Proc. Nat. Acad. Sci. USA Vol. 72, No. 7, pp. 2631-2634, July 1975
Biochemistry
Separation of "estrogen-induced" protein from phosphoprotein phosphatase activity of immature rat uterus (steroids/hormone/cyclic AMP/cyclic GMP)
ALVIN M. KAYE, MICHAEL D. WALKER, AND DALIA SOMJEN Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel
Communicated by I. Edelman, May 2, 1975
ABSTRACT Preparations of the "induced protein" which appears in the rat uterus within 40 min of estradiol administration have recently been reported to contain phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity. We found that these two proteins distribute differently on ammonium sulfate fractionation of uterine cytosol. Preparative cellulose acetate electrophoresis afforded complete (>99.9%) separation of phosphoprotein phosphatase activity from the induced protein. The specific activity of phosphoprotein phosphatase in uterine cytosol was unchanged 1, 4, 12, or 24 hr after estradiol administration. These results are incompatible with the view that the induced protein mediates estrogen action by virtue of an inherent phosphoprotein phosphatase activity.
Administration of estradiol-17fl to immature rats, or mature ovariectomized rats, results in an increased rate of synthesis of a uterine protein which has been designated "induced protein" (IP) by Notides and Gorski (1). Synthesis of estrogen-induced protein can be detected within 40 min of estradiol injection into rats (2) or incubation of surviving uteri in physiological concentrations of estrogens (3). This cytosol protein (4) has a molecular weight of approximately 4 X 104 and an isoelectric point of approximately 4.5 (5-7). Because the appearance of IP represents one of the earliest effects of estradiol on the uterus, Baulieu et al. (8) have proposed that IP is the "key intermediate protein" in the stimulation of bulk RNA synthesis by estradiol. However, until a recent report by Vokaer et al. (9), no experimental data on the possible function of IP were available. These au-
thors reported that phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (PPPase) is associated with purified preparations of IP. The proposal of Liu and Greengard (10), that increases in PPPase activity may modulate the response of toad bladder to the steroid hormone aldosterone, lends plausibility to the idea that an increase in PPPase activity may also be involved in the mechanism of estrogen action. In view of the conceptual importance of the identification of the function of IP and the practical advantage of having an enzymic activity for the quantitation of IP in our uterine cell-free synthetic system (11), we investigated the relationship between IP and PPPase. We studied the distribution of IP and PPPase during fractionation by ammonium sulfate precipitation and electrophoresis on cellulose acetate gels and found it possible to separate IP and PPPase activity. MATERIALS AND METHODS Animals. Rats of the departmental colony, derived from Wistar stock, were used and cared for as previously de-
scribed (4). Abbreviations: IP, estrogen-induced protein; PPPase, phosphoprotein phosphatase; EDTA, ethylenediaminetetraacetic acid; EGTA,
ethyleneglycol-bis-(3-aminoethyl ether)N,N'-tetraacetic acid.
2631
Biochemicals. [r-32P]ATP(1-2 Ci/mmol), [3H]leucine (58 Ci/mmol) and [14C]leucine (0.34 Ci/mmol) were obtained from the Radiochemical Centre, Amersham. "Cellogel," gelatinized cellulose acetate analytical strips and preparative blocks were purchased from Chemetron, Milan. Protamine sulfate was from Fluka. ATP, GTP, ethyleneglycol-bis-(j3-aminoethyl ether)N,N'tetraacetic acid (EGTA), Tris, and theophylline were obtained from Sigma; cyclic AMP, cyclic GMP and glucose 6-phosphate from Boehringer; ADP from Pabst Labs; f-glycerophosphate from Fisher; and sodium barbitone (Veronal) from British Drug Houses. Estradiol-173[1,3,5(10)-estratriene-3, 173-diol] was a product of Organon; it was stored in ethanol at 40 at a concentration of 0.5 mg/ml. Preparation of Protein Kinase. Partially purified protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was prepared from a homogenate of muscle of 7-month-old male rats using the procedure of Kuo and Greengard (12) through the stages of acid precipitation and ammonium sulfate fractionation. Preparation of Protamine Phosphate. The substrate for the assay of PPPase activity was protamine phosphate prepared by phosphorylation of protamine sulfate using rat muscle protein kinase in the presence of [-y-32P]ATP. The incubation mixture contained 50 mM sodium acetate buffer, pH 6.0, 10 mM magnesium acetate, 2 mM theophylline, 0.3 mM EGTA, 10 mM sodium fluoride, 5 ,uM cyclic AMP, 10 ,kM [32P]ATP (about 400 cpm/pmol), 1 mg/ml of protamine sulfate, and protein kinase preparation (150 ,ug of protein per ml). Routinely, an incubation volume of 15 ml was used. After 30 min at 300, ATP (0.1 ml of 350 mM) and 0.9 ml of 100 mM sodium pyrophosphate were added and the reaction was stopped by addition of 25% (w/v) trichloroacetic acid to yield a final concentration of 20%. After centrifugation at 10,000 X g for 10 min, the pellet was extracted with 16 ml of 100 mM sodium pyrophosphate-0.5 mM ATP, and dissolved proteins were reprecipitated in 20% trichloroacetic acid. The centrifugation,. solubilization, and precipitation were repeated. The resulting pellet was extracted with distilled water and the extract was centrifuged to remove insoluble material before being used as substrate. No significant phosphorylation of protein in the enzyme extract was detectable in protamine-free incubations. Phosphoprotein Phosphatase Assay. The standard assay mixture was modified from that of Vokaer et al. (9) by the addition of ethylenediaminetetraacetic acid (EDTA) and sodium barbitone. It contained, in a volume of 0.2 ml: 0.1 M Tris-HCl pH 7.2, 0.1 mg of disodium EDTA-2H20, 3.5 ,jg of protamine [32P]phosphate (10-20,000 cpm), 20 mM sodium barbitone, together with an amount of PPPase adjusted so that less than 25% of the substrate was degraded. Sodium barbitone (20 mM) was included when necessary as a control
2632
Proc. Nat. Acad. SC{. USA 72 (1975)
Biochemistry: Kaye et al. I
I
.+
>
,',
-
E 8
.0.4 0.3
E' 0.
5'
0)
0.2~
v25
0.
010
8
16
E
0
X
75 50 02
25-
0
assay.
RESULTS PPPase activity of uterine cytosol No significant variations were observed between the PPPase specific activity of cytosol from control and estradiol-treated uteri at 1, 4, 12, or 24 hr after treatment (Fig. 1). Seven paired measurements were made of PPPase specific activity of control and treated uteri after 1 hr and showed an aver-
0.1
C~~~~~~~0C a.~~~~~~~~~0
Time (hr) FIG. 1. Phosphoprotein phosphatase specific activity of uterine cytosol from 19- to 21-day-old rats after estradiol treatment. For each time point, extracts were made from uteri of four rats given an injection of 5lug of estradiol-17# (closed circles) or control solution (open circles). Assays were performed as described in Materials and Methods. Activity is expressed as nmol of inorganic [32P]phosphate released per mg of cytosol protein per 10 min
for the amount added in extracts from preparative gel electrophoresis (see below). Incubations were carried out at 300 for either 10 or 30 min; 0.1 ml of 1% bovine serum albumin was introduced and the reaction was stopped by addition of 3 ml of 25% trichloroacetic acid. After 5 min at 00, samples were centrifuged for 10 min at 6000 X g and the 32P in the supernatant solution was assayed directly by its Cerenkov radiation (12) in a Packard model 3390 scintillation spectrometer at an efficiency of approximately 45%. The phosphomolybdate-alcohol extraction procedure for inorganic phosphate described by Maeno and Greengard (14) was applied to selected samples following incubation; it was found that more than 90% of the radioactivity soluble in trichloroacetic acid was extractable by this procedure, showing that the activity observed was not attributable to protease action. Fractionation of Uterine Cytosol and Determination of IP (4). Immature female rats (19-21 days old) were injected intraperitoneally with 5 jg of estradiol-17# in 0.5 ml of 2% ethanol or with 0.5 ml of 2% ethanol (controls). One hour later, animals were killed, and uteri were excised and incubated (6/ml) in phosphate-buffered saline (15) in the presence of 25 MACi of [3H]leucine per ml (estradiol-treated) or 5 MCi of ['4C]leucine per ml (controls). The uteri were homogenized as previously described (4) in 0.25 M sucrose in TKM (16). Following centrifugation at 198,000 X g for 50 min at 40, the supernatant solution was fractionated by treatment with solid ammonium sulfate (4). Determination of induced protein was performed using electrophoresis on analytical Cellogel strips as previously described (4). Both total and fractionated cytosol preparations were analyzed by electrophoresis (6) on preparative Cellogel blocks (11 X 6 X 0.25 cm) in an apparatus cooled to 00. Up to 500 Ml of sample was applied to the Cellogel block. After 2 hr electrophoresis using 20 mA constant current (approximately 14 V/cm) the block was sliced into 20 strips (0.4 X 6.0 cm). The fluid in each strip (approximately 0.3 ml) was expressed through a 1 ml syringe fitted with a 27 gauge needle. Suitable volumes of these fractions (5-100,Ml) were then assayed for PPPase activity (see above) and for protein concentration by the method of Lowry et al. (17).
t
25~~~~~~~~~N
24
0.5
1.0
Mobility (RA)
FIG. 2. Electrophoretic distribution of phosphoprotein phosphatase of uterine cytosol. Ten 19- to 21-day-old rats were given an injection of 5 fig of estradiol-17g# (E) or control solution (C). Rats were killed 1 hr after injection and uterine cytosol was subjected to preparative cellulose acetate (Cellogel) electrophoresis as described in Materials and Methods. Closed circles, PPPase activity (32P released/30 min per 100 Ml of fraction). Open circles, protein concentration. The mobility (RA) is expressed as migration relative to that of bovine serum albumin 1.0. =
of 3% less activity in estrogen-stimulated uteri. No significant change in PPPase or in protein distribution was observed on preparative Cellogel electrophoresis of such uterine extracts (Fig. 2). PPPase migrates as a single peak with a mobility relative to bovine serum albumin of approximately 0.5. No PPPase activity (
Biochemistry
Separation of "estrogen-induced" protein from phosphoprotein phosphatase activity of immature rat uterus (steroids/hormone/cyclic AMP/cyclic GMP)
ALVIN M. KAYE, MICHAEL D. WALKER, AND DALIA SOMJEN Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel
Communicated by I. Edelman, May 2, 1975
ABSTRACT Preparations of the "induced protein" which appears in the rat uterus within 40 min of estradiol administration have recently been reported to contain phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity. We found that these two proteins distribute differently on ammonium sulfate fractionation of uterine cytosol. Preparative cellulose acetate electrophoresis afforded complete (>99.9%) separation of phosphoprotein phosphatase activity from the induced protein. The specific activity of phosphoprotein phosphatase in uterine cytosol was unchanged 1, 4, 12, or 24 hr after estradiol administration. These results are incompatible with the view that the induced protein mediates estrogen action by virtue of an inherent phosphoprotein phosphatase activity.
Administration of estradiol-17fl to immature rats, or mature ovariectomized rats, results in an increased rate of synthesis of a uterine protein which has been designated "induced protein" (IP) by Notides and Gorski (1). Synthesis of estrogen-induced protein can be detected within 40 min of estradiol injection into rats (2) or incubation of surviving uteri in physiological concentrations of estrogens (3). This cytosol protein (4) has a molecular weight of approximately 4 X 104 and an isoelectric point of approximately 4.5 (5-7). Because the appearance of IP represents one of the earliest effects of estradiol on the uterus, Baulieu et al. (8) have proposed that IP is the "key intermediate protein" in the stimulation of bulk RNA synthesis by estradiol. However, until a recent report by Vokaer et al. (9), no experimental data on the possible function of IP were available. These au-
thors reported that phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (PPPase) is associated with purified preparations of IP. The proposal of Liu and Greengard (10), that increases in PPPase activity may modulate the response of toad bladder to the steroid hormone aldosterone, lends plausibility to the idea that an increase in PPPase activity may also be involved in the mechanism of estrogen action. In view of the conceptual importance of the identification of the function of IP and the practical advantage of having an enzymic activity for the quantitation of IP in our uterine cell-free synthetic system (11), we investigated the relationship between IP and PPPase. We studied the distribution of IP and PPPase during fractionation by ammonium sulfate precipitation and electrophoresis on cellulose acetate gels and found it possible to separate IP and PPPase activity. MATERIALS AND METHODS Animals. Rats of the departmental colony, derived from Wistar stock, were used and cared for as previously de-
scribed (4). Abbreviations: IP, estrogen-induced protein; PPPase, phosphoprotein phosphatase; EDTA, ethylenediaminetetraacetic acid; EGTA,
ethyleneglycol-bis-(3-aminoethyl ether)N,N'-tetraacetic acid.
2631
Biochemicals. [r-32P]ATP(1-2 Ci/mmol), [3H]leucine (58 Ci/mmol) and [14C]leucine (0.34 Ci/mmol) were obtained from the Radiochemical Centre, Amersham. "Cellogel," gelatinized cellulose acetate analytical strips and preparative blocks were purchased from Chemetron, Milan. Protamine sulfate was from Fluka. ATP, GTP, ethyleneglycol-bis-(j3-aminoethyl ether)N,N'tetraacetic acid (EGTA), Tris, and theophylline were obtained from Sigma; cyclic AMP, cyclic GMP and glucose 6-phosphate from Boehringer; ADP from Pabst Labs; f-glycerophosphate from Fisher; and sodium barbitone (Veronal) from British Drug Houses. Estradiol-173[1,3,5(10)-estratriene-3, 173-diol] was a product of Organon; it was stored in ethanol at 40 at a concentration of 0.5 mg/ml. Preparation of Protein Kinase. Partially purified protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was prepared from a homogenate of muscle of 7-month-old male rats using the procedure of Kuo and Greengard (12) through the stages of acid precipitation and ammonium sulfate fractionation. Preparation of Protamine Phosphate. The substrate for the assay of PPPase activity was protamine phosphate prepared by phosphorylation of protamine sulfate using rat muscle protein kinase in the presence of [-y-32P]ATP. The incubation mixture contained 50 mM sodium acetate buffer, pH 6.0, 10 mM magnesium acetate, 2 mM theophylline, 0.3 mM EGTA, 10 mM sodium fluoride, 5 ,uM cyclic AMP, 10 ,kM [32P]ATP (about 400 cpm/pmol), 1 mg/ml of protamine sulfate, and protein kinase preparation (150 ,ug of protein per ml). Routinely, an incubation volume of 15 ml was used. After 30 min at 300, ATP (0.1 ml of 350 mM) and 0.9 ml of 100 mM sodium pyrophosphate were added and the reaction was stopped by addition of 25% (w/v) trichloroacetic acid to yield a final concentration of 20%. After centrifugation at 10,000 X g for 10 min, the pellet was extracted with 16 ml of 100 mM sodium pyrophosphate-0.5 mM ATP, and dissolved proteins were reprecipitated in 20% trichloroacetic acid. The centrifugation,. solubilization, and precipitation were repeated. The resulting pellet was extracted with distilled water and the extract was centrifuged to remove insoluble material before being used as substrate. No significant phosphorylation of protein in the enzyme extract was detectable in protamine-free incubations. Phosphoprotein Phosphatase Assay. The standard assay mixture was modified from that of Vokaer et al. (9) by the addition of ethylenediaminetetraacetic acid (EDTA) and sodium barbitone. It contained, in a volume of 0.2 ml: 0.1 M Tris-HCl pH 7.2, 0.1 mg of disodium EDTA-2H20, 3.5 ,jg of protamine [32P]phosphate (10-20,000 cpm), 20 mM sodium barbitone, together with an amount of PPPase adjusted so that less than 25% of the substrate was degraded. Sodium barbitone (20 mM) was included when necessary as a control
2632
Proc. Nat. Acad. SC{. USA 72 (1975)
Biochemistry: Kaye et al. I
I
.+
>
,',
-
E 8
.0.4 0.3
E' 0.
5'
0)
0.2~
v25
0.
010
8
16
E
0
X
75 50 02
25-
0
assay.
RESULTS PPPase activity of uterine cytosol No significant variations were observed between the PPPase specific activity of cytosol from control and estradiol-treated uteri at 1, 4, 12, or 24 hr after treatment (Fig. 1). Seven paired measurements were made of PPPase specific activity of control and treated uteri after 1 hr and showed an aver-
0.1
C~~~~~~~0C a.~~~~~~~~~0
Time (hr) FIG. 1. Phosphoprotein phosphatase specific activity of uterine cytosol from 19- to 21-day-old rats after estradiol treatment. For each time point, extracts were made from uteri of four rats given an injection of 5lug of estradiol-17# (closed circles) or control solution (open circles). Assays were performed as described in Materials and Methods. Activity is expressed as nmol of inorganic [32P]phosphate released per mg of cytosol protein per 10 min
for the amount added in extracts from preparative gel electrophoresis (see below). Incubations were carried out at 300 for either 10 or 30 min; 0.1 ml of 1% bovine serum albumin was introduced and the reaction was stopped by addition of 3 ml of 25% trichloroacetic acid. After 5 min at 00, samples were centrifuged for 10 min at 6000 X g and the 32P in the supernatant solution was assayed directly by its Cerenkov radiation (12) in a Packard model 3390 scintillation spectrometer at an efficiency of approximately 45%. The phosphomolybdate-alcohol extraction procedure for inorganic phosphate described by Maeno and Greengard (14) was applied to selected samples following incubation; it was found that more than 90% of the radioactivity soluble in trichloroacetic acid was extractable by this procedure, showing that the activity observed was not attributable to protease action. Fractionation of Uterine Cytosol and Determination of IP (4). Immature female rats (19-21 days old) were injected intraperitoneally with 5 jg of estradiol-17# in 0.5 ml of 2% ethanol or with 0.5 ml of 2% ethanol (controls). One hour later, animals were killed, and uteri were excised and incubated (6/ml) in phosphate-buffered saline (15) in the presence of 25 MACi of [3H]leucine per ml (estradiol-treated) or 5 MCi of ['4C]leucine per ml (controls). The uteri were homogenized as previously described (4) in 0.25 M sucrose in TKM (16). Following centrifugation at 198,000 X g for 50 min at 40, the supernatant solution was fractionated by treatment with solid ammonium sulfate (4). Determination of induced protein was performed using electrophoresis on analytical Cellogel strips as previously described (4). Both total and fractionated cytosol preparations were analyzed by electrophoresis (6) on preparative Cellogel blocks (11 X 6 X 0.25 cm) in an apparatus cooled to 00. Up to 500 Ml of sample was applied to the Cellogel block. After 2 hr electrophoresis using 20 mA constant current (approximately 14 V/cm) the block was sliced into 20 strips (0.4 X 6.0 cm). The fluid in each strip (approximately 0.3 ml) was expressed through a 1 ml syringe fitted with a 27 gauge needle. Suitable volumes of these fractions (5-100,Ml) were then assayed for PPPase activity (see above) and for protein concentration by the method of Lowry et al. (17).
t
25~~~~~~~~~N
24
0.5
1.0
Mobility (RA)
FIG. 2. Electrophoretic distribution of phosphoprotein phosphatase of uterine cytosol. Ten 19- to 21-day-old rats were given an injection of 5 fig of estradiol-17g# (E) or control solution (C). Rats were killed 1 hr after injection and uterine cytosol was subjected to preparative cellulose acetate (Cellogel) electrophoresis as described in Materials and Methods. Closed circles, PPPase activity (32P released/30 min per 100 Ml of fraction). Open circles, protein concentration. The mobility (RA) is expressed as migration relative to that of bovine serum albumin 1.0. =
of 3% less activity in estrogen-stimulated uteri. No significant change in PPPase or in protein distribution was observed on preparative Cellogel electrophoresis of such uterine extracts (Fig. 2). PPPase migrates as a single peak with a mobility relative to bovine serum albumin of approximately 0.5. No PPPase activity (
