167s
Biochemical SocietyTransactions ( 1 992) 20 Purification
Human
of
Foetal
Hepatic
Bile
Acid
S u l p h n t r a n s f e r a s e (RAST).
(500 u l ) c o l l e c t e d f r o m each s t a g e was a p p l i e d o n t o
a Superosel2
( 3 0 ~ 1 . 0 cm,
FPLC,
Pharmacia
Ltd.),
f i g u r e ?. MEHRAN MAGHSOUDLOO, MALCOLM J . P . M.
Gastroenterology
MURPHY.
Medicine &
Division o f
acid
(LCA)
component o f
acid
faecal
LCA o r i t s t a u r i n e
is
in
man
formed
in
the
distal
7a-dehydroxylation o f and
b i l e acid.
is
a
major
Administration
of
Sp. Ac. p u r i f i c a t i o n
16.66
16.75
MonoQ
5.0
11.0
Affinity
0.06
52.6
Cytosol
1.0
1.0 2.2
2.2
862
862.3
c o n j u g a t e t o r a t s and hamsters However,
accumulation
Three
it i s postulated
of
attempts
to
in
LCA
i s prevented
enterohepatic c i r c u l a t i o n sulphation.
Table 1: Prot.(mg) pmol/min
(COCA)
r e s u l t s i n cholestasis. that
of
Guy's
UMDS,
U.K.
human i n t e s t i n e by b a c t e r i a l chenodeoxycholic
Division
Biochemistry,
H o s p i t a l , London, S E I SRT, Lithocholic
H I G G I N S and GERARD
Unit,
As a p r e l i m i n a r y s t e p i n t h e assessment o f t h e b i l e a c i d s u l p h a t i o n and i t s c o n t r o l i n human l i v e r we
by h e p a t i c
have attempted t o i s o l a t e and c h a r a c t e r i s e t h e BAST
from
enzyme. A p p l i c a t i o n o f c y t o s o l on t h e ion-exchange
purify
human l i v e r have been made [ 1 , 2 , 3 ] .
the
BAST
I t was shown
column gave r i s e t o a m a j o r broad band a p p e a r i n g
estrone,
a f f i n i t y chromatography, an enhancement o f 862 was
dehydroepiandrosterone
[ l ] . However
the
procedure
(DHEA) and phenol,
used t o
isolate
the
achieved.
NaCl
gradient
Following
t.hat. t h e ' p u r i f i e d ' p r o t e i n was a l s o a c t i v e towards
after the
wt.
Mol.
of
the
fold
compared a g a i n s t a s e r i e s o f standards.
inactivat.ion
was
a
suggesting t h a t
major
problem.
enzyme
estrone,
testosterone,
or
[2].
phenol
Enzyme i s o l a t i o n r e s u l t e d i n 760 f o l d p u r i f i c a t i o n with
an
overall
yield
of
15% o f
by
approx.
gel
55,000
18
nmol/(mg
1.
Loof L. & H j e r t e n S.
Acta,
Chen L.
2.
( 1 9 8 0 ) , Biochem.
Biophys.
617, 192-204. &
Segel
(19851,
I.H.
Arch.
Biochem.
Biophys., 241,371-373.
A s i n g l e band on SDS/PAGE was shown t o
prot/min).
have a m o l e c u l a r w e i g h t o f 65000. R e c e n t l y , claimed,
that
[3]
a
purified
i t was
dehydro-
e p i a n d r o s t e r o n e (DHEA) ST from human l i v e r c y t o s o l liver
(16
weeks)
was
homogenised
Radominska A.,
Comer
K.,
Zimniak
in
a
P.
et
al.
(1990), Biochem. J., 272,597-604. NaC1 ( m M )
15 0 400
i
dithiothreitol
m i x t u r e o f t r i e t h a n o l a m i n e ( 1 0 mM), (1.5mM) and g l y c e r o l (10% v/v)
3.
I n o u r s t u d y human
was a l s o r e a c t i v e towards LCA. foetal
be
Another
p r e p a r a t i o n o f BAST was shown t o have no a c t i v i t y towards
to
peak
enzyme was a s s o c i a t e d w i t h a v e r y low r e c o v e r y (31 i n purity),
estimated
major
filtration
increase
was
reached IOOmM.
a t pH 7.5 - b u f f e r A. 300
The homogenate was spun (@I1OOOg) f o r t e n mins. and the
resulting
supernatant
was
further
spun
(a
100,OOOg) f o r 60 mins. T h i s c y t o s o l i c f r a c t i o n (2.0 ml)
was
applied
1:nto
exchange
resin(FPLC,
ml/min),
followed
MonoO,
a
strong
Pharmacia L t d . ) ,
by
buffer
A
200
anion
column (0.5
(6.0ml).
100
Proteins
were e l u t e d by a two-stage NaCl g r a d i e n t i n b u f f e r A.
Fract,ions (1.0ml)
BAST
activity
(LCA)
F r a c t i o n s 15 -18
0
were c o l l e c t e d and assayed f o r as
a substrate,
figure
1.
from t h e MonoQ column were pooled
3.0
B>A
agarose a f f i n i t y column (7x50 mm)
which had been
I
2.0
1.5
p r e v i o u s l y washed and e q u i l i b r a t e d i n b u f f e r A . The column was washed (5.0 m l ) w i t h b u f f e r A c o n t a i n i n g NaCl (50 mM), and t h e n e l u t e d w i t h (2.5ml) containing
u n l a b e l l e d PAPS
f30
o r d e r t o e s t i m a t e t h e mole. w t .
UM),
table
1.
In
I
!i
1.04
buffer A
o f RAST, an a l i q u o t
-
- IW!.
was
t h e n a p p l i e d o n t o an adenosine 3 ' , 5 ' - d i p h o s p h a t e -
15
10
20
25
Fraction (rnl)
( 4 . 0 m l ) , mixed and d i a l y s e d a g a i n s t b u f f e r A a t 4
deg. C o v e r n i g h t . The d i a l y s e d m a t e r i a l (2.5ml)
5
c.5 0
i 1
o0c
0Cytonol
a uorrup I Affinity
30
Biochemical SocietyTransactions ( 1 992) 20 Purification
Human
of
Foetal
Hepatic
Bile
Acid
S u l p h n t r a n s f e r a s e (RAST).
(500 u l ) c o l l e c t e d f r o m each s t a g e was a p p l i e d o n t o
a Superosel2
( 3 0 ~ 1 . 0 cm,
FPLC,
Pharmacia
Ltd.),
f i g u r e ?. MEHRAN MAGHSOUDLOO, MALCOLM J . P . M.
Gastroenterology
MURPHY.
Medicine &
Division o f
acid
(LCA)
component o f
acid
faecal
LCA o r i t s t a u r i n e
is
in
man
formed
in
the
distal
7a-dehydroxylation o f and
b i l e acid.
is
a
major
Administration
of
Sp. Ac. p u r i f i c a t i o n
16.66
16.75
MonoQ
5.0
11.0
Affinity
0.06
52.6
Cytosol
1.0
1.0 2.2
2.2
862
862.3
c o n j u g a t e t o r a t s and hamsters However,
accumulation
Three
it i s postulated
of
attempts
to
in
LCA
i s prevented
enterohepatic c i r c u l a t i o n sulphation.
Table 1: Prot.(mg) pmol/min
(COCA)
r e s u l t s i n cholestasis. that
of
Guy's
UMDS,
U.K.
human i n t e s t i n e by b a c t e r i a l chenodeoxycholic
Division
Biochemistry,
H o s p i t a l , London, S E I SRT, Lithocholic
H I G G I N S and GERARD
Unit,
As a p r e l i m i n a r y s t e p i n t h e assessment o f t h e b i l e a c i d s u l p h a t i o n and i t s c o n t r o l i n human l i v e r we
by h e p a t i c
have attempted t o i s o l a t e and c h a r a c t e r i s e t h e BAST
from
enzyme. A p p l i c a t i o n o f c y t o s o l on t h e ion-exchange
purify
human l i v e r have been made [ 1 , 2 , 3 ] .
the
BAST
I t was shown
column gave r i s e t o a m a j o r broad band a p p e a r i n g
estrone,
a f f i n i t y chromatography, an enhancement o f 862 was
dehydroepiandrosterone
[ l ] . However
the
procedure
(DHEA) and phenol,
used t o
isolate
the
achieved.
NaCl
gradient
Following
t.hat. t h e ' p u r i f i e d ' p r o t e i n was a l s o a c t i v e towards
after the
wt.
Mol.
of
the
fold
compared a g a i n s t a s e r i e s o f standards.
inactivat.ion
was
a
suggesting t h a t
major
problem.
enzyme
estrone,
testosterone,
or
[2].
phenol
Enzyme i s o l a t i o n r e s u l t e d i n 760 f o l d p u r i f i c a t i o n with
an
overall
yield
of
15% o f
by
approx.
gel
55,000
18
nmol/(mg
1.
Loof L. & H j e r t e n S.
Acta,
Chen L.
2.
( 1 9 8 0 ) , Biochem.
Biophys.
617, 192-204. &
Segel
(19851,
I.H.
Arch.
Biochem.
Biophys., 241,371-373.
A s i n g l e band on SDS/PAGE was shown t o
prot/min).
have a m o l e c u l a r w e i g h t o f 65000. R e c e n t l y , claimed,
that
[3]
a
purified
i t was
dehydro-
e p i a n d r o s t e r o n e (DHEA) ST from human l i v e r c y t o s o l liver
(16
weeks)
was
homogenised
Radominska A.,
Comer
K.,
Zimniak
in
a
P.
et
al.
(1990), Biochem. J., 272,597-604. NaC1 ( m M )
15 0 400
i
dithiothreitol
m i x t u r e o f t r i e t h a n o l a m i n e ( 1 0 mM), (1.5mM) and g l y c e r o l (10% v/v)
3.
I n o u r s t u d y human
was a l s o r e a c t i v e towards LCA. foetal
be
Another
p r e p a r a t i o n o f BAST was shown t o have no a c t i v i t y towards
to
peak
enzyme was a s s o c i a t e d w i t h a v e r y low r e c o v e r y (31 i n purity),
estimated
major
filtration
increase
was
reached IOOmM.
a t pH 7.5 - b u f f e r A. 300
The homogenate was spun (@I1OOOg) f o r t e n mins. and the
resulting
supernatant
was
further
spun
(a
100,OOOg) f o r 60 mins. T h i s c y t o s o l i c f r a c t i o n (2.0 ml)
was
applied
1:nto
exchange
resin(FPLC,
ml/min),
followed
MonoO,
a
strong
Pharmacia L t d . ) ,
by
buffer
A
200
anion
column (0.5
(6.0ml).
100
Proteins
were e l u t e d by a two-stage NaCl g r a d i e n t i n b u f f e r A.
Fract,ions (1.0ml)
BAST
activity
(LCA)
F r a c t i o n s 15 -18
0
were c o l l e c t e d and assayed f o r as
a substrate,
figure
1.
from t h e MonoQ column were pooled
3.0
B>A
agarose a f f i n i t y column (7x50 mm)
which had been
I
2.0
1.5
p r e v i o u s l y washed and e q u i l i b r a t e d i n b u f f e r A . The column was washed (5.0 m l ) w i t h b u f f e r A c o n t a i n i n g NaCl (50 mM), and t h e n e l u t e d w i t h (2.5ml) containing
u n l a b e l l e d PAPS
f30
o r d e r t o e s t i m a t e t h e mole. w t .
UM),
table
1.
In
I
!i
1.04
buffer A
o f RAST, an a l i q u o t
-
- IW!.
was
t h e n a p p l i e d o n t o an adenosine 3 ' , 5 ' - d i p h o s p h a t e -
15
10
20
25
Fraction (rnl)
( 4 . 0 m l ) , mixed and d i a l y s e d a g a i n s t b u f f e r A a t 4
deg. C o v e r n i g h t . The d i a l y s e d m a t e r i a l (2.5ml)
5
c.5 0
i 1
o0c
0Cytonol
a uorrup I Affinity
30
