Enzymatic conversion of Δ8,14-cholestadien-3β-ol to cholesterol

Vol. 33, No. 3, 1968

BlOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ENZYMATIC CONVERSION OF ’ 4-CBOLESTADIEN-3&OL

A83

TO

CHOLESTEROL1

Barry

and ChmicaZ

N. Lutsky

and G. J.

Division of Biochemistry, Engineering, University

Schroepfer,

Department of Illinois,

Jr.

of

chemistry Urbana, ItI.

61801

Received October 2, 1968

The of

nature

of

cholesterol

vious

has

as

double

A5,7-

and

,

removal

As.

of

the

led

two

role

of

A*(14)-sterols

is

this of

further

finding

1.

have

laboratory

to

to

(Lee

enzymatic

This work National

removal

was Heart

of

supported Institute.

and

group

a grant

492

the

enzymatic

of

lanosterol

intermediary

cholesterol.

in

Fried,

rat

liver

of

1968)

also

homo-

preparations

been

consideration

methyl

14

AT-,

possible

Schroepfer,

in

by

with

sterols

4,4-dimethyT-68(14)-

similar

has

the

of

of

of that

with

significance

1967))

atom

cholesterol

A8(14)-cholesten-3B-ol of

carbon the

shown

studies

Schroepfer,

A8-,

mechanism

biosynthesis

convertible In

the

investigate the

(1968)

preparations.

version

the

in

Pre-

positions:

attached to

and

a number

of

group

formation

laboratories. (Frantz

following

Considerations

and Brown

in

the

enzymatic

several

intermediates

laboratories

cholesten-36-01

liver

in

methyl

have

Dudowjtz,

in

the

elsewhere

probable

bonds

in

studied

reviewed

implicated

nuclear

intermediates

been

publications,

have

genate

the

the

(HE-09501)

facile

con-

demonstrated. of

attached

rat

the to

from

A

mechanism carbon

the

atom

of 14

Vol.

33,

No.

of

BIOCHEMICAL

3, 1968

lanosterol

is

that

the

to

cholesterol.

recent

158-hydrogen

As by

the

an

this

our

its

of

preparations.

to

We wish of

Synthesis

this

of

9 infrared

scopy,

mass

these

impregnated

of

techniques:

very

152’)

by

and

silica

1567),

gel

acetone (E

with

prepared

oxidation purified

from

potassium

by

were

ultraviolet

spectro-

resonance

spectro-

on

chromato-

plates

of

(Kammereck,

neutral

Lee,

chromatographic

(m.p. from

Paliokas

analysis

(3%

of

(Fieser

and

the

acetate

prisms,

chromium column

trioxide

chromatography

99.5

-

101.5’;

X

A5,7-cholestadien-3B-ol acetic

by The

m.p.

free

113.5

on

493

under

-

pyridine alumina,

anhydride,

reflux

crystal 0

114.5

;

prepared at

with 1 ized

XEtOH from

4’. and

l51-

Aa,14-cholestadien-

sterol

was in

acetic

1953). heating

EtOH (m.p.

acid,

Ourisson,

Aa,14-cholestadien-3-one with

homogenate

thin-layer

and

gas-liquid

hydroxide.

elongated

18,000).

as-

enzymatic

magnetic

nitrate

a mixture

acid

in

to

liver

point,

G plates

prepared

treatment

methanolic

prepared

intermediates

analysis,

silver and

18,lQQ))was

was

have

Q).

hydrochloric

38-01

rat

efficient

melting

Aa~14-cholestadienyl-3f3-acetate (E

we

following

nuclear

with

gas-chrom

mu

stimulated

hydrogen by

the

elemental

on

Schroepfer,

250

al.

conversion

cholesterol.

purity

spectroscopy,

alumina

on

--et

(1368)

enzymatic

clearly

isotopic

the

spectroscopy,

analysis

QF-1

to

and

graphic

and

and

al.

Aa~14-choZestadien-3-one

by

scopy

work

COMMUNICATIONS

et

upon

cholesterol

report

sterol

Authenticity determined

to

lost

with

RESEARCH

Canonica

Canonica

labeled

convertibility

conversion

is

previous

observation

A*,14-cholestadien-3b-ol certain

by

lanosterol

of

important

BIOPHYSICAL

observation

of

extension

AND

from

250 the

The crystallization

15%

mu alcohol

ketone

by was

Vol. 33,No.

3, 1968

from

BIOCHEMICAL

methanol-ether

yielding

AEtoH 250 mu

133.5O;

Synthesis

250

liquid

mu

aluminum

(E

18,100);

by and

nitrate

(2)

digitonide

The

specific

in

in

propylene x

g

3 hours

in

air

sterols

were

extraction

further

and

was

39.9

mc/mmole.

of

96%

plates

of

lithium

silica

basis

an

from

methanol.

radiopurity

of

was

analysis

G and

by

aiumina-

1968),

ization The

gel

on

Schroepfer,

recrystall

the

gas-

A8~14-

by

thin-layer

gas-liquid

- A8*14-cholestadien-3G-ol (100

supernatant at

37’

isolated

chroma-

from

they

nitrate

(98~2) is

as

shown

radioactivity

characterized

and the

the

eluting

34

cm)

of

I.

by

purification

494

of

a for

by

incubated

radio-

on

an

of

chloroform

alumina-

resulting 75%

of

chromatographically in

107

The

the

a mixture

Approximately

associated

ml

medium

chromatography

The

x

homogenate 1968).

solvent.

Figure

such

liver

2.1 30

incubation

using

cholesterol as

rat

Schroepfer,

to

x

labeled

a

pg; with

(88% recovery

(1

in

incubated

saponified

subjected

was The

was of

ether

were

column

~1)

(Paliokas

to choZestero2

(148.5

fraction

petroleum

and

acetone

A83 14-choleetadien-3~-ol preparations.

glycol

with

activity)

cholesterol.

Lee,

on

from

chromatography

(3)

homogenate

10,000

covered

(1)

- 114.2’; and

of [~CX- 34 -

130- 3Hl

chromatogram

-

.

liver

silver

137

thin-layer

tritium-labeled

and

excess on

Comersion

cpm)

by

on

prepared

with

(Paliokas,

activity be

was

purified

formation,

to

by rat

m.p.

(m.p. 113.5

component

reduction

column

chromatography

and

single analyses)

hydride

tog raphy

needles,

A8*14-cholestadien-3@-ol

-

cholestadien-j-one

judged

compact

18,200).

(E

chromatographic

silver

short,

RESEARCH COMMUNICATIONS

of L3c.x- 3Hl - A8, 14-cholestadien-3&3-ol

[3a - 3H] hEtOH

AND BIOPHYSICAL

the with

fractions

62-75 by

re-

way

of

was the

dibromide

Vol.

33,

No.

BIOCHEMICAL

3, 1968

AND

FRACTION

Figure

2.

BIOPHYSICAL

RESEARCH

NUMBER

chxomatogrwn showing enzymatic

Column

13a - “HI - A83 14-chdesten-3fho~ A-d, OV, radioactivity; colorimetricaZZy;

COMMUNICATIONS

of

conversion

to cholesterol.

measured

choZe.sterol Aa, ’ 4-cho~estcdien-

A-A,

38-ol measured coZorimetricalZy.

(Paliokas

and

terol

.

were

67,000

the

radioactivity

column of

The

mobility

activities 67,800

a

less

material

These to

the

biosynthesis

the

peak

has

not

after

dilution

before

and

and studies

by

were suggests

negative. a

of

after

this

choles-

purification

tritium)

emerged

3.5-45).

determined.

amount

The ‘27

from

precise

-sterols

of

the nature

with

similar

Aa(14)-cholesten-3b-ol,

cholestan-3b-01.

establish

cholesterol

unlabeled

A significant

recovered (fractions

been

with

respectively.

ha-cholesten-3b-01,

controls

conversion

of

polar

A7-cholesten-3b-01,

enzyme

cpm/mg,

(10%

include

vertible

1368)

specific and

in

this

Schroepfer,

possible

rat

that

Aa,14-cholestadien-3b-ol

liver

homogenate

The

cholesterol.

495

preparations.

efficient

intermediary

is

nature role

of

of this

conBoi led

the

observed

compound

in

Vol. 33, No. 3, 1968

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

References

Canonica,

L., Grossi 90, L.F., I.D.,

Fieser, Frantz,

Fiecchi, A., Galli Paoletti, E., and 3597 (1968). and Ourisson, G., Jr., and Schroepfer,

Kienle, Paoletti,

M., R.,

Scala, J.

A., Am.

Galli, G., Cbm. Sot.,

J. Am. Chem. Sot., 15, 4404 (1953). G.J., Jr., Ann. Review Biochem.,

36, 691 (1967). Fried,

J.,

Dudowitz,

A.,

Commun., 2, Kammereck, Lee,

W.,

Paliokas,

R.,

Lee,

and

Brown,

J.W.,

Biochem.

Biophys.

W.,

Paliokas,

A.,

and

Schroepfer,

G.J.,

J. lipid? Res., g, 282 (1967). and Schroepfer, G.J., Jr., Biochem. Biophys. 32, 635 (1968). A.M.,

Res.

568 (1968).

Lee,

W.,

and

Schroepfer,

G.J.,

Jr.,

Jr.,

Res. Comnun., J.

Lipid

Res.,

2, 143 (1968). Pal iokas,

A.M.,

and

Schroepfer,

G.J.,

(1968).

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