Solubilisation and reconstitution of acylcoenzyme A:estradiol-17β acyltransferase

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 144, No. 2, 1987

Pages 559-575

April 29, 1987

SOLUBILISATION ACYLCOENZYME

AND R E C O N S T I T U T I O N

A:ESTRADIOL-17~

F.T.

Lee a n d J.B.

OF

ACYLTRANSFERASE

Adams

School of Biochemistry, U n i v e r s i t y of N e w South Wales, Sydney, N.S.W. 2033, A u s t r a l i a Received February 26, 1987

SUMMARY: Acylcoenzyme A:estradiol-17~ acyltransferase in m i c r o s o m e s of b o v i n e p l a c e n t a c o t y l e d o n s was s t r o n g l y m e m b r a n e bound. The e n z y m e was s o l u b i l i s e d f r o m m i c r o s o m e s b y s o d i u m cholate a n d was r e c o n s t i t u t e d into p h o s p h o l i p i d vesicles. The a p p a r e n t Km for e s t r a d i o l - 1 7 ~ was II ~M w h i c h was close to the value of 8 ~M p r e v i o u s l y found with the m e m b r a n e - b o u n d enzyme. T e s t o s t e r o n e was also a s u b s t r a t e for the r e c o n s t i t u t e d enzyme (apparent Km 62 ~ M ) a n d was a c o m p e t i t i v e i n h i b i t o r (Ki 74 ~M) of the acylation of estradiol-17~. Although various long-chained f a t t y a c y l C o A s a c t e d as a c y l d o n o r s , these proved to h a v e widely differing apparent Km values with palmitoleoyl C o A h a v i n g the h i g h e s t a f f i n i t y (Km 24 ~M) and a r a c h i d o n o y l CoA the lowest a f f i n i t y (Km 330 ~M). © 1987 AcademicP r e .... Inc.

Hochberg

and

estradiol-17~ the

derivatives

17-position

fatty

acids

bovine

there

tissue

example,

(1,2)

have

in w h i c h

long-chain

(2,3)

fatty

and

differences

formed (4,5,8),

whilst

in or

acids

the

mammary

arachidonate

formed

in

bovine

tissues,

in m a m m a r y

cancer

tissue

situ

exposure

in

culture

of h u m a n

undergoes

mammary

hydrolytic

cancer

these

E2-L, AEAT,

fatty

mammary

lines

was

in

(3).

(7,8).

the

major

a

lesser

E2-L , formed

cells once

via

formed

and

was

ester

cells.

cleavage

Abbreviations: E 2, 17~-estradiol; acid esters of 17~-estradiol; estradiol-17~ acyltransferase.

569

this

and

of

tissues cell

of

Unsaturated

cotyledons

cancer

component

family

of E 2 - L

nature

bovine

component

upon

(E2-L) .

placenta in

a

is e s t e r i f i e d

components

the

estradiol

identified

estradiol

to be the m a i n

are

components

cancer For

to

appear

endometrium

However, acid

coworkers

i__nn

to e s t r a d i o l the

cells

long-chained fatty acyl

are

fatty CoA:

0006-291X/87 $1.50 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 144, No. 2, 1987

placed

in

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

medium

established

lacking

that

the

estradiol

esterified

selective

turnover

These

substrates

for

enable

them

to the

esterase

receptor

to

(9)

biological

the

and

tissue

effects

have

solubilisation vesicles

is

be

possess

to

not

cleaved

ensue.

placenta

reported and

reconstitution

reported

acyl C o A substrates

(3,8).

and

its

culture be

through

cotyledons In this

which

the

free

steroid

estrogen

of

in

acyl

mammary

communication,

towards

for

microsomal

and h u m a n

AEAT

specificity

better

with

in

(8).

membranes

properties (AEAT)

of

of

undergoes

properties

the

The

comprised

perhaps

combine to

been

in

rapidly

acyltransferase

from bovine been

or

also

acids,

cells may

more

has

E2-L,

esters

E2-L does

must

It

fatty

mammary

esterase,

CoA:estradiol-17~ preparations

human

transported

site.

of

essential

estradiol

be

(7).

subfraction with

in

particular

estradiol

the

phospholipid steroid

and

examined.

MATERIALS

AND METHODS: [2,4,6,7-3H] Estradiol (i01 Ci/mmol) and [I,2,6,7-3H] testosterone (91.7 Ci/mmol) were obtained from the Radiochemical Centre (Amersham, U.K.) . Crude soybean type II phospholipids, sodium cholate, unlabelled s t e r o i d s and f a t t y acyl C o A t h i o e s t e r s w e r e from Sigma C h e m i c a l Co. (St. Louis, Mo). M i c r o s o m e s were p r e p a r e d as p r e v i o u s l y described (3). Briefly, 20g of m i n c e d b o v i n e p l a c e n t a was h o m o g e n i s e d in 4 vols of 50 m M Tris/HCL, pH 7.4 containing imM EDTA and ImM dithiothreitol (buffer A) . Homogenates were centrifuged sequentially at 8 0 0 g (I0 min), 1 0 , 0 0 0 g (15 min) and f i n a l l y at 1 0 0 , 0 0 0 g (60 min) to collect the m i c r o s o m a l pellet. S o d i u m C a r b o n a t e Treatment: Microsomes (8.5mg protein) were s t i r r e d g e n t l y in 5ml of 0 . 1 M s o d i u m c a r b o n a t e for 1 hr at 4°C. The r e s u l t i n g m i x t u r e was c e n t r i f u g e d at 1 0 0 , 0 0 0 g for 1 hr a n d the p e l l e t a n d s u p e r n a t a n t e x a m i n e d for A E A T activity. P r o t e i n was d e t e r m i n e d by the m e t h o d of B r a d f o r d (10).

$olubilisation and Reconstitution of Enzyme: Soybean phospholipid (1.6 ml of a 25% w/v s o l u t i o n in chloroform) was d r i e d by a s t r e a m of nitrogen, r e d i s s o l v e d in 0.5 ml ether and d r i e d a g a i n u n d e r nitrogen, i0 ml of b u f f e r A was a d d e d to m a k e up a 40 m g / m l p h o s p h o l i p i d s u s p e n s i o n . The m i x t u r e was s o n i c a t e d w i t h a B r a n s o n s o n i c a t o r microprobe, s e t t i n g 3 for 3 x 3 min periods with cooling. Microsomal pellets were s o l u b i l i s e d in 6% w / v s o d i u m cholate. The m i x t u r e c o n t a i n i n g 6 mg m i c r o s o m a l p r o t e i n / m l of b u f f e r A was s t i r r e d g e n t l y for 1 hr at 4°C a n d c e n t r i f u g e d at 1 0 0 , 0 0 0 g for 1 hr. The supernatant was r e m o v e d a n d the p e l l e t was r e s u s p e n d e d in b u f f e r A. Enzyme was r e c o n s t i t u t e d in two w a y s : - (a) cholate 4 i l u t i o n (Ii); d e t e r g e n t - p r o t e i n e x t r a c t s were d i l u t e d into i0 v o l s of the a b o v e p h o s p h o l i p i d suspension, a n d (b) c h o l a t e dialysis; extracts were diluted into 2 vols of the p h o s p h o l i p i d s u s p e n s i o n a n d d i a l y s e d e x t e n s i v e l y in b u f f e r A. For (a), r e c o n s t i t u t e d enzyme was i n c u b a t e d on ice for 20 min

570

Vol. 144, No. 2, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with occasional determined.

stirring

before

enzyme

activity

was

Assay: E n z y m e a c t i v i t y was d e t e r m i n e d b y a m o d i f i c a t i o n of the p r e v i o u s l y de~cribedj m e t h o ~ (3). The i n c u b a t i o n m i x t u r e c o n t a i n e d i0 ~M [ H ] E 2 (5 x i0 dpm) a d d e d in 2 ~i ethanol, 0.i m M o l e o y l CoA, 1 m M EDTA, 1 mM dithiothreitol, 0.i M s o d i u m a c e t a t e buffer, p H 5.0. The total v o l u m e was 0.2 ml. After incubation for 30 m i n at 37°C, 1.2 ml of 82% a q u e o u s m e t h a n o l was a d d e d to stop the r e a c t i o n a n d the m i x t u r e was e x t r a c t e d w i t h 1.4 ml of h e x a n e p r e v i o u s l y s a t u r a t e d w i t h 70% v/v aqueous methanol. After centrifugation, a 1 ml a l i q u o u t of the h e x a n e was r e m o v e d a n d b a c k w a s h e d w i t h 0.25 ml of 70% aqueous methanol. An a l i q u o u t of t h e h e x a n e fraction was counted for r a d i o a c t i v i t y . A u n i t of e n z y m e a c t i v i t y was d e f i n e d as that p r o d u c i n g 1 nmol [3H]E2-L/hr.

RESULTS

AND

treat

membranes

mild

alkali

membrane total the

DISCUSSION:

Sodium

derived

subcellular

sheets

protein

was

in

the

did

not

several

the

that

and

simple

and a detergent

These sodium

has

systems

different

to p r o t e i n

remained

membrane using

Methods),

supernatant

was

may

to

(data

ratio The

bound.

of

of

shown

longer

to

in e f f o r t s

to

PX, was

dialysis.

571

of

of

speed

the

enzyme

cholate

effective

for

reconstituted

in

i,

of

enzyme

supernatant

whilst

of

78%

enzyme

cholate

in

recovery

cholate

Microsomes

solubilised

method

A

sodium

most

Table

activity

Lower

exposure

in

mixtures

reconstituting

adopted.

was

Triton strongly

shown). for

i0 was

high

When

enzyme

recovered.

carbonate bound

Lubrol

not

latter

an a l t e r n a t i v e

46%

tested

concentrations

in the

during

was

activity

strongly

Enzyme

then

recoverable

reconstituted

were

was

activity

was

was

used

As

due

sodium

the into

activity

Since

been

(ii),

solubilisation.

be

of

released

no

original

included

inhibition

which

vesicles.

(see

was

76%

of d e t e r g e n t - m i c r o s o m e

phospholipid

4%

Some

the

cholate.

dilution

this

with

enzyme

of d e t e r g e n t s

and

method,

treated

the

enzyme.

membrane

enzyme

of

Reconstitution:

a variety

relieve

dilution

were

and

Digitonin

inhibited

flat

in the pellet.

revealed

solubilise X-100,

This

into

Whilst

65%

to

(12).

preparation

some

used

vesicles

proteins.

centrifugation.

supernatant,

recovered

microsomes,

unbound

was

fractions

sealed

microsomal

after

Solubilisation treatment

converts

releasing

of the

supernatant

found

from

treatment

carbonate

the in

to

was

dialysis

high

speed

this

instance

sodium

cholate

Vol. 144, No. 2, 1987

Table i.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Solubilisation and Reconstitution of Enzyme Activity

Fraction

Vol (ml)

Total Activity (units)

Recovery (%)

Microsomes

2.5

34.5

I00

Reconstituted cholate supernatant

4.8

26.9

78.0

Resuspended pellet

5.0

1.5

4.3

Placenta microsomes were solubilised with sodium cholate and reconstituted by dilution into phospholipid vesiqles as described under Methods. The concentration of [~H]E 2 was i0 ~M and that of oleoyl CoA 0.i mM.

Acyl-donor were

Specificity:

examined

using

for t h e i r

dialysed

obtained (3).

reconstituted

Oleoyl

were poor

their

donors

high

Km

membrane behaviour

Whereas

the

was

the

values

towards

high

concentrations

of o l e o y l little

difference

which

may

thioesters marked mM

may

(13).

inhibition

(Fig.l) .

actual

Despite

the of

by HPLC,

estradiol-essential palmitoyl intact

CoA

estradiol fraction

was

membrane

to

the

known

0.4

was

an

data

cotyledons

revealed

high

acid as

f o r m e d by b o v i n e

a

minor

tissues.

572

was

CoA. at

reconstituted

phospholipids of

acyl

in

Table

incubation

CoA

whereby

observed

at

0.4

2,

the

of

[3H]

or e n d o m e t r i u m

(2,3)

concentrations

esters. acyl

acyl

exception

was

relative

reconstituted was

difference

concentration.

of

shown by

had

inhibited

(3), mM

CoAs

enzyme

of

effect

enzyme

formed

effective

palmitate

mM

to

placenta

fatty

or

0.2

by

a c i d CoAs

acyl

strongly

presence

CoA

kinetic E2-L

fatty

notable

detergency

reconstituted

with bovine

and a n a l y s e d

due

up

Palmitoleoyl of

was

were

membranes

reconstituted

CoA beyond

the

composition

estradiol

enzyme

formation

followed

particular A

CoAs

results

donor,

concentrations

inhibition

be

alleviate

similar

2).

and

acyl

[3H]E2-L

Essential

These

(Table

fatty

microsomal

acyl

CoA.

enzyme

bound

then

intact

best

bound

enzyme The

with

(Fig i).

membrane

showed

to i n c r e a s e

enzyme,

found

C o A and p a l m i t o y l

acyl

uniformly between

CoA

long-chained

ability

as p r e v i o u s l y

palmitoleoyl

When

Again,

donor, enzyme, component

both

of

although with

the

nevertheless of

the

E2-L

Vol. 144, No. 2, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2.5

o 2.0

C

$

O

1.5 Cn

E p E C

1.0

PO

,-I

0

I

I

I

I

I

0.1

0.2

0.3

0,4

0.5

Acyl CoA (raM) Fig.l. Effect of concentration of fatty acyl CoAs on [3H]E2-L synthesis. The concentration of [3H]E 2 was i0 ~M. Values are means of triplicate assays. O, oleoyl CoA; PO, palmitoleoyl CoA; P, p a l m i t o y l CoA; Le, l i n o l e n o y l CoA; L, l i n o l e o y l CoA a n d Ar, a r a c h i d o n o y l CoA. No fatty acyl CoA was added in the control.

Apparent dialysed

Km

of

Estradiol:

reconstituted

Table 2.

Acyl CoA

oleoyl palmitoleoyl palmitoyl linoleoyl linolenoyl arachidonoyl

The

enzyme

apparent was

found

Km to

of

estradiol

be

ii

Kinetic Data Obtained with Reconstituted

Apparent (~M)

Km

~LM

using (Fig.2)

Enzyme

Umax

nmol/hr/mg protein

50 24 39 80 140 330

2.7 1.9 1.6 0.6 0.4 0.7

Data obtained from Fig.l by construction of double reciprocal plots. In the case of linol~oyl CoA, l i n o l e n o y l CoA and arachidonoyl CoA, the value of [ H]E2-L obtained in the absence of added acyl CoA was substracted for each concentration used. The concentration of estradiol was i0 ~M.

573

Vol

144, No 2, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

3.0

r 1.0

©

c~

.0

=

1.0

f-

0 ~D

E

-~

0.5

_.1

I

I 0.1

-0.1

®

I

I 0.2

/

I -0.05

ESTRADIOL (jJM)-1

/

I

Q

I 0.05

I

0.1

TESTOSTERONE (jJM)-1

Fig. 2. Competitive inhibition of the acylation of estradiol ~ £es-terone. Double reciprocal plots of [H3]E2-L synthesis in the absence (close circles) or presence of 75 ~M testosterone (open circles). Dialysed reconstituted enzyme was assayed as described in Methods except that the concentration of estradiol was varied as indicated. Each point represents the mean of triplicate assays.

Fig. 3. Double reciprocal plot of [3H] t e s t o s t e r o n e - e s t e r formation. The concentration of [3HI testosterone was varied as i n d i c a t e d and that of oleoyl CoA was 0.i mM. Enzyme activity was determined as for AEAT described under Methods. Each point represents the mean of triplicate assays.

which for

was the

in

close

membrane

reconstitution affinity was the

cholate

due

to

the

the to

effect

and In

was

the

addition,

indicates

showed and of

respectively

the data

did The

to Km

fact

74 ~M, enzyme

estradiol We

were

with

that

is not

values that

574

8 ~M

most

by

likely

dialysed

acylation

substrate

of

its

prepared

The

the

and

estradiol

was

testosterone

Fig.2)

the

62 ~ M was

acylation

absolutely

of

a of

specific

with the membrane-bound

competitive

Ki

conclude

is

cholate.

this

obtained

of

alter

for

enzyme

catalyse

for

not

Km

difference

sodium

value

Solubilisation

dehydroepiandrosterone,

testosterone

(3).

This of

the

Previous that

(3).

reconstituted

able

(Ki =

that

observed

apparently

apparent

inhibitor

for e s t r a d i o l .

acylation

76 ~ M u s i n g

enzyme

the

estradiol.

method.

competitive

17~-diol

enzyme

substrate

dilution

testosterone

estradiol

the

with

enzyme

detergency

reconstituted

(Fig.3) .

bound

of

for

increased

enzyme

agreement

of

5-androstene-3~, inhibitors 46,

solubilised

39

and

for

the

79

~M,

reconstituted

Vol. 144, No. 2, 1987

AEAT

possesses

enzyme. obtained

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

similar

properties

Solubilisation which

indicate

has

that

to

enabled the

the

membrane

kinetic

enzyme

cotyledons has widely differing affinities

data

from bovine

bound to

be

placenta

for fatty acyl CoAs

and is not absolutely specific for estradiol.

REFERENCES

i. 2. 3. 4. 5 6 7 8 9 i0 ii. 12. 13.

Schatz, F., and Hochberg, R.B. (1981) E n d o c r i n o l o g y 109, 697-703. M e l l o n - N u s s b a u m , S.H., Ponticorvo, L., Schatz, F., and Hochberg, R.B. (1982) J. Biol. Chem. 257, 5678-5684. Martyn, P., Smith, D.L., and Adams, J.B. J. Steroid Biochem. a. Submitted. Abul-Hajj, Y.J. (1982) Steroids 40, 144-156. Larner, J.M., Eisenfeld, A.J., and Hochberg, R.B. (1985) J. Steroid Biochem. 23, 637-641. Abul-Hajj, Y., and Nurieddin, A. (1983) Steroids 42, 417-426. Adams, J.B., Hall, R.T., and Nott, S. (1986) J. Steroid Biochem. 24, 1159-1162. Martyn, P., Smith, D.L., and Adams, J.B. J. S t e r o i d Biochem. b. Submitted. Janocko, L., Larner, J.M., and Hochberg, R.B. (1984) Endocrinology 114, 1180-1186. Bradford, M.M. (1976) Analytical Biochem. 72, 248-254. Racker, E., Chien, T.F., and Kandrach, A. (1975) FEBS Lett. 57, 14-19. Fujiki, Y., Hubbard, A.L., Fowler, S., and Lazarow, P.B. (1982) J. Cell. Biol. 93, 97-102. Spector, A.A., Mathur, S.N., and Kaduce, T.L. (1979) Prog. Lipid Res. 18, 31-53.

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