Activation of Δ5-3-ketosteroid isomerase of bovine adrenal microsomes by serum albumins

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 13, 1979

Pages 1158-1166

ACTIVATION

OF A5-3-KETOSTEROID

ISOMERASE OF BOVINE

ADRENAL MICROSOMES BY SERUM ALBUMINS ARTHUR BERTOLINO,

ANN M. BENSON, and PAUL TALALAY'

Department of Pharmacology and Experimental Therapeutics, University School of Medicine, Baltimore, Maryland Received

The Johns Hopkins 21205, U.S.A.

May 7, 1979

SUMMARY. The A5-3-ketosteroid isomerase (EC 5.3.3.1) of bovine adrenal microsomes is activated as much as lo- to 20-fold by micromolar concentrations of bovine serum albumin. Comparable activations are observed with the serum albumins of 10 other mammalian species, but are not seen with ovalbumin Evidence that the activation is attributable to the serum or conalbumin. rather than to a small, firmly-bound ligand, is based on: albumins, (1) Failure to remove the stimulatory activity from the albumin by chloroform extraction, dialysis, or gel filtration; (2) Destruction of the activity by heating or by trypsin digestion; (3) Precipitation of the stimulatory activiBovine serum albumin inty of bovine serum albumin by specific antibody. duces small decreases in the Michaelis constant for A5-androstene-3,17-dione, but most of the activational effect reflects an increase in the maximum velocity. Low concentrations of Triton X-100, which are without effect on the isomerase activity, prevent the activation by bovine serum albumin. INTRODUCTION The conversion one)

to A 4 -androstene-3,17-dione

key steps

a nicotinamide

(EC 1.1.1.145)

steroidogenic

Several

studies

reaction

have dealt

microsome

weight

isomerase

cortex,

with

"activators"

activity

is

(EC 5.3.3.1).

are rich

the enhancement

and specifically

0006-291X/79/111158-09$01.00/0 1158

in

membrane

sources

and by serum proteins. profoundly

The dehydro-

extensively

of these

1To whom correspondence should be addressed. Abbreviations: BSA, bovine serum albumin; isomerase, isomerase.

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

dehydrogenase

in the microsomal which

are

Each transformation

has been investigated

and especially

adrenal

to progesterone

3S-hydroxysteroid isomerase

sequence (l),

and rat

by low molecular

hormones.

nucleotide-linked

tissues

of bovine

of steroid

and a A5-3-ketosteroid

genase-isomerase

(3$-hydroxy-A5-androsten-17-

and of pregnenolone

in the biosynthesis

involves

tion

of dehydroepiandrosterone

frac-

of these

enzymatic Bovine stimulated

A5-3-ketosteroid

enzymes.

activities adrenal by low

Vol. 88, No. 3, 1979

- 0.5

(0.1

pM) levels

catalytic tively

BIOCHEMICAL

function

of NAD or NADH (2).

by cyclic in beef

0.5 - 2.0 M CaC12 (or that

the

phospholipid-protein demonstration

that with

microsomal

lipids

beef

activity;

cited), by rat

tional

effect

bovine

adrenal

MATERIALS

(4)

microsomal

isomerase

a

is competi-

examined

isomer-

by treatment

with

at pH 7, and suggested containing

for

who observed

this

a divalent

view

cation-

was afforded

by the

was destroyed

activity

be restored

describes of various

was observed that

microsomes

(and possibly

The stimulatory

paper

aggregate

phenomenon

adrenal

the isomerase

This

--et al.

salts)

A, and could

activation

human serum albumins ovalbumin.

cation

involve

by NAD(H)

obtained

Some support

adrenal

does not

by addition

of total

(5).

and references

to progesterone

fragments

as a micellar

phospholipase

An interesting (6,

divalent

complex.

effect

Geynet

(3).

microsome

other

enzyme existed

by treatment

3',5'-AMP

adrenal

This

The activation

of the nucleotides.

inhibited

ase activity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

effect reaction

conversion

was stimulated

by lactalbumin), was on the was not

and examines mammalian

the

by Hamilton

was not

3S-hydroxysteroid

affected

on the

bovine,

and

stimulated

by

dehydrogenase

(6).

the mechanism

albumins

of pregnenolone by rat,

but

--et al.

of the profound isomerase

activity

activaof

microsomes.

AND METHODS

Materials. A5-Androstene-3,17-dione and A5-pregnene-3,20-dione were synthesized (7). Solutions of crystalline bovine plasma albumin (Armour) and serum albumins from other species (U.S. Biochemicals) were neutralized with NaOH. although in fact All albumins were assumed to have the mol. wt. of 68,000, Conalbumin (mol. wt. 89,000) was minor differences in mol. wt. exist (8). from SchwarzfMann; and chicken ovalbumin (mol. wt. 45,000) and whole bovine serum were from Pentex. Anti-bovine albumin was a rabbit IgG fraction (Cappel Laboratories) which contained 4.2 mg of antibody protein and 16.4 mg of total protein per ml. Trypsin (twice crystallized) was obtained from Worthington. Triton X-100 was from Sigma. Concentrations of Triton X-100 were determined spectrophotometrically at 275 nm (2.153 ml mg-lcm-1), and based on an average mol. wt. of 636 (9). Two types of tissue were used in these studies: Preparation of Microsomes. whole frozen calf adrenals and fresh bovine adrenal gortex (defatted and All operations were conducted at O-4 . Tissues were homodemedullated). genized in a Waring Blendor with 3 volumes of 50 mM TriE HCl, 250 mM sucrose, with HCl to 7.0 at 4 >. Successive 5 mM MgC12, 25 mM KC1 (pH adjusted centrifugations at 600 x g, 6,000 x g, 20,000 x g, and finally at 105,000 x Calf adrenal microsomes were g for 2 hours, yielded the microsomal pellets.

1159

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

suspended in the above medium at a protein concentration of 17.9 mg/ml. Beef adrenal cortical microsomes were washed once by centrifugation and resuspended in the medium at a protein concentratign of 33 mg/ml. These preparations were frozen rapidly and stored at -80 . Aliquots were thawed Protein concenand diluted lo-fold with distilled water just prior to use. trations were determined by the method of Lowry --et al. (10). Enzyme Assays. The standard spectrophotometric assay system for A5-3ketosteroid isomerase activity contained 100 mM potassium phosphate at pH 7.0, and either 68 pM A5-androstene-3,17-dione and 0.67% (v/v) methanol, or 10 UM A5-pregnene-3,20-dione and 3.3% methanol. The microsomal suspension The initial velocity of the formation was added to initiate the reaction. of the product, A4-androstene-3,17-dione (aM = 16,300 M-lcm-1) or A4pregnene-3,20-dione (aM = 17,000 M-lcm-l) was measured at 248 nm at 25' Corrections against a blank containing all components except the steroid. for nonenzymatic isomerization rates. were made, when necessary, RESULTS AND DISCUSSION Properties preparations and diluted of steroid the presence

of the

Steroid

retained aliquots

full lost

isomerization and absence

Isomerase;

Effect

isomerase no activity

activity

for

at least

in 10 hours

at 0 .

was proportional of BSA.

CONCENTRATION

of Serum Albumins.

0

Microsomal

1 month Initial

to enzyme concentration

At pH 7.0,

the

OF PROTEIN

rate

of isomerization

at -BOO, velocity both

in of

(MM)

of isomerization of A5-androstene-3,17-dione (open Fig. 1. Velocity symbols) and A5-pregnene-3,20-dione (solid symbols) by calf adrenal microof the concentrations of BSA (o and l ), human serum somes, as a function albumin (A and A), conalbumin (0 and I), and chicken ovalbumin (V and1 ). These proteins had been dialyzed against 0.1 M potassium phosphate at pH 7.0. The assa systems contained 0.1 M potassium phosphate, pH 7.0, and either 65 uM A3 -androstene-3,17-dione 0.67% methanol, and 11.9 ug of microsomal protein per ml, or 10 uM A 5 -pregnene-3,20-dione, 3.3% methanol, and 3.0 ug of microsomal protein per ml.

1160

BIOCHEMICAL

Vol. 88, No. 3, 1979

A5-androstene-3,17-dione 6 UM BSA,

and that

BSA (Fig.

1).

activities,

adrenal

Human serum albumin

cortex

microsomes

Effect

of pH.

(Table Figure

similar

of ten other

Effect

assay

species

of Albumins by

on the

Beef

Adrenal

by beef

Isomerization Cortex

adrenal

of

cortex

A5-Androstene-3,

activity

systema

-1

-lof mg protein)

89 116

albumins: Rat

391

Human

at b

394 Pig

461

Bovineb

487

Horse

498

Sheep

508

Goat

508

Rabbitb

528

Rabbit

537

Pig

545

Dog Chicken

556 569

standard

assay

a concentration

Crystalline

serum

system of

several-

profile

Microsomes

Conalbumin

aThe

were

adrenal

of BSA on the pa-activity

ovalbumin

Guinea

PM

enhanced

119

Serum

by l-2 these

by beef

(nmol min microsomal

Chicken

by

and conalbumin,

Specific to

in

lo-fold

I).

17-dione

Addition

20-fold

of A5-androstene-3,17-dione

of A 5 -androstene-3,17-dione

I.

was increased

increases

ovalbumin

2A shows the effect

of the isomerization

TABLE

proteins,

Serum albumins

of isomerization

RESEARCH COMMUNICATIONS

was enhanced

produced

two unrelated

ineffective.

the rate

microsomes

of A5-pregnene-3,20-dione

whereas

virtually fold

by calf

AND BIOPHYSICAL

was

supplemented

with

the

specified

5 NM.

albumins.

Others

were

1161

Cohn

Fraction

V.

proteins

mic-

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

PH

Fig. 2A. Velocity of isomerization of A5-androstene-3,17-dione by beef adrenal cortex microsomes as a function of pH, in the absence of BSA (A) and in the presence of 5 ~.IM gSA (0). Assays were conducted in Tris-potassium phosphate buffers at 25 , with 58 uM A5-androstene-3,17-dione. Fig. 2B. Velocity of isomerization A5-androstene-3,17-dione (0) and A5pregnene-3,20-dione (s) by calf adrenal microsomes as a functio; of pH. Assays were conducted in Tris-potassium phosphate buffers at 35 , with 2.5 uM BSA, and either 60 PM A5-androstene-3,17-dione or 5.3 PM A -pregnene3,20-dione. rosomes. with

As has been observed

increasing

ase

Maximal

levels

(Fig.

2A).

by calf

microsomes

A5-androstene-3,17-dione activities

in the

(Fig. assay

the absence

of specific

the adrenal

isomerase

pH 7, at which marked

of the

2B).

isomerization

most

increased

pH 8.5-9

absence.

of the

isomer-

of A5-pregnene-3,20-

of BSA resembles

Some instability

in the

The activa-

in the pH optimum

was apparent

information

activity

at about

pH in its

in the presence

systems

--in vivo,

isomerase

observed

to a shift

The pH dependence adrenal

(ll),

higher

by BSA is not due primarily

dione

were

of BSA, and at a slightly

presence tion

pH.

previously

of these

at pH values

above

that

of

enzymatic 8.5.

In

as to the pH of the microenvironment of our

pH the enzyme activities,

studies

while

stability. 1162

not

have been performed optimal,

exhibited

of at

Vol. 88, No. 3, 1979

TABLE II.

BIOCHEMICAL

Effect

AND BIOPHYSICAL RESEARCH COA4MUNlCATlONS

of BSA on the Kinetic

Parameters

for

Isomerization

of

A'-Androstene-3,17-dione source of microsomes

BSA

Beef adrenal cortex

Kinetics kinetic

constants

shown 0.5

of the Activation

in Table

for II.

pM) elicited

absence

of BSA did

a decrease

progressively

enhanced

such effects

0.1

0.51

41.9 +- 5.1

63

+

4

0.82

40.0 -+ 2.2

134

+

4

1.23

38.0 -+ 2.7

189

+-

7

2.05

41.9 +- 4.4

235

_+ 13

4.11

39.5 -+ 3.7

269

+ 12

6.16

35.8 -+ 5.1

268

_+ 20

8.22

38.5 +- 0.4

296

+-

1

10.27

39.2 -+ 1.7

311

+-

6

0

61.5 -+ 11.7

215

t 21

0.10

25.7 +-

3.9

268

2 13

0.25

19.4 +-

5.9

378

+- 31

0.50

21.4 -+

5.6

452

-+ 34

5.00

23.0 +-

2.0

655

+ 18

lowest

The effects

concentrations

solubility are not

on the

of the values in V max'

in Km, but were

to account

for

1163

the

aqueous

in the

concentrations

the Vmax values

might

large

(0.1

observed

Higher

increased.

in the

are

of BSA tested

A5-androstene-3,17-dione

of the steroid likely

40-70%

changes

as the BSA concentrations for

of BSA concentration

of A5-androstene-3,17-dione

as an increase

in further

of BSA on the Km values from

27.8 f-

in Km (to

as well

not result

54.6 -+ 0.4

isomerization

Even the

of albumin),

0

by BSA.

the

Vma* -+ S.E. (nmol min -1 mg-1 )

(d.0

Cd.0

Calf adrenal

+ S.E.

Km

rose

The small

effects

possibly

result

system. increases

However, in Vmax

Vol. 88, No. 3, 1979

which

are

tion

BIOCHEMICAL

primarily

responsible

of BSA required

for

androstene-3,17-dione not

appear

15.5

- 62 MM.

Triton

by Triton X-100

activity

of calf

efficiently

prevented

This

or calf

of the

the assay

was 1.57

of the steroid is

over

PM and did

the range

to be controlled

of

by the

first

these

is

not

affect

levels

the

isomerase

X-100

by 1 UM BSA (Fig. effect

was not observed incubated

related

as

of Triton

the activational

uM) and then

X-100

did

detergent

A5-androstene-3,17-dione

produced

X-100

were (372

- 1.62

of BSA, the nonionic

system)

with

reduced

of Triton

of Triton

unlikely

of A5-

complex.

the activation

detergent

effect

microsomes

in the same system,

microsomes

The concentra-

of isomerization

In the absence

X-100

by BSA.

activation

rate

microsomes

effect

adrenal

Thus the

reaction

X-100.

as 11 PM Triton

one-half.

levels

the

adrenal

However,

activation

the concentration

(3 - 186 )-M in the assay

substrate.

little

adrenal

of a steroid-BSA

Inhibition

the

half-maximal

with

Thus,

concentration

for

by calf

to vary

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

diluted

relatively

BSA

high

the assay

final

As

of BSA by

when either

into

to its

3).

concentration

system. in

system.

0

1 50

TRITON

1 loo

X-100

I

150

1

200

(LIPA)

Fig. 3. Effect of5Triton X-100 on the BSA-mediated activation of the isomerization of A -androstene-3,17-dione by calf adrenal microsomes. The assay systems contained 0.1 M potassium phosphate at pH 7.0, 68 uM A5androstene-3,17-dione, 0.67% methanol, 6.0 ug of microsomal protein per ml, and either 1 uM BSA (0) or no BSA (0).

1164

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ANTI-BSA

(ma)

of activator by rabbit anti-BSA IgG. A Fig. 4. Irmnunoprecipitation series of solutions containing 0.1 mg of BSA and either 0, 0.5, 1.0, or 1.4 mg of antibody protein in 0.4 n$ of 0.15 M NaCl-0.1 M potassium phosphate at pH 7.1, were incubated at 4 for 16 hours. After centrifugation, equal portions of each of the supernatant fractions (sufficient to yield a maximal concentration of 0.2 uM BSA in the assay system) were tested for their ability to enhance the rate of isomerization of A5-androstene-3,17-dione by beef adrenal cortex microsomes (0). A parallel series of incubations which contained antibody (and no BSA) served as a control (0).

Examination

of the Nature

activation

of the microsomal

or to a contaminant tor

could

not

in 88% loss for

with

G-150.

activator steroid activator

potency.

from

in

at the void

antibody

fraction

(Fig.

with

serum,

which

to gel

filtration

volume

of the

in loss with

1165

also

filtration

at 25'

of the

activated

the microsomal

on Sephadex

all

on

10 min resulted

loss

column.

BSA itself,

solution

(5%, w/w)

total

of activator

essentially

for

trypsin

and finally

resulted 4),

or by gel

the

The activa-

of an aqueous

of BSA at 100°

progressive

was subjected

was eluted

was considered.

by dialysis,

a solution

of whether

was due to the

BSA by extraction

Whole bovine

isomerase,

isomerase

and incubation

resulted

BSA by specific supernatant

Heating

The question

preparations,

chloroform,

of activity,

26 hours

steroid

in these

be removed

of the protein Sephadex

of the Activator.

G-25;

Precipitation potency

of the

from

activator

the of the

Vol. 88, No. 3, 1979

being

removed

BSA.

We conclude

the microsomal

BIOCHEMICAL

at an antibody that steroid

BSA is

protein directly

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

concentration

of 14 mg per mg of

responsible

for

the activation

of

isomerase.

ACKNOWLEDGMENTS These studies National Institutes GM 7309.

were supported by Research of Health, and by Medical

Grant AM 07422 from the Scientist Training Grant

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Beyer, K.F., and Samuels, L.T. (1956) J. Biol. Chem. 219, 69-76. 5, 715-724. Oleinick, N.L., and Koritz, S.B. (1966) Biochemistry Chem. McCune, R.W., Roberts, S., and Young, P.L. (1970) J. Biol. 245, 3859-3867, 1970. A. (1972) Eur. J. Biochem. 31, Geynet, P., Gallay, P., and Alfsen, 464-469. Geynet, P., De Paillerets, C., and Alfsen, A. (1976) Eur. J. Biochem. 71, 607-612. Hamilton, M.A., McCune, R.W., and Roberts, S. (1972) J. Endocrinol. 54, 297-315. F.S. (1962) Methods in Enzymology, 5, 527-532. Kawahara, (H. Neurath, ed.) F.W. Putnam, The Proteins III, 153-267, 1965. Academic Press, New York, N.Y. Ford, H.C., and Engel, L.L. (1974) J. Biol. Chem. 249, 1363-1368. Lowry, O-H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Murota, S., Fenselau, C.C., and Talalay, P. (1971) Steroids 17, 2537.

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