Vol.
166,
No.
January
30,
EVIDENCE
2, 1990
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
FOR THE PRESENCE INHIBITOR OF INTESTINAL
A. MARTIN, Department
of
OF AN ENDOGENOUS FUCOSYLTRANSFERASE
D. RUGGIERO-LtiPEZ,
General
December
15,
M.C.
1024-1031
CYTOSOLIC ACTIVITIES
PROTEIN
and P. LOUISOT**
BIOL*,
Medical Biochemistry, BP 12, 69921 OULLINS
and
School,
NkdiCdl
Received
AND
1990
INSERM-CNRS U 189, Cedex, FKANCE
Lyon-Sud
1989
Soluble endogenous inhibitory activities for glycoprotein : a (l-2) and a (l-3) fucosyltransferases are demonstrated in rdt small intestinal cytosol. These inhibitors are retained on DEAE-cellulose and are eluted as two fractions A and B. Fraction B is non dialyzable, heat stable and pronase-resistant and consists probably of polynucleotides. Fraction A is also non-dialyzable, but is thermolabile and pronase-sensitive, suggesting that it contains proteins. The inhibition of fucosyltransferase activity by fraction A is competitive for GDP-fucose and non-competitive for the Inhibition is not due to interfering enzymatic activities @ycoprotein substrate. (glycosyl-nucleotidt pyrophosphatases, glycosidases or proteases) and is reversible. This protein inhibitor, with a molecular weight of 60,000, is found only in the intestine and the pancreas and appears to be different from the previously reported inhibitors of brain glycolipid glycosyltransferases. Q isgo Academic PWS, 1~. Glycoprotein l),
which
biosynthesis
involves
nucleotide-sugar
reticulum
proteolysis
(3). The
many
and
effecters,
reports
have
inhibitors have
for
appeared,
of some glucosyl
(4,5),
been
of total
led us to
of intestinal
found and
divalent
in which
the
activities,
the
demonstrate
fucosyltransferase
or
Copyright All rights
to
known enzyme
and
presence
of endogenous
of some
protein
(sialyltransferase
(61, N-acetylgalactosaminyltransferase
course
of the purification
purification
purified
the existence
fraction
of the intestinal
step greatly
enhanced
(8). The pursuit
of a powerful
(7))
endogenous
the
of this protein
activities.
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
but
partial
quantity
Moreover,
in brain
rough
and depends
to the
nucleotides.
especially
see and
apparatus,
perhaps
sensitive
* Charge de Recherches au Centre National de la Recherche ** To whom all correspondence should be addressed. 0006-291X/90
review, in the
Golgi
due
partially
characterization
activities,
the first
in the partially
cations
partial
localized
availability, to be
(for
glycosidases)
and
of the state,
is only
substrate
is known
example
pathway
principally
a soluble
of such a pathway activity
During
are
compartments in
galactosyltransferase
described.
activity
different
glycosyltransferases
and
fucosyltransferase
the
metabolic
(glycosyltransferases
systems
are
as enzyme
such
Glycosyltransferase
various
enzymes These
regulation
factors,
activity.
has
(2).
glycosyltransferases
sorne
is a sophisticated
specific
carriers
endoplasrnic
on
Inany
1024
Scientifique.
soluble recovery
observation inhibitor
Vol.
166, No. 2, 1990
MATERIALS
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
AND METHODS
Preparation of fucosyltransferase activities. The purification of fucosyltransferase has been already reported (8). Briefly, intestinal cytosol (200,000 g supernatant of the mucosa homogenate obtained from 15 Sprague l)awley male rats, weighing 220-240 g) in 10 mM Tris, 10 mM KCI, 10 mM MgC12, 250 mM sucrose, pH 7.4, was incubated with 15 g of OEAE-cellulose (Whatman DE521 equiliorated in 10 mM Tris pH 7.6, for 30 nrin at + 4’C. After filtration on a glass filter, the filtrate was adjusted to pH 5.5 and incubated for 20 rnin with 10 g of CM-cellulose (Whatman CIVICS), equilibrated in a 10 mM MES buffer pH 5.5. After rinsing with MES buffer, fucosyltransferase activity was eluted with 0.2M KC1 in MES buffer. This partially purified preparation (x 200, as compared to cytosol) showed no loss of activity upon storage at - 20°C and was used for routine determinations of the inhibitory activity. It contain5 a mixture of oc (l-2jfucosyltransferase (acting on asialofetuin) and of a (l-3) fucosyltransferase (acting on asialoserotransferrin) (8). The separation of the two transferases by Sephaoex Cl5 chromatography after CUP-Sepharose ana Concanavalin A -Sepharose was achieved as previously reported (8). Microsomal fucosyltransferases were solubilized by 0.5 % Triton X-100 and processed in the same way (9). Preparation of the inhibitor. The material which was retained on DEAE-cellulose during fucosyltransferase Purification contained the inhibitory activity. The cellulose was poured into a column (2.0 x 10.0 cm) and was rinsed with 10 mivi MES buffer pH 5.5, then with 0.2 M KC1 in the same buffer. The column was then eluted either witn a continuous or a discontinuous gradient between 0.2 ana 0.7 M KCl. Fractions were dialyzea against water with changes every 2 hours, then overnight against MES buffer when a discontinuous gradient was used for elution. For continuous gradients, fractions were desalted by passing through small columns of Sepnadex G 25 (Pi, 10, Pharrnacia). In one pilot experiment, labeled GDP-fucose was added to the cytosol and the radioactivity in the fractions was monitored. When investigating the presence of inhibitory activity in intestinal rnicrosomes or in the cytosol from other organs, the same experimental design was applied to the microsomes (after soluoilization by Triton X-100), or to the cytosol of the liver, the lung, the pancreas and tne brain. Enzymatic cieterminations. For the deterllunation of fucosyltransferase activity with or without the inhibitor, the incubation mixtures contained : 40 Pl of partially purified fucosyltransferase, either 160 pl of the itiES buffer after dialysis (controls) or of dialyzed frdCtiOnS diluted with the saine buffer in order to adjust protein concentrations to the same values (about 1 - 5 pg by assay). Then, 0.2 kBq of COP--fucose (from New England Nuclear, sp. act. 8.8 C;Bq/rnmol) and 0.2 rnv of asialofetuin (for a (I-2)fucosyltransferase) or asialoserotrdnsferrin (for 0: (l-39 fucosyltrdnsferase) were added (X,9); incubatiotrs were carried out for 10 min at 23” C ana tne reaction stopped by 1 ml 20 % trichloroacetic acid. Trichloroacetic precipitates were filtered on Whatman C;F/B gldss filters and their radioactive content was determined by liquid scintillation counting. Determinations on each fraction were carried out at several protein concentrations, in order to assess the linearity of the inhibition. One innibitor unit was defined as the arnount of protein giving 50 % inhibition in this standardized assay (4-7). Controls. Glycosidase activities were determined with the corresponding paranitrophenyl-sugars according to Levvy and Conchie (10). Proteolytic activities were estimated with 3H-acetylhemoglobin according to Hille et al. (11). Labelled fucosylated asialofetuin was prepared by incubation of asialofemith fucosyltransferase, then purified by chromatography on a Sephadex Cl5 column. GDP-fucose breakdown Proteins were was assessed by high performance liquid chromatography (12). deterlnined using the arnido-black staining method of Schaffner and Weissman (13). The presence of guanine-nucleotide binding proteins (G-proteins) was monitored using New England Nuclear, sp. act. 48.8 the fixation of labelleo GTP- y -35s (from TBq/mmol) according to Moirii et al. (14). The Ki of guanine nucleotides for the fucosyltransferase in the absence or the presence of the protein inhibitor was calculated according to the method of Dixon (15). 1025
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS Identification
of
inhibitory
activities
for
fucosyHransferase
activity.
During
the
course of fucosyltransferase purification, the incubation of intestinal cytosol with DEAE-cellulose greatly enhanced the recovery of total activity of fucosyltransferase activity
(which
activity found
after
was
not
the DEAE-cellulose
in the cytosol
separate
experiments).
microsomal
retained
which
on the
step was multiplied
had not undergone
In contrast,
fucosyltransferase
(8). Total
cellulose)
by 6.8 + 1.8 compared
the DEAE-cellulose
the DEAE-cellulose
(9) increased
total
fucosyltransferase
step performed
activity
to that
step
(twelve
on solubilized
only by a factor
1.2 + 0.6
(8 experiments). The proteins, retained on the cellulose, were eluted with a KC1 gradient up to 0.7 M (figure 1). After elimination of the KCl, the inhibitory activity was found in two peaks about 0.27 M (fraction They were free of glycosylnucleotide-pyrophosphatase binding capacity. solubilized
No inhibitory
microsomal
KC1 (fraction
activity
preparations
A) contained
absorption
Similar
was detected
the major proportion
such conditions
under
of fucosyltransferase.
(Table I). The second peak (fraction rnaximurn
A) and 0.45 M KC1 (fraction 8). activity and also of GTP-Y-S The peak eluting at 0.27 M
of total
B) was of low
for the
activity
protein
and of proteins
content,
but exhibited
at 254 nm.
experirnents,
carried
out on various
organs,
indicated
that
such
inhi-
bitory activity was found only in the pancreas, but not in the liver, brain or lung. Controls. The inhibitory activity of fraction A was not due to the presence of interfering reactions. fraction (glycosidases, be significant
Indeed, the activity of several degradative enzymes in this pyrophosphatases and proteases) was very low aIla appeared to
only after
10 min incubation.
: Elution
When determinations
were
carried
pattern of the DEAE-cellulose column (linear gradient 200-700 mM activity is directly determined after desalting the fractions on The tubes corresponding to fractions A and B are Sephadex G25 PD 10 columns. pooled, as indicated oy horizontal bars, for further characterization.
1026
out
Vol.
166,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TABLE I Separation
of tuo soluble innibitory by E&E-cellulose
Fraction
total
proteins (n-d
specific activity (units/q)
activity (units)
722 22.5 5.3
Cell sap Fraction A Fraction B
activities of fucosyltransferase chraratography
23,248 9,439 4,024
purification factor
yield %
A.0
32.2 419.5 759.2
100 40.6 17.3
23.6
The tubes corresponding to fractions A and B from Figure 1 are pooled (as indicated in Figure I), dialyzed and the inhibitory activities are determined using the stanuarciized assay. One unit is defined as the amount of protein giving 50 % inhioition in this assay.
with of
saturating
concentrations
1.6 unit
cosamine
of inhibitory
activity,
was respectively
14,
asialofetuin
was only
per
minute.
As
tion
with
In order
to confirm
tory
activity,
the inhibitory
modify
of the
value
the
is
pmol/min.
galactose
The
and that
degradation
N-acetylglu-
of 14C-fucose-
of 3H-acetylhemoglobin
only
was not cleaved
l-h
enzymes
fucosyltransferase
after
This
with
was
0.1 % incuba-
with
where
the
but
radioactivity
up to
on the
was almost content
the
other not
preincubation.
the
inhibitor
was
leading
to
indicating
eluate,
radioactive
either
3 h, did
without
the
with of
DEAE-cellulose, in
retained
the innibitor
dialysis
of
activity
in the inhioi-
addition
fucosyltransferase,
column
partially
the
to controls
the
fucosyltransferase
involved
preincubation,
as compared
In the experiment
eluted
not
was preincubated
before
reaction.
inhibitor
radioactivity
were
A (1.6 unit/assay)
on a small
reversible.
since
of fucose,
GDP-fucose
inhibition
of
after
or
degradative
or
of the
was
release 19
fraction
of the
cytosol,
GDP-fucose dialysis
that
fucosylation
restoration
inhibition
and
by HPLC,
chromatography
by
complete
5
in the presence
fraction.
the
preincubation
removed
to
controlled
asialofetuin
constituents
the
of each paranitrophenyl-sugar
0.2 % per minute
the inhibitory
GDP-fucose,
After
(5 mM)
a
that
GDP-fucose
was added
DEAE-cellulose
(40
completely
%).
eliminated
was less than
by
2 % of initial
radioactivity. Characterization activity
of the
were
B was heat 50”
(3 for
non dialyzable stable
fraction
A had content
ribonuclease
fraction
rnodified of
of fraction Tl
(E.C.
activity
two stable
15 min.
75,
48,
insensitive 3.1.27.5)
only might
fractions
and
depend
for several
at 23O C and
In contrast,
18 and and its to pronase
to pronase.
A and B containing
at - 20°C
by incubation
B was low
B was
A was sensitive
the inhibitory
C for
a half-life
at 254 nm. Fraction
The
and were
ana not
1 h and at 75”
proteinic of
inhibitors.
at these
2 min action
together,
on the presence 1027
C for
same
3 h, at
In addition, was
and sensitive these
Fraction
temperatures,
spectrum II
deoxyribonuclease
Taken
months. 37’
respectively.
absorption
inhibitory
(E.C
to the action while
3.1.21.11,
results
of polynucleotides
the
maximum
suggest for
that
fraction
Vol.
166, No. 2, 1990
BIOCHEMICAL
10
AND BIOPHYSICAL
20
RESEARCH COMMUNICATIONS
40
30
50
fraction
number
Figure 2 : Pattern of inhibitory activity of fraction A (from Figure 1) on a Sephacry1 HR - 100 S column (2 x 100 cm) equilibrated in MES 10 mM pH 5.5. The column is calibratea with standards from Pharmacia : dextran blue (exclusion volume),
albumin
(a,
MW
67,000),
ovalbumin
(b,
MW
43,000)
and
chymotrypsinogen
H
(c, MW 25,000).
B and
of
proteins
was performed After graphed single
for
fraction
on the protein dialysis,
Figure
with of
competitive
HK-100
a
for
II
inhibitor
and
the
guanine
activrty.
The
affinity for
S. The
GDP-fucose
Table
pattern
GDP,
nucleotides
for
(nM”)
was the for
of the
and
inhibitory 60,000
concentrations
nucfeotide
in the presence
GDP-fucose
the
about It
non-competitive there
of guanine
of
activity.
that
of the
by lyophilization
weight
of two
and
indicate
characterization
innibitor
A.
concentrated
(l-2)fucosyltransferase
in
the
fraction
molecular
influence
the
reported
particularly
A was
an apparent
3 shows
parameters
containing
fraction
on Sephacryl peak,
A. Therefore,
is
activity
(figure
on the
clear
inhibition
for
that
asialofetuin. between of
the
inhibitor.
kinetic was
The
results
the
protein
is decreased, The
inhibitor
was
l/G
Figure) 3 : Influence of the inhibitor (fraction A) on the kinetic 0: 1-2 fucosyitransferase activity, determined with asialofetuin. 1028
a
fucosyltransferase
the fucosyltransferase protein
showed
2).
of inhioitor
competitron inhioition
chromato-
parameters
of
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
Ki (UM) of various
nticleotides
the
presence
absence
or
the
Without
1Nucleotide
fraction
c&P
31
aP cm WP-mannose
0.9 3.3 49
active
to
the it
substrates;
ferasrs
same
for
a(l-2)
the
proteinic
A
fucosyltransferase inhioitor
With
activity
A (1.6
fraction
unit/assay)
A
69 31 7.3 326
extent
either
with
active
on the
purified
was also
separated
of
COMMUNICATIONS
II
THBLE
in
RESEARCH
by Sephadex
Gl5
asialofetuin
or
oc (l-2)
and
cnromatography
after
asialoserotransferrin oc (l-3)
the
affinity
as
fucosyltrans-
chromatography.
DISCUSSION The existence ferase
activities
activity
after
was first partial
DEAE-cellulose. on
the
radioactivity
are
Thus,
after
cannot
dialysis) Intestinal
parameters
such
the
the
protein
concentration,
negligible
controls in the
fucosyltransferase
very
low
intestinal are
eluted
deterrninations
and
are
negligible
glycosyl-nucleotide at
sensitive
use
and
physico-chemical pH
(8,9).
of the dialysis
buffer
Alternatively,
use of rat
concentration
Thus
for
used
in the
up to 50 ug/assay
prepa-
desalting
results.
The
and bovine
a
is achieved
the use of buffers
satisfactory
of
substrate.
This
G25 and
traces (determined
activity.
of Sephadex give
to
strength
innibitory
on the
2 % of the initial
very
fractions.
experiments,
of inhibitory
the glycoprotein
rate,
less than
only
radioactive
the
at concentrations
dialysis,
of the
ionic
by the
of endogenous
dilution
of inhibitory
of protein
ana indirect
source
nucleotide-sugar,
range
for
with
retained
fractions
true
dilutions
treatment
of these
is
and
by
of total
serum assay
by
similarly effect
of
albumins, since
these
do no inhibit
the
activity.
direct
as a possible
the
after
fucosyltrans-
recovery
is partially
activity
concentration,
and
proteins
accounting
to measure
in the
to the fixation
However,
by isotopic
as confirmed
binding
attributed
inhibitory
soluble
transferase
Gi)P-fucose
activity
columns
increase
soluble
Ldbeied
of the fractions
on small
for
From
important
is needea
treated
the
K(31 gradient.
ana for dilutions
chromatography
are
the
as protein
controls
of
be explained
dialysis
non specific
(8).
intestinal
by the drastic
in the fractions,
the
assay
exhaustive
ring
with
inhibiting
was tentatively
fucosyltransferase
standardized by
effect
found
radioactivity.
suggested
cellulose
and elutes
factor
purification
This
Zi)P-fucose cellulose
of an endogenous
a lower are
interfering These
acceptor
or the enzyme,
compared
confirmed
strength by indirect
reactions,
to the
pyrophosphatases ionic
reactions
activity.
than
are
degree
can be eliminated
which
can destroy
are found of inhibition.
retained
at a
Moreover,
on DEAE-cellulose,
fractions
A and
B (16).
experiments.
Indeed,
preincubation
1029
the
to occur
These
but direct experi-
Vol.
166,
No.
ments
2, 1990
confirm
Further,
that
the
existence protein
action
enzyme.
This
itself
by
fact
apparent
It
to be very activities.
the
enzyme
inhibitor the
is
reversible.
either
binding
a soluble A from
donor,
site
G-protein the
for
the
with
of the
the sugar-nucleo-
on the
and
glycofor
nucleotides
fucosyltransferase.
: GTP- y -S
DEAE-cellulose
the
confirmation
and
guanine
interacts
Therefore,
competitive
of
process.
is established.
is non
affinity
the inhibitor
COMMUNICATIONS
in the inhibitory
activity
sugar-nucleotide
fraction
in the brain,
These
binding does
activity
is
exhibit
not
from
were
reported
inhibitor
studied
(7). They
characterized
preparation
nor
several
subunits
inhibitory
the
activity
any
of
for
brain
Albarracin with
the
its
(5). This These
and
reacts
appears
(4,6)
asialofetuin
or soluble
than
inhibiting organs, is about for
polysaccharides
The
best
sialyltrans-
weight also
inhibitory
(5,7).
in other
are
:
UDP-N-acetyl-
degrades
protein,
inhioitors
with
of one of the
and
molecular
sulfated
inhibitor
in
: glycosyltransferases
is non competitive
protein
this
likely
and
glycolipid
stable
apparent
inhibitor
Thus,
exception
in the brain
abundant
of fucosyltransferase
of glycosyltransferase
labile
heat
of
inhibitors (5) also
is heat
protein
in the intestine
or the liver.
membrane-bound
intestine;
heparin
only
and
reported
(5) is an acid
pointed
have important
intestinal
either
inhiiiitor
labile
but it has 70-80,000
the sugar
different
nucleofrom
described
for
the brain
(17).
As already on
are
acceptor.
fucosyltransferase
Studies
which
more
in the
for
stable,
(luiroga,
It is
tested
by
are heat by
galactosamine
(4) with
the lung
inhibitors They
activity.
state,
previously
describea
been
in a soluble
as a heat
is a true
and
the
the
sialyltransferase.
is characterizea
60,000
but
not
paper
about
only
though
ferase
in this
weight
different
activities
true
involved
not
the sugar-nucleotide in
is found
the pancreas,
could
for
that
A described
molecular
activities.
tide
the
RESEARCH
activity.
Fraction
not
that
is not
before
are
on the
decrease
the
BIOPHYSICAL
of fucosyltransferase
suggests
inhibitor
eluted
(4-7),
the
with
the
inhibitory
inhibitor
and competitive
or
However,
the
indicate
provided
clearly
of
AND
enzymes
inhibitor
studies
acceptor is
tide
degradative
of a true
Kinetic this
BIOCHEMICAL
the
out (5,6),
consequences
mechanism
fucosyltransferase
of
the existence
of such
in the regulation developmental
activities
must
(18) take
glycosyltransferase
inhibitors
of glycosyltransferase or nutritional into
account
(19)
activities. regulation
the presence
of
of this
inhibitor.
ACKNOWLEDGMENTS The skillful technical assistance of I&e HUGUENY was greatly appreciated. We thank Dr Jerry QUASH for critical revision of the manuscript. This work was supported by the Institut National de la San& et de la Recherche Mhdicale, the Centre National de la Recherche Scientifique and the Universitb de Lyon. REFERENCES I. 2.
I
166,
No.
January
30,
EVIDENCE
2, 1990
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
FOR THE PRESENCE INHIBITOR OF INTESTINAL
A. MARTIN, Department
of
OF AN ENDOGENOUS FUCOSYLTRANSFERASE
D. RUGGIERO-LtiPEZ,
General
December
15,
M.C.
1024-1031
CYTOSOLIC ACTIVITIES
PROTEIN
and P. LOUISOT**
BIOL*,
Medical Biochemistry, BP 12, 69921 OULLINS
and
School,
NkdiCdl
Received
AND
1990
INSERM-CNRS U 189, Cedex, FKANCE
Lyon-Sud
1989
Soluble endogenous inhibitory activities for glycoprotein : a (l-2) and a (l-3) fucosyltransferases are demonstrated in rdt small intestinal cytosol. These inhibitors are retained on DEAE-cellulose and are eluted as two fractions A and B. Fraction B is non dialyzable, heat stable and pronase-resistant and consists probably of polynucleotides. Fraction A is also non-dialyzable, but is thermolabile and pronase-sensitive, suggesting that it contains proteins. The inhibition of fucosyltransferase activity by fraction A is competitive for GDP-fucose and non-competitive for the Inhibition is not due to interfering enzymatic activities @ycoprotein substrate. (glycosyl-nucleotidt pyrophosphatases, glycosidases or proteases) and is reversible. This protein inhibitor, with a molecular weight of 60,000, is found only in the intestine and the pancreas and appears to be different from the previously reported inhibitors of brain glycolipid glycosyltransferases. Q isgo Academic PWS, 1~. Glycoprotein l),
which
biosynthesis
involves
nucleotide-sugar
reticulum
proteolysis
(3). The
many
and
effecters,
reports
have
inhibitors have
for
appeared,
of some glucosyl
(4,5),
been
of total
led us to
of intestinal
found and
divalent
in which
the
activities,
the
demonstrate
fucosyltransferase
or
Copyright All rights
to
known enzyme
and
presence
of endogenous
of some
protein
(sialyltransferase
(61, N-acetylgalactosaminyltransferase
course
of the purification
purification
purified
the existence
fraction
of the intestinal
step greatly
enhanced
(8). The pursuit
of a powerful
(7))
endogenous
the
of this protein
activities.
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
but
partial
quantity
Moreover,
in brain
rough
and depends
to the
nucleotides.
especially
see and
apparatus,
perhaps
sensitive
* Charge de Recherches au Centre National de la Recherche ** To whom all correspondence should be addressed. 0006-291X/90
review, in the
Golgi
due
partially
characterization
activities,
the first
in the partially
cations
partial
localized
availability, to be
(for
glycosidases)
and
of the state,
is only
substrate
is known
example
pathway
principally
a soluble
of such a pathway activity
During
are
compartments in
galactosyltransferase
described.
activity
different
glycosyltransferases
and
fucosyltransferase
the
metabolic
(glycosyltransferases
systems
are
as enzyme
such
Glycosyltransferase
various
enzymes These
regulation
factors,
activity.
has
(2).
glycosyltransferases
sorne
is a sophisticated
specific
carriers
endoplasrnic
on
Inany
1024
Scientifique.
soluble recovery
observation inhibitor
Vol.
166, No. 2, 1990
MATERIALS
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
AND METHODS
Preparation of fucosyltransferase activities. The purification of fucosyltransferase has been already reported (8). Briefly, intestinal cytosol (200,000 g supernatant of the mucosa homogenate obtained from 15 Sprague l)awley male rats, weighing 220-240 g) in 10 mM Tris, 10 mM KCI, 10 mM MgC12, 250 mM sucrose, pH 7.4, was incubated with 15 g of OEAE-cellulose (Whatman DE521 equiliorated in 10 mM Tris pH 7.6, for 30 nrin at + 4’C. After filtration on a glass filter, the filtrate was adjusted to pH 5.5 and incubated for 20 rnin with 10 g of CM-cellulose (Whatman CIVICS), equilibrated in a 10 mM MES buffer pH 5.5. After rinsing with MES buffer, fucosyltransferase activity was eluted with 0.2M KC1 in MES buffer. This partially purified preparation (x 200, as compared to cytosol) showed no loss of activity upon storage at - 20°C and was used for routine determinations of the inhibitory activity. It contain5 a mixture of oc (l-2jfucosyltransferase (acting on asialofetuin) and of a (l-3) fucosyltransferase (acting on asialoserotransferrin) (8). The separation of the two transferases by Sephaoex Cl5 chromatography after CUP-Sepharose ana Concanavalin A -Sepharose was achieved as previously reported (8). Microsomal fucosyltransferases were solubilized by 0.5 % Triton X-100 and processed in the same way (9). Preparation of the inhibitor. The material which was retained on DEAE-cellulose during fucosyltransferase Purification contained the inhibitory activity. The cellulose was poured into a column (2.0 x 10.0 cm) and was rinsed with 10 mivi MES buffer pH 5.5, then with 0.2 M KC1 in the same buffer. The column was then eluted either witn a continuous or a discontinuous gradient between 0.2 ana 0.7 M KCl. Fractions were dialyzea against water with changes every 2 hours, then overnight against MES buffer when a discontinuous gradient was used for elution. For continuous gradients, fractions were desalted by passing through small columns of Sepnadex G 25 (Pi, 10, Pharrnacia). In one pilot experiment, labeled GDP-fucose was added to the cytosol and the radioactivity in the fractions was monitored. When investigating the presence of inhibitory activity in intestinal rnicrosomes or in the cytosol from other organs, the same experimental design was applied to the microsomes (after soluoilization by Triton X-100), or to the cytosol of the liver, the lung, the pancreas and tne brain. Enzymatic cieterminations. For the deterllunation of fucosyltransferase activity with or without the inhibitor, the incubation mixtures contained : 40 Pl of partially purified fucosyltransferase, either 160 pl of the itiES buffer after dialysis (controls) or of dialyzed frdCtiOnS diluted with the saine buffer in order to adjust protein concentrations to the same values (about 1 - 5 pg by assay). Then, 0.2 kBq of COP--fucose (from New England Nuclear, sp. act. 8.8 C;Bq/rnmol) and 0.2 rnv of asialofetuin (for a (I-2)fucosyltransferase) or asialoserotrdnsferrin (for 0: (l-39 fucosyltrdnsferase) were added (X,9); incubatiotrs were carried out for 10 min at 23” C ana tne reaction stopped by 1 ml 20 % trichloroacetic acid. Trichloroacetic precipitates were filtered on Whatman C;F/B gldss filters and their radioactive content was determined by liquid scintillation counting. Determinations on each fraction were carried out at several protein concentrations, in order to assess the linearity of the inhibition. One innibitor unit was defined as the arnount of protein giving 50 % inhibition in this standardized assay (4-7). Controls. Glycosidase activities were determined with the corresponding paranitrophenyl-sugars according to Levvy and Conchie (10). Proteolytic activities were estimated with 3H-acetylhemoglobin according to Hille et al. (11). Labelled fucosylated asialofetuin was prepared by incubation of asialofemith fucosyltransferase, then purified by chromatography on a Sephadex Cl5 column. GDP-fucose breakdown Proteins were was assessed by high performance liquid chromatography (12). deterlnined using the arnido-black staining method of Schaffner and Weissman (13). The presence of guanine-nucleotide binding proteins (G-proteins) was monitored using New England Nuclear, sp. act. 48.8 the fixation of labelleo GTP- y -35s (from TBq/mmol) according to Moirii et al. (14). The Ki of guanine nucleotides for the fucosyltransferase in the absence or the presence of the protein inhibitor was calculated according to the method of Dixon (15). 1025
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS Identification
of
inhibitory
activities
for
fucosyHransferase
activity.
During
the
course of fucosyltransferase purification, the incubation of intestinal cytosol with DEAE-cellulose greatly enhanced the recovery of total activity of fucosyltransferase activity
(which
activity found
after
was
not
the DEAE-cellulose
in the cytosol
separate
experiments).
microsomal
retained
which
on the
step was multiplied
had not undergone
In contrast,
fucosyltransferase
(8). Total
cellulose)
by 6.8 + 1.8 compared
the DEAE-cellulose
the DEAE-cellulose
(9) increased
total
fucosyltransferase
step performed
activity
to that
step
(twelve
on solubilized
only by a factor
1.2 + 0.6
(8 experiments). The proteins, retained on the cellulose, were eluted with a KC1 gradient up to 0.7 M (figure 1). After elimination of the KCl, the inhibitory activity was found in two peaks about 0.27 M (fraction They were free of glycosylnucleotide-pyrophosphatase binding capacity. solubilized
No inhibitory
microsomal
KC1 (fraction
activity
preparations
A) contained
absorption
Similar
was detected
the major proportion
such conditions
under
of fucosyltransferase.
(Table I). The second peak (fraction rnaximurn
A) and 0.45 M KC1 (fraction 8). activity and also of GTP-Y-S The peak eluting at 0.27 M
of total
B) was of low
for the
activity
protein
and of proteins
content,
but exhibited
at 254 nm.
experirnents,
carried
out on various
organs,
indicated
that
such
inhi-
bitory activity was found only in the pancreas, but not in the liver, brain or lung. Controls. The inhibitory activity of fraction A was not due to the presence of interfering reactions. fraction (glycosidases, be significant
Indeed, the activity of several degradative enzymes in this pyrophosphatases and proteases) was very low aIla appeared to
only after
10 min incubation.
: Elution
When determinations
were
carried
pattern of the DEAE-cellulose column (linear gradient 200-700 mM activity is directly determined after desalting the fractions on The tubes corresponding to fractions A and B are Sephadex G25 PD 10 columns. pooled, as indicated oy horizontal bars, for further characterization.
1026
out
Vol.
166,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TABLE I Separation
of tuo soluble innibitory by E&E-cellulose
Fraction
total
proteins (n-d
specific activity (units/q)
activity (units)
722 22.5 5.3
Cell sap Fraction A Fraction B
activities of fucosyltransferase chraratography
23,248 9,439 4,024
purification factor
yield %
A.0
32.2 419.5 759.2
100 40.6 17.3
23.6
The tubes corresponding to fractions A and B from Figure 1 are pooled (as indicated in Figure I), dialyzed and the inhibitory activities are determined using the stanuarciized assay. One unit is defined as the amount of protein giving 50 % inhioition in this assay.
with of
saturating
concentrations
1.6 unit
cosamine
of inhibitory
activity,
was respectively
14,
asialofetuin
was only
per
minute.
As
tion
with
In order
to confirm
tory
activity,
the inhibitory
modify
of the
value
the
is
pmol/min.
galactose
The
and that
degradation
N-acetylglu-
of 14C-fucose-
of 3H-acetylhemoglobin
only
was not cleaved
l-h
enzymes
fucosyltransferase
after
This
with
was
0.1 % incuba-
with
where
the
but
radioactivity
up to
on the
was almost content
the
other not
preincubation.
the
inhibitor
was
leading
to
indicating
eluate,
radioactive
either
3 h, did
without
the
with of
DEAE-cellulose, in
retained
the innibitor
dialysis
of
activity
in the inhioi-
addition
fucosyltransferase,
column
partially
the
to controls
the
fucosyltransferase
involved
preincubation,
as compared
In the experiment
eluted
not
was preincubated
before
reaction.
inhibitor
radioactivity
were
A (1.6 unit/assay)
on a small
reversible.
since
of fucose,
GDP-fucose
inhibition
of
after
or
degradative
or
of the
was
release 19
fraction
of the
cytosol,
GDP-fucose dialysis
that
fucosylation
restoration
inhibition
and
by HPLC,
chromatography
by
complete
5
in the presence
fraction.
the
preincubation
removed
to
controlled
asialofetuin
constituents
the
of each paranitrophenyl-sugar
0.2 % per minute
the inhibitory
GDP-fucose,
After
(5 mM)
a
that
GDP-fucose
was added
DEAE-cellulose
(40
completely
%).
eliminated
was less than
by
2 % of initial
radioactivity. Characterization activity
of the
were
B was heat 50”
(3 for
non dialyzable stable
fraction
A had content
ribonuclease
fraction
rnodified of
of fraction Tl
(E.C.
activity
two stable
15 min.
75,
48,
insensitive 3.1.27.5)
only might
fractions
and
depend
for several
at 23O C and
In contrast,
18 and and its to pronase
to pronase.
A and B containing
at - 20°C
by incubation
B was low
B was
A was sensitive
the inhibitory
C for
a half-life
at 254 nm. Fraction
The
and were
ana not
1 h and at 75”
proteinic of
inhibitors.
at these
2 min action
together,
on the presence 1027
C for
same
3 h, at
In addition, was
and sensitive these
Fraction
temperatures,
spectrum II
deoxyribonuclease
Taken
months. 37’
respectively.
absorption
inhibitory
(E.C
to the action while
3.1.21.11,
results
of polynucleotides
the
maximum
suggest for
that
fraction
Vol.
166, No. 2, 1990
BIOCHEMICAL
10
AND BIOPHYSICAL
20
RESEARCH COMMUNICATIONS
40
30
50
fraction
number
Figure 2 : Pattern of inhibitory activity of fraction A (from Figure 1) on a Sephacry1 HR - 100 S column (2 x 100 cm) equilibrated in MES 10 mM pH 5.5. The column is calibratea with standards from Pharmacia : dextran blue (exclusion volume),
albumin
(a,
MW
67,000),
ovalbumin
(b,
MW
43,000)
and
chymotrypsinogen
H
(c, MW 25,000).
B and
of
proteins
was performed After graphed single
for
fraction
on the protein dialysis,
Figure
with of
competitive
HK-100
a
for
II
inhibitor
and
the
guanine
activrty.
The
affinity for
S. The
GDP-fucose
Table
pattern
GDP,
nucleotides
for
(nM”)
was the for
of the
and
inhibitory 60,000
concentrations
nucfeotide
in the presence
GDP-fucose
the
about It
non-competitive there
of guanine
of
activity.
that
of the
by lyophilization
weight
of two
and
indicate
characterization
innibitor
A.
concentrated
(l-2)fucosyltransferase
in
the
fraction
molecular
influence
the
reported
particularly
A was
an apparent
3 shows
parameters
containing
fraction
on Sephacryl peak,
A. Therefore,
is
activity
(figure
on the
clear
inhibition
for
that
asialofetuin. between of
the
inhibitor.
kinetic was
The
results
the
protein
is decreased, The
inhibitor
was
l/G
Figure) 3 : Influence of the inhibitor (fraction A) on the kinetic 0: 1-2 fucosyitransferase activity, determined with asialofetuin. 1028
a
fucosyltransferase
the fucosyltransferase protein
showed
2).
of inhioitor
competitron inhioition
chromato-
parameters
of
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
Ki (UM) of various
nticleotides
the
presence
absence
or
the
Without
1Nucleotide
fraction
c&P
31
aP cm WP-mannose
0.9 3.3 49
active
to
the it
substrates;
ferasrs
same
for
a(l-2)
the
proteinic
A
fucosyltransferase inhioitor
With
activity
A (1.6
fraction
unit/assay)
A
69 31 7.3 326
extent
either
with
active
on the
purified
was also
separated
of
COMMUNICATIONS
II
THBLE
in
RESEARCH
by Sephadex
Gl5
asialofetuin
or
oc (l-2)
and
cnromatography
after
asialoserotransferrin oc (l-3)
the
affinity
as
fucosyltrans-
chromatography.
DISCUSSION The existence ferase
activities
activity
after
was first partial
DEAE-cellulose. on
the
radioactivity
are
Thus,
after
cannot
dialysis) Intestinal
parameters
such
the
the
protein
concentration,
negligible
controls in the
fucosyltransferase
very
low
intestinal are
eluted
deterrninations
and
are
negligible
glycosyl-nucleotide at
sensitive
use
and
physico-chemical pH
(8,9).
of the dialysis
buffer
Alternatively,
use of rat
concentration
Thus
for
used
in the
up to 50 ug/assay
prepa-
desalting
results.
The
and bovine
a
is achieved
the use of buffers
satisfactory
of
substrate.
This
G25 and
traces (determined
activity.
of Sephadex give
to
strength
innibitory
on the
2 % of the initial
very
fractions.
experiments,
of inhibitory
the glycoprotein
rate,
less than
only
radioactive
the
at concentrations
dialysis,
of the
ionic
by the
of endogenous
dilution
of inhibitory
of protein
ana indirect
source
nucleotide-sugar,
range
for
with
retained
fractions
true
dilutions
treatment
of these
is
and
by
of total
serum assay
by
similarly effect
of
albumins, since
these
do no inhibit
the
activity.
direct
as a possible
the
after
fucosyltrans-
recovery
is partially
activity
concentration,
and
proteins
accounting
to measure
in the
to the fixation
However,
by isotopic
as confirmed
binding
attributed
inhibitory
soluble
transferase
Gi)P-fucose
activity
columns
increase
soluble
Ldbeied
of the fractions
on small
for
From
important
is needea
treated
the
K(31 gradient.
ana for dilutions
chromatography
are
the
as protein
controls
of
be explained
dialysis
non specific
(8).
intestinal
by the drastic
in the fractions,
the
assay
exhaustive
ring
with
inhibiting
was tentatively
fucosyltransferase
standardized by
effect
found
radioactivity.
suggested
cellulose
and elutes
factor
purification
This
Zi)P-fucose cellulose
of an endogenous
a lower are
interfering These
acceptor
or the enzyme,
compared
confirmed
strength by indirect
reactions,
to the
pyrophosphatases ionic
reactions
activity.
than
are
degree
can be eliminated
which
can destroy
are found of inhibition.
retained
at a
Moreover,
on DEAE-cellulose,
fractions
A and
B (16).
experiments.
Indeed,
preincubation
1029
the
to occur
These
but direct experi-
Vol.
166,
No.
ments
2, 1990
confirm
Further,
that
the
existence protein
action
enzyme.
This
itself
by
fact
apparent
It
to be very activities.
the
enzyme
inhibitor the
is
reversible.
either
binding
a soluble A from
donor,
site
G-protein the
for
the
with
of the
the sugar-nucleo-
on the
and
glycofor
nucleotides
fucosyltransferase.
: GTP- y -S
DEAE-cellulose
the
confirmation
and
guanine
interacts
Therefore,
competitive
of
process.
is established.
is non
affinity
the inhibitor
COMMUNICATIONS
in the inhibitory
activity
sugar-nucleotide
fraction
in the brain,
These
binding does
activity
is
exhibit
not
from
were
reported
inhibitor
studied
(7). They
characterized
preparation
nor
several
subunits
inhibitory
the
activity
any
of
for
brain
Albarracin with
the
its
(5). This These
and
reacts
appears
(4,6)
asialofetuin
or soluble
than
inhibiting organs, is about for
polysaccharides
The
best
sialyltrans-
weight also
inhibitory
(5,7).
in other
are
:
UDP-N-acetyl-
degrades
protein,
inhioitors
with
of one of the
and
molecular
sulfated
inhibitor
in
: glycosyltransferases
is non competitive
protein
this
likely
and
glycolipid
stable
apparent
inhibitor
Thus,
exception
in the brain
abundant
of fucosyltransferase
of glycosyltransferase
labile
heat
of
inhibitors (5) also
is heat
protein
in the intestine
or the liver.
membrane-bound
intestine;
heparin
only
and
reported
(5) is an acid
pointed
have important
intestinal
either
inhiiiitor
labile
but it has 70-80,000
the sugar
different
nucleofrom
described
for
the brain
(17).
As already on
are
acceptor.
fucosyltransferase
Studies
which
more
in the
for
stable,
(luiroga,
It is
tested
by
are heat by
galactosamine
(4) with
the lung
inhibitors They
activity.
state,
previously
describea
been
in a soluble
as a heat
is a true
and
the
the
sialyltransferase.
is characterizea
60,000
but
not
paper
about
only
though
ferase
in this
weight
different
activities
true
involved
not
the sugar-nucleotide in
is found
the pancreas,
could
for
that
A described
molecular
activities.
tide
the
RESEARCH
activity.
Fraction
not
that
is not
before
are
on the
decrease
the
BIOPHYSICAL
of fucosyltransferase
suggests
inhibitor
eluted
(4-7),
the
with
the
inhibitory
inhibitor
and competitive
or
However,
the
indicate
provided
clearly
of
AND
enzymes
inhibitor
studies
acceptor is
tide
degradative
of a true
Kinetic this
BIOCHEMICAL
the
out (5,6),
consequences
mechanism
fucosyltransferase
of
the existence
of such
in the regulation developmental
activities
must
(18) take
glycosyltransferase
inhibitors
of glycosyltransferase or nutritional into
account
(19)
activities. regulation
the presence
of
of this
inhibitor.
ACKNOWLEDGMENTS The skillful technical assistance of I&e HUGUENY was greatly appreciated. We thank Dr Jerry QUASH for critical revision of the manuscript. This work was supported by the Institut National de la San& et de la Recherche Mhdicale, the Centre National de la Recherche Scientifique and the Universitb de Lyon. REFERENCES I. 2.
I
