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A novel sulfotransferase sulfates tyrosine-containing peptides and proteins
voi. I 40, No. 1, 1986 October
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
15. 1986
Pages
38-42
A NOVEL SULFOTRANSFERASE SULFATES TYROSINE-CONTAINING PEPTIDES AND PROTEINS
Kyoichi
Kobashi
and Dong-Hyun
Kim
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama-shi, Toyama 930-01, JAPAN Received August 14, 1986 novel sulfotransferase (M.W. 315 kDa) obtained from A-44 catalyzed the sulfation of tyrosine residues of peptides and proteins such as kyotorphin, enkephalin, cholecystokinin-8 (non-sulfated form), trypsin inhibitor and insulin. Also, the enzyme sulfated tyrosine residues of protein fractions purified from Eubacterium A-44. 0 1986 Academic PRSS, 1~. The
Eubacterium
Among
various
protein
(l-3),
posttranslational
phosphorylation
residues
of
regulatory
such as cell
events
growth
(4-8).
tyrosine-O-sulfate protein?, fibronectin,
is generally about
modification.
catalyzes peptides
the
not
enzyme(s)
from the and the
sulfate
and proteins,
here
(9-13)
number of
and the role Also,
understood. for
a novel
type
this of
bacterium,
the properties reaction
and the role
A-44. 0006-291X/86$1.50 Copyright 0 1986 by Academic Press, Inc. All rights of reproducfion in any form reserved.
implied
in
38
of
G
and
of tyrosine
very
little
is
posttranslatioal
arylsulfotransferase A-44
Eubacterium
the
to tyrosine of the
that
secretary
such as immunoglobulin
responsible
transfer
been
studies
in a large
human intestinal
report
has
studied.
have shown
which,
yet
extensively
recent
occur
We discovered
obtained (14),
of
proteins
and hormone-induced
have been identified
sulfation known
some
of
transformation
However, residues
only
has been
tyrosine
Phosphorylation
cell
modifications
enzyme, residue(s)
enzyme in
Eubacterium
which of
Vol.
140,
No.
1, 1986
BIOCHEMICAL
AND
MATERIALS
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Insulin, trypsin inhibitor, albumin and p-nitrophenyl sulfate (PNS) were purchased from Sigma Chem. Co. Tyrosine from Ltd. methylester and tyramine were Nakarai Chem. (Leu)-enkephalin and Kyotorphin was from Funakoshi Chem. Co. cholecystokinin-8 (non-sulfated form) (CCK-8-NS) were kindly donated from Dr. T. Morikawa, Fuji Chemical Industries, Ltd. (35S) PNS was synthesized accordfng to the previous method (15).
Materials.
Enzyme activity assay. Sulfotransferase activity was determined for 15 min at 37°C of the reaction mixture by incubation containing 0.03 ml of 50 mM (35S) PNS or cold PNS, 0.2 ml of 1 mM acceptor (except 0.1 mM albumin), 0.18 ml of enzyme (specific activity 13.7 units/mg protein) purified by the previous method (stage 3) 0.02 ml of 0.25 mM MgC12 and 0.2 ml of 0.1 M (15), glycine-NaOH buffer, pH 8.5. Determination
of Lowry
of
et al
protein.
(16)
with
Protein was determined by the bovine serum albumin as standard.
method
Purification Eubacterium
of a protein fraction and its sulfation. Cultivated A-44 was collected, washed with saline, sonicated. centrifugated and applied to DEAE-cellulose column chromatography equilibrated with 0.1 M acetate buffter, pH 5.7. And then the column was eluted with 0.1 M acetate buffer, pH 5.7 containing 0.15 M KCl. The eluted fraction (free of sulfotransferase activity) was concentrated by ultrafiltration through a UM-10 membrane. The concentrated solution was dialyzed against 0.1 M glycine-NaOH buffer, pH 8.5, overnight (9.49 mg protein/ml). The sulfation reaction was performed for 100 min at 37°C under the following conditions: The reaction mixture (total volume 1.2 ml) contained purified 0.38 ml of the protein fraction, 0.38 ml of 0.1 M glycine-NaOH buffer, pH 8.5, and 0.02 ml of 0.25 M MgC12 in the presence of (a) 0.38 ml of an enzyme solution (total activity 2.66 units, sp. act. 13.7 units/mg protein) and 0.04 ml of 50 mM (35S)PNS (3.1 x lo6 CPM/w), (b) 0.38 ml of the enzyme solution and 0.04 ml of 50 mM (35S)Na SO (1.5 x lo7 CPM/mg), (c) 0.38 ml of the enzyme solution previo&l$ inactivated by heating at 100°C for 10 min and 0.04 ml of 50 mM (35S)PNS and (d) 0.04 ml of 50 mM (35 S)PNS and 0.38 ml of 0.1 M glycine-NaOH buffer, pH 8.5. The reaction mixture was applied to Sephadex G-50 coarse column chromatography at 4"C, and the column eluted with 0.1 M Tris-HCl buffer, pH 7.0 (fraction volume, The radioactivity for the eluted fractions was 2 ml). determined with a liquid scintillation counter.
RESULTS
The known The
novel
mammal
present
wow
bacterial
ian
enzymes
sulfotransferase in the
enzyme catalyzed
from
phenolic
AND DISCUSSIONS
substrate
the
sulfate
transfer esters
phosphoadenosine
5'-phosphosulfate(PAPS)
compound.
studied
We
acceptor 39
substrate
differed
from
specificity reaction
(14,15). of a
but
not
to
another specificity
the
sulfate
from
3'-
phenolic for
some
Vol. 140. No. 1, 1986
BIOCHEMICAL
Table
I.
The
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Specificity
of Acceptor
Substrate
Activitya (nmol/min/mg
Substrates Tyrosine methylester Tyramine Kyotorphin (Leul-enkephalin Cholecystokinin-8-nonsulfate(CCK-8-NS) Insulin Trypsin inhibitor Albumin a b
(Table was
active
I).
terminal
more
than is
hardly
containing
The enzyme activity the
inorganic when the A-44
donor sulfate
35
was incubated
an
with
the
G-50 coarse
and
chromatography,
only
and PNS (Fig.
the reaction
kyotorphin, was a better
tyrosine novel
active
weight contains
residues
of
sulfotransferase.
active site
peptides
and
may be regulated
acceptor
novel in
ten
media,
from
sulfotransferase
mixture sulfation
in the
reaction
mixture
1).
When, alkaline
presence
hydrolysates
Eubacterium
by
protein
containing
by
heat-
(35S)PNS
fractionated of the
not
or its of
by
addition,
In
fraction
fold
but
substrate.
the
40
approximately culture
protein
preparation,
S)Na2S04,
observed
by
by
N-
A-44.
to the
sulfotransferase-free
inactivated (
by this
was increased
or
the
and albumin
buried
Eubacterium
substrate
at
the molecular
inhibitor
at the
substrate
is
inhibitor
physiologically
tyrosine in
followed
although
Thus,
that
by the enzyme present
adding
trypsin
sulfated
suggest
PNS as a donor
each acceptor
Trypsin
residues.
results
proteins
than
using
whose tyrosine
and albumin,
lower
were
These
pH for
inhibitor.
insulin
tyrosine
proteins
optimal
was most effective,
CCK-8-NS and trypsin
insulin
the
3 hr.
and proteins
(Leu)-enkephalin
position
substrate
determinations. incubated for
peptides
Although
different,
of
3640 3600 35.3 218.2 8.1 0.12b 0.24b 0.07b
Average value of triplicate The reaction mixture was
physiologically
protein)
Sephadex
fraction active
of the
or
was enzyme
sulfated
Vol.
140,
No.
BIOCHEMICAL
1, 1986
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
200
-z $ .-s> .-s
100
.-$ -0 2 ul s
10
0
30
20
40
Fraction Fig.
1. Sulfation
ferase. buffer (b,
of
The and
0)
MgCl2
the
(SSS)PNS, described
in
protein
the
mixture
in the
enzyme
and
and
0)
(d,
chromatography intense
authentic It
presence
of
(35S)PNS
sulfated tyrosine-protein
and
n
(c, only.
by
the
protein
the
novel
sulfotrans-
fraction,
) active 0)
The
17-20)
enzyme
glycine-NaOH and
inactivated
reaction
be of to
of
analyzed
by cellulose
radioactive
spot
tyrosine-O-sulfate will
the (a,
70
No. fraction
(35S)Na2S04,
(No. were
an
protein contained
60
( 35S)PNS, enzyme
conditions
and
were
Text.
fractions
(14),
purified
reaction
50
(data interest
determine
to the
Sephadex plate
G-50
electrophoresis
coincided not
shown).
identify
the
physiological
coarse
with
proteins role
of
that
of
which
are
the
novel
sulfotransferase.
FUWERENCES
1. 2. 3. 4. 5.
LJy, R., and Wold, F. (1977) Science 198, 890-896. Wold, F. A. (1981) Rev. Biochem. 50, 783-814. Cohen, P. (1982) Nature 296, 613-620. Hunter, T. (1980) Cell 22, 647-648. Hunter, T., and Sefton, B. M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1131-1315. 6. Kasuga, M., Karlsson, F. A., and Kahn, C. R. (1982) Science 215, 185-187. 7. Cooper, J. A.. Reiss, N. A., Schwartz, R. J. and Hunter, T. (1983) Nature 302, 218-223. 41
Vol. 140, No. 1, 1986
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
8. White, M. F., Maron, R., and Kahn, C.R. (1985) 183-186. 9. Huttner, W. B. (1982) Nature 299, 273-276. 10. Lee, R. W. H. and Hutter, W. B. (1983) J. Biol.
Nature
318,
Chem. 258,
11326-11334. 11.
12. 13. 14. 15. 16.
Baeuerle, P. A. and Huttner, W. B. (1984) EMBO J. 3, 2209-2215. Liu, M. C. and Lipmann, F. (1985) Pro. Natl. Acad. Sci. U.S.A. 82, 34-37. Liu, M. C., Yu, S., Sy, J., Redman, C. M., and Lipmann, F. (1985) Pro. Natl. Acd. Sci. U.S.A. 82, 7160-7164. Kobashi. K., Fukaya, Y., Kim, D.-H., Akao, T., and Takebe, S. (1986) Arch. Biochem. Biophys. 245, 537-539. Kim, D.-H., Konishi, L., and Kobashi, K. (1986) Biochim. Acta 872, 33-41. Biophys. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275.
42
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
15. 1986
Pages
38-42
A NOVEL SULFOTRANSFERASE SULFATES TYROSINE-CONTAINING PEPTIDES AND PROTEINS
Kyoichi
Kobashi
and Dong-Hyun
Kim
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama-shi, Toyama 930-01, JAPAN Received August 14, 1986 novel sulfotransferase (M.W. 315 kDa) obtained from A-44 catalyzed the sulfation of tyrosine residues of peptides and proteins such as kyotorphin, enkephalin, cholecystokinin-8 (non-sulfated form), trypsin inhibitor and insulin. Also, the enzyme sulfated tyrosine residues of protein fractions purified from Eubacterium A-44. 0 1986 Academic PRSS, 1~. The
Eubacterium
Among
various
protein
(l-3),
posttranslational
phosphorylation
residues
of
regulatory
such as cell
events
growth
(4-8).
tyrosine-O-sulfate protein?, fibronectin,
is generally about
modification.
catalyzes peptides
the
not
enzyme(s)
from the and the
sulfate
and proteins,
here
(9-13)
number of
and the role Also,
understood. for
a novel
type
this of
bacterium,
the properties reaction
and the role
A-44. 0006-291X/86$1.50 Copyright 0 1986 by Academic Press, Inc. All rights of reproducfion in any form reserved.
implied
in
38
of
G
and
of tyrosine
very
little
is
posttranslatioal
arylsulfotransferase A-44
Eubacterium
the
to tyrosine of the
that
secretary
such as immunoglobulin
responsible
transfer
been
studies
in a large
human intestinal
report
has
studied.
have shown
which,
yet
extensively
recent
occur
We discovered
obtained (14),
of
proteins
and hormone-induced
have been identified
sulfation known
some
of
transformation
However, residues
only
has been
tyrosine
Phosphorylation
cell
modifications
enzyme, residue(s)
enzyme in
Eubacterium
which of
Vol.
140,
No.
1, 1986
BIOCHEMICAL
AND
MATERIALS
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Insulin, trypsin inhibitor, albumin and p-nitrophenyl sulfate (PNS) were purchased from Sigma Chem. Co. Tyrosine from Ltd. methylester and tyramine were Nakarai Chem. (Leu)-enkephalin and Kyotorphin was from Funakoshi Chem. Co. cholecystokinin-8 (non-sulfated form) (CCK-8-NS) were kindly donated from Dr. T. Morikawa, Fuji Chemical Industries, Ltd. (35S) PNS was synthesized accordfng to the previous method (15).
Materials.
Enzyme activity assay. Sulfotransferase activity was determined for 15 min at 37°C of the reaction mixture by incubation containing 0.03 ml of 50 mM (35S) PNS or cold PNS, 0.2 ml of 1 mM acceptor (except 0.1 mM albumin), 0.18 ml of enzyme (specific activity 13.7 units/mg protein) purified by the previous method (stage 3) 0.02 ml of 0.25 mM MgC12 and 0.2 ml of 0.1 M (15), glycine-NaOH buffer, pH 8.5. Determination
of Lowry
of
et al
protein.
(16)
with
Protein was determined by the bovine serum albumin as standard.
method
Purification Eubacterium
of a protein fraction and its sulfation. Cultivated A-44 was collected, washed with saline, sonicated. centrifugated and applied to DEAE-cellulose column chromatography equilibrated with 0.1 M acetate buffter, pH 5.7. And then the column was eluted with 0.1 M acetate buffer, pH 5.7 containing 0.15 M KCl. The eluted fraction (free of sulfotransferase activity) was concentrated by ultrafiltration through a UM-10 membrane. The concentrated solution was dialyzed against 0.1 M glycine-NaOH buffer, pH 8.5, overnight (9.49 mg protein/ml). The sulfation reaction was performed for 100 min at 37°C under the following conditions: The reaction mixture (total volume 1.2 ml) contained purified 0.38 ml of the protein fraction, 0.38 ml of 0.1 M glycine-NaOH buffer, pH 8.5, and 0.02 ml of 0.25 M MgC12 in the presence of (a) 0.38 ml of an enzyme solution (total activity 2.66 units, sp. act. 13.7 units/mg protein) and 0.04 ml of 50 mM (35S)PNS (3.1 x lo6 CPM/w), (b) 0.38 ml of the enzyme solution and 0.04 ml of 50 mM (35S)Na SO (1.5 x lo7 CPM/mg), (c) 0.38 ml of the enzyme solution previo&l$ inactivated by heating at 100°C for 10 min and 0.04 ml of 50 mM (35S)PNS and (d) 0.04 ml of 50 mM (35 S)PNS and 0.38 ml of 0.1 M glycine-NaOH buffer, pH 8.5. The reaction mixture was applied to Sephadex G-50 coarse column chromatography at 4"C, and the column eluted with 0.1 M Tris-HCl buffer, pH 7.0 (fraction volume, The radioactivity for the eluted fractions was 2 ml). determined with a liquid scintillation counter.
RESULTS
The known The
novel
mammal
present
wow
bacterial
ian
enzymes
sulfotransferase in the
enzyme catalyzed
from
phenolic
AND DISCUSSIONS
substrate
the
sulfate
transfer esters
phosphoadenosine
5'-phosphosulfate(PAPS)
compound.
studied
We
acceptor 39
substrate
differed
from
specificity reaction
(14,15). of a
but
not
to
another specificity
the
sulfate
from
3'-
phenolic for
some
Vol. 140. No. 1, 1986
BIOCHEMICAL
Table
I.
The
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Specificity
of Acceptor
Substrate
Activitya (nmol/min/mg
Substrates Tyrosine methylester Tyramine Kyotorphin (Leul-enkephalin Cholecystokinin-8-nonsulfate(CCK-8-NS) Insulin Trypsin inhibitor Albumin a b
(Table was
active
I).
terminal
more
than is
hardly
containing
The enzyme activity the
inorganic when the A-44
donor sulfate
35
was incubated
an
with
the
G-50 coarse
and
chromatography,
only
and PNS (Fig.
the reaction
kyotorphin, was a better
tyrosine novel
active
weight contains
residues
of
sulfotransferase.
active site
peptides
and
may be regulated
acceptor
novel in
ten
media,
from
sulfotransferase
mixture sulfation
in the
reaction
mixture
1).
When, alkaline
presence
hydrolysates
Eubacterium
by
protein
containing
by
heat-
(35S)PNS
fractionated of the
not
or its of
by
addition,
In
fraction
fold
but
substrate.
the
40
approximately culture
protein
preparation,
S)Na2S04,
observed
by
by
N-
A-44.
to the
sulfotransferase-free
inactivated (
by this
was increased
or
the
and albumin
buried
Eubacterium
substrate
at
the molecular
inhibitor
at the
substrate
is
inhibitor
physiologically
tyrosine in
followed
although
Thus,
that
by the enzyme present
adding
trypsin
sulfated
suggest
PNS as a donor
each acceptor
Trypsin
residues.
results
proteins
than
using
whose tyrosine
and albumin,
lower
were
These
pH for
inhibitor.
insulin
tyrosine
proteins
optimal
was most effective,
CCK-8-NS and trypsin
insulin
the
3 hr.
and proteins
(Leu)-enkephalin
position
substrate
determinations. incubated for
peptides
Although
different,
of
3640 3600 35.3 218.2 8.1 0.12b 0.24b 0.07b
Average value of triplicate The reaction mixture was
physiologically
protein)
Sephadex
fraction active
of the
or
was enzyme
sulfated
Vol.
140,
No.
BIOCHEMICAL
1, 1986
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
200
-z $ .-s> .-s
100
.-$ -0 2 ul s
10
0
30
20
40
Fraction Fig.
1. Sulfation
ferase. buffer (b,
of
The and
0)
MgCl2
the
(SSS)PNS, described
in
protein
the
mixture
in the
enzyme
and
and
0)
(d,
chromatography intense
authentic It
presence
of
(35S)PNS
sulfated tyrosine-protein
and
n
(c, only.
by
the
protein
the
novel
sulfotrans-
fraction,
) active 0)
The
17-20)
enzyme
glycine-NaOH and
inactivated
reaction
be of to
of
analyzed
by cellulose
radioactive
spot
tyrosine-O-sulfate will
the (a,
70
No. fraction
(35S)Na2S04,
(No. were
an
protein contained
60
( 35S)PNS, enzyme
conditions
and
were
Text.
fractions
(14),
purified
reaction
50
(data interest
determine
to the
Sephadex plate
G-50
electrophoresis
coincided not
shown).
identify
the
physiological
coarse
with
proteins role
of
that
of
which
are
the
novel
sulfotransferase.
FUWERENCES
1. 2. 3. 4. 5.
LJy, R., and Wold, F. (1977) Science 198, 890-896. Wold, F. A. (1981) Rev. Biochem. 50, 783-814. Cohen, P. (1982) Nature 296, 613-620. Hunter, T. (1980) Cell 22, 647-648. Hunter, T., and Sefton, B. M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1131-1315. 6. Kasuga, M., Karlsson, F. A., and Kahn, C. R. (1982) Science 215, 185-187. 7. Cooper, J. A.. Reiss, N. A., Schwartz, R. J. and Hunter, T. (1983) Nature 302, 218-223. 41
Vol. 140, No. 1, 1986
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
8. White, M. F., Maron, R., and Kahn, C.R. (1985) 183-186. 9. Huttner, W. B. (1982) Nature 299, 273-276. 10. Lee, R. W. H. and Hutter, W. B. (1983) J. Biol.
Nature
318,
Chem. 258,
11326-11334. 11.
12. 13. 14. 15. 16.
Baeuerle, P. A. and Huttner, W. B. (1984) EMBO J. 3, 2209-2215. Liu, M. C. and Lipmann, F. (1985) Pro. Natl. Acad. Sci. U.S.A. 82, 34-37. Liu, M. C., Yu, S., Sy, J., Redman, C. M., and Lipmann, F. (1985) Pro. Natl. Acd. Sci. U.S.A. 82, 7160-7164. Kobashi. K., Fukaya, Y., Kim, D.-H., Akao, T., and Takebe, S. (1986) Arch. Biochem. Biophys. 245, 537-539. Kim, D.-H., Konishi, L., and Kobashi, K. (1986) Biochim. Acta 872, 33-41. Biophys. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275.
42