Effect of membrane lipid environment on the activity of bovine adrenal 3-oxo-Δ5-steroid isomerase

EFFECT OF MEMBRANE LIPID ENVIRONMENT ON THE A C T M T Y OF BOVINE ADRENAL3-OXO-AS-STEROID ISOMERASE Janna

P. W e h r l e #

and R a l p h M.

Pollack*

$ D e p a r t m e n t of R a d i o l o g y and R a d l o l o g l c a l S c i e n c e The J o h n s H o p k i n s S c h o o l of M e d i c i n e B a l t i m o r e , M a r y l a n d 21205 * L a b o r a t o r y for C h e m i c a l D y n a m i c s D e p a r t m e n t of C h e m i s t r y U n i v e r s i t y of M a r y l a n d B a l t i m o r e C o u n t y Baltimore, Maryland 21228 R e c e i v e d M a r c h 25, 1986 R e v i s e d N o v e m b e r 7, 1986 ABSTRACT

The 3-oxo-A5-steroid isomerase (EC 5 . 3 . 3 . 1 ) activity from b o v i n e a d r e n a l c o r t e x m i c r o s o m e s can be e x t r a c t e d in s o l u b l e f o r m by the use of a p p r o p r i a t e d e t e r g e n t s , a l t h o u g h r e c o v e r y of e n z y m e a c t i v i t y is low (ca. 2%). A c t i v i t y is r e s t o r e d u p o n r e m o v a l of d e t e r g e n t and r e c o n s t i t u t i o n of the e n z y m e 5into p h o s p h o l l p l d vesicles. Both K and V of 3 - o x o - A - s t e r o i d i s o m e r a s e of i n t a c t m l• c r o s o m me s i n c r ~ ? e as the pH is r a i s e d from 7.5 to 9.5, w i t h a p a r t i c u l a r l y sharp increase (6to 8-fold) above pH 8.5. The kinetic p a r a m e t e r s of a d e t e r g e n t - s o l u b i l l z e d isomerase preparation show l i t t l e increase from pH 7.5 to 9.0, but isomerase reconstituted into artificial phospholipid vesicles d e m o n s t r a t e s a 6- to 1 0 - f o l d i n c r % % s e in b o t h K and V over this pH range. A d d i t i o n of Ca (i mM) e n h a n c e s t h e m ~ d e p e n d e n c e of b o t h K and V of the m e m b r a n e - b o u n d isomerase, c a u s i n g a s l i g h ~ rise ~ X V m a x / K m.

INTRODUCTION

The

3-oxo-A5-sterold

physiologically hormonal

irreversible

steroids.

4-positlon increases ase

is

found

in

peripheral

mlcrosomal

planarlty in m a m m a l s

the

of the both

protein

(EC 5 . 3 . 3 . 1 )

of

the

double

formed

steroid in

catalyzes

in the b i o s y n t h e s i s

newly

steroldogenic

membrane

S T E R O I D S 47/2-3

step

Conjugation

stabilizes the

isomerase

the

(1,3),

3-oxo

A ring.

adrenal

tissues the

bond

(2).

cortex An

isomerase

February-March 1986 (115-130)

of all to

group The

a

the and

isomer(i)

and

integral is

inactl-

115

I16

Wehrle and Pollack

vated (4) of

by

disruption

or by two

treatment

distinct

activity

of

(i,I0,ii),

and

one

factors

in b o v i n e

for

A

(5).

adrenal

pregnenedione,

serum

albumin

the

activity

Ca ++

of

the

of P s e u d o m o n a s inherent

by

detergents

The

existence

cortex,

has

(12),

one

been

None

of

for

suggest-

localization role

of

mammalian

isomerase,

adrenal

detergent-solubilized phospholipld

however,

may

the

the

membrane

enzymes be

compared

glands: and

in

three

may

be

from

due

to

mammal~an upon To

behavior forms

(13), affects

enzyme. the

pH

isomerase

dependent

mammalian

the

alkaline

nucleotides

variations

of

enzyme,

including

these,

the

they

we have

increase

3-oxo-A5-steroid

These

or

to

pyridine

between

sources,

the

artificial

isomerase,

(14).

difference

bovine

reported

water-soluble

establish

from

been

testosteroni.

bacterial

membrane

have

microsomal

millimolar

ase

phospholipase

the

and

and

with

membrane

(6-9). Several

an

the m i c r o s o m a l

isomerases

androstenedione ed

of

of

of

the help the

isomer-

native

microsomal

enzyme,

enzyme

reconstituted

into

vesicles.

MATERIALS

AND

METHODS

Bovine adrenal glands were obtained frozen from Pel-~/~.z and stored at -70°C until used. Zwittergent 3-16 was obtained from Calbiochem-Behring Corp. Substrates (5-androstene-3,17-dione and 5-pregnene-3,20-dione) were p r e p a r e d and p u r i f i e d as d e s c r i b e d p r e v i o u s l y for 5-androstene-3,17-dione (15), from dehydroepiandrosterone and p r e g n e n o l o n e r e s p e c t i v e l y . The f o l l o w i n g c o m p o u n d s were obtained from Sigma and used without purification: L-=-phosphatidylcholine, b o v i n e b r a i n (P9137); L - ~ - p h o s p h a tidylinositol, soybean (P5766) ; L-a-phosphatidylserine, b o v i n e b r a i n (P6441); cholesterol (CH-S); and b o v i n e s e r u m albumin (BSA) which was essentially fatty acid-free

LIPID CONTROL OF ADRENAL A~-ISOMERASE

117

(0.005%). Blobeads SM2 w e r e o b t a i n e d f r o m B i o - R a d Corp., and a s o l e c t i n (mixed soy p h o s p h o l l p i d ) was obtained from Associated Concentrates. B e f o r e use a s o l e c t i n was p a r t i a l l y p u r i f i e d as f o l l o w s : Acetone-soluble m a t e r i a l was r e m o v e d by f i l t r a t i o n after stirring under nitrogen for 24 h at 20°C. The r e s i d u e was e x t r a c t e d with chloroform:methanol (2:1) a n d the s o l u b l e m a t e r i a l w a s h e d w i t h 50 mM KCI at 4°C and d r i e d over m a g n e s i u m sulfate. S o l v e n t was r e m o v e d u n d e r vacuum, the l i p i d s d i s s o l v e d to 150 mg/mL in c h l o r o f o r m and stored frozen under nitrogen. All other reagents were o b t a i n e d f r o m c o m m e r c i a l s o u r c e s and u s e d as p r o v i d e d . Adrenal mlcrosomes were p r e p a r e d at 0°C in i s o l a t i o n medium consisting of 250 mM s u c r os e , i0 mM potassium phosphate, 1 mM dithiothreitol, 10% (v/v) glycerol, 1.0 mg/mL BSA, pH 8.0. Whole adrenal glands were thawed, t r i m m e d of fat a~d c o n n e c t i v e tissue, m i n c e d , and h o m o g e n ized in a W a r l n g b l e n d e r w i t h i s o l a t i o n m e d i u m and f r o z e n isolation medium. The p o s t - m i t o c h o n d r i a l supernatant (20 m i n at 2 0 , 0 0 0 x g) was c e n t r i f u g e d for 60 m i n at I 0 0 , 0 0 0 x g. The pellets (microsomes) were homogenized in i00 mM potassium phosphate, pH 8, and c e n t r i f u g e d for 60 m i n at i 0 0 , 0 0 0 x g. W a s h e d m i c r o s o m e s w e r e r e s u s p e n d e d by h o m o g e n ization in i s o l a t i o n m e d i u m to a p r o t e i n c o n c e n t r a t i o n of 2 5 - 3 0 m g / m L and s t o r e d in 1 mL a l i q u o t s at -20°C. Solubillzation of i s o m e r a s e a c t i v i t y was a c c o m p l i s h e d by d i l u t i n g w a s h e d m i c r o s o m e s in i s o l a t i o n m e d i u m at 0°C and adding, in order, sodium deoxycholate and Z w i t t e r g e n t 3-16 so that the f i n a l c o n c e n t r a t i o n s w e r e 4.5 mg/mL of p r o t e i n , 0.5% (w/v) d e o x y c h o l a t e , and 0.5% (w/v) Z w i t t e r g e n t 3-16. A f t e r 5 m i n at 0°C the s o l u b i l i z e d m a t e r i a l was c e n t r i f u g e d for 90 m i n at 2 0 0 , 0 0 0 x g. The s u p e r n a t a n t (solubilized isomerase) could be stored at -20°C without loss of activity. Reconstitution of s o l u b i l l z e d isomerase into p h o s p h o l i p i d v e s i c l e s was a c c o m p l i s h e d by the f o l l o w i n g p r o c e d u r e : The d e s i r e d p h o s p h o l i p ~ d s (5 mg total) in c h l o r o f o r m w e r e a d d e d to a 30 mL C o r e x " tube, d r i e d as a thin l a y e r u n d e r n i t r o g e n , r e d i s s o l v e d in a p p r o x i m a t e l y 1 m L a n h y d r o u s ether, dried under nitrogen, then e v a c u a t e d for 20 mln. Sodium phosphate (300 ~L of I0 mM, pH 7), was added, and the m i x t u r e was v o r t e x e d , t h e n s o n i c a t e d u n d e r n i t r o g e n for I0 m i n at 25*C in a b a t h - t y p e sonlcator. To this s u s p e n s i o n was a d d e d 600 pL of s o l u b i l i z e d mlcrosomes and i00 p L of b o v i n e s e r u m a l b u m i n (i0 m g / m L ) . The m i x t u r e was a l l o w e d to s t a n d for 30 m i n on ice, f o l l o w e d by the a d d i t i o n of suffic i e n t damp ~ i o b e a d s SM2 to r e a c h the s u r f a c e of the liquid. A f t e r an a d d i t i o n a l 60 m l n on ice the m i x t u r e was f i l t e r e d t h r o u g h g l a s s w o o l in a s y r i n g e . The v e s i c l e s u s p e n s i o n was d i l u t e d w i t h 500 pL of 10 mM s o d i u m p h o s p h a t e (pH 7) and centrifuged for 90 m i n at 1 5 0 , 0 0 0 x g (4"C). The s u p e r n a rant was r e m o v e d , and the v e s i c l e s w e r e g e n t l y r e s u s p e n d e d

Wehrle and Pollack

118

by h a n d in 600 f r o z e n at -20°C

#L of i s o l a t i o n for s e v e r a l days

medium. without

V e s i c l e s c o u l d be loss of a c t i v i t y .

I s o m e r a s e a c t i v i t y was a s s a y e d at 25°C by m e a s u r i n g the i n i t i a l rate_lof _ ~ s o m e r i z a t i o n of 5 - a n d r o s t e n e - 3 , 1 7 - d l o n e (E .16,100 M cm ) or 5 - p r e g n e n e - 3 ,20- d i o n e (E 17,600 -I M cm ) at 248 nm. Except where indicated, assays were p e r f o r m e d at pH 8.5 in 2.0 mL total v o l u m e c o n t a i n i n g 100 mM KCI, I0 mM EPPS ( N - [ 2 - h y d r o x y e t h y l ] - p l p e r a z l n e - N ' - 3 - p r o p a n e s u l f o n i c acid, p o t a s s i u m salt), 3 mM CaCI2, 0.I m g / m L BSA, and 2.5% (v/v) m e t h a n o l . All rate c o n s t a n t s are c o r r e c t e d for the rate of n o n - e n z y m a t l c i s o m e r i z a t i o n of the s u b s t r a t e u n d e r i d e n t i c a l c o n d i t i o n s of m e d i u m c o m p o s i t i o n , substrate concentration, and pH. Control experiments with appropriate mixtures of llpids , d e t e r g e n t s , and other reagents were p e r f o r m e d to m a t c h each type of p r e p a r a t i o n used. Vesicular preparations were sonicated for i0 see in a bath-type s o n i c a t o r p r i o r to assay. A s s a y s were l i m i t e d to s u b s t r a t e concentrations at w h i c h the s p o n t a n e o u s isomerization rate was low. Kinetic parameters were obtained from Lineweaver-Burk p l o t s u s i n g nine s u b s t r a t e concentrations, r a n g i n g from 3-80 ~M for a n d r o s t e n e d i o n e and 1 . 2 - 1 2 . 8 #M for pregnenedione. Values of the kinetic parameters were g e n e r a l l y r e p r o d u c i b l e to + 10%. -

Protein concentration was d e t e r m i n e d by the m e t h o d of L o w r y (16) on t r i c h l o r o a c e t i c acld-precipitated or i c e - c o l d acetone-precipitated protein.

RESULTS Adrenal consisting BSA, gave the To

and

mlcrosomes

of sucrose,

were

potassium

dlthlothreitol

consistently kinetic

avoid

at

acceptable

parameters

parameters

pH

due were

in

phosphate 8.0.

isolation buffer,

Although

activity,

slight

apparent

between

were

complications

of k i n e t i c

prepared

to

this

always

within

glycerol,

this

method

differences

in

preparations.

variability,

made

medium

comparisons

the

same

batch

of enzyme. A wide were

variety

examined

steroid

for

isomerase

of d e t e r g e n t s , their

ability

activity

of

to

both

ionic

solubillze

bovine

adrenal

and n o n - l o n l c , the

3 - o x o - A 5-

cortex

micro-

LIPID CONTROL OF ADRENAL A°-ISOMERASE somes.

These

lecithin,

included

Triton

n-octylglucoside,

X i00,

CHAPS

3-16,

cholate,

and

effective

for m e m b r a n e

activity

was

deoxycholate resulted both

in

(0.5% recovery

pregnenedione

activity

caused

reversible

upon

phospholipid (Table

At

solubilization,

w/v) of

and by

low. and

about

A

of

3-16

1-2%

activity

the

of

however,

thorough

sodium

(0.1%

Loss

reconstitution

with

of i s o m e r a s e

Zwittergent of

3-14,

concentrations

combination

solubilization,

vesicles

lyso-

3-8,

recovery

androstenedione.

successful

soy

Zwittergents

deoxycholate.

consistently

and

(3-[(3-cholamidopropyl)di-

methylammonio]-l-propanesulfonate), and

egg

119

w/v)

towards

isomerase

is

largely

of the e n z y m e

removal

of

into

detergent

i). TABLE

i

K i n e t i c P a r a m e t e r s for V a r i o u s Isomerase Preparations K i n e t i c p a r a m e t e r s were d e t e r m i n e d as at 25°C in I00 mM KCI, i0 mM EPPS, CaCI2, pH 8.5.

described 0.I m g / m L

Pregnenedione

Androstenedione Preparation

KM,

Vmax,

Vmax/KM,

KM,

~M

nmol/ (min.mg)

mL/ (mln.mg)

~M

nmol/ (min.mg)

10

5300

80

70

i0

2400

Intact microsomes

40

900

Solubilized microsomes

30

i0

Reconstituted isomerase a

45

500

23

0.3

ii

a R e c o n s t i t u t e d u s i n g 5 mg a s o l e c t i n p r o t e i n s o l u b i l i z e d w i t h 0.5% (w/v) Z w i t t e r g e n t 3-16.

in M e t h o d s BSA, 3 mM

Vmax,

Vmax/KM, mL/ (min.mg)

530

0.9

240

plus 0.8 mg m i c r o s o m a l d e o x y c h o l a t e plus 0.5%

120

Wehrle and Pollack Factors

s tud y was

of

affecting

the

not

reconstitution.

phospholipid

undertaken, Modifications

necessary

in o r d e r

lipid

is

important of

factor

remaining

for

reliable

the

The

several

may

specific the

highest

isomerase

fraction

of

The

due

of

of

are in

low

necessary

presence that we

For

probably

of so~e

do

not

enzyme

the

same

relative unwarrant-

Methods

restores

activity

as

have

activity

removal

reconstituted

is s u i t a b l e

which

are

reason

characteristics

enzyme

most

relatively

androstenedlone

isomerase

studies, very

this

possible.

activity

the

that

the

is

detergent

suggesting

the

value

functional

the

that

For

to be

Methods)

each

removal

the

found

are

phospho-

The

assayed

essential,

described

reconstituted

to

and

for

added

appears

have (see

comparisons

as

purification

diff.icult

2

activity

the

were

protocol

detergent

not

remain.

procedure

important

enzyme.

SM

absolutely

quantitative

substantial

and

is

pregnenedlone

ed.

Biobeads

residue

represent

reason

have

reconstitution,

albumin

consider

to

of

mixtures

unsuccessful.

in our p r e p a r a t i o n s , but

detergent

to

We

unless

activity

complete

of a c t i v i t y

before is

in r e s t o r i n g

detergent.

amounts

serum

recovery

a

reconstltutlon

reconstltution

solubillzed

reconstitution

residual

large

the

In p a r t i c u l a r ,

entirely

initiated,

in

for

phospholipid

to o p t i m i z e

combination.

lipid

requirements

several

tested.

Although

well

as

of

the

for

functional

previously of

a

native

been

the

solubilized

preparation

asolectln

enzyme. The

mixed

soy

phospholipid

LIPID CONTROL OF ADRENAL AS-ISOMERASE provides

a

activity. mately

consistent A mixture

native

serine,

of p u r i f i e d

microsomal

33%

choline,

reconstitution

the

Km

this

for

and

purified

economy.

Omission

Sphingomyelin,

phospholiplds,

concentration

described lacking of

by

microsomal

of

the

phosphatldyl-

et

rate

added

despite

al

proteins and

17,

(19),

also

enzymatic

to

increased

not the

recovery

tested in mixture

of

of

activity

its c h a r a c t e r i s t i c a l l y

cortex

mlcrosomes.

phospholipld

catalyze

pregnenedione,

of

refs.

of c h o l e s t e r o l

when

adrenal

Blomquist

androstenedlone

less

5%

cholesterol,

decreases

in

approxi-

phosphatidyl-

( p r e g n e n e d l o n e was

and so was r o u t i n e l y omitted, high

of

A s o l e c t i n was r o u t l n e l y used for

androstenedione

system).

10%

specific

(47%

phosphatidylethanolamlne,

18) gives slmilar results.

high

phosphollplds

proportions

5% p h o s p h a t l d y l i n o s l t o l ,

convenience

of

121

the

As

vesicles

isomerization

but

at

i0%

catalysis

in

the

- 15%

or

standard

assay. one p r e p a r a t i o n of Isomerase

For example, with

asolectin

dione

in

equivalent activity

the

standard

volume

to

phospholipid except

reconstituted nmol/min-mg.

2

a specific

assay

of pure

equivalent

equivalent

above,

exhibited

to

90% in

840

activity

I00

without

was

observed

The

rate

while

had

an

Isomerization

an

for

An

activity

a

synthetic

phosphatldylethanolamine

mixture of

pregnene-

had a specific

nmol/mln.mg.

phosphatidylcholine), this

for

nmol/mln.mg,

asolectln vesicles

nmol/min.mg

mixture

of

reconstituted

while activity by

(as

Isomerase of

290

phospholipld

122

Wehrle end Pollack

vesicles

alone

is

catalysis

by

activators

serum

uH

buffer

albumin

activators

by

The

K

the

assay

buffer

both

K

and

V

the

from

7.5

3-

to

however, in

this

Vma x

to

catalytic the n a t i v e

but

microsomal

Activators. differ

of

reaction.

the

tially

the

max

pH

c r e a s e d (Table

3).

pregnenedlone

could

of

bovine

Ca ++

serum

increases

the

the

pH

(The

for

K

V

bovine

max

on

raising

buffer

Trls

values

In e a c h

case,

with

of K m

activity

is

After

pH and

det~r-

successful

recovers

not

only

characteristic

mlcrosomal on the

serum

of

pH

isomerase dependence

albumin

substan-

androstenedione

both of

K

m

the

and

at

V

max

as is

isomerization

reliably

comparison.) only

the

IC). of

measured

and

phosphate

sensitivity

effect

of

pH, In

of

absence

slightly

dependence

kinetics be

EPPS.

vss B).

(Figure

in the

Intermediate

isomerase

pH

dependence

and

the

Isomerization

cationic

only

at e a c h pH w i t h

not

is

isomerase

10-fold

isomerase

in t h e i r

albumin m

to

buffer

A

the

Although V

(as

the

2).

seen.

activators

markedly

increases

substrate,

1,

enzyme

Two

(12,14)

in

characteristic

however,

activity

7-

varies

(Figure

reconstltution,

pH

strength,

(Table

are

when

for

max

ionic

whereas

entirely

gent-solubillzed

by

Isomerase

medium

Vmax/K m

The

disappears

V

zwltterionlc

ratio

range.

to

increases

in the

the

not

increase

max

9.5,

4-fold

are o b s e r v e d

and

m

sensitive

of

only

but

the

or Ca ++ .

composition

pH

increasing

3-oxo-AS-sterold

are

m

by

alone),

Deuendence.

androstenedlone of

increased

In

in

the

pH

deof

absence

contrast,

elevated

the

and

~ mM has

LIPID C O N T R O L

little the

or

high

small

no pH

effect

these

stimulation

effect

tic

dibucaine,

the

pH

Neither

on

on

the

at

BSA

nor

ratio

Vmax/K m .

although

both

has

any

TABLE

at

addition

V

pH

of

has

max

As

has

local

K

stimulating

only

effect

are

m

with a

anesthe-

little

and

123

7.5.

Ca ++

The

concentration,

Ca ++

Variation

AS-ISOMERASE

parameters

itself,

low

dependence,

OF ADRENAL

on

reduced.

effect

on

the

2

in I s o m e r a s e K i n e t i c P a r a m e t e r s with Buffer Composition

Kinetic parameters for the isomerization of androstenedione by microsomal isomerase were determined at 25°C as described in Methods with a~say medium and pH as indicated. i00 mM KP i

i00 mM EPPS

25

15

25

20

8.5

40

30

35

50

9.5

120

i00

i00

80

7.5

40

35

65

40

8.5

70

85

115

80

9.5

265

340

380

145

Parameter

pH

Kin, #M

7.5

Vma x , nmol/(min-mg)

Vmax/K m, mL/(min, mg)

activity

of

conditions the both BSA

(pH

Ca ++

(2.5-fold

1.6

2.3

2.6

2.0

8.5

1.8

2.8

3.3

1.6

9.5

2.2

3.4

3.8

1.8

8.5,

57

activity (3-fold at

I00 mM TrisCl

7.5

solubilized

isomerase by

I0 mM KP i

0.3

isomerase #M

of at

under

androstenedione).

reconstituted 2

mg/mL,

standard

mM pH

Ca ++, 8.5).

pH

In

enzyme 8.5,

is no

assay

contrast, stimulated

BSA)

and

by

124

Wehrle and Pollack

A.

B.

NATIVE M I CROSOMES

SOLUB ILl ZED

e2.0

$~ 1.o &>~ o

~

1.0

"~E

~>

300

300

,

A

0

E

300

•

~00.r-

e-

~"

200

2oo-~

I=

~- 2oo c

c :1<

100

loo ~"

loo

>

7.5

8.5

>

9.5

7.5

pH C.

®~E2.0

8.5

9.5

pH

RECONSTITUTED VES I CLES

A

900

900 • = •m E

::L ° 600

600

v'

,

300

300 >

.7.5

8.5 pH

9.5

Fieu~e i. oH Devendence of kinetic oaramete~s. Kinetic parameters were determined as described in M e t h o d s at 2 5 . 0 ° C in 50 m M K P i (A a n d B) or b O O m M T r i s C I ( C ) at t h e i n d i c a t e d

pH:

Km

(X)', Vm x

(')"

Vma /Km

(A). Walues o~ V /K ~ave max been normalized for eac~ type of i s o m e r a s e preparation, with the value at p H 8.5 s e t e q u a l co i. A. Native microsomes. B. Solubilized microsomes. C. Isomerase reconstituted as described in Methods into phosphollpid vesicles (asolectin).

LIPID CONTROL OF ADRENAL AS-ISOMERASE

125

TABLE 3 Modulators of Mierosomal Isomerase

Kinetic parameters for isomerlzatlon of androstenedione by mierosomal isomerase were determined as described in Methods in i00 mM KCI, I0 mM EPPS, 25°C, with additions and pH as indicated.

Parameter

No Addition

pH

Kin, /JM

Vma x, nmol/(min.mg)

Vmax/K m, mL/(min- mg)

+ Ca++ + Dibucaine + BSA (I mM) (50 ~M) (0.I mg/mL)

+ BSA + Ca++

7.5

40

40

30

70

70

8.5

80

70

60

70

80

9.5

240

500

200

110

200

7.5

I00

90

70

550

530

8.5

200

200

150

1300

1200

9.5

780

2000

450

1800

2300

7.5

2.5

2.3

2.3

7.9

7.6

8.5

2.5

2.9

2.5

19

15

9.5

3.3

4.0

2.3

16

12

DISCUSSION

Although an

integral

solubillzed using

a

adrenal

microsomal

3-oxo-A5-steroid

protein

(non-sedimentable

combination

detergents (8).

bovine

of

employed

Solubilized

by

(i). after

detergents Gallay

isomerase

et

shows

enzyme 90

mln

similar al

in

isomerase

activity at to

200,000 the

their

properties

can

is be

x

g)

mixture

of

purification which

differ

126

Wehrle and Pollack

substantially provide

from

an

mammalian original

the

native

appropriate isomer~se.

activity

system In

is

retained

of the s o l u b i l l z e d

however,

regenerates

tions

necessary Geynet

for

et al

treatment

somes,

A.

enzyme

of

the

than

2%

form.

the n a t i v e intact

and

form.

These

phosphollpld of

of i s o m e r a s e

the

dispersions In v i e w

the

of

total

reconstituted

addltlon-detergent

the e n z y m e

does not

activity.

with

not

able

of

to

aqueous

A-treated

micTo-

llplds

did

the

isomerase

asolectln

vesicles

removal

require

is

isomerase

that

into

observa-

after

mlcrosomal

of our f i n d i n g

a

activity

al w e r e

phosphollpase

in

bilayer

isomerase

3-oxo-AS-sterold

to

Recon-

results

introduction

detergent that

less

by

successfully

appears

the

activity

can

a

of

et

activity.

using

investigation

Geynet

restore be

not

Although

dispersions

aqueous

does

activity

a loss

thus

into a s o l e o t l n v e s i c l e s ,

expression

mlcrosomal

phospholipase

asolectin

proper

and

solubillzed

enzyme

an

(5) o b s e r v e d

of

regenerate

that

the

in

enzymatic

closely mimics

demonstrate

for

addition,

stltutlon

system which

enzyme

protocol,

specific

it

mlcrosomal

lipids. vH-Rate parameters tO

a

is

and

max

significant

(Tables more

V

vrofile.

also

for

the m l c r o s o m a l

et

of al

upon

the

For example,

in p h o s p h a t e

true

DePaillerets

m

variation

degree

2 and 3).

marked

K

The

buffer

with

pH

of

the k i n e t i c

isomerase

composition

of

the p H - d e p e n d e n c e than

the

reconstituted

(20)

have

found

it

that

is

the

the

medium

of V

in Trls.

enzyme,

depends

not

apparent

max

is

(This shown.) pK a of

LIPID CONTROL OF ADRENAL AS-ISOMERASE

Vma x

is

8.7

temperature-dependent,

at

13°C

in

DePaillerets that

an

9_~ al also

V m a x is r e l a t i v e l y

9.2

in

the

pH-dependence

their

substrate,

system.

to

above

In increase

neither

of

in

the

with I).

Vmax/K m

is

Although

a detailed

Km

a

can

alkaline isomerase effect s tat e the

pH

be to

is

of pH of

exhibited

by

the

seen

the

other protein

when

Reconstltutlon

an of

8.5 and examined of

which

conditions.

With

ourselves

to

a

K

and

m

to

rate

the

with

the

constant in

profiles

of

rate

of V

data,

pK a of

max

Isomerase

variations

pH-rate

V

it

about

conversion

pH. max is 8.5 of

to p r o d u c t s . from

Figure

either

solubillzed. on

pH

mlcrosomal

group

in

results,

profile

second-order

a

Trls.

profiles.

precision

of

to

concentration

confine

the

of the

increase

increase

some

isomerase

the

mM

have

tested~ b o t h

for

analysis

complex

will

36"C

our

a pH-rate

insensitive

deprotonatlon

the M i c h a e l l s It

pH

to

one

Vmax/K m

systems

contrast,

substantial

only

the p H - r a t e

buffer

unwarranted

that

causes

of

at

20

(1,11,21)

gives

we

8.3

to pH b e t w e e n

at

nor

mind,

relatively

is

clear

In

contrast

workers

Vma x

increasing

(Figure

and

in

isomerase

from

containing

insensitive

discussion

all

found,

unfortunately

results

qualitative

medium

Other

of

which

corresponds the

assay

shifting

127

membrane

that V

is

max

ability

lost

suggests

be 'due

to

may

since bilayer

into

the

result

component,

itself,

Isomerase

or

m

This

isomerase

intact

K

I

the

the

rather effect

membrane

asolectln

of

when

the

that

the

protonatlon than

of pH is

vesicles

that

of

is only present. restores

128

Wehrle and Pollack

not

only

substantial

alkaline

pH

to

should

be

system

depends

tution, Poor of

increase

noted

here upon

the

reconstitution

activity,

strong

dependence.

Two bovine

serum

ase

only

the

enzyme

albumin

is

seems

alkaline

pH.

Thls

higher

than

lipid

mlcrosomal

effect

on K m

in

opposite

to

the

which

than is

(23).

is

Also,

fluidity

as

pH

is

lowered

the

isomerase

it

2-fold

(14)

the

only

and

isomer-

only

when

effect

pH

of ~y 7 " 5,

9.5.

at

levels

certainly

an

enzyme

dibucaine,

which

has

on

a

the

an

which

been

a function

kinetic

with

at pH

than

8.5

high

at

Ca ++ ,

from

of

ions

almost

has

levels

or V m a x

(22),

of

low

caused

anesthetic

that

of

BSA.

changes

rather

bilayers

and

The

observed

physiological,

membrane

when

over

affected.

little

fluidity

fluidity

altered

the

the

reconsti-

isomerase

enhance

structure,

for

to a f f e c t

to

bilayer

reconstituted

calcium

appear

Ca ++ , w h i c h

It

associated

membrane.

Significantly,

isomerase

reduce

of

IC).

Recovery

always

more

(Figure

used

the

parameters

of

recovery

stimulates

in

ability

phospholipids

isomerase,

Each

be

both

on l i p i d

e nh a n c e s

range

to

is

the

the

sensitivity.

also

embedded

effect

directly.

the

(12),

It has

increases

effect

the

indirectly.

calcium

much

of

of

in the

however,

modulators

of

conditions

result pH

but

V max

behavior

level

little

s pec i f i c pH

the

the

and

Km

exact

can

with

activity,

both

that

particularly

isomerase

but

specific

effect

on

tends

to

reported

that

of

temperature

to

7.5

parameters

(24),

is the

ar~e m o s t

LIPID CONTROL OF ADRENAL Aa-ISOMERASE Bovine effector has et

no al

of

the

activity

been

shown

systems

by

similar

degraded

(25).

Its

role

even could

at all to

reduce free

in

components

in v l t r o

studies

BSA

in

(12),

Bertolino non~onic increases

affects Serum

other

the

albumin membrane

lysophosphatldes,

which may

e_tt a l

the

examined.

acids,

positive

the

block

"leakiness"

fatty

membrane

of

calcium,

pH v a l u e s

a

Indeed,

amounts

completely

Unlike

as

Bertollno

enzyme.

small

BSA.

removing

the b i l a y e r It

by

described by

solubilized

X-100

induced

first

isomerase

that

Triton

isomerase has

on

reported

Vma x

albumin,

mlcrosomal

effect

detergent in

serum

129

be

act

and

as

detergents

simply

to r e s t o r e

structure.

is

clear

from

investigators

(4,5,24)

crucial

for

the

Although

the

solubilized

characteristics reconstituted

that

results

a proper

and

those

membrane

of

functioning

of

the

can

solubilized,

its

activity

is from

be

low the

and

shows

activity

both

is

isomerase.

substantially in

other

environment

optimum

enzyme state

these

in

the

different native

and

forms. ACKNOWLEDGMENTS

This w o r k was s u p p o r t e d by G r a n t s CA 2 4 4 1 0 and GM 33059 a w a r d e d by the D e p a r t m e n t of H e a l t h and H u m a n S e r v i c e s . The a u t h o r s w i s h to t h a n k Ms. P a t r l c l a B o u n d s for m a n y h e l p f u l discussions.

REFERENCES

i. 2.

Murota, S., Fenselau, C.C., and T a l a l a y , 17, 25-37 (1971). Samuels, L.T., Helmrlch, M.L., Lasater, Reich, H. S C I E N C E 113, 4 9 0 - 4 9 1 (1951).

P.

STEROIDS

M.B.,

and

130

3 4 5 6 7 8 9 10

ii 12 13 14 15 16 17 18. 19. 20.

21. 22. 23. 24.

25.

Wehrle and Pollack

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