Cll,

P.450 via

1966).

determine

least

for

These hydroxylation

of a flavoprotein

to bring

at

(hydroxylase)

Health

Vol. 43, No. 4, 1971

enzymes.

BIOCHEMICAL

These alternatives

cytochrome

P.450

describes

the

from bovine

will

is obtained

'be difficult

in soluble

solubilization

adrenal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to resolve

form.

and properties

This

until

communication

of cytochrome

P.450

cortex. EXPERIMENTAL PROCEDURE

Cytochrome Horie

(1969)

supernate

up to and including

was treated

The precipitate (pH 7.6) for

with

(Pi)

and a supernate

(called

S2-35)

(0.1 M pH 7.0)

against

for

at least

centrifugation

P.450 was eluted

x g for

was dialysed

potassium

preparation

is

Side-chain

called

'before soluble

cleavage

mM

S2-80).

against

potassium (0.1 mM); the

S2-80 was dissolved The solution

overnight.

at O" and also

phosphate

0.1 mM pH 7.6.

0.1 mM dithioerythritol

night

phosphate

and dithioerythritol against

0.1

it

remained

after

120 minutes.

to calcium

by potassium

P2 was

containing

to the naked eye;

7 days on standing

S2-80 was applied

a precipitate

(NH4)2S04 in two

experiments.

the same buffer

at 105,000

was

dithioerythritol

red and clear

dialysis

and 35-80%(called over

containing

P.450 was used in these

with

phosphate

studies,

(0.1 M, pH 7.0)

in and dialysed

of S2-80 was brownish clear

phosphate

O-35% sat.

to give

use in these

S2 was precipitated

in and dialysed

at pH 7.0.

After

the suspension

Before

(S2).

S2-35 was suspended

dialysed

and 1 mM EDTA.

60 minutes

and

The middle

(NH4)2S04 to 60% saturation

x g for

suspended in potassium dithioerythritol.

to Mitani

step 8.

the same buffer,

at 105,000

phosphate

their

10 mM cysteine

3 hours against

fractions:

according

was suspended in 0.05 M potassium

containing

centrifuged (P,)

P.450 was prepared

gel from which

0.3 M containing

The eluate

phosphate

(0.1

(soluble M pH 7.0)

use in the present

studies;

EDTA 1 mM P.450) containing this

P.450.

(cholesterol-3

748

H +

pregnenolone-3H)

iol.43,No.4,

BIOCHEMICAL

1971

and lip-hydroxylation

et

al.,

C) were measured as described

according

DOC-14C +

elsewhere

Amounts of P.450 were calculated

1970).

heme content

14 C i.e.

(ll-deoxycorticosterone-

14

corticosterone-

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(Young

on the basis

of

to the method of Omura (1967). RESULTS

Substrate-Induced

Difference

P2 and S2-35 show a positive with

DOC, soluble

this

substrate.

P.450 Moreover

Table

Spectra:

spectral

shift

shows a positive the

soluble

1 shows that

while

at 390 nm (type

shift

I)

at 420 nm (type

P.450 shows

a

1I)wi

peak at

small

TABLE 1 SUBSTRATE-INDUCED DIFFERENCE SPECTRA FOR P.450 FRACTIONS

Substrate

Spectral

Difference

390 - 420 Soluble P.450: Cholesterol DOC 20~OH Cholesterol Pregnenolone Corticosterone s2-35: Cholesterol DOC 20a-OH Cholesterol Pregnenolone Corticosterone

+ 0.043 0 0 0

P2: Cholesterol DOC 2Oa-OH Cholesterol Pregnenolone Corticosterone

+ 0.050 0 0 0

(nm)

(AA/pmole 420

390

+ + -t + +

0.005 0.022 0.145 0.091 0.018

+ +

0 0 0.190 0.092

0

0

-

P.450) (nm)

+ 0.003 0 0 + 0.079

+ 0.045 0

Substrate difference spectra were measured by adding steroid substrates dissolved in propylene glycol to cytochrome P.450. Final concentration of substrates was 5 w. Spectra were read after standing for 30 minutes. 749

Vol. 43, No. 4, 1971

BIOCHEMICAL

420 nm (type

II)

with

demonstrable

difference

cholesterol

shows a small 52-35 but

II) type

not with

additional

P2.

preparations

Enzyme Activity:

It

with

P2 and S2-35 give cholesterol.

show well-marked

with II

while

spectrum

and 20a-hydroxycholesterol at 420 nm (type

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

all

three

difference

positive

preparations; spectrum

These findings

Both

with

no pregnenolone

spectral

shifts

corticosterone soluble

were confirmed

P.450 and with

two

of P.450. is clear

from Table

2 that

the soluble

TABLE 2 ENZYME ACTIVITY Fraction

Preparation

OF P.450 FRACTIONS

Side-Chain Cleavage (% Conve;s~;;<~mole .

lip-Hydroxylation (nmoles/~mole P.450/ 20 min)

1

p2

0

0

s2 - 35

0

0

45.1

0

p2

0

0

s2 - 35

0

0

P.450

26.2

0

P2 + s2 - 35

0

48.7

Soluble Preparation

Soluble

P.450 2

P.450 fractions were incubated with TPNH, non-heme iron, diaphorase and cholesterol-7a-3H or DOC-14C for 20 minutes at Side-chain cleavage and lip-hydroxylation were measured 370. Zero means as described elsewhere (Young et al., 1970).
BIOCHEMICAL

Vol. 43, No. 4, 1971

P.450 possesses

side-chain

activity

(( 0.01

contrast

P2 and 52-35

11(3-hydroxylase

but

lip-hydroxylation

P.450/20

However,

has"been

cleavage

cleavage

side-chain

By nor

S2-35 showed

activity

that

soluble

cleavage

found with

no lip-hydroxylase

minutes).

P2 plus

The observation

of supporting

but

side-chain

not side-chain

No. 2).

RESEARCH COMMUNICATIONS

activity

show neither

activities.

capable

cleavage

nmoles product/pmole

lip-hydroxylase

(preparation

AND BIOPHYSICAL

P.450 is

activity

'but not

five

separate

enzyme

three

important

preparations. DISCUSSION The present of cytochrome Firstly,

P.450

at least

solubilised remains

findings from bovine

one species

to the extent

clear

for

centrifugation

at

at

preparation

least

acid

the

cytochrome

cholesterol

but

not

shows both

II).

(Young et al., with

both

during

purification

specifically

will

one part

The best remains

during

previous

in suspension

support

or that

cleavage

Although soluble

a peak at

and Horie

I difference

of

shows a

DOC, giving

of Mitani

from the of

interesting

The solution

type

and DOC.

or site

side-chain

and lip-hydroxylase

and gives

needed for

solution

sediment

shows the following

and with

cleavage

activity

that

P.450

The preparation

cholesterol

either

2 hours.

1969) only

cholesterol

1970)

lip-hydroxylase

x g for

lip-hydroxylation.

side-chain

straw-coloured

7 days and does not

soluble

with

P.450 has been

is present.

properties:

420 nm (type

the pale

features

mitochondria.

of mitochondrial

and Horie,

Secondly,

shift

adrenocortical

that

105,000

(Mitani

as long as cholic

spectral

reveal

(1969)

activities spectrum

the absence of preparation

might

mean

the enzyme has been damaged

some (?) protein

ll@hydroxylation,

751

hydroxylase

factor,

has been removed,

the

Vol. 43, No. 4, 1971

fact but

that

P2

BIOCHEMICAL

S2-35 are together

plus

not of side-chain

of two distinct and one for the

fact

with

cytochromes

that

of this

is reported

(soluble

+0.043

no interaction

+0.050

11 spectrum

is soluble

showing enzymatic

activity

observed

is

reported

to show a type

Horie,

type

II

is capable These findings

prepare

of the P-450 associated and encourage supporting being

attempts

with

pursued

in this

fractions

for

of these DOC (A 390:

soluble

P.450 shows a

is

the only

three

crude

has been

P.450

fractions, (Table

the way for

further

to solubilise

fraction

(Mitani

only

characterisation

cleavage

of cholesterol

a second P.450

Such endeavours

capable

are at present

laboratory.

Yu and Gunsalus,

Commun., 40,

I.C.,

Biochem.

1431 (1970). 752

one of

1).

REFERENCES Chang-an,

and

20a-hydroxycholesterol

cleavage

side-chain

shift

the spectrum

Cholesterol

with

all

ll@-hydroxylation.

DOC

and Horie,

mixture

substrate;

Furthermore

of side-chain

For example,

to show a spectral

1 and 2).

with

enzyme and

extravagant

(Mitani

hand,

this

I spectrum

spectrum

spectrum

1).

with

1970).

against

I spectrum

P.450 which

(Table

Young et al.,

shows a type which

II

I difference

of the soluble

e.g.

one of our fractions

cholesterol

with

cholesterol.

an equimolar

DOC (Table

cleavage

consistent

'between our three

On the other

with

The only with

I spectrum

show type

-0.022).

side-chain

phenomenon.

P2 and S2-351,

would probably

with

as a caution

occurs

of the existence

is perhaps

spectra

empirical

llfl-hydroxylation

in favour

show type

spectrum

to show a type

P.450,

fractions

This

serve

of

- one for

the difference

interpretation

1969) ; if

P.450

no difference

of P2 and S2-35,

capable

argues

P2 and S2-35 always

Thirdly,

type

cleavage,

11P-hydroxylation.

DOC but

those

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Biophys.

Res.

of

BIOCHEMICAL

Vol. 43, No. 4, 1971

Lu,

A.Y.H.

Mitani,

and Coon, M.J.,

F. and Horie,

Omura, T. and Sato, page 556, Omura, T., Rosenthal,

AND BIOPHYSICAL

J. Biol.

S.,

J.

RESEARCH COMMUNICATIONS

Chem., 243,

of Biochemistry,

R.,

Methods

E.,

Estabrook,

1331 (1968).

65,

in Enzymology,

269 (1969).

Volume X,

1967. Sanders, O.,

Arch.

Young,

D.G.

and Hall,

Young,

D.G.,

Holroyd,

Res. Commun., 38,

Biochem. P.F., J.

R.W., Biophys.,

Biochemistry,

and Hall,

184 (1970).

753

P.F.,

Cooper, 117,

D.Y.

and

660 (1966).

In Press -Biochem.

(1971). Biophys.