- Email: [email protected]
Multiple forms of alkaline phosphatase in untreated and 5-bromo-2′-deoxyuridine-treated choriocarcinoma cells
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 191, No. 2, December, pp. 782-791,1978
Multiple Forms of Alkaline Phosphatase in Untreated and 5-Bromo-2’deoxyuridine-treated Choriocarcinoma Cells’ TAKESHI
SAKIYAMA,
J. C. ROBINSON,
AND
JANICE
YANG
CHOU’
Section on Developmental Enzymology, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Received May 7, 1978; revised July 25, 1978 The alkaline phosphatases present in choriocarcinoma cells, either untreated or treated with 5-bromo-2’-deoxyuridine (BrdUrd), were purified and characterized. Three forms of phosphatase [I, IIa (or IIIa), and IIb (or IIIb)] were isolated from both the untreated and BrdUrd-treated cells. Although BrdUrd induced the synthesis of all three forms of alkaline phosphatase in these cells, the synthesis of forms IIa and IIb was, however, preferentially stimulated. The forms of phosphatase in choriocarcinoma cells resembled each other in their kinetic properties and thermal lability, but differed in their molecular weights and in their electrophoretic mobilities in nondenaturing polyacrylamide gels. All three phosphatases were inactivated by antiserum to term-placental alkaline phosphatase. The alkaline phosphatases from choriocarcinoma cells differed, however, from the enzyme from term placentas in several physicochemical properties. The phosphatases from choriocarcinoma cells had a lower K, value for p-nitrophenyl phosphate, were more sensitive to inhibition by L-leucine, levamisole, l-p-bromotetramisole, and EDTA, and were more heat-labile. Phosphatase I comigrated with term-placental alkaline phosphatase on nondenaturing polyacrylamide electrophoretic gels, but phosphatases IIa and IIb migrated more slowly. The apparent molecular weights of phosphatase forms I, IIa, and IIb were estimated by gel filtration and polyacrylamide gel electrophoresis to be 115,000,240,000, and 510,000, respectively. Although three molecular forms of alkaline phosphatase occurred in choriocarcinoma cells, the subunit molecular weight of these phosphatases appeared to be identical to each other and to the subunit of term-placental alkaline phosphatase (63,000 MW). The alkaline phosphatase in choriocarcinoma cells therefore exists in the dimeric, tetrameric, and octameric forms.
The placental isoenxyme of alkaline phosphatase (EC 3.1.3.1) is normally found in maternal serum during pregnancy (1). Ectopic synthesis of this enzyme occurs, however, in many nontrophoblastic tumors and tumor-derived cell lines (2-10). The similarities between these tumor phosphatases and term-placental alkaline phosphatase suggest that all these phosphatases are encoded by the same gene and that malignant transformation derepresses the gene for this embryonic protein (11). Although alkaline phosphatases from term placenta and many nontrophoblastic tumors have been characterized (7, 10, 12-16), little is 1 This work was supported in part by National Institute of Child Health and Human Development Contract NOl-HD-6-2864. ’ To whom all correspondence should be sent.
known about this enzyme in human placentas following malignant transformation. Two types of alkaline phosphatase have been implied from heat inactivation studies in crude extracts of choriocarcinoma cells, malignant cells derived from cancer of placenta (17), but these have not been characterized. Alkaline phosphatase activity is lower in choriocarcinoma cells than in term placenta (18). Enzyme activity in choriocarcinoma cells can be induced by BrdUrd3 (18, 19). To study the regulation of genes for alkaline phosphatase in choriocarcinoma cells, it is necessary to investigate the relationship between the phosphatases in untreated and BrdUrd-treated choriocarcinoma cells and 3 Abbreviation dine. 782
0003-9861/78/1912-0782$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
used: BrdUrd,
5-bromo-2’-deoxyuri-
CHORIOCARCINOMA
ALKALINE
normal placenta. This paper presents the purification and characterization of alkaline phosphatases from choriocarcinoma cells grown either in the absence or in the presence of BrdUrd. Three molecular forms of alkaline phosphatase have been identified in these cells; all of these phosphatases are inactivated by antiserum to term-placental alkaline phosphatase. They differ, however, from the term-placental enzyme in several physicochemical properties. EXPERIMENTAL
PROCEDURES
cells Cell culture. Cloned JEG-3 choriocarcinoma (20) were grown in Ham’s F12 medium supplemented with 10% fetal calf serum (Flow Laboratories), penicillin (100 U/ml), and streptomycin (100 pg/ml). BrdUrd-treated choriocarcinoma cells were obtained by incubating JEGJ cells in medium containing 10 rg/ml of BrdUrd for 7 days. Medium was changed every other day. Enzyme assay and immunoassay. Alkaline phosphatase activity was measured by the release of pnitrophenol from p-nitrophenyl phosphate at pH 10.5 and 37°C (18). A double-antibody technique was used in the immunochemical assay for alkaline phosphatase. The enzyme was first allowed to react with the rabbit antiserum against term-placental alkaline phosphatase in 10 mre Tris-HCl (pH 7.4) containing 0.9% NaCl and 0.1% bovine serum albumin. After incubation at 37°C for 2 h and 4°C for 16 h, the enzyme-antibody complex was then precipitated with sheep anti-rabbit y-globulin serum. This immunoprecipitation was quantitative. Any residual enzyme activity in the supernatant fraction was enzyme that was unable to react with the specific antiserum. Polyacylamide gel electrophoresis. Analytical gel electrophoresis was carried out in 5% acrylamide gels modified from the method of Davis (21). Phosphatase activity was determined by incubating the gels in 50 mM Tris-HCl, pH 9.0, containing a-naphthyl acid phosphate, polyvmylpyrrolidone iodide, and fast blue 2B (8). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was carried out according to the method of O’Farrell (22). The size of native alkaline phosphatase was estimated in nondenaturing polyacrylamide electrophoretie gels according to the method of Hedrick and Smith (23). The mobilities of alkaline phosphatases and standard proteins were determined using seven different gel concentrations between 4 and 10%. The ratio of N,N’-methylenebisacrylamide to acrylamide (1:38) was maintained constant. A plots of log R, (ratio of protein migration to dye migration) versus gel concentration resulted in straight lines for each of the proteins used. A graph of the slopes of these lines
PHOSPHATASE
783
versus the molecular weights of the protein standards was used to estimate the molecular weights of the phosphatases. /3-Galactosidase, phosphorylase a, fumarase, yglobulin, lactic dehydrogenase (LDH), bovine serum albumin, ovalbumin, a-chymotrypsinogen (all from Sigma Chemical Co.), catalase (Worthington), apoferritin (Mann) glucose-g-phosphate dehydrogenase (GGPD), and glutamate dehydrogenase (GDH) (both from Boehringer Mannheim) were used as molecular weight standards. Neuraminidase treatment. Alkaline phosphatase with neuraminidase preparations were treated (Worthington NEUP, 1.6 IU/ml) for 16 h at 37°C (24). The resulting mixtures were analyzed by means of polyacrylamide gel electrophoresis. f*P]Phosphate binding. Purified choriocarcinoma and term-placental alkaline phosphatases were labeled by incubation with carrier-free Ha3’P04 (New England Nuclear) at pH 5.0 for 10 min at O’C following the technique of Milstein (25). The 32P-labeled phosphatases were precipitated with acidified acetone, washed successively with acidified acetone and ether, and dried. The proteins were dissolved in sample buffer containing 5% /?-mercaptoethanol and 1% sodium dodecyl sulfate and heated 2 min at lOO”C, and used for electrophoresis in a 10% polyacrylamide slab gel containing sodium dodecyl sulfate. After electrophoresis, the gel was dried and used for autoradiography with x-ray film (Kodak SB-5). RESULTS
Enzyme Purification Step 1: Butanol extraction and acetone precipitation. Approximately 100 g of choriocarcinoma cells and 60 g of BrdUrdtreated choriocarcinoma cells were used in this study. Cells were homogenized (Sorval Omni-mixer) in 4 volumes of 10 mM Tris-HCl, pH 7.4, for 2 min. 1-Butanol was added to the homogenate (final concentration 30%, v/v), and the resulting mixture was first stirred at 37°C for 1 h, then at 4°C overnight. The aqueous and butanol phases were separated by centrifugation at 10,000 g for 15 min, the aqueous phase was removed, and the butanol phase was re-extracted with additional buffer. The resulting aqueous phase was combined with that from the first extraction. The phosphatase in the combined aqueous phase was then precipitated overnight with acetone (final concentration 60%, v/v). The acetone precipitate was suspended in 10 mu Tris-HCl, pH 7.4, and dialyzed overnight versus the same buffer.
784
SAKIYAMA,
ROBINSON, AND CHOU
Step 2: DEAE-cellulose chromatography. The dialyzed acetone precipitate was applied to a column (2.5 x 20 cm) of DEAEcellulose previously equilibrated with 10 mu Tris-HCl, pH 7.4, at 4’C. The column was eluted. at 4’C with the same buffer containing four consecutive linear sah gradients [0-0.0s M NaCl (1 liter); 0.08-0.15 NaCl (2.5 liters); 0.X-0.4 M NaCl (1.5 liters); 0.4-l M NaCl(l liter)]. Alkaline phosphatase activities appeared in three peaks, I, II, and III, in untreated choriocarcinoma cells and in five peaks, I, II, III, IV, and V, in BrdUrd-treated cells (Fig. 1). Treatment of choriocarcinoma cells with BrdUrd therefore not only increased the synthesis
FIG. 1. DEAE-cellulose column chromatography of alkaline phosphatases from untreated and BrdUrdtreated choriocarcinoma cells. The fractions (10 ml) were eluted from the column (2.5 x 20 cm) with four consecutive linear gradients (O-0.08 M Nac1; 0.08-0.15 M NaCh 0.X-0.4 M NaC1; and 0.4-1.0 M NaCl) in 10 mu Tris-HCl, pH 7.4. Horizontal arrows indicate the fractions that were pooled. (-), OD2so;(- - - - -), NaCl gradient (conductivity); and (M), alkaline phosphatase activity @mol/ml/min).
of pre-existing choriocarcinoma enzymes but induced the synthesis of new forms of alkaline phosphatase. The amounts of enzyme activity in each phosphatase separated by the DEAE-cellulose column are shown in Table I. BrdUrd increased the activities in all three phosphatases, but phosphatases II and III were preferentially induced. Step 3: Gel filtration. The most active fractions of each peak of alkaline phosphatase were pooled and concentrated by ultrafiltration in an Amicon cell. Each concentrated pool (I to III, from choriocarcinoma cells and I to V from BrdUrd-treated choriocarcinoma cells) was applied separately to a Bio-Gel A-0.5m column (2.5 x 100 cm) equilibrated at 4°C with 10 mu Tris-HCl (pH 7.4) containing 0.14 M NaCl and eluted with the same buffer. Phosphatase I from untreated and BrdUrd-treated choriocarcinema cells appeared in a single peak and eluted at the same position (Fig. 2). Phosphatases II and III from both cell types appeared in two peaks each. Peaks IIa and IIIa were in the included volume and were eluted at approximately the same position. Likewise peaks IIb and IIIb were both partially excluded and were eluted at approximately the same position. The active fractions in peaks IIa, IIb, IIIa, and IIIb from untreated and BrdUrd-treated choriocarcinema cells were separately pooled, concentrated, and applied either to a Sephadex G-200 column (Sephacryl S200, 2.5 X 100 cm; IIa and IIIa) or to a Sepharose 4B column (2 x 60 cm, IIb and IIIb). Both columns were equilibrated and eluted with 10 mu Tris-HCl, pH 7.4, containing 0.14
TABLE1 COMPAREKIN OFTHERELATIVJXAMOUNTSOFALKALW~PHOSPHATASESIN UNTREATEDANDB~~U~~TREATED CHOFU~~ARCINOMA CELLS EL~JTED FROM DEAEXELLULOSE COLUMN
Alkaline phosphatase
Fraction number
I II III IV V
(101-155) (156-260) (346375)
choriocarcinoma CeIls Total activity/ g cells (pol/min) 0.23 0.17 0.027
%oftotaI activity
BrdUrd-treated choriocarcinoma cells Fraction number
53.9 39.8
(106-140) (165-285)
6.3
(326368) (376390) (406-420)
Total activity/ g cells tpmol/min) 1.7 2.5 0.5
0.09 0.12
%oftotal activity
34.8 51.1 10.2 1.8 2.0
CHOBIOCABCINOMA
ALKALINE
BrdUrd
0.8
= 0.16 E E E 0.08
0.4
F? -i >
-. FRACTION
PHOSPHATASE
785
when analyzed by electrophoresis both in nondenaturing and in denaturing polyacrylamide gel systems, and hence is considered to be pure. Molecular Weight Determination The molecular weights of the choriocarcinema alkaline phosphatases were estimated both by gel filtration (Bio-Gel A0.5m column for I, IIa, and IIIa and Sepharose 4B column for IIb and IIIb; Fig. 3) and by polyacrylamide gel electrophoresis (Fig. 4) according to the method of Hedrick and Smith (23). Both methods gave similar results. The apparent molecular weight for phosphatase I was 115,000, that for phosphatases IIa and IIIa was 240,000, and that for phosphatases IIb and IIIb was 510,060. Choriocarcinoma cells, therefore, have at least three forms of alkaline phosphatases, I, IIa (IIIa), and IIb (IIIb).
NUMBER
Polyacrylamide Gel Electrophoresis of Choriocarcinoma Alkaline Phosphatase In a nondenaturing 5% acrylamide gel system phosphatases I, IIa (or IIIa), and IIb (or IIIb) of untreated and BrdUrdtreated choriocarcinoma cells migrated into the gel. Phosphatase I has the same electrophoretic mobility as the term-placental alkaline phosphatase (R, = 0.50), whereas phosphatase IIa and IIb migrated slower (Rf = 0.36 and 0.08, respectively). Phosphatases IV and V from BrdUrd-treated choriocarcinoma cells did not migrate into the M MaCl. Fractions IV and V from BrdUrdtreated cells appeared in a broad peak that gel. All choriocarcinoma alkaline phosphawas partially excluded from the Bio-Gel tases (I, IIa, IIb), like the enzyme from term column. Since the amounts of enzyme ac- placenta, reacted with neuraminidase, intivity in Fractions IV and V were low, no dicating that these enzymes contain sialic further purification was carried out with acid residues. A summary of the Rf values these phosphatases. A summary of purifi- of the choriocarcinoma phosphatases in the absence and in the presence of neuraminication is presented in Tables II and III. dase is presented in Table IV. Purification of Term-Placental Alkaline Phosphatase Subunit Structure of Choriocarcinoma Alkaline Phosphatases Term-placental alkaline phosphatase was purified using the same procedures as [32P]Phosphate has been shown to bind above: butanol extraction, acetone precipi- tightly to the active site of alkaline phostation, chromatography on DEAE-celluphatase (25). Choriocarcinoma alkaline lose, and gel filtration through Bio-Gel A- phosphatases were labeled with [32P]phos0.5m and Sephadex G-200. After these pro- phate at pH 5.0 and electrophoresed in a cedures, the preparation of placental slka- polyacrylamide gel containing sodium doline phosphatase had one protein band decyl sulfate. Autoradiography of the dried
FIG. 2. Gel filtration of alkaline phosphatases from untreated and BrdUrd-treated choriocarcinoma cells. The most active fractions from each of the phosphatase peaks obtained with the DEAE-cellulose column were pooled, concentrated and applied separately to a Bio-Gel A-0.5m column (2.5 X 100 cm) equilibrated with 10 mu Tris-HCl (pH 7.4) containing 0.14 M NaCl. The column was eluted with the same buffer; the fraction size was 3.2 ml. The arrow marked Vo indicates the void volume. Horizontal arrows indicate the fractions which were pooled. (W), alkaline phosphatase activity Q.unol/ml/min); and (. . . . .), ODzao.
786
SAKIYAMA,
ROBINSON,
AND
TABLE II SUMMARYOFPURIFICATIONOFALRALINEPHOSPHATASE Procedure
Whole homogenate Butanol extract Acetone pellet DEAE-cellulose I II III Bio-Gel A-0.5m I IIa IIb IIIb Sephacryl S-200 I IIa Sepharose 4B IIb IIIb
Total activity (pmol/min)
13,414 4,680 1,023
75 62 75
0.006 0.013 0.073
14.4 14.3 1.3
1.17 0.36 0.2
19 19 1.7
195 60 33
2.8 1.3 0.75 0.44
8.7 7.1 1.6 0.4
3.1 5.5 2.1 0.9
12 10 2.1 0.5
517 917 350 150
0.16 0.59
3.0 4.9
18.8 8.3
4.0 6.5
3,133 1,383
0.13 0.17
0.9 0.2
6.9 1.2
1.2 0.3
1,150 200
12.3 40.2 6.4
Specific activity (fimol/min/mg protein)
TABLE
Whole homogenate Butanol Extract Acetone pellet DEAE-cellulose I II III IV V Bio-Gel A-0.5m I IIa IIb IIIb IV V Sephacryl S-209 I IIa Sepharose 4B IIb IIIb
FROMCHORIOCARCINOMACELLS
Total protein (mid
SUMMARYOFPURIFICATIONOFALRALINE Procedure
CHOU
Total protein (me) 8,014 1,697 1,496
Purification (-fold) 100 83 100
1 2 12
III
PHOSPHATASEFROMB~~U~~-TREATEDCHORIOCARCINOMACELLS
Total activity (w-wmin)
Specific activity (ymol/min/mg protein)
638 555 635
0.08 0.33 0.42
Purification (-fold) 100 87 loo
1 4 5
64 132 22 3.1 4.2
20.9 11.7 2.7 0.13 0.12
10 21 3.4 0.5 0.7
1.1 0.50 0.69 0.50 0.27 0.22
35 26 24 3.1 0.6 0.5
31.8 52.0 34.8 6.2 2.2 2.3
5.5 4.1 3.8 0.5 0.1 0.1
398 650 435 78 28 28
0.22 0.19
22.9 10.1
104 53.2
3.6 1.6
1300 665
0.23 0.29
17.1 2.5
74.3 8.6
2.7 0.4
925 108
3.1 11.3 8.2 23.6 33.8
slab gel showed that [“2P]phosphate &o binds tightly to the choriocarcinoma phosphatases and that each of the choriocarcinoma phosphatases was composed of just
261 146 34 1.6 1.5
one subunit. The electrophoretic mobility of the subunit was the same for each of the choriocarcinoma phosphatases; likewise the mobility of this subunit was the same
CHORIOCARCINOMA
ALKALINE
as that of the subunit of term-placental alkaline phosphatase. The data for phosphatases I, IIa, and IIb from BrdUrdtreated cells are shown in Fig. 5. The active form of term-placental alka-
787
PHOSPHATASE
line phosphatase is a dimer (26). It appears that in the choriocarcinoma cells, in addition to the dimeric phosphatase, the active alkaline phosphatases occurred mainly in the tetrameric (IIa and IIIa) and octameric (IIb and IIIb) forms.
Characterization of Choriocarcinoma Alkaline Phosphatases
LLLLL 0.5
1
i ---,-J 2 3 2 MOLECULARWElGHT ix 10 51
Over 90% of all the choriocarcinoma phosphatases was precipitated with the specific antiserum to alkaline phosphatase of term placenta (Fig. 6). The immunological determinants of the choriocarcinoma phosphatases are therefore similar to those of term-placental alkaline phosphatase. Furthermore, the choriocarcinoma and term-placental alkaline phosphatases have the same pH optimum (pH 11.0). Although the choriocarcinoma phosphatases were similar to each other in heat stability, they were considerably less stable to heat denaturation than the term-placental alkaline phosphatase (Fig. 7). The K, for p-nitrophenyl phosphate in 2-amino-2methyl-1-propanol buffer, pH 11, was 0.7 mu for all the choriocarcinoma phosphatases and was 1.8 mu for term-placental alkaline phosphatase. The various isoenzymes of human alkaline phosphatase can also be distinguished by differences in their sensitivity to inhibition by L-amino acids (27, 28), levamisole (29), e-p-bromotetramisole (30), and EDTA (31). Choriocarcinoma and placental alkaline phosphatases were similarly inhibited by L-phenylalanine and L-homoarginine (Table V). Choriocarcinema phosphatases were, however, more sensitive to inhibition by L-leucine, levamisole, /-p-bromotetramisole, and EDTA
34187
FIG. 3. Elution volume as a function of molecular we&&t on Bio-Gel A-0.5m and Sepharose 4B columns.
bovine serum albumin
1
I 10
I
I
I
I
J
50 30 40 MOLECULAR WEIGHT x 1O-4 20
FIG. 4. The slope molecular weight relation of standard proteins, choriocarcinoma alkaline phosphatases, and term-placental alkaline phosphatase. The relative mobilities, Rf, of these proteins were determined using seven (4 to 10%) gel concentrations. The slopes of the log Rf versus gel concentration were plotted against the molecular weights of the protein standards.
TABLE IV COMPARISON OF Rf VALUES OF ALKALINE PHOSPHATASES Treatment
Rf values” Choriocarcinoma alkaline phosphatase
None Neuraminidase
BrdUrd-treated choriocarcinoma alkaline phosphatase
I
IIa
IIb
I
IIa
IIb
0.49
0.36 0.24
0.06 0.04
0.50 0.37
0.35 0.24
0.09 0.04
0.36
DRf is the ratio of protein migration
to bromophenol
blue dye migration.
Term-placental alkaline phosphatase
0.50 0.37
CHORIOCARCINOMA
ALKALINE
789
PHOSPHATASE
I
I I
I 0.1
0.2
0.3
0.4
ANTIBODY $111
FIG. 6. Immunoprecipitation of choriocarcinoma alkaline phosphatases by specific antiserum to termplace&l alkaline phosphatases. The double-antibody immunoassay for alkaline phosphatase was performed as described under Experimental Procedure. Residual activity in the supernatant fraction represents the excess of enzyme for each amount of the specific antiserum added. The amount of phosphatase activity present in the immunoassay was 9 nmol/min for all phosphatases used. The background activity in the absence of phosphatase was 3.4 nmol/min and was substracted from all samples. (u), term-placental alkaline phosphatase; (O--O), I; (A-A), IIa; and (M), IIb. BrdUrd-treated choriocarcinoma phosphatases were used for the experiment shown here. Similar results were obtained with the control choriocarcinoma phosphatases.
than was term-placental tase.
alkaline phospha-
DISCUSSION
Three forms of aIkaIine phosphatases have been purified and characterized from choriocarcinoma cells grown in the absence and in the presence of BrdUrd. The three choriocarcinoma phosphatases contained an apparently identical monomeric unit (apparent molecular weight = 63,000) which comigrates with the monomer of term-placental alkaline phosphatase in polyacrylamide gels containing sodium do-
11 10
I i 20 TIME Imin)
I 10
1
I 20
FIG. 7. Heat stabilities of alkaline phosphatases from untreated and BrdUrd-treated choriocarcinoma cells. Alkaline phopshatases were heated at 56°C in 0.3 M 2-amino-2-methyl-1-propanol, pH 11, containing 1 mg per ml of bovine serum albumin. Phosphatase activities (nmol/min) before heat denaturation were: 2.5 for term-placental enzyme; 1.8, 2.4, and 1.3 for I, IIa, and IIb of control choriocarcinoma cells; 2.2, 2.9, and 2.4 for I, IIa, and IIb of BrdUrd-treated choriocarcinoma cells. (O---O), term-placental alkaline phosphatase; (C--O), I; (A-A), IIa; and (C-----U), IIb.
decyl sulfate. The apparent molecular weights for phosphatases I, IIa (IIIa), and IIb (IIIb) estimated by gel filtration and polyacrylamide gel electrophoresis were 115,000, 240,000, and 510,000, respectively. Although we could not rule out possible conjugation of phosphatases IIa (IIIa) and IIb (IIIb) with lipid or carbohydrate, the molecular weight estimation of these phosphatases suggests that phosphatase I, IIa (IIIa), and IIb (IIIb) probably existed in dimeric, tetrameric, and octameric forms, respectively. The dimeric phosphatase I con&rated with the native term-placental alkaline phosphatase in polyacrylamide electrophoretic gels. The tetramer IIa and octamer IIb migrated into the gel but more slowly than the enzyme from term placenta. The existence of large amounts of tetramerit and octameric forms of alkaline phos-
FIG 5 [32P]Phosphate labeling of alkaline phosphatases. Autoradiography procedures were described under Experimental Procedures. 32P-Labeled alkaline phosphatases were electrophoresed in a 10% polyacrylamide gel containing sodium dodecyl sulfate. Gels 1, 2, 3, and 4 are term-placental alkaline phosphatase, I, IIa, and IIb, respectively. BrdUrd-treated choriocsrcinoma phosphatases were used for the experiment shown here. Similar results were obtained with the control choriocarcinoma phosphatases.
790
SAKIYAMA,
EFFECT OF L-PHENYLALANINE,
ROBINSON,
CHOU
TABLE V L-LEUCINE, L-HOMOARGININE, LEVAMISOLE, /‘-p-BROM~TETRAMIS~LE, EDTA ON ALKALINE PHOSPHATASE ACTIVITIES
Compounds”
AND
Percentage of control activity” Choriocarcinoma alkaline phosphatase
None L-PhenyIaIanine L-Leucine L-Homoarginine Levamisole e-p-Bromotetramisole EDTA
AND
BrdUrd-treated choriocarcinoma alkaline phosphatase
I
Ha
IIb
I
IIa
IIb
109 37 45 93 62 58 44
199 40 43 86 77 66 39
loo 37 38 85 66 78 40
loo 36 39 88 66 73 45
loo 35 42 85 75 69 37
109 37 39 86 63 65 53
Term-placental alkaline phosphatase
109 42 85 95 94 loo 88
a The concentrations of the inhibitors were: L-phenylanine, L-leucine, and L-homoarginine (all from Calbiochem) 5 nue; levamisole, 0.5 mu, and /-p-bromotetramisole, 0.05 mu (both from Aldrich Chemical Co.), EDTA (from Sigma Chemical Co.), 5 mu. ’ Phosphatase activities (nmol/min) in the untreated samples were: 2.5 for the term-placenta) enzyme#2.2, 1.5, and 1.9 for I, IIa. and IIb of untreated choriocarcinoma cells; and 2.1, 1.2, and 1.5 for I, Ha, and IIb of BrdUrd-treated choriocarcinoma cells.
phatase in choriocarcinoma cells contrasts with the distribution in term placenta, where over 90% of the enzyme exists in the dimeric form. Aggregated forms of alkaline phosphatase have been found in firsttrimester and term placentas and in nontrophoblastic tumors (Sakiyama and Chou, unpublished results) (1, 32, 33). The amount of highly aggregated alkaline phosphatase relative to the dimqic form decreases during placental development from approximately 40% in first-trimester placentas to less than 10% in term placentas (Sakiyama and Chou, unpublished results). The importance of these aggregated forms is not known. Induction of alkaline phosphataae activity in choriocarcinoma cells by BrdUrd increased the synthesis of all three forms of phosphatase. The highly aggregated enzymes were, however, preferentially induced. Phosphatases IV and V in BrdUrd-treated choriocarcinoma cells might represent alkaline phosphatases of even higher degrees of aggregation or might be conjugated to lipids or carbohydrates. Although the choriocarcinoma phosphatases occurred in different aggregated states, they resembled each other in kinetic and thermal denaturation properties. The choriocarcinoma and term-placental enzymes had the same monomeric size, pH
optimum, and immunologic reactivity. The choriocarcinoma alkaline phosphatases differed, however, from the term-placental enzyme in several physicochemical properties. First, the Kmvalue for p-nitrophenyl phosphate was 0.7 mu for the choriocarcinoma phosphatases, yet was 1.8 mu for the termplacental enzyme. Second, the choriocarcinoma enzymes were more sensitive to inhibition by L-leucine, levamisole, /-p-bromotetramisole, and EDTA. Third, the choriocarcinoma phosphatases were more heatlabile. It appears that malignant transformation in placenta results in the synthesis of alkaline phosphatases with altered structures. The role of increased production of highly aggregated phosphatases in the cancer cells is unknown. ACKNOWLEDGMENTS The authors thank Dr. C. Edwards for helpful suggestions. REFERENCES 1. BOYER, S. H. (1961) Science 134, 1992-1904. 2. STOLBACH, L. L., KRANT, M. J., AND FISHMAN, W. H. (1969) N. Engl. J. Med. 281,757-762. 3. ELSON, H. A., AND Cox, R. P. (1969) Biochem.
Genet. 3,549-561. 4. FISHMAN, W. H. (1969) Ann. N.Y. Acad. Sci. 166, 745-759.
CHORIOCARCINOMA
ALKALINE
5. WARNOCK, M. L., AND REISMAN, R. (1969) Clin.
Chim. Actu 24,5-H. 6. NAKAYAMA, T., YOSHIDA, M., AND KITAMURA, M. (1970) Clin. Chim. Acta 30,546-548. 7. GHOSH, N. J., RUKENSTEIN, A., BALTIMORE, R., AND Cox, R. P. (1972) Biochim. Biophys. Acta 286, 175-185. 8. HIGAHINO, K., HASHINOTSUME, M., KANG, K-Y., TARAHASHI, Y., AND YAMAMURA, Y. (1972) Clin. Chim. Acta 40, 67-81. 9. SINGER, R. M., AND FISHMAN, W. H. (1974) J.
Cell. Biol. 60, 777-786. 10. LUDUENA, M. A., AND SUSSMAN, H. H., (1976) J. Biol. Chem. 251.2620-2628. 11. FISHMAN, W. H., AND SELL, S. V. (eds.) (1976) Onto-Developmental Gene Expression, Academic Press, New York. 12. HARKNESS, D. R. (1968) Arch. Biochem. Biophys. 126,503-512. 13. SUSSMAN, H. H., SMALL, P. A. JR., AND COTLOVE, E. (1968) J. Biol. Chem. 243, 160-166. 14. SUSSMAN, H. H., AND GOTTLIEB, A. J. (1969) Biochim. Biophys. Acta 194,170-179. 15. SINGER, R. M., AND FIQHMAN, W. H. (1975) in Isozymes III. Developmental Biology (Markert, C. L., ed.), pp. 753-774, Academic Press, New York. 16. GREENE, P. J., AND SUSSMAN, H. H. (1973) Proc.
Nat. Acad. Sci. USA 70, 2936-2940. 17. SPEEG, K. V. JR., A~IZKHAN, J. C., AND STROMBERG, K. (1977) Exp. Cell Res. 106, 199-206. 18. EDLOW, J. B., OTA, T., RELACION, J., KOHLER, P.
PHOSPHATASE
791
O., AND ROBINSON, J. C. (1975) Amer. J. Obstet. Gynecol. 121,674~681. 19. CHOU, J. Y., AND ROBINSON, J. C. (1977) In Vitro 13,450-460. 20. KOHLER, P. O., AND BRIDSON, W. E. (1971) J.
Clin. Endocrinol. Metab. 32, 683-687. 21. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-416. 22. O’FARRELL, P. H. (1975) J. Biol. Chem. 250, 4007-4021. 23. HEDRICK, J. L., AND SMITH, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164. 24. ROBINSON, J. C., AND PIERCE, J. E. (1964) Nature 204,472-473. 25. MILSTEIN, C. (1964) Biochem. J. 92,410-421. 26. GOTTLIEB, A. J., AND SUSSMAN, H. H. (1968) Biochim. Biophys. Acta 160, 167-171. 27. FISHMAN, W. H., INGLIS, N. R., AND GHOSH, N. K. (1968) Clin. Chim. Actu 19, 71-79. 28. FISHMAN, W. H., AND SINGER, R. M. (1975) Ann.
N.Y. Acud. Sci. 259,261-272. 29. CHAN, A. W.-L., AND KELLEN, J. A. (1975) Clin.
Chim. Acta 60,91-96. 30. VAN BELLE, H., DE BROE, M. E., AND WIEME, R. J. (1977) Clin. Chem. 23, 454-459. 31. Cox, R. P., ELSON, N. A., Tu, S., AND GRIFFIN, M. J. (1971) J. Mol. Biol. 58, 198-215. 32. ROBSON, E. B., AND HARRIS, H. (1965) Nature 207, 1257-1259. 33. DOELLGAST, G. J., SPIEGEL, J., GUENTHER, R. A., AND FISHMAN, W. H. (1977) Biochim. Biophys.
Acta 484, 59-78.
Multiple Forms of Alkaline Phosphatase in Untreated and 5-Bromo-2’deoxyuridine-treated Choriocarcinoma Cells’ TAKESHI
SAKIYAMA,
J. C. ROBINSON,
AND
JANICE
YANG
CHOU’
Section on Developmental Enzymology, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Received May 7, 1978; revised July 25, 1978 The alkaline phosphatases present in choriocarcinoma cells, either untreated or treated with 5-bromo-2’-deoxyuridine (BrdUrd), were purified and characterized. Three forms of phosphatase [I, IIa (or IIIa), and IIb (or IIIb)] were isolated from both the untreated and BrdUrd-treated cells. Although BrdUrd induced the synthesis of all three forms of alkaline phosphatase in these cells, the synthesis of forms IIa and IIb was, however, preferentially stimulated. The forms of phosphatase in choriocarcinoma cells resembled each other in their kinetic properties and thermal lability, but differed in their molecular weights and in their electrophoretic mobilities in nondenaturing polyacrylamide gels. All three phosphatases were inactivated by antiserum to term-placental alkaline phosphatase. The alkaline phosphatases from choriocarcinoma cells differed, however, from the enzyme from term placentas in several physicochemical properties. The phosphatases from choriocarcinoma cells had a lower K, value for p-nitrophenyl phosphate, were more sensitive to inhibition by L-leucine, levamisole, l-p-bromotetramisole, and EDTA, and were more heat-labile. Phosphatase I comigrated with term-placental alkaline phosphatase on nondenaturing polyacrylamide electrophoretic gels, but phosphatases IIa and IIb migrated more slowly. The apparent molecular weights of phosphatase forms I, IIa, and IIb were estimated by gel filtration and polyacrylamide gel electrophoresis to be 115,000,240,000, and 510,000, respectively. Although three molecular forms of alkaline phosphatase occurred in choriocarcinoma cells, the subunit molecular weight of these phosphatases appeared to be identical to each other and to the subunit of term-placental alkaline phosphatase (63,000 MW). The alkaline phosphatase in choriocarcinoma cells therefore exists in the dimeric, tetrameric, and octameric forms.
The placental isoenxyme of alkaline phosphatase (EC 3.1.3.1) is normally found in maternal serum during pregnancy (1). Ectopic synthesis of this enzyme occurs, however, in many nontrophoblastic tumors and tumor-derived cell lines (2-10). The similarities between these tumor phosphatases and term-placental alkaline phosphatase suggest that all these phosphatases are encoded by the same gene and that malignant transformation derepresses the gene for this embryonic protein (11). Although alkaline phosphatases from term placenta and many nontrophoblastic tumors have been characterized (7, 10, 12-16), little is 1 This work was supported in part by National Institute of Child Health and Human Development Contract NOl-HD-6-2864. ’ To whom all correspondence should be sent.
known about this enzyme in human placentas following malignant transformation. Two types of alkaline phosphatase have been implied from heat inactivation studies in crude extracts of choriocarcinoma cells, malignant cells derived from cancer of placenta (17), but these have not been characterized. Alkaline phosphatase activity is lower in choriocarcinoma cells than in term placenta (18). Enzyme activity in choriocarcinoma cells can be induced by BrdUrd3 (18, 19). To study the regulation of genes for alkaline phosphatase in choriocarcinoma cells, it is necessary to investigate the relationship between the phosphatases in untreated and BrdUrd-treated choriocarcinoma cells and 3 Abbreviation dine. 782
0003-9861/78/1912-0782$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
used: BrdUrd,
5-bromo-2’-deoxyuri-
CHORIOCARCINOMA
ALKALINE
normal placenta. This paper presents the purification and characterization of alkaline phosphatases from choriocarcinoma cells grown either in the absence or in the presence of BrdUrd. Three molecular forms of alkaline phosphatase have been identified in these cells; all of these phosphatases are inactivated by antiserum to term-placental alkaline phosphatase. They differ, however, from the term-placental enzyme in several physicochemical properties. EXPERIMENTAL
PROCEDURES
cells Cell culture. Cloned JEG-3 choriocarcinoma (20) were grown in Ham’s F12 medium supplemented with 10% fetal calf serum (Flow Laboratories), penicillin (100 U/ml), and streptomycin (100 pg/ml). BrdUrd-treated choriocarcinoma cells were obtained by incubating JEGJ cells in medium containing 10 rg/ml of BrdUrd for 7 days. Medium was changed every other day. Enzyme assay and immunoassay. Alkaline phosphatase activity was measured by the release of pnitrophenol from p-nitrophenyl phosphate at pH 10.5 and 37°C (18). A double-antibody technique was used in the immunochemical assay for alkaline phosphatase. The enzyme was first allowed to react with the rabbit antiserum against term-placental alkaline phosphatase in 10 mre Tris-HCl (pH 7.4) containing 0.9% NaCl and 0.1% bovine serum albumin. After incubation at 37°C for 2 h and 4°C for 16 h, the enzyme-antibody complex was then precipitated with sheep anti-rabbit y-globulin serum. This immunoprecipitation was quantitative. Any residual enzyme activity in the supernatant fraction was enzyme that was unable to react with the specific antiserum. Polyacylamide gel electrophoresis. Analytical gel electrophoresis was carried out in 5% acrylamide gels modified from the method of Davis (21). Phosphatase activity was determined by incubating the gels in 50 mM Tris-HCl, pH 9.0, containing a-naphthyl acid phosphate, polyvmylpyrrolidone iodide, and fast blue 2B (8). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was carried out according to the method of O’Farrell (22). The size of native alkaline phosphatase was estimated in nondenaturing polyacrylamide electrophoretie gels according to the method of Hedrick and Smith (23). The mobilities of alkaline phosphatases and standard proteins were determined using seven different gel concentrations between 4 and 10%. The ratio of N,N’-methylenebisacrylamide to acrylamide (1:38) was maintained constant. A plots of log R, (ratio of protein migration to dye migration) versus gel concentration resulted in straight lines for each of the proteins used. A graph of the slopes of these lines
PHOSPHATASE
783
versus the molecular weights of the protein standards was used to estimate the molecular weights of the phosphatases. /3-Galactosidase, phosphorylase a, fumarase, yglobulin, lactic dehydrogenase (LDH), bovine serum albumin, ovalbumin, a-chymotrypsinogen (all from Sigma Chemical Co.), catalase (Worthington), apoferritin (Mann) glucose-g-phosphate dehydrogenase (GGPD), and glutamate dehydrogenase (GDH) (both from Boehringer Mannheim) were used as molecular weight standards. Neuraminidase treatment. Alkaline phosphatase with neuraminidase preparations were treated (Worthington NEUP, 1.6 IU/ml) for 16 h at 37°C (24). The resulting mixtures were analyzed by means of polyacrylamide gel electrophoresis. f*P]Phosphate binding. Purified choriocarcinoma and term-placental alkaline phosphatases were labeled by incubation with carrier-free Ha3’P04 (New England Nuclear) at pH 5.0 for 10 min at O’C following the technique of Milstein (25). The 32P-labeled phosphatases were precipitated with acidified acetone, washed successively with acidified acetone and ether, and dried. The proteins were dissolved in sample buffer containing 5% /?-mercaptoethanol and 1% sodium dodecyl sulfate and heated 2 min at lOO”C, and used for electrophoresis in a 10% polyacrylamide slab gel containing sodium dodecyl sulfate. After electrophoresis, the gel was dried and used for autoradiography with x-ray film (Kodak SB-5). RESULTS
Enzyme Purification Step 1: Butanol extraction and acetone precipitation. Approximately 100 g of choriocarcinoma cells and 60 g of BrdUrdtreated choriocarcinoma cells were used in this study. Cells were homogenized (Sorval Omni-mixer) in 4 volumes of 10 mM Tris-HCl, pH 7.4, for 2 min. 1-Butanol was added to the homogenate (final concentration 30%, v/v), and the resulting mixture was first stirred at 37°C for 1 h, then at 4°C overnight. The aqueous and butanol phases were separated by centrifugation at 10,000 g for 15 min, the aqueous phase was removed, and the butanol phase was re-extracted with additional buffer. The resulting aqueous phase was combined with that from the first extraction. The phosphatase in the combined aqueous phase was then precipitated overnight with acetone (final concentration 60%, v/v). The acetone precipitate was suspended in 10 mu Tris-HCl, pH 7.4, and dialyzed overnight versus the same buffer.
784
SAKIYAMA,
ROBINSON, AND CHOU
Step 2: DEAE-cellulose chromatography. The dialyzed acetone precipitate was applied to a column (2.5 x 20 cm) of DEAEcellulose previously equilibrated with 10 mu Tris-HCl, pH 7.4, at 4’C. The column was eluted. at 4’C with the same buffer containing four consecutive linear sah gradients [0-0.0s M NaCl (1 liter); 0.08-0.15 NaCl (2.5 liters); 0.X-0.4 M NaCl (1.5 liters); 0.4-l M NaCl(l liter)]. Alkaline phosphatase activities appeared in three peaks, I, II, and III, in untreated choriocarcinoma cells and in five peaks, I, II, III, IV, and V, in BrdUrd-treated cells (Fig. 1). Treatment of choriocarcinoma cells with BrdUrd therefore not only increased the synthesis
FIG. 1. DEAE-cellulose column chromatography of alkaline phosphatases from untreated and BrdUrdtreated choriocarcinoma cells. The fractions (10 ml) were eluted from the column (2.5 x 20 cm) with four consecutive linear gradients (O-0.08 M Nac1; 0.08-0.15 M NaCh 0.X-0.4 M NaC1; and 0.4-1.0 M NaCl) in 10 mu Tris-HCl, pH 7.4. Horizontal arrows indicate the fractions that were pooled. (-), OD2so;(- - - - -), NaCl gradient (conductivity); and (M), alkaline phosphatase activity @mol/ml/min).
of pre-existing choriocarcinoma enzymes but induced the synthesis of new forms of alkaline phosphatase. The amounts of enzyme activity in each phosphatase separated by the DEAE-cellulose column are shown in Table I. BrdUrd increased the activities in all three phosphatases, but phosphatases II and III were preferentially induced. Step 3: Gel filtration. The most active fractions of each peak of alkaline phosphatase were pooled and concentrated by ultrafiltration in an Amicon cell. Each concentrated pool (I to III, from choriocarcinoma cells and I to V from BrdUrd-treated choriocarcinoma cells) was applied separately to a Bio-Gel A-0.5m column (2.5 x 100 cm) equilibrated at 4°C with 10 mu Tris-HCl (pH 7.4) containing 0.14 M NaCl and eluted with the same buffer. Phosphatase I from untreated and BrdUrd-treated choriocarcinema cells appeared in a single peak and eluted at the same position (Fig. 2). Phosphatases II and III from both cell types appeared in two peaks each. Peaks IIa and IIIa were in the included volume and were eluted at approximately the same position. Likewise peaks IIb and IIIb were both partially excluded and were eluted at approximately the same position. The active fractions in peaks IIa, IIb, IIIa, and IIIb from untreated and BrdUrd-treated choriocarcinema cells were separately pooled, concentrated, and applied either to a Sephadex G-200 column (Sephacryl S200, 2.5 X 100 cm; IIa and IIIa) or to a Sepharose 4B column (2 x 60 cm, IIb and IIIb). Both columns were equilibrated and eluted with 10 mu Tris-HCl, pH 7.4, containing 0.14
TABLE1 COMPAREKIN OFTHERELATIVJXAMOUNTSOFALKALW~PHOSPHATASESIN UNTREATEDANDB~~U~~TREATED CHOFU~~ARCINOMA CELLS EL~JTED FROM DEAEXELLULOSE COLUMN
Alkaline phosphatase
Fraction number
I II III IV V
(101-155) (156-260) (346375)
choriocarcinoma CeIls Total activity/ g cells (pol/min) 0.23 0.17 0.027
%oftotaI activity
BrdUrd-treated choriocarcinoma cells Fraction number
53.9 39.8
(106-140) (165-285)
6.3
(326368) (376390) (406-420)
Total activity/ g cells tpmol/min) 1.7 2.5 0.5
0.09 0.12
%oftotal activity
34.8 51.1 10.2 1.8 2.0
CHOBIOCABCINOMA
ALKALINE
BrdUrd
0.8
= 0.16 E E E 0.08
0.4
F? -i >
-. FRACTION
PHOSPHATASE
785
when analyzed by electrophoresis both in nondenaturing and in denaturing polyacrylamide gel systems, and hence is considered to be pure. Molecular Weight Determination The molecular weights of the choriocarcinema alkaline phosphatases were estimated both by gel filtration (Bio-Gel A0.5m column for I, IIa, and IIIa and Sepharose 4B column for IIb and IIIb; Fig. 3) and by polyacrylamide gel electrophoresis (Fig. 4) according to the method of Hedrick and Smith (23). Both methods gave similar results. The apparent molecular weight for phosphatase I was 115,000, that for phosphatases IIa and IIIa was 240,000, and that for phosphatases IIb and IIIb was 510,060. Choriocarcinoma cells, therefore, have at least three forms of alkaline phosphatases, I, IIa (IIIa), and IIb (IIIb).
NUMBER
Polyacrylamide Gel Electrophoresis of Choriocarcinoma Alkaline Phosphatase In a nondenaturing 5% acrylamide gel system phosphatases I, IIa (or IIIa), and IIb (or IIIb) of untreated and BrdUrdtreated choriocarcinoma cells migrated into the gel. Phosphatase I has the same electrophoretic mobility as the term-placental alkaline phosphatase (R, = 0.50), whereas phosphatase IIa and IIb migrated slower (Rf = 0.36 and 0.08, respectively). Phosphatases IV and V from BrdUrd-treated choriocarcinoma cells did not migrate into the M MaCl. Fractions IV and V from BrdUrdtreated cells appeared in a broad peak that gel. All choriocarcinoma alkaline phosphawas partially excluded from the Bio-Gel tases (I, IIa, IIb), like the enzyme from term column. Since the amounts of enzyme ac- placenta, reacted with neuraminidase, intivity in Fractions IV and V were low, no dicating that these enzymes contain sialic further purification was carried out with acid residues. A summary of the Rf values these phosphatases. A summary of purifi- of the choriocarcinoma phosphatases in the absence and in the presence of neuraminication is presented in Tables II and III. dase is presented in Table IV. Purification of Term-Placental Alkaline Phosphatase Subunit Structure of Choriocarcinoma Alkaline Phosphatases Term-placental alkaline phosphatase was purified using the same procedures as [32P]Phosphate has been shown to bind above: butanol extraction, acetone precipi- tightly to the active site of alkaline phostation, chromatography on DEAE-celluphatase (25). Choriocarcinoma alkaline lose, and gel filtration through Bio-Gel A- phosphatases were labeled with [32P]phos0.5m and Sephadex G-200. After these pro- phate at pH 5.0 and electrophoresed in a cedures, the preparation of placental slka- polyacrylamide gel containing sodium doline phosphatase had one protein band decyl sulfate. Autoradiography of the dried
FIG. 2. Gel filtration of alkaline phosphatases from untreated and BrdUrd-treated choriocarcinoma cells. The most active fractions from each of the phosphatase peaks obtained with the DEAE-cellulose column were pooled, concentrated and applied separately to a Bio-Gel A-0.5m column (2.5 X 100 cm) equilibrated with 10 mu Tris-HCl (pH 7.4) containing 0.14 M NaCl. The column was eluted with the same buffer; the fraction size was 3.2 ml. The arrow marked Vo indicates the void volume. Horizontal arrows indicate the fractions which were pooled. (W), alkaline phosphatase activity Q.unol/ml/min); and (. . . . .), ODzao.
786
SAKIYAMA,
ROBINSON,
AND
TABLE II SUMMARYOFPURIFICATIONOFALRALINEPHOSPHATASE Procedure
Whole homogenate Butanol extract Acetone pellet DEAE-cellulose I II III Bio-Gel A-0.5m I IIa IIb IIIb Sephacryl S-200 I IIa Sepharose 4B IIb IIIb
Total activity (pmol/min)
13,414 4,680 1,023
75 62 75
0.006 0.013 0.073
14.4 14.3 1.3
1.17 0.36 0.2
19 19 1.7
195 60 33
2.8 1.3 0.75 0.44
8.7 7.1 1.6 0.4
3.1 5.5 2.1 0.9
12 10 2.1 0.5
517 917 350 150
0.16 0.59
3.0 4.9
18.8 8.3
4.0 6.5
3,133 1,383
0.13 0.17
0.9 0.2
6.9 1.2
1.2 0.3
1,150 200
12.3 40.2 6.4
Specific activity (fimol/min/mg protein)
TABLE
Whole homogenate Butanol Extract Acetone pellet DEAE-cellulose I II III IV V Bio-Gel A-0.5m I IIa IIb IIIb IV V Sephacryl S-209 I IIa Sepharose 4B IIb IIIb
FROMCHORIOCARCINOMACELLS
Total protein (mid
SUMMARYOFPURIFICATIONOFALRALINE Procedure
CHOU
Total protein (me) 8,014 1,697 1,496
Purification (-fold) 100 83 100
1 2 12
III
PHOSPHATASEFROMB~~U~~-TREATEDCHORIOCARCINOMACELLS
Total activity (w-wmin)
Specific activity (ymol/min/mg protein)
638 555 635
0.08 0.33 0.42
Purification (-fold) 100 87 loo
1 4 5
64 132 22 3.1 4.2
20.9 11.7 2.7 0.13 0.12
10 21 3.4 0.5 0.7
1.1 0.50 0.69 0.50 0.27 0.22
35 26 24 3.1 0.6 0.5
31.8 52.0 34.8 6.2 2.2 2.3
5.5 4.1 3.8 0.5 0.1 0.1
398 650 435 78 28 28
0.22 0.19
22.9 10.1
104 53.2
3.6 1.6
1300 665
0.23 0.29
17.1 2.5
74.3 8.6
2.7 0.4
925 108
3.1 11.3 8.2 23.6 33.8
slab gel showed that [“2P]phosphate &o binds tightly to the choriocarcinoma phosphatases and that each of the choriocarcinoma phosphatases was composed of just
261 146 34 1.6 1.5
one subunit. The electrophoretic mobility of the subunit was the same for each of the choriocarcinoma phosphatases; likewise the mobility of this subunit was the same
CHORIOCARCINOMA
ALKALINE
as that of the subunit of term-placental alkaline phosphatase. The data for phosphatases I, IIa, and IIb from BrdUrdtreated cells are shown in Fig. 5. The active form of term-placental alka-
787
PHOSPHATASE
line phosphatase is a dimer (26). It appears that in the choriocarcinoma cells, in addition to the dimeric phosphatase, the active alkaline phosphatases occurred mainly in the tetrameric (IIa and IIIa) and octameric (IIb and IIIb) forms.
Characterization of Choriocarcinoma Alkaline Phosphatases
LLLLL 0.5
1
i ---,-J 2 3 2 MOLECULARWElGHT ix 10 51
Over 90% of all the choriocarcinoma phosphatases was precipitated with the specific antiserum to alkaline phosphatase of term placenta (Fig. 6). The immunological determinants of the choriocarcinoma phosphatases are therefore similar to those of term-placental alkaline phosphatase. Furthermore, the choriocarcinoma and term-placental alkaline phosphatases have the same pH optimum (pH 11.0). Although the choriocarcinoma phosphatases were similar to each other in heat stability, they were considerably less stable to heat denaturation than the term-placental alkaline phosphatase (Fig. 7). The K, for p-nitrophenyl phosphate in 2-amino-2methyl-1-propanol buffer, pH 11, was 0.7 mu for all the choriocarcinoma phosphatases and was 1.8 mu for term-placental alkaline phosphatase. The various isoenzymes of human alkaline phosphatase can also be distinguished by differences in their sensitivity to inhibition by L-amino acids (27, 28), levamisole (29), e-p-bromotetramisole (30), and EDTA (31). Choriocarcinoma and placental alkaline phosphatases were similarly inhibited by L-phenylalanine and L-homoarginine (Table V). Choriocarcinema phosphatases were, however, more sensitive to inhibition by L-leucine, levamisole, /-p-bromotetramisole, and EDTA
34187
FIG. 3. Elution volume as a function of molecular we&&t on Bio-Gel A-0.5m and Sepharose 4B columns.
bovine serum albumin
1
I 10
I
I
I
I
J
50 30 40 MOLECULAR WEIGHT x 1O-4 20
FIG. 4. The slope molecular weight relation of standard proteins, choriocarcinoma alkaline phosphatases, and term-placental alkaline phosphatase. The relative mobilities, Rf, of these proteins were determined using seven (4 to 10%) gel concentrations. The slopes of the log Rf versus gel concentration were plotted against the molecular weights of the protein standards.
TABLE IV COMPARISON OF Rf VALUES OF ALKALINE PHOSPHATASES Treatment
Rf values” Choriocarcinoma alkaline phosphatase
None Neuraminidase
BrdUrd-treated choriocarcinoma alkaline phosphatase
I
IIa
IIb
I
IIa
IIb
0.49
0.36 0.24
0.06 0.04
0.50 0.37
0.35 0.24
0.09 0.04
0.36
DRf is the ratio of protein migration
to bromophenol
blue dye migration.
Term-placental alkaline phosphatase
0.50 0.37
CHORIOCARCINOMA
ALKALINE
789
PHOSPHATASE
I
I I
I 0.1
0.2
0.3
0.4
ANTIBODY $111
FIG. 6. Immunoprecipitation of choriocarcinoma alkaline phosphatases by specific antiserum to termplace&l alkaline phosphatases. The double-antibody immunoassay for alkaline phosphatase was performed as described under Experimental Procedure. Residual activity in the supernatant fraction represents the excess of enzyme for each amount of the specific antiserum added. The amount of phosphatase activity present in the immunoassay was 9 nmol/min for all phosphatases used. The background activity in the absence of phosphatase was 3.4 nmol/min and was substracted from all samples. (u), term-placental alkaline phosphatase; (O--O), I; (A-A), IIa; and (M), IIb. BrdUrd-treated choriocarcinoma phosphatases were used for the experiment shown here. Similar results were obtained with the control choriocarcinoma phosphatases.
than was term-placental tase.
alkaline phospha-
DISCUSSION
Three forms of aIkaIine phosphatases have been purified and characterized from choriocarcinoma cells grown in the absence and in the presence of BrdUrd. The three choriocarcinoma phosphatases contained an apparently identical monomeric unit (apparent molecular weight = 63,000) which comigrates with the monomer of term-placental alkaline phosphatase in polyacrylamide gels containing sodium do-
11 10
I i 20 TIME Imin)
I 10
1
I 20
FIG. 7. Heat stabilities of alkaline phosphatases from untreated and BrdUrd-treated choriocarcinoma cells. Alkaline phopshatases were heated at 56°C in 0.3 M 2-amino-2-methyl-1-propanol, pH 11, containing 1 mg per ml of bovine serum albumin. Phosphatase activities (nmol/min) before heat denaturation were: 2.5 for term-placental enzyme; 1.8, 2.4, and 1.3 for I, IIa, and IIb of control choriocarcinoma cells; 2.2, 2.9, and 2.4 for I, IIa, and IIb of BrdUrd-treated choriocarcinoma cells. (O---O), term-placental alkaline phosphatase; (C--O), I; (A-A), IIa; and (C-----U), IIb.
decyl sulfate. The apparent molecular weights for phosphatases I, IIa (IIIa), and IIb (IIIb) estimated by gel filtration and polyacrylamide gel electrophoresis were 115,000, 240,000, and 510,000, respectively. Although we could not rule out possible conjugation of phosphatases IIa (IIIa) and IIb (IIIb) with lipid or carbohydrate, the molecular weight estimation of these phosphatases suggests that phosphatase I, IIa (IIIa), and IIb (IIIb) probably existed in dimeric, tetrameric, and octameric forms, respectively. The dimeric phosphatase I con&rated with the native term-placental alkaline phosphatase in polyacrylamide electrophoretic gels. The tetramer IIa and octamer IIb migrated into the gel but more slowly than the enzyme from term placenta. The existence of large amounts of tetramerit and octameric forms of alkaline phos-
FIG 5 [32P]Phosphate labeling of alkaline phosphatases. Autoradiography procedures were described under Experimental Procedures. 32P-Labeled alkaline phosphatases were electrophoresed in a 10% polyacrylamide gel containing sodium dodecyl sulfate. Gels 1, 2, 3, and 4 are term-placental alkaline phosphatase, I, IIa, and IIb, respectively. BrdUrd-treated choriocsrcinoma phosphatases were used for the experiment shown here. Similar results were obtained with the control choriocarcinoma phosphatases.
790
SAKIYAMA,
EFFECT OF L-PHENYLALANINE,
ROBINSON,
CHOU
TABLE V L-LEUCINE, L-HOMOARGININE, LEVAMISOLE, /‘-p-BROM~TETRAMIS~LE, EDTA ON ALKALINE PHOSPHATASE ACTIVITIES
Compounds”
AND
Percentage of control activity” Choriocarcinoma alkaline phosphatase
None L-PhenyIaIanine L-Leucine L-Homoarginine Levamisole e-p-Bromotetramisole EDTA
AND
BrdUrd-treated choriocarcinoma alkaline phosphatase
I
Ha
IIb
I
IIa
IIb
109 37 45 93 62 58 44
199 40 43 86 77 66 39
loo 37 38 85 66 78 40
loo 36 39 88 66 73 45
loo 35 42 85 75 69 37
109 37 39 86 63 65 53
Term-placental alkaline phosphatase
109 42 85 95 94 loo 88
a The concentrations of the inhibitors were: L-phenylanine, L-leucine, and L-homoarginine (all from Calbiochem) 5 nue; levamisole, 0.5 mu, and /-p-bromotetramisole, 0.05 mu (both from Aldrich Chemical Co.), EDTA (from Sigma Chemical Co.), 5 mu. ’ Phosphatase activities (nmol/min) in the untreated samples were: 2.5 for the term-placenta) enzyme#2.2, 1.5, and 1.9 for I, IIa. and IIb of untreated choriocarcinoma cells; and 2.1, 1.2, and 1.5 for I, Ha, and IIb of BrdUrd-treated choriocarcinoma cells.
phatase in choriocarcinoma cells contrasts with the distribution in term placenta, where over 90% of the enzyme exists in the dimeric form. Aggregated forms of alkaline phosphatase have been found in firsttrimester and term placentas and in nontrophoblastic tumors (Sakiyama and Chou, unpublished results) (1, 32, 33). The amount of highly aggregated alkaline phosphatase relative to the dimqic form decreases during placental development from approximately 40% in first-trimester placentas to less than 10% in term placentas (Sakiyama and Chou, unpublished results). The importance of these aggregated forms is not known. Induction of alkaline phosphataae activity in choriocarcinoma cells by BrdUrd increased the synthesis of all three forms of phosphatase. The highly aggregated enzymes were, however, preferentially induced. Phosphatases IV and V in BrdUrd-treated choriocarcinoma cells might represent alkaline phosphatases of even higher degrees of aggregation or might be conjugated to lipids or carbohydrates. Although the choriocarcinoma phosphatases occurred in different aggregated states, they resembled each other in kinetic and thermal denaturation properties. The choriocarcinoma and term-placental enzymes had the same monomeric size, pH
optimum, and immunologic reactivity. The choriocarcinoma alkaline phosphatases differed, however, from the term-placental enzyme in several physicochemical properties. First, the Kmvalue for p-nitrophenyl phosphate was 0.7 mu for the choriocarcinoma phosphatases, yet was 1.8 mu for the termplacental enzyme. Second, the choriocarcinoma enzymes were more sensitive to inhibition by L-leucine, levamisole, /-p-bromotetramisole, and EDTA. Third, the choriocarcinoma phosphatases were more heatlabile. It appears that malignant transformation in placenta results in the synthesis of alkaline phosphatases with altered structures. The role of increased production of highly aggregated phosphatases in the cancer cells is unknown. ACKNOWLEDGMENTS The authors thank Dr. C. Edwards for helpful suggestions. REFERENCES 1. BOYER, S. H. (1961) Science 134, 1992-1904. 2. STOLBACH, L. L., KRANT, M. J., AND FISHMAN, W. H. (1969) N. Engl. J. Med. 281,757-762. 3. ELSON, H. A., AND Cox, R. P. (1969) Biochem.
Genet. 3,549-561. 4. FISHMAN, W. H. (1969) Ann. N.Y. Acad. Sci. 166, 745-759.
CHORIOCARCINOMA
ALKALINE
5. WARNOCK, M. L., AND REISMAN, R. (1969) Clin.
Chim. Actu 24,5-H. 6. NAKAYAMA, T., YOSHIDA, M., AND KITAMURA, M. (1970) Clin. Chim. Acta 30,546-548. 7. GHOSH, N. J., RUKENSTEIN, A., BALTIMORE, R., AND Cox, R. P. (1972) Biochim. Biophys. Acta 286, 175-185. 8. HIGAHINO, K., HASHINOTSUME, M., KANG, K-Y., TARAHASHI, Y., AND YAMAMURA, Y. (1972) Clin. Chim. Acta 40, 67-81. 9. SINGER, R. M., AND FISHMAN, W. H. (1974) J.
Cell. Biol. 60, 777-786. 10. LUDUENA, M. A., AND SUSSMAN, H. H., (1976) J. Biol. Chem. 251.2620-2628. 11. FISHMAN, W. H., AND SELL, S. V. (eds.) (1976) Onto-Developmental Gene Expression, Academic Press, New York. 12. HARKNESS, D. R. (1968) Arch. Biochem. Biophys. 126,503-512. 13. SUSSMAN, H. H., SMALL, P. A. JR., AND COTLOVE, E. (1968) J. Biol. Chem. 243, 160-166. 14. SUSSMAN, H. H., AND GOTTLIEB, A. J. (1969) Biochim. Biophys. Acta 194,170-179. 15. SINGER, R. M., AND FIQHMAN, W. H. (1975) in Isozymes III. Developmental Biology (Markert, C. L., ed.), pp. 753-774, Academic Press, New York. 16. GREENE, P. J., AND SUSSMAN, H. H. (1973) Proc.
Nat. Acad. Sci. USA 70, 2936-2940. 17. SPEEG, K. V. JR., A~IZKHAN, J. C., AND STROMBERG, K. (1977) Exp. Cell Res. 106, 199-206. 18. EDLOW, J. B., OTA, T., RELACION, J., KOHLER, P.
PHOSPHATASE
791
O., AND ROBINSON, J. C. (1975) Amer. J. Obstet. Gynecol. 121,674~681. 19. CHOU, J. Y., AND ROBINSON, J. C. (1977) In Vitro 13,450-460. 20. KOHLER, P. O., AND BRIDSON, W. E. (1971) J.
Clin. Endocrinol. Metab. 32, 683-687. 21. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-416. 22. O’FARRELL, P. H. (1975) J. Biol. Chem. 250, 4007-4021. 23. HEDRICK, J. L., AND SMITH, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164. 24. ROBINSON, J. C., AND PIERCE, J. E. (1964) Nature 204,472-473. 25. MILSTEIN, C. (1964) Biochem. J. 92,410-421. 26. GOTTLIEB, A. J., AND SUSSMAN, H. H. (1968) Biochim. Biophys. Acta 160, 167-171. 27. FISHMAN, W. H., INGLIS, N. R., AND GHOSH, N. K. (1968) Clin. Chim. Actu 19, 71-79. 28. FISHMAN, W. H., AND SINGER, R. M. (1975) Ann.
N.Y. Acud. Sci. 259,261-272. 29. CHAN, A. W.-L., AND KELLEN, J. A. (1975) Clin.
Chim. Acta 60,91-96. 30. VAN BELLE, H., DE BROE, M. E., AND WIEME, R. J. (1977) Clin. Chem. 23, 454-459. 31. Cox, R. P., ELSON, N. A., Tu, S., AND GRIFFIN, M. J. (1971) J. Mol. Biol. 58, 198-215. 32. ROBSON, E. B., AND HARRIS, H. (1965) Nature 207, 1257-1259. 33. DOELLGAST, G. J., SPIEGEL, J., GUENTHER, R. A., AND FISHMAN, W. H. (1977) Biochim. Biophys.
Acta 484, 59-78.