EndocrinolJapon

1992, 39 (1), 93-101

Purification Placenta

and Properties

of Steroid

Sulfatase

TAKASHI SUZUKI, KUMIKO HIRATO, TAKUMI YANAIHARA, TSUYOKI KADOFUKU*, TSUNEO SATO*, MINORU HOSHINO**

from Human

AND

NOBORU YANAIHARA**

Departmentof Obstetricsand Gynecology, and *SecondDepartment of Biochemistry, ShowaUniversity,SchoolofMedicine,Tokyo142, and **Departnientof PharmaceuticalScience , Universityof Shizuoka,Shizuoka422, Japan Abstract.

Steroid

sulfatase

was purified

microsomes and properties procedure interaction weight kDa

included solubilization chromatography on

of 500-600 on

sulfatase Triton

kDa

microsomes

The

obtained

chromatofocusing. defect

in

plancental for

result

membrane-enzyme

sulfatase

did

not

(E1-S)

substrate

were

50.6ƒÊM

specificity

and

inhibitors.

Key

words:

Steroid

PSD

contain

cysteine

residue.

sulfate

(DHA-S)

were

0.33nmol/min,

purified

sulfatase,

Placenta,

but

The

enhance

sulfatase

The

and

studies

Dehydroepiandrosterone

number

of

(sterol sulfate sulfohydrolase; enzyme which hydrolyzes a sulfates.

reported to be ovary, fibroblast

This

en-

present in liver, and especially in

normal related

human placenta. Steroid sulfatase and its physiological effects are important for

steroid

metabolism

in the

feto-placental

unit.

In

precursors Steroid attempts

17, 1992

cine, 1-5-8 Hatanodai,

Takashi Showa

the

be done

to purify

purified steroid

sulfatase

those

for

kinetic

study

suggest

with

different

Estrone

sulfate,

a

human

while

SUZUKI, University,

Shinagawa-ku,

Department of School of Medi-

Tokyo 142, Japan.

the enzyme

estrone the

substrates

Placental

enzyme,

have been

as to molecular mass and properties enzyme. In this study, the steroid

purified clarify

1991

Correspondence to: Dr. Obstetrics and Gynecology,

of

of the

and than

and

reported

by several investigators [1-5], with varying results

Received:

Accepted: January

values

the

rather

to estrogens. sulfatase is a microsomal

microsomes

18,

of 2%

in the placental

are hydrolyzed by steroid sulfatase and this step is essential for further metabolism of sulfated steroid

estrogen synthesis, the sulfo-conjugated steroids October

of 73

of steroid

(Endocrinol Japon 39: 93-101, 1992)

3ƒÀ-hydroxysteroid

zyme has been kidney, adrenal,

that

sulfate,

sulfatase deficiency.

STEROID sulfatase EC 3.1.6.2) is an

activity

in the enzyme

Vmax

should

mass

point

(PSD) after solubilization

revealed

results

hydrophobic a molecular

as a molecular

0.56nmol/min,

The

placental

gel in the presence

of a defect

Km and

and showed

the enzyme

analysis

7.8ƒÊM

human

isoelectric

deficiency

result

acid

further

migrated

in polyacrylamide

respectively.

enzyme,

normal

chromatofocusing, purified sulfatase

enzyme

not

is the

Amino

and

of the

did

structure.

dehydroepiandrosterone

sulfate

that

from

The major steps in the purification

electrophoresis.

focusing

with placental

indicates

the

gel

of phosphatidylcholine

from two patients This

the

whereas

polyacrylamide

to be 6.7 by isoelectric

addition

170-fold

X-100, column CL-4B. The

gel filtration,

sulfate

was estimated X-100.

with Triton phenylsepharose

on HPLC

sodium..dodecyl

approximately

of the enzyme were investigated.

same from

of

normal

and kinetic the enzyme procedure patients

human

of the purified sulfatase from placenta

studies were performed properties. In addition,

was to the

was applied to placentas obtained clinically diagnosed as having

placental sulfatase deficiency (PSD) [6].

SUZUKI

94

Materials

and

et al.

Protein

Methods

Chemicals

Protein

Polybuffer CL-4B,

exchanger

PBE

Polybuffer

purchased

from

74,

94, Phenylsepharose

and

Pharmacia

Ampholine LKB

radioactive

steroids

Chemical.

were

were

Tritium

labeled

steroids,

from

Sigma.

dehydroepian-

drosterone sulfate (DHA-S) ammonium salt [7-3H(N)] SA 851 GBq/mmol and estrone sulfate (E1-S) ammonium salt [6, 7-3H(N)] SA 1.77 TBq/ were purchased from New England Nuclear For the enzymatic studies, radioactive ster-

oids were purified by thin layer chromatography before use. Other chemicals were reagent grade. Reagents for electrophoresis were of specialreagent

grade

reagent

grade

for

electrophoresis

as *described

or

previously

analytical

were

stored

placentas in human after delivery and and vascular tissues

at -80•Ž

until

were after they

Bovine

mixed

[8]. with

imately

enzyme

of

in a 50mM

incubated

was terminated

suspension

DHA-S

3•~104dpm

dissolved and

The 10ƒÊM

at

tritium

labeled

Davis [10]. Sodium dodecyl sulfate (SDS) polyacrylamide

gel

for

by adding

20

min.

The

of an equal

were

extracted

into

1ml of toluene.

toluene extract (0.5ml) was then transferred

a

method of Laemmli [1 1]. Isoelectric focusing was carried on a 4% polyacrylamide gel containing 2% Triton X-100 and

2% Ampholine

(pH range 3.5-10.0) as described

HPLC gel filtration The

molecular G-3000

weight

of purified

by the LKB

SW

column

HPLC

(0.8•~30

phosphate

buffer

sulfa-

system

with a

cm)

equilibrated

(pH 7.0). An aliquot

measured

for

molecular

7.4),

sulfatase

steroid

was

each

weight

lin (670kDa), min (44kDa),

was

mixed

fraction

with

a small and was

(0.3ml/tube)

was calculated

with

and

thyroglobu-

gamma globulin (158kDa), ovalbumyoglobin (17kDa) and cyanocoba-

lamin (1.35kDa) as calibration standard proteins. of Amino

acid

analysis

The

to a

Approximately sulfatase

sured with an Aloka ter. Enzyme activity

containing

units (IU).

on

the

scintillation counting vial and added to 10ml of a Bray scintillation solution. Radioactivity was meaARC-301B is reported

performed

of calibration standard proteins with the same buffer. Enzyme activity

reaction

volume

was

amount eluted

0.1N NaOH. The unconjugated products of the reaction

electrophoresis

10% cylindrical polyacrylamide gel (5mm I.D.) by

of and

DHA-S)

(pH

as a standard

purified

approx-

buffer

used

of the

(20ƒÊl)

(containing

Tris-HCl

37•Ž

by desulfation by Burstein

was

Disc electrophoresis was carried out on a 7.5% cylindrical polyacrylamide gel (5mm I.D.) containing 2% Triton X-100 according to the method of

with 0.1M

activity was measured as reported first

albumin

Electrophoresis

TSK

Dorfman

serum

tase was estimated

studied.

Assay of enzyme activity Sulfatase 3H -DHA-S

with a commercial

previously [7]. Gels were stained overnight in 0.05% Coomassie Brilliant Blue R-250 in methanol-acetic acid-water (50:7:43, v/v), and destained in methanol-acetic acid-water (30:7:63, v/v).

[7].

Materials Normal and PSD obtained immediately removal of membranes

was estimated

protein.

Biotechnology.

obtained

content

dye reagent (Tonein TP kit, Otsuka Pharmaceuticals) according to the procedure of Bradford [9].

Tris, ethylenediaminetetraacetic acid (EDTA), Triton X-100, dithioerythritol (DTE), phenylmethylsulfonyl fluoride (PMSF), and non-

mmol Corp.

determination

scintillation counin international

pressure

3ƒÊg

was

hydrolyzed

for

20

h

1% phenol

in

of

the

at

110•Ž

6

N

purified

steroid

under

reduced

hydrochloric

with a Waters

acid

PICO-TAG

work station. Amino acids were analyzed Hitachi L-8500 amino acid analyzer.

with

a

STEROID

SULFATASE

IN HUMAN

Preparation of microsomes

95

PLACENTA

mM DTE,

0.05%

PMSF and

0.5% Triton

pH 4.0) and enzyme containing Frozen placentas (approximately 1,200g, wet weight) were thawed and all subsequent steps were

collected.

An aliquot

determine

steroid

carried

out

at

content.

minced

and

homogenized

4•Ž.

with 3 volumes

The

placental

tissues

in an

of buffer

were

electrical

I (10mM

also obtained

at delivery

procedure

described

homogenate

was

min.

The

for

referred

was

1h.

to as the

The

by the

The

at

for

finally

centrifuged

105,000•~g

pellets

The eluate were pooled column

resulting

10,000•~g

microsome

was taken

activity

and

sulfatase

to

protein

Hydrophobic interaction chromatography

Tris-acetate,

prepared

above.

centrifuged

supernatant

105,000•~g

and

were

fraction

mixer

0.25M sucrose and 5mM EDTA, pH 7.0). Placentas from the patients diagnosed as PSD [6] were

of each

X-100,

fractions

20

at are

(1•~5cm)

The column buffer II,

containing were

preparation.

fractions containing sulfatase activity and applied to a phenylsepharose equilibrated

was washed then eluted

1.25% Triton

collected

purified

with

with with

and

steroid

used

the

buffer

X-100.

Active

for

experiments

sulfatase

II.

10 bed volumes of the same buffer all

fractions as

preparations.

Solubilization of microsomes Microsomal

pellets

were

resuspended

in buffer

II (10mM Tris-HCl, 1mM EDTA, 1mM DTE, 0.05% PMSF and 0.5% Triton X-100, pH 7.5). The

suspension

wasgently

mixed

with

stirrer for 1 h at room temperature tion. The suspension was centrifuged g

for

1

h

at

4•Ž

to

remove

Results

Purification of steroidsulfatase

a magnetic Homogenates,

for solubilizaat 105,000

insoluble

microsomes

and

solubilized

mic-

rosomal fractions were prepared from eight normal term placentas. The solubilization followed by

materials.

chromatofocusing chromatography resulted in a

and hydrophobic interaction of normal placental microsomes 167-fold increase of its specific

supernatant.

activity

1). Solubilized

Chromatofocusing

applied to a PBE 94 column with buffer II. The column

Under

these

imately

70% of the

sulfatase

The 94

(pH

experimental

in the

resulting

column

8.0).

volumes fractions

original

microsomes

of steroid

was applied

equilibrated

thorough

of the same were eluted

approx-

activity was released

supernatant

(1•~38cm)

After

conditions,

with

washing

buffer, at 30

sulfatase milli with

into

the

to a PBE buffer

with

II

2 bed

containing 500ml of

buffer III (12.5% polybuffer 74, 1mM EDTA, 1 Table

1.

Purification

of steroid

(Table

microsomes

were

equilibrated to pH 8.0 was then washed with

the same buffer and 5 m/-fractions were collected. Sulfatase activity, protein content and the pH of each fraction were recorded. Figure 1 shows the elution pattern obtained solubilized microsomes. normal

placentas

were

in chromatofocusing The active fractions eluted

from

pH

of in

7.0 to 6.6

(Fig. 1a). They were pooled and were applied to a

sulfatase

from

human

placenta

96

SUZUKI

Fig.

1.

Elution ized

pattern

from

microsomes

collected

et al.

a chromatofocusing

were

applied

for determination

to

a PBE

94

column

of solubilized

column

(1•~38cm),

of pH, sulfatase

activity

microsomes. and

and protein

5ml

Solubil-

fractions

content.

were

In the left

panel, microsomes from normal placentas (1a). In the right panel, microsomes from PSD placentas (1b).

Fig. 2.

Elution

pattern

pooled

chromatofocusing

from

a phenylsepharose fractions

column were

of chromatofocusing

applied

to

a

column

pool. The (1•~5cm)

of

phenylsepharose CL-4B equilibrated with buffer II containing 0.5% Triton X-100. 2ml fractions were collected. Sulfatase was eluted with 1.25% Triton X-100 in the same buffer. The sulfatase activity of each fraction was assayed. Column fractions from 1 to 20 were collected and pooled as fraction A; 21-26, B; 27-37, C. Sulfatase activity was present in fraction C.

phenylsepharose buffer II. The

CL-4B column equilibrated with column was then washed with 40ml

cedure,

of

and

somes with approximately

buffer

II

containing

eluted

1.25% Triton

with

the

same

X-100. Fractions

buffer

preparations.

With

this experimental

1.7mg 1,764mg

of

steroid

of normal

sulfatase human

was

purified

placental

micro-

a 15.8% yield (Table 1).

contain-

ing steroid sulfatase activity (fraction numbers 27-37 in Fig. 2) were pooled and used as purified enzyme

from

pro-

PSD The

microsomes same

purification

steps

were

performed

on

STEROID

SULFATASE

IN HUMAN

PLACENTA

97

A;

Fraction

A;

Fraction

No 1-20

B;

Fraction

No 21-26

B;

Fraction

No 21-26

C; Fraction

No 27-37

Fraction

No 27-37

C;

Fig. 3.

Polyacrylamide numbers 1-20; and C, fraction

Std;

gel electrophoresis

. A, fraction B, fraction numbers 21-26; numbers 27-37 collected by

phenylsephaVose column. Enzyme activity of C gel was measured with DHA-S as substrate on 2mm gel slices.

Fig. 5.

Fig. 4.

No 1-20

5 Calibration

protein

SDS polyacrylamide

gel electrophoresis.

kDa), phosphorylase b (97.4kDa), glutamate dehydrogenase (55.4kDa), lactate dehydrogenase (36.5kDa) and trypsin inhibitor (20.1kDa).

Molecular weight determination of steroid sulfatase by HPLC. By means of a LKB HPLC system with a TSK G-3000 SW column, the purified sulfatase was mixed with a small amount of calibration standard proteins and eluted with 0.1

M phosphate enzyme

activity

buffer (pH 7.0). Fractions of 0.3ml of each

fraction

A, fraction

numbers 1-20; B, fraction numbers 21-26; and C, fraction numbers 27-37 collected by phenylsepharose column. Marker proteins were cr2-macroglobulin (170

was measured.

were collected and the Marker

proteins

were

1,

thyroglobulin (670kDa); 2, gamma globulin (158kDa); 3, ovalbumin (44kDa); 4, myoglobin (17kDa); and 5, cyanocobalamin (1.35kDa).

SUZUKI

98

2 PSD placentas. No sulfatase activity was found in microsomes. solubilized microsomes or after chromatofocusing

of solubilized

shows the elution of PSD placental addition

microsomes.

pattern from solubilized

buffer

following

the

to the

method

of

McNaught

and

Electrophoretic analysis purified

enzyme

phenylsepharose analyzed rylamide When

preparation

sulfatase

was calculated

on HPLC gel filtration

to be 500-600

obtained

by

amino

acid composition

approximately

3

preparation.

The

protein

contained

glycine

and leucine Table

2.

eu,g of results

Amino

was examined

the

purified

are given

relatively residues, acid analysis

large

of purified

sulfatase

CL-4B column (fraction C) was

sulfatase

electrophoresis,

the

activity

was measured

activity

after

corresponded

well

with the major protein band (Fig. 3). On the other hand, the sulfatase migrated according to a molecular mass

polyacrylamide

as a single band of 73 kDa on SDS

gql (Fig. 4).

Estimation of isoelectric point The estimated

isoelectric to

polyacrylamide

point be

6.7

of the steroid on

isoelectric

sulfatase

was

focusing

gel in the presence

in

of 2% Triton

X-100.

Estimation of molecular weight The

apparent

molecular

Fig. 6.

mass

of

Lineweaver-Burk purified

steroid

the

purified

graphs sulfatase.

illustrating

the effect

of the DHA-S concentration

on

with

sulfatase

in Table

and small

by disc electrophoresis on a 7.5% polyacgel in the presence of 2% Triton X-100.

steroid

kDa

(Fig. 5).

Amino acid analysis The

incubation

France [12] did not yield any sulfatase activity.

The

steroid

Figure 1b

chromatofocusing microsomes. The

of phosphatidylcholine

et al.

2. This

amounts amounts steroid

of of

STEROID

Fig. 7.

methionine, interest

tyrosine that

there

and

no

residues.

It is of

cysteine.

Kinetic

experiments steroid

substrates. protein

were

sulfatase

10ƒÊl

of

concentration)

7.8ƒÊM

those

for

with

and

the

E 1-S as

the effect

99

of the E 1-S concentration

The molecular weight assessed to be 500-600kDa

incubated

at

37•Ž

(Fig.

50.6ƒÊM

and

6),

0.33

previously data

are

McNaught

(3ƒÊg/ml for

incubator in a room air Vmax values for DHA-S

0.56nmol/min

were

out

DHA-S preparation

was

and

E1-S

carried

and

enzyme

20 min in a metabolic atmosphere. Km and were

PLACENTA

of steroid sulfatase was by HPLC gel filtration

on a TSK G-3000 SW column. The molecular mass of steroid sulfatase found by means of the gel filtration technique was reported as 166-440 kDa

Kinetic experiments

purified

IN HUMAN

Lineweaver-Burk graphs illustrating on purified steroid sulfatase.

histidine

was

SULFATASE

while

nmol/min,

respectively (Fig. 7).

with DHA-S as the substrate in

agreement

and France

with

trophoresed on SDS polyacrylamide gel, the sulfatase migrated as a single band with a molecular of 73kDa.

Similar

values

m icrosorne with Triton X-100, column and phenylsepharose

purified of the

chromatofocusing column chromato-

by other

[1-5, 13], 3 to 5 chromatographic used

to

obtain

homogeniety

amide gel electrophoresis. have used only a 2 step

laboratories

on

SDS

polyacrylstudy

weight may be due to used for enzyme preparation.

results

enzyme steroid

may

studied. sulfatase

reported

et al. (78

we

also be due

the various The diffe-

to the structure

Previous reports indicate is composed of identical

[1, 2, 4, 5]. McNaught and France [12] reported was no steroid

separations were

In the present procedure.

molecular methods rent

graphy. The purified enzyme revealed a single band on both disc and SDS polyacrylamide gel electrophoresis indicating apparent homogeneity. reported

have been

[1], Vaccaro

kDa) [2], Noel et al. (62kDa) [3], Burns (74kDa) [4], Loos et al. (63.7kDa) [5] and Moriyasu et al. (72 kDa) [13]. The different results with respect to the

steroid sulfatase has been 170- fold by solubilization

studies

of

two peaks

of sulfatase activity after sepharose 4B chromatography with molecular weights of 1,000kDa and 600kDa. When the purified enzyme was elec-

mass

Discussion

In previous

report

[12] who found

by Dibbelt and Kuss (64kDa)

The placental approximately

[1-5]. Our

the

microsomes. tidylcholine solubilized hypothesis

sulfatase

However, enhanced

activity

of the that the subunits

that there

in untreated

the addition the sulfatase

PSD

of phosphaactivity of the

preparation. They proposed that the biochemical basis of PSD

the may

SUZUKI

100

involve a defect in the membrane-enzyme ture which blocks normal function of the In the present the microsomes tase

activity

sulfatase as the enzyme preparation, and Vmax values for these substrates were 0.56nmol/min

strucenzyme.

study, following solubilization of and chromatofocusing, no sulfa-

was detected

although

et al.

phosphatidyl-

and 0.33nmol/min, have

also reported

steroid

sulfatase

respectively. the

Noel et al. [3]

Km for DHA-S

of purified

to be 14 AM M. Evidence

has

been

choline The sulfatase

was added to each preparation. amino acid composition of the steroid was calculated for the first time from the

presented that arylsulfatase C and steroid sulfatase is ranked, among the microsomal type I arylsulfatases. However, it is not yet clear whether this

sample found

of purified enzyme that cysteine was

group species

preparation, and it was not contained in this

enzyme. This bond in the

suggests that conformation

Further

is required

study

sequence zyme.

and

specificity

there is no disulfide of steroid sulfatase.

to clarify

higher-order

the amino

structure

this

acid

of this

really consists of several or of only one sulfatase

en-

[16]. Further

problem

purified S, E1-S,

by

solubilized

steroid

DHA-S

purified

as the

sulfatase

were

substrate.

arylsulfg.tase

by Vaccaro

reported

to solve

activity

of

the

esters such as DHAas substrates and

7.8,u,M

The

a Km

properties

for DHA-S

of

of 50,u,M

Km values for DHA-S and E 1-S

and

50.6ƒÊM

with

purified

Acknowledgements

to

C (ASC) have been reported

et al. with

[2]. In our study, were

the

inhibitors.

be 100 au,M and 25,u,M [12], and 1.4 pdM [14, 15], with

enzyme substrate

study is required

determining

enzyme with sulfate and p-nitrophenylsulfate

A kinetic study of the purified steroid sulfatase was performed with DHA-S and E 1-S as substrates. In previous reports, the Km of microsomal and

different of low

steroid

We are Pittsburgh,

indebted to Dr. P. Troen, Pittsburgh, Pennsylvania,

and correcting the manuscript. supported in part by Grant-in-Aid Research

No.

Education,

01570940

from

Univesity of for reading

This work was for Scientific the

Ministry

of

Science and Culture of Japan.

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purification,

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rat

liver

STEROID

microsomes.

14.

Biochem

92:

IN HUMAN

sulfatase

1189-1195.

101

PLACENTA

solubilized

ity to glycosidases. Hoppe-Seyter's Z Physiot Chem

the

in-vitro

hydroxydehydroepiandrosterone Seyler's

Z Physiol

hydrolysis

3-sulfate

Chem

364:

and

of

of

365:

16ƒ¿-

3-sulfate. Hoppe187-191.

Dibbelt L, Kuss E (1984) Human placental steroid-

16.

properties

and

derivative:

sulfatase

of

kinetic

a cholic-acid

Dibbelt L, Kuss E (1983) Human placental steroidkinetics

mass,

with

molecular

dehydroepiandrosterone

15.

SULFATASE

susceptibil-

1145-1153.

Rose FA (1982) The mammalian sulphatase and placental sulphatase deficency in man. J Inher Metab 5: 145-152.