Vol. 70, No. 1, 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PHOSPHOPROTEIN-PHOSPHATASE ACTIVITY ASSOCIATED WITH HUMAN PLACENTAL ALKALINE PHOSPHATASE Kuo-Ping Huang, J. C. Robinson, and Janice Y. Chou on Developmental Enzymology, Laboratory of Biomedical Sciences National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20014
Section
Received
March
8,1976
SUMMARY. Human placental alkaline phosphatase, a marker protein for some nontrophoblastic neoplasms, was found to have phosphoprotein @osphatase activity. This was demonstrated by the dephosphorylation of P-labeled histones, protamine, glycogen synthetase, casein, and phosvitin at various pH values. Unlike the general phosphoprotein phosphatase, the placental alkaline phosphatase does not have phosphorylase a phosphatase activity. Placental trophin, the
proteins
and alkaline
phosphatase
serum of pregnant
man or nonpregnant tumors It
such as placental
derepression synthesis
suggested of the
that
phosphatase
derepression carcinoma its
of the
interesting
of placental
gene expression alkaline
phosphatase
In the is
tumors, activity.
a significant (5).
of other
indeed
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
in
On the
other
Induction
of the
hand, derived
of placental by addition
to the culture
repression
as related we examined
the
in cells
compounds,
and
to choriothis
enzyme for
Phosphoprotein-phosphatase
role
in regulating
present
study,
a phosphoprotein 186
of a
proteins.
can be achieved
phosphatase
and many nontrophoblastic
to play
(3,4).
phenomenon
alkaline
phosphoprotein-phosphatase
thought
Copyright All rights
or a variety
placental
is repressed
cells
in
in the serum
results (2).
of placenta
in choriocarcinoma
of 5-bromodeoxyuridine, Because
phosphatase
a neoplasm
these
proteins
present
Many nontrophoblastic
transformation
these
alkaline
(1).
gonado-
normally
presence
synthesize
malignant
chorionic
are
their
neoplasm
lines
genome for
choriocarcinoma,
(3,4).
However,
cell
of placental
alkaline
(EC. 3.1.3.1)
woman indicates
and tumor-derived
has been
from
women.
lactogen,
cellular
we report
metabolism that
phosphatase.
placental
is and
VoL70,No.
1,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Calf thymus histone, type II-A (histone mixture) and type III-S (lysine-rich fraction); protgvine; and phosvitin were purchased from Sigma Chemical Company. [yP]ATP was obtained from New England Nuclear Corporation. Vitamin-free casein (Nutritional Biochemicals) was suspended in water according to the method of Reimann, --et al. (6). Human placental alkaline phosphatase was purified and assayed according to the method of Sussman, --et al. (7). Cyclic AMP-dependent protein kinase from rabbit muscle was purified according to the method of Yamamura, et al. (8); the muscle protein-kinase B1 fraction was used. Phosvitin kinac from the same tissug2was prepared according to the P-Labeled proteins were prepared method of Goldstein and Hasty (9). by incubation of the pt$ein substrates3yith cyclic AMP-dependent protein kinase (for preparing 32P-histones and 3!-protamine) or phosvitln kinase &or preparing P-casein) as described previously P-phosvitin and 'P-Labeled glycogen synthetase was prepared by incubation of I(10). form enzyme wisj both cyclic AMP-dependent protein kinase and phosvitin kinase (11). P-Phosphorylase a was kindly provided by Dr. F. W. Huang of this laboratory. Phosphoprotgin phosphatase activity was measured at 37' in a reaction mixture of 25 ul containing 200 mM sodium acg$ate, Tris-Cl, or 2-amino-2-methylql-propanol; 0.04 to 0.8 mg/ml of Plabeled protein (2 to 6 x 10 CPM); and 0.2 to 2 pg of purified alkaline phosphatase (specific activity of 400 U/mg with e-nitrophenylphosphate). The sfaction was stopped by 3) e addition of 5 ul glacial acetic acid, by the ITLC-chromatography P. was separated from P-protein ii",hod af Huang and Robinson (10). At alkaline
RESULTS AND DISCUSSION hydrolyzes
a variety
(12).
However,
values
for
(Fig. 32
reactions
The optimum
the hydrolysis extending
of the
between
protein,
pH for
was 11.5,
alkaline the
phosphatase
substrate
may reflect
and the
state
ranging of the 5 and 6.
pH for which
acidic
to alkaline
The optimum
pH for
broad,
hydrolysis
of a very
to the optimum The wide
protein
variation
substrates
of the
of the
pH
proteins,
was similar
the accessibility
of ionization
acid
was very the
of various
linkage the optimum
from
was used as substrate. hydrolysis
phosphatase
as substrate,
32P-histories,
The optimum
32 P-protamine,
the
broadly,
proteins,
6 and 10.
pH for
served
was between
basic
alkaline
the phosphomonoester
the hydrolysis
pH when e-nitrophenylphosphate in the optimum
having
varied
and 32P-casein,
P-phosvitin
basic
of compounds
when phosphoproteins
these
1).
pH placental
substrate
by
phosphate at various
on pH
values. 32 P-Phosphorylase phosphoprotein tested,
resistant
a which
phosphatase, to alkaline
is
was,
readily in contrast
phosphatase
187
hydrolyzed
by rabbit
muscle
to the phosphoproteins throughout
the entire
pH range
VoL70,No.
BIOCHEMICAL
1,1976
o-
Fig.
(from
1.
associated
with
It
seems that
placental
including
of 32 P-glycogen
synthetase
lation
by divalent by alkaline
In order
phosphorylase
with
Fig.
is not that
alkaline
2 shows coincides
activities.
all
the major alkaline
The extent phosphatase
is
protein
the
the
with
all
phosphatase
indeed
in Fig.
is the
metal
These
dephosphory-
ions. activity
enzyme was subjected for
the
to disc hydrolysis
the phosphoprotein-phosphatase activities
were
experiments
associated
show that
has phosphoprotein-phosphatase of 32 -labeled
3.
whereas
activity
of
hydrolysis
phosphatase
by these
enzyme
the
a variety the
and Cat'),
the
that
of dephosphorylation illustrated
hydrolyzes
phosphoprotein-phosphatase
band of the gel.
phosphatase
from
Furthermore,
affected
phosphatase,
of e-nitrophenylphosphate Furthermore,
Mgtt,
activity
different
phosphoprotein
(Mntt,
phosphatase
is
which 5.
by general
cations
electrophoresis.
placental
12
phosphoprotein-phosphatase
(13,14)
to demonstrate
is associated
with
10
phosphatase
phosphoprotein-phosphatase
activated
8 PH
the
alkaline
phosphoproteins
gel
6
RESEARCH COMMUNICATIONS
Dependence of phosphoprotein phosphatase activity on pH. The buffers used were sodium acetate (from pH 3.5 to 6.5), TrisCl (from pH 7.0 to 9.0), and 2-amino-2-mgthyl-1-propanol (51; pH 9.5 to 12.0). The substrates "'"s2p-hp;~~~~i~-(;O~~~ @stone mixture (-I -)j21ysine-rich P-phosvitin (- 0 -); P-glycogen synthetase (- A-); 32?protamine (-A -); p--nitrophenyl phosphate (- 4 -).
3.5 to 12).
general
4
AND BIOPHYSICAL
After
188
proteins
prolonged
activity.
by alkaline
incubation,
over
VoL70,No.
Fig.
1, 1976
2.
BIOCHEMICAL
Disc gel electrophoresis of the purified placental alkaline phosphatase and the measurement of phosphatase activities in The electrophoresis was carried out the fractionated gel. according to the method of Davis (15). After electrophoresis the gel was fractionated into l-mm fractions in a gel slicer, and the fractions were assayed for phosphatase activities at the optimum pH for each substrate. The duplicate gel was stained for protein with Coomassie blue. The symbols for the phosphatase activities are the same as those shown in Fig. 1.
80 percent synthetase,
of the phosphate and phosvitin
of 32 Pi was released (0.1 for those
N HCl at 100" 15 min)
of the
phosphates
of serine phosphorylase
for
were
and threonine
&may
be in a conformation
shown
alkaline
constants in Table
1.
serine
it
N NaOH at
and concluded
that
is
unaccessible
189
for
for the
protein
be dephosphorysite
to the
a variety
only
Although
cannot
The phosphate
of the phosphatase The Km values
it
100"
characteristic
be hydrolyzed.
phosphate,
phosphatase. that
(0.1
60 percent
lability
but acid-stable, could
glycogen
approximately
the acid
lability
proteins
alkali-labile
phosphates,
by placental
Kinetic
of these
histones,
only
We analyzed
and alkali
contains
protamine,
however,
32P-casein.
phosphate
5 also
P-labeled
was released;
15 min)
which
32
from
from
lated
are
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on phosphorylase
phosphatase.
of substrates substrates
were
in
Vol. 70, No. 1, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
*rF s
190
VoL70,No.
1, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TIME
Fig.
3.
Extent of dephosphorylation of 32 P-labeled proteins by purified alkaline phosphatase. The reactions were carried out under standard assay conditions in a volume of 0.125 ml with 20 ug of enzyme at the optimum pH for each substrate. At timed jytervals lo-v1 samples were withdrawn for the determination of P. released. Arrow indicates that 10 pq phosphatase was agaln added to the reaction mixtures. The symbols for the 32Pprotein substrates are the same as those shown in Fin. 1.
the uM range. "P-glycogen purified
alkaline cance
The Vmax values synthetase
phosphoprotein Since
histones phosphatase
in
mammalian
(mid
the regulation
of the phosphatase
as substrates phosphatase
and glycogen
are from
comparable rabbit
synthetase
at physiological of glycogen
using
pH, this synthesis
to those
muscle
are
32 P-histone
and
of partially
(13).
substrates enzyme might
of placental be of signifi-
and gene transcription
in
tissue.
REFERENCES 1. 2. 3. 4. 5.
Rosen, S.W., Weintraub, B.O., Vaitukaitis, J.L., Sussman, H.H., Hershman, J.M., and Muggia, F.M. (1975) Ann. Intern. Med. --) 82 71-8.3. Lipsett, M.B. (1965) Cancer Res. 25, 1068-1073. Edlow, J.B., Ota, T., Relation, J., Kohler, P-O., and Robinson, J.C. (1975) Am. J. Obstet. Gynecol. 121, 674-681. Chou, J.Y., and Robinson, J.C. (in preparation). Meisler, M.H., and Langan, T.A. (1969) J. Biol. Chem. 244, 4961-4968.
191
Vol.
70, No.
6. 7. 8. 9. 10. 11. K: 14. 15.
1, 1976
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Chem. Reimann, E.M., Walsh, D.A., and Krebs, E.G. (1971) J. Biol. 246, 1986-1995. and Catlove, E. (1968) J. Biol. Chem. Sussman, H-H., Small, P.A. Jr., 243, 160-166. Yamamura, H., Nishiyama, K., Shimonura, R., and Nishizuka, Y. (1973) Biochemistry 12, 856-862. Goldstein, J.L, and Hasty, M.A. (1973) J. Biol. Chem. 248, 6300-6307. Huang, K.-P., and Robinson, J.C. (1976) Anal. Biochem. (in press). Huang, K.-P., Huang, F.L., Glinsmann, W.H., and Robinson, J.C. (1975) Biochem. Biophys. Res. Commun. 65, 1163-1169. Harkness, D.R. (1968) Arch. Bio%em. Biophys. 126, 513-523. Kato, K., and Bishop, J.S. (1972) J. Biol. Chem. 247, 7420-7429. Brandt. H., Capulong, Z.L., and Lee, E.Y.C. (1975). Biol. Chem. 250, 8038-8044. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427.
192
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PHOSPHOPROTEIN-PHOSPHATASE ACTIVITY ASSOCIATED WITH HUMAN PLACENTAL ALKALINE PHOSPHATASE Kuo-Ping Huang, J. C. Robinson, and Janice Y. Chou on Developmental Enzymology, Laboratory of Biomedical Sciences National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20014
Section
Received
March
8,1976
SUMMARY. Human placental alkaline phosphatase, a marker protein for some nontrophoblastic neoplasms, was found to have phosphoprotein @osphatase activity. This was demonstrated by the dephosphorylation of P-labeled histones, protamine, glycogen synthetase, casein, and phosvitin at various pH values. Unlike the general phosphoprotein phosphatase, the placental alkaline phosphatase does not have phosphorylase a phosphatase activity. Placental trophin, the
proteins
and alkaline
phosphatase
serum of pregnant
man or nonpregnant tumors It
such as placental
derepression synthesis
suggested of the
that
phosphatase
derepression carcinoma its
of the
interesting
of placental
gene expression alkaline
phosphatase
In the is
tumors, activity.
a significant (5).
of other
indeed
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
in
On the
other
Induction
of the
hand, derived
of placental by addition
to the culture
repression
as related we examined
the
in cells
compounds,
and
to choriothis
enzyme for
Phosphoprotein-phosphatase
role
in regulating
present
study,
a phosphoprotein 186
of a
proteins.
can be achieved
phosphatase
and many nontrophoblastic
to play
(3,4).
phenomenon
alkaline
phosphoprotein-phosphatase
thought
Copyright All rights
or a variety
placental
is repressed
cells
in
in the serum
results (2).
of placenta
in choriocarcinoma
of 5-bromodeoxyuridine, Because
phosphatase
a neoplasm
these
proteins
present
Many nontrophoblastic
transformation
these
alkaline
(1).
gonado-
normally
presence
synthesize
malignant
chorionic
are
their
neoplasm
lines
genome for
choriocarcinoma,
(3,4).
However,
cell
of placental
alkaline
(EC. 3.1.3.1)
woman indicates
and tumor-derived
has been
from
women.
lactogen,
cellular
we report
metabolism that
phosphatase.
placental
is and
VoL70,No.
1,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Calf thymus histone, type II-A (histone mixture) and type III-S (lysine-rich fraction); protgvine; and phosvitin were purchased from Sigma Chemical Company. [yP]ATP was obtained from New England Nuclear Corporation. Vitamin-free casein (Nutritional Biochemicals) was suspended in water according to the method of Reimann, --et al. (6). Human placental alkaline phosphatase was purified and assayed according to the method of Sussman, --et al. (7). Cyclic AMP-dependent protein kinase from rabbit muscle was purified according to the method of Yamamura, et al. (8); the muscle protein-kinase B1 fraction was used. Phosvitin kinac from the same tissug2was prepared according to the P-Labeled proteins were prepared method of Goldstein and Hasty (9). by incubation of the pt$ein substrates3yith cyclic AMP-dependent protein kinase (for preparing 32P-histones and 3!-protamine) or phosvitln kinase &or preparing P-casein) as described previously P-phosvitin and 'P-Labeled glycogen synthetase was prepared by incubation of I(10). form enzyme wisj both cyclic AMP-dependent protein kinase and phosvitin kinase (11). P-Phosphorylase a was kindly provided by Dr. F. W. Huang of this laboratory. Phosphoprotgin phosphatase activity was measured at 37' in a reaction mixture of 25 ul containing 200 mM sodium acg$ate, Tris-Cl, or 2-amino-2-methylql-propanol; 0.04 to 0.8 mg/ml of Plabeled protein (2 to 6 x 10 CPM); and 0.2 to 2 pg of purified alkaline phosphatase (specific activity of 400 U/mg with e-nitrophenylphosphate). The sfaction was stopped by 3) e addition of 5 ul glacial acetic acid, by the ITLC-chromatography P. was separated from P-protein ii",hod af Huang and Robinson (10). At alkaline
RESULTS AND DISCUSSION hydrolyzes
a variety
(12).
However,
values
for
(Fig. 32
reactions
The optimum
the hydrolysis extending
of the
between
protein,
pH for
was 11.5,
alkaline the
phosphatase
substrate
may reflect
and the
state
ranging of the 5 and 6.
pH for which
acidic
to alkaline
The optimum
pH for
broad,
hydrolysis
of a very
to the optimum The wide
protein
variation
substrates
of the
of the
pH
proteins,
was similar
the accessibility
of ionization
acid
was very the
of various
linkage the optimum
from
was used as substrate. hydrolysis
phosphatase
as substrate,
32P-histories,
The optimum
32 P-protamine,
the
broadly,
proteins,
6 and 10.
pH for
served
was between
basic
alkaline
the phosphomonoester
the hydrolysis
pH when e-nitrophenylphosphate in the optimum
having
varied
and 32P-casein,
P-phosvitin
basic
of compounds
when phosphoproteins
these
1).
pH placental
substrate
by
phosphate at various
on pH
values. 32 P-Phosphorylase phosphoprotein tested,
resistant
a which
phosphatase, to alkaline
is
was,
readily in contrast
phosphatase
187
hydrolyzed
by rabbit
muscle
to the phosphoproteins throughout
the entire
pH range
VoL70,No.
BIOCHEMICAL
1,1976
o-
Fig.
(from
1.
associated
with
It
seems that
placental
including
of 32 P-glycogen
synthetase
lation
by divalent by alkaline
In order
phosphorylase
with
Fig.
is not that
alkaline
2 shows coincides
activities.
all
the major alkaline
The extent phosphatase
is
protein
the
the
with
all
phosphatase
indeed
in Fig.
is the
metal
These
dephosphory-
ions. activity
enzyme was subjected for
the
to disc hydrolysis
the phosphoprotein-phosphatase activities
were
experiments
associated
show that
has phosphoprotein-phosphatase of 32 -labeled
3.
whereas
activity
of
hydrolysis
phosphatase
by these
enzyme
the
a variety the
and Cat'),
the
that
of dephosphorylation illustrated
hydrolyzes
phosphoprotein-phosphatase
band of the gel.
phosphatase
from
Furthermore,
affected
phosphatase,
of e-nitrophenylphosphate Furthermore,
Mgtt,
activity
different
phosphoprotein
(Mntt,
phosphatase
is
which 5.
by general
cations
electrophoresis.
placental
12
phosphoprotein-phosphatase
(13,14)
to demonstrate
is associated
with
10
phosphatase
phosphoprotein-phosphatase
activated
8 PH
the
alkaline
phosphoproteins
gel
6
RESEARCH COMMUNICATIONS
Dependence of phosphoprotein phosphatase activity on pH. The buffers used were sodium acetate (from pH 3.5 to 6.5), TrisCl (from pH 7.0 to 9.0), and 2-amino-2-mgthyl-1-propanol (51; pH 9.5 to 12.0). The substrates "'"s2p-hp;~~~~i~-(;O~~~ @stone mixture (-I -)j21ysine-rich P-phosvitin (- 0 -); P-glycogen synthetase (- A-); 32?protamine (-A -); p--nitrophenyl phosphate (- 4 -).
3.5 to 12).
general
4
AND BIOPHYSICAL
After
188
proteins
prolonged
activity.
by alkaline
incubation,
over
VoL70,No.
Fig.
1, 1976
2.
BIOCHEMICAL
Disc gel electrophoresis of the purified placental alkaline phosphatase and the measurement of phosphatase activities in The electrophoresis was carried out the fractionated gel. according to the method of Davis (15). After electrophoresis the gel was fractionated into l-mm fractions in a gel slicer, and the fractions were assayed for phosphatase activities at the optimum pH for each substrate. The duplicate gel was stained for protein with Coomassie blue. The symbols for the phosphatase activities are the same as those shown in Fig. 1.
80 percent synthetase,
of the phosphate and phosvitin
of 32 Pi was released (0.1 for those
N HCl at 100" 15 min)
of the
phosphates
of serine phosphorylase
for
were
and threonine
&may
be in a conformation
shown
alkaline
constants in Table
1.
serine
it
N NaOH at
and concluded
that
is
unaccessible
189
for
for the
protein
be dephosphorysite
to the
a variety
only
Although
cannot
The phosphate
of the phosphatase The Km values
it
100"
characteristic
be hydrolyzed.
phosphate,
phosphatase. that
(0.1
60 percent
lability
but acid-stable, could
glycogen
approximately
the acid
lability
proteins
alkali-labile
phosphates,
by placental
Kinetic
of these
histones,
only
We analyzed
and alkali
contains
protamine,
however,
32P-casein.
phosphate
5 also
P-labeled
was released;
15 min)
which
32
from
from
lated
are
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on phosphorylase
phosphatase.
of substrates substrates
were
in
Vol. 70, No. 1, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
*rF s
190
VoL70,No.
1, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TIME
Fig.
3.
Extent of dephosphorylation of 32 P-labeled proteins by purified alkaline phosphatase. The reactions were carried out under standard assay conditions in a volume of 0.125 ml with 20 ug of enzyme at the optimum pH for each substrate. At timed jytervals lo-v1 samples were withdrawn for the determination of P. released. Arrow indicates that 10 pq phosphatase was agaln added to the reaction mixtures. The symbols for the 32Pprotein substrates are the same as those shown in Fin. 1.
the uM range. "P-glycogen purified
alkaline cance
The Vmax values synthetase
phosphoprotein Since
histones phosphatase
in
mammalian
(mid
the regulation
of the phosphatase
as substrates phosphatase
and glycogen
are from
comparable rabbit
synthetase
at physiological of glycogen
using
pH, this synthesis
to those
muscle
are
32 P-histone
and
of partially
(13).
substrates enzyme might
of placental be of signifi-
and gene transcription
in
tissue.
REFERENCES 1. 2. 3. 4. 5.
Rosen, S.W., Weintraub, B.O., Vaitukaitis, J.L., Sussman, H.H., Hershman, J.M., and Muggia, F.M. (1975) Ann. Intern. Med. --) 82 71-8.3. Lipsett, M.B. (1965) Cancer Res. 25, 1068-1073. Edlow, J.B., Ota, T., Relation, J., Kohler, P-O., and Robinson, J.C. (1975) Am. J. Obstet. Gynecol. 121, 674-681. Chou, J.Y., and Robinson, J.C. (in preparation). Meisler, M.H., and Langan, T.A. (1969) J. Biol. Chem. 244, 4961-4968.
191
Vol.
70, No.
6. 7. 8. 9. 10. 11. K: 14. 15.
1, 1976
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Chem. Reimann, E.M., Walsh, D.A., and Krebs, E.G. (1971) J. Biol. 246, 1986-1995. and Catlove, E. (1968) J. Biol. Chem. Sussman, H-H., Small, P.A. Jr., 243, 160-166. Yamamura, H., Nishiyama, K., Shimonura, R., and Nishizuka, Y. (1973) Biochemistry 12, 856-862. Goldstein, J.L, and Hasty, M.A. (1973) J. Biol. Chem. 248, 6300-6307. Huang, K.-P., and Robinson, J.C. (1976) Anal. Biochem. (in press). Huang, K.-P., Huang, F.L., Glinsmann, W.H., and Robinson, J.C. (1975) Biochem. Biophys. Res. Commun. 65, 1163-1169. Harkness, D.R. (1968) Arch. Bio%em. Biophys. 126, 513-523. Kato, K., and Bishop, J.S. (1972) J. Biol. Chem. 247, 7420-7429. Brandt. H., Capulong, Z.L., and Lee, E.Y.C. (1975). Biol. Chem. 250, 8038-8044. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427.
192
