Proc. Nati. Acad. Sci. USA Vol. 74, No. 6, pp. 2269-2272, June 1977 Biochemistry
a-Fetoprotein is not a component of the estradiol receptor of the rat uterus (estrogen receptors/estrone/diethylstilbestrol/sex steroid binding plasma protein/differential dissociation of binding complexes)
CHRISTINE RADANYI, CHRISTINE MERCIER-BODARD, CLAUDE SECCO-MILLET, ETIENNE-EMILE BAULIEU, AND HELtNE RICHARD-FOY* Institut National de la Sante et de la Recherche Medicale, Unite de Recherches sur le Metabolisme Moleculaire et la Physio-Pathologie des Steroides,
Departeinent de Chimie Biologique, Faculte de MWdecine de Bicetre, 94-Bicetre, Francet Communicated by Elwood V. Jensen, March 14, 1977
ABSTRACT In high-salt medium, cytosol from immature rat uteri displays two main high-affinity estradiol-binding peaks after ultracentrifugation in a sucrose gradient. The two components are the estradiol receptor which has a sedimentation coefficient of 5.5 S, and the a-fetoprotein which sediments at 4.5 S. The dissociation rate constants (k-1) of plasma a-fetoprotein-estradiol complexes measured at O0 in the absence or presence of 0.4 M KCI were found to be 7 X 10-5 and 8 X 10-5 sec 1, respectively. The half-time of dissociation of these hormone-plasma protein complexes is 100-200 times more rapid than that of the estradiol-receptor complexes. These data led to the use of two "differential dissociation" methods for the measurement of the hormone-binding protein complexes. In a high-salt cytosol, the charcoal technique measured selectively the receptor binding sites; the hydroxylapatite technique measured the sum of the a-fetoprotein plus receptor binding sites. Under these conditions, binding specificity studies provided evidence that a-fetoprotein is not a subunit of the receptor. This was confirmed by binding specificity studies in high-salt medium of the receptor separated from a-fetoprotein by ultra-
centrifugation. In cytosol from immature rat uteri, two macromolecules that bind estradiol (E2) with high affinity are present concurrently: estradiol receptor, an intracellular soluble protein, and a-fetoprotein, a circulating a-globulin (1). The two proteins can be distinguished by differences of their physicochemical properties. In low-salt medium, the sedimentation coefficients are about 8 S and 4.1-4.5 S for the receptor and the a-fetoprotein, respectively (2-5). Conversely, in a high-salt medium, the two proteins cosediment in the region of 4.5 S (1, 6-8). The apparent equilibrium dissociation constant (KD) is approximately 0.5 X 10-1o M for the E2-receptor complex (9) and 2.5 X 10-8 M for the a-fetoprotein-E2 complex (5, 10-12). The binding specificity of the two proteins is very different. The receptor has similar affinities for E2 and diethylstilbestrol (DES), a synthetic nonsteroidal estrogen, whereas its affinity for estrone (E1), an estradiol metabolite, is lower (13, 14). The affinity of a-fetoprotein for E1 is higher than for E2, and it binds DES weakly (4, 12, 15). Recently, a new model for the structure of the receptor was proposed (16). It implies that a-fetoprotein is a, if not the, hormone-binding subunit of the receptor and can be obtained from the "8S" receptor by salt treatment. This dissociation would result in the observed changes in physicochemical properties of the two macromolecules. This paper reports some properties of the immature rat uterine E2 receptor and a-fetoprotein in high-salt medium. These studies suggest that the receptor and a-fetoprotein are two distinct proteins present simultaneously in immature rat Abbreviations: E2, estradiol; DES, diethylstilbestrol; E1, estrone; TE buffer, 50 mM Tris-HCI, pH 7.4/1.5 mM EDTA.
* Preceding papers published under the name of Truong. t Postal address: Lab Hormones, 94270-Bicetre, France.
2269
cytosol and that a-fetoprotein is not a hormone-binding subunit of the receptor.
MATERIALS AND METHODS Female Wistar rats 21-25 days old were used in Animals. all experiments. Cytosol. The animals were decapitated, the blood was collected, and the uteri were quickly removed and rinsed in cold 50 mM Tris-HCl, pH 7.4/1.5 mM EDTA (TE buffer). The uteri were homogenized in the TE buffer (three uteri/ml), in a glass-glass homogenizer at 0°, and all subsequent operations were performed at 0-4°. The homogenate was centrifuged at 200,000 X g for 1 hr, and the resulting supernatant was taken as the cytosol. Measurement of Binding Activity. Cytosol was incubated with 7 nM radioactive E2 ([3H]E2, 45 Ci/mmol; CEA France) for at least 2 hr. For competition studies, unlabeled compounds at 70 nM were added to the radioactive E2 prior to incubation. This concentration was chosen in order to be able to distinguish easily between competitors of different affinities. The background binding (nonsaturable) was estimated by isotopic dilution of [3H]E2 in the presence of 3 AM unlabeled E2. High-affinity binding of E2 was measured by "differential dissociation" techniques, either by charcoal/dextran (17) or by hydroxylapatite (18) methods, as previously described (19). Charcoal removes free hormone, including that released during exposure to the adsorbent. The duration of treatment with the adsorbent is critical, considering the rate of dissociation of hormone-protein complexes (17). Hydroxylapatite adsorbs hormone-receptor and hormone-a-fetoprotein complexes, and free hormone is eliminated by an extensive and rapid wash with buffer. Ultracentrifugation through Sucrose Gradient. For analytical studies, samples (0.2-0.3 ml) containing 10 jig of peroxidase, as an internal standard [S = 3.6 (20)], were layered on a 5-20% linear sucrose gradient in TE buffer containing 0.4 M KCI and run for 16-17 hr at 48,000 rpm in an SW 60 rotor (Beckman). Two-drop fractions were collected, and sedimentation coefficients were calculated according to Martin and Ames (21). For preparative studies, samples were layered on a 5-20% linear sucrose gradient in TE buffer and run for 15 hr at 37,000 rpm in an SW 41 rotor. Radioactivity Measurement. Aliquots (0.5 ml) were assayed in 10 ml of Bray's solution and 0.2-ml aliquots were counted in ethanol/toluene (3:10, vol/vol) Omnifluor mixture, with a 3H efficiency of 27% and 30%, respectively.
RESULTS Sedimentation Coefficients of E2-Binding Macromolecules in Cytosol at High Ionic Strength. On the basis of their sedimentation coefficient in low-salt medium, it is easy to dif-
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Biochemistry: Radanyi et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
C',)
0~~~~~~~
g' 5.25]
~~~~~~~~~0
I 5.00
100 5
1
200 Time, min
FIG. 3. Dissociation of the a-fetoprotein-E2 complex as a function of time. B is the concentration of bound E2 expressed in cpm/ml. 0, with TE buffer; 0, with TE/0.4 M KCl buffer.
2.5
5
7.5
S
Fw.. 1. Sucrose gradient ultracentrifugation of [:H]E2-receptor and aY-fetoprotein complexes from immature rat uterine cytosol and [3HJE.-a-fetoprotein complexes from immature rat plasma in highsalt medium. Cytosol (0) (0.3 ml) and plasma (*) (0.2 ml) samples were separately ultracentrifuged at 48,000 rpm for 16 and 17 hr, respectively. In both experiments, S values were calculated according to Martin. and Ames (21).
ferentiate a-fetoprotein from E2 receptor in a cytosol. However, at high ionic strength, the sedimentation coefficients are similar (about 4-5 S). The 4-5S peak can be partially resolved into two peaks, 4.5 and 5.5 S, respectively (Fig. 1).Two sets of data indicated that the 4.5S peak is a-fetoprotein. a-Fetoprotein from plasma of 21- to 24-day old rats has a sedimentation coefficient of 4.5 S (Fig. 1). In presence of E2, a marked decrease in the binding of [3H]E2 was observed in the two peaks, indicating that both binding systems are saturable; however, DES depressed the [3H]E2 binding of the 5.5S peak, more than that of the 4.5S peak (Fig. 2). These ultracentrifugation experiments gave some qualitative indications, but they also illustrated the difficulty in obtaining quantitative results by this method. More precise information may be obtained from binding measurements performed by "differential dissociation" techniques. With the latter, highaffinity ligand macromolecule complexes are selectively
S
FIG. 2. Sucrose gradient ultracentrifugation of cytosol after incubation with [3HJE2 7 nM, alone (0), plus DES 70 nM (0), or plus E2 70 nM (U). Samples (0.3 ml) were layered and the tubes were centrifuged for 16 hr at 48,000 rpm. The small differences in the sedimentation coefficient values observed in the absence or presence of E2 and DES are explainable by the higher amount of free [3H]E2 in the competition experiments.
measured because they dissociate more slowly than the "nonsaturable" and "nonspecific" complexes at 40. Dissociation Rate Constant of the E2-a-Fetoprotein Complexes. In order to work under optimal conditions for measuring binding by differential dissociation techniques, it is necessary to know the dissociation rate constant of the hormone-protein complexes. For a-fetoprotein, the affinity for the hormone is known, but the dissociation rate of the complex is not. This dissociation rate constant (kL1) was measured in the absence or presence of 0.4 M KC1. The measurement of k-1 was performed with plasma of 21-day-old rats, diluted 1:10 in TE or TE/0.4 M KC1 buffer and incubated for 17 hr with 20 nM [3H]E2. At zero time, charcoal/dextran, 1:1 (vol/vol), suspension was added to the incubated plasma. At different times (0-0 min), aliquots were centrifuged at 700 X g for 10 min. The radioactivity of the supernatant represents the a-fetoprotein-E2 complexes. Fig. 3 shows the dissociation of the complexes as a function of time, in the presence or absence of 0.4 M KCL. The half-lives of the complexes at 00 were 160 min with k-1 = 7 X 10-5 sec-1 and 140 min with k.1 = 8 X 10-5 sec-1, respectively. These results are different from those obtained for the E2receptor complex-ti/2 = 10-20 days and k-1 = 7.3 X 10-7 sec-1 (22, 23), and no change in the presence of 0.4 M KCI (24, 25). Comparison of Estrogen Binding to Plasma a-Fetoprotein Measured by Charcoal and Hydroxylapatite Techniques. From the preceding results, one can predict that selective measurement of receptor is possible in a cytosol containing both a-fetoprotein and receptor, if exposure of the extract to the charcoal is performed over a long period of time (17 hr). On the contrary, by a technique such as hydroxylapatite method with a short washing time (30 min), one-measures simultaneously E2 binding to both a-fetoprotein and receptor. It was verified that, with the charcoal technique, the contribution of a-fetoprotein to the high-affinity E2 binding is not measured. Plasma from 21- to 25-day old rats diluted 1:10 with TE/0.4 M KCl buffer was incubated with the hormone and competitors. The highaffinity binding was measured in parallel by the charcoal and the hydroxylapatite techniques. Table 1 shows that no specific binding was detected with the charcoal technique. On the contrary, the hydroxylapatite technique measured the a-fetoprotein-[3H]E2 complex with the expected specificity [already observed with the pure protein (5)] E1 being more potent than E2, and DES not competing. This confirms the prediction based on the difference of the dissociation rate constants for the two protein-E2 complexes. High-Affinity Binding of E2 to Cytosol Proteins. The comparative evaluation of the binding activity in cytosol by the two adsorbent techniques may be a good tool for discriminating between the contribution of the two proteins to total estrogen binding. The charcoal technique gives a measurement of the
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Radanyi et al. Table 1. Binding of estradiol to plasma a-fletoprot~in ithe of competitors: Comparison of hydroxylapatite (HAP) and charcoal methods.
presence
Binding activity, cpm/ml HAP [3H]E2 [:3H]E2 + E2 [3H]E2 + E1 [:PH]E2 + DES
Binding activity, cpm/ml Charcoal* HAP*
%
HAP
0
100
49,700 28,900 26,900 49,300
Table 2. High-affinity binding of estradiol to salt-treated cytosol proteins in the presence of competitors: Comparison of hydroxylapatite (HAP) and charcoal methods.
Residual activity,
Charcoal 0
58
0
54
0
99
binding of the receptor, and the hydroxylapatite technique, the total high-affinity binding. Cytosol containing 0.4 M KCI was incubated with E2 and competitors. Table 2 shows that, as measured by the charcoal technique, the binding affinities with the receptor are in the order E2 DES > El. On the contrary, with the hydroxylapatite technique, the observed order E2 > DES E1 suggests binding of ligands to both receptor and a-fetoprotein. If, as recently published (16), in high-salt medium the receptor dissociates to yield a-fetoprotein, the charcoal measurement would be irtipossible. The dissociated part of the receptor would escape detection after the 17-hr exposure to charcoal. In fact, the binding activity of the cytosol measured by the charcoal technique was found to be the same at low and high ionic strengths, 16,500 and 16,400 cpm/ml, respectively. These results are in agreement with the data previously published, indicating the same dissociation rate constant of the E2-receptor complex from rat at low and high ionic strengths (26). -
Binding Specificity of the Receptor in Absence of a-Fetoprotein at High Ionic Strength. The comparison of the
high-affinity binding specificity obtained by the two techniques in the cytosol at high ionic strength suggests that the receptor does not dissociate to a-fetoprotein in the presence of salt. More direct evidence would be obtained if receptor free of a-fetoprotein and treated by KCI showed the same specificity as the receptor in low-salt medium. To obtain this evidence, lowionic-strength cytosol was run through a sucrose gradient and the fractions corresponding to the top and the heavier side of the "8S" peak were pooled. A tube containing cytosol labeled with radioactive E2 served as a marker for locating the "8S" peak. At low ionic strength, a-fetoprotein migrated in the 4.5S region and the "8S" peak contained the receptor. To the "8S" pool, KCI was added to a final concentration of 0.4 M, and then this was incubated with estrogens. Binding was measured by the hydroxylapatite technique. The data presented in Table 3 show that DES competes strongly with [3H]E2 and that it competes more strongly than E1 (compare also Table 2, charcoal column). These results clearly indicate that a-fetoprotein is not a subunit of the receptor. DISCUSSION
Recently, a new model for the structure of the cytosol estrogen receptor of the immature rat uterus was proposed (16). This model postulated that the "8S" receptor (2), which is dissociated into "4-5S" subunits by salt treatment (0.4 M kCl) (6-8), is mainly if not entirely composed of a-fetoprotein. Evidence for this model was drawn from the following experimental and theoretical considerations. A low-salt-buffered cytosol, containing the "8S" receptor, was treated with an anti-a-fetoprotein immunoadsorbent in order to remove free a-fetoprotein. The nonadsorbed fraction was then exposed to high-salt conditions (0.4 M KCI) in order to dissociate the "8S" receptor into the "4-5S" subunits. The salt-treated cytosol was then divided
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Residual activity, %
HAP
Charcoal
100 100 16,400 41,160 [3H]E2 19 13 3,160 [3H]E2+ E2 5,260 74 54 12,200 22,160 [3H]E2 + E1 29 49 [3H]E2 + DES 19,960 4,770 The 29% residual activity observed in presence of DES is due to the relatively low concentration of nonradioactive competitor. Under these conditions, the "nonspecific" binding, higher for DES than for E2, cannot be neglected and explains this value. * The data shown were obtained with two different cytosol preparations.
in two parts. One part was subjected to an anti-IgG immunoadsorbent and served as the control (high-salt control); the concentration of a-fetoprotein left after adsorbtion was 1.84 ,ug/ml. The other part was subjected to an anti-a-fetoprotein immunoadsorbent, and the concentration of a-fetoprotein in the nonadsorbed fraction was 0.14 /ig/ml. The authors were unable to detect any receptor activity in the latter fraction. They concluded that the "4-5S" E2-binding peak obtained by high-salt treatment consisted entirely of a-fetoprotein, and that a-fetoprotein must be present in the "85" receptor peak at low-salt concentrations. They suggested that, under these conditions, a-fetoprotein is not detected by immunological methods because the antigenic determinants of the a-fetoprotein are masked when it is present in an oligomeric form. In fact, the data presented in the above-mentioned paper (16) can be reevaluated as follows. Their "8S" receptor peak (their figure 1, curve A) corresponds to a concentration of binding sites of approximately 0.5 pmol/ml. After incubation with 5 nM [3H]E2, the labeling of this peak is due to the high affinity of the receptor for E2 (KD -10-10 M). Under these conditions, no radioactive a-fetoprotein peak is visible in the "4-5S" region, although, according to the authors, the concentration of afetoprotein in this area is 1.84 ,tg/ml-that is to say, 25 pmol/ ml. From the data of their figure 2, this last result could be due to the competition, by the receptor, with E2 binding to a-fetoprotein. The authors assumed that, after KCI treatment, afetoprotein had been released from the "85" and was present in the "4-5S" peak of their figure 1, curve B. Since "free" afetoprotein has an affinity no more than 5% of that of the "8S" receptor (cited in table 1 of their paper), it is difficult to understand how the transfer of bound [3H1E2 from the "8S" to the "4-5S" region (in which the protein has much lower affinity for the hormone) can give a radioactive peak of approximately the same magnitude. We therefore conclude that the "4-5S" peak of their figure 1 curve B is a-fetoprotein. In consequence, it is not surprising that immunoadsorbtion with anti-a-fetoprotein antibodies resulted in the disappearance of this peak Table 3. Specific binding of radioactive estradiol to 0.4 M KCltreated estradiol receptor obtained from "8S" fraction of immature rat uterine cytosol
[3H]E2 [3H]E2 + E2 [3HJE2 + E1
[3H1E2 + DES
Binding activity,
Residual
cpm/ml
activity, %
6650 810 4100
100 12 62 13
860
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Biochemistry: Radanyi et al.
(curveC). It remains to be explained what happened to the receptor in B and C and the reason for the presence of a "4-5S" a-fetoprotein peak in curve B but not in curve A. We suggest that, for the most part, receptor had not resisted the KCl treatment followed by immunoadsorbtion with anti-IgG as well as with anti-a-fetoprotein because, in both cases, protective proteins can be removed nonspecifically. It is known that estrogen receptor is more fragile in 0.4 M KCI and especially in the absence of E2, the experimental conditions of the reported immunoadsorbtion procedure (16). An important decrease of receptor binding activity would suppress the competitive effect of E2 binding to a-fetoprotein, and therefore it can be understood that the a-fetoprotein peak is absent in curve A but is revealed in curve B. The results presented here show that it is possible to distinguish between a-fetoprotein and receptor present concurrently in immature rat uterine high-salt cytosol by using different approaches. First, this can be obtained on the basis of their sedimentation coefficients, 4.5 and 5.5 S. Both peaks can be saturated with E2. DES competes strongly with E2 for the binding to the 5.5S receptor peak and only slightly for binding to the 4.5S a-fetoprotein peak. Second, precise data concerning the binding specificity of estrogens to the cytosol high-affinity binding proteins can be obtained with differential dissociation methods. Because of the different dissociation rates of the two E2-protein complexes at 00 (receptor-E2, t1/2 = 20 days; afetoprotein-E2, t 1/2 = 140 min), selective measurement of the receptor was achieved by incubating the extract with a charcoal/dextran suspension for 17 hr. The high-affinity binding specificity was E2 - DES > El, which should be expected for receptor specificity. It was observed (26) that salt treatment does not change the concentration of E2 binding sites measured by the charcoal method. Conversely, when receptor and a-fetoprotein were measured simultaneously by the hydroxylapatite technique, the binding specificity was E2 El > DES, suggesting the contribution of both proteins to estrogen binding. Third, when a-fetoprotein is removed by ultracentrifugation, the binding specificity of the receptor, dissociated into subunits -by salt, is typical of the low-salt receptor, with the order E2 DES > E1. The present studies clearly demonstrate that a-fetoprotein is not a component of the receptor. It is difficult to imagine that a-fetoprotein, which binds E2 with high affinity in only two animal species (rat and mouse) and which disappears almost completely in the mature animal, could be a component of the Ea receptor. In any case, attempts to demonstrate binding of a-fetoprotein-E2 complexes to DNA-cellulose (27) or to uterine nuclei (A. M. Kaye, personal communication) were unsuccessful, contrary to corresponding controls with E2-receptor complexes. No a-fetoprotein has been detected in the uterine nuclear fraction (16). It has been observed that the estrogen receptors from somatic cells of all studied species (mammalian, avian,-amphibian) exhibit similar physicochemical properties (28), and they are of major physiological importance in mature animals. A possible role of a-fetoprotein is the protection of the fetus against the circulating steroids coming from the mother. During fetal life in most species, this role may be played by sex-steroid-binding plasma protein (29), and it is just in the rat and in the mouse that this protein has not been found. Note Added in Proof. Unpublished results from B. Attardi and E. Ruoslahti of studies with the mouse uterine cytosol and specific afetoprotein antibody confirm the lack of a-fetoprotein component in KCI-dissociated receptor preparation. The authors thank G. Redeuilh for his help, E. Eigenmann for his active participation in the writing of the manuscript, and F. Trautsch and S. Bellorini for secretarial assistance. This work was partially
Proc. Natl. Acad. Sci. USA 74 (1977) supported by the Centre National de la Recherche Scientifique, the Delegation Generalea la Recherche Scientifique et Technique, and
the Ford Foundation. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1. Michel,G., Jung, I., Baulieu, E. E., Aussel, C. & Uriel, J. (1974) Steroids 24, 437-449. 2. Toft, D., Shyamala, G. & Gorski, J. (1967) Proc. Natl. Acad. Sci. USA 57, 1740-1745. 3. Jensen, E. V., De Sombre, E. R., Hurst, D. J., Kawashima, T. & Jungblut, P. W. (1967) Arch. Anat. Microsc. Morphol. Exp. (Suppl.) 56,547-569. 4. Soloff, M. S., Morrisson, M. J. & Swartz, T. L. (1972) Steroids 20, 597-608. 5. Aussel, C., Uriel, J. & Mercier-Bodard, C. (1973) Biochimie 55, 1431-1437. 6. Jensen, E. V., Suzuki, T., Kawashima, T., Stumpf, W. E., Jungblut, P. W. & De Sombre, E. R. (1968) Proc. Natl. Acad. Sci. USA 59, 632-638. 7. Erdos, T. (1968) Biochem. Biophys. Res. Commun. 32, 338343. 8. Rochefort, H. & Baulieu, E. E. (1968), C. R. Hebd. Seances Acad. Sci. 267, 662-665. 9. Raspe, G., ed. (1971) Advances in the Biosciences (Pergamon Press-Vieweg, Oxford), Vol. 7. 10. Ekka, E. & De Herthog, R. (1976) J. Steroid Biochem. 7,241247. 11. Savu, L., Crepy, O., Guerier, M. A., Nunez, E., Engelmann, F., Benassayag, C. & Jayle, M. F. (1972) FEBS Lett. 22, 113-116. 12. Raynaud, J. P., Mercier-Bodard, C. & Baulieu, E. E. (1971) Steroids 18, 767-787. 13. Baulieu, E. E., Alberga, A. & Jung, I. (1967) C. R. Hebd. Seances Acad. Sci. 265,354-357.
14. Geynet, C., Millet, C., Truong, H. & Baulieu, E. E. (1972) Gy-
necol. Invest. 3,2-29. 15. Nunez, E., Savu, L., Engelmann, F., Benassayag, C., Crepy, 0. & Jayle, M. F. (1971) C. R. Hebd. Seances Acad. Sci. 276, 242-245. 16. Uriel, J., Bouillon, D., Aussel, C. & Dupiers, M. (1976) Proc. Natl. Acad. Sci. USA 73, 1452-1456. 17. Milgrom, E. & Baulieu, E. E. (1969) Biochim. Biophys. Acta 194, 602-605. 18. Erdos, T., Best-Belpomme, M. & Bessada, R. (1970) Anal. Biochem. 37,244-252. 19. Truong, H., Geynet, C., Millet, C., Soulignac, O., Bucourt, R., Vignau, M., Torelli, V. & Baulieu, E. E. (1973) FEBS Lett. 35, 289-294. 20. Shannon, L. M., Kay, E. & Lew, J. Y. (1966) J. Biol. Chem. 241, 2166-2172. 21. Martin, R. G. & Ames, B. N. (1962) J. Biol. Chem. 236, 13721379. 22. Best-Belpomme, M., Fries, J. & Erdos, T. (1970) Eur. J. Biochem. 17,425, 432. 23. Geynet, C., Millet, C., Truong, H. & Baulieu, E. E. (1972) C. R. Acad. Sci. Paris, 275, 1551-1554. 24. Soulignac, 0. (1974) These de doctorat en M6decine, Universite de Paris XI. 25. Salat-Trepat, J. & Reti, E. (1974) Biochim. Biophys. Acta 338, 92-103. 26. Rochefort, H. & Baulieu, E. E. (1971) Biochimie 53,893-907. 27. Fox, T. 0. (1975) Nature 258,441-444. 28. Baulieu, E. E., Atger, M., Best-Belpomme, M., Corvol, P.,
Courvalin, J. C., Mester, J., Milgrom, E., Robel, P., Rochefort, H. & de Catalogne, D. (1975) Vitam. Horm. (NY) 33, 649731. 29. Mercier-Bodard, C., Renoir, J . & Baulieu, E. E. (1976) Symposium au V Congre's International d'Endocrinologie, Ham-
bourg, 18-24 Juillet 1976 (Excerpta Medica, Amsterdam), in press.
a-Fetoprotein is not a component of the estradiol receptor of the rat uterus (estrogen receptors/estrone/diethylstilbestrol/sex steroid binding plasma protein/differential dissociation of binding complexes)
CHRISTINE RADANYI, CHRISTINE MERCIER-BODARD, CLAUDE SECCO-MILLET, ETIENNE-EMILE BAULIEU, AND HELtNE RICHARD-FOY* Institut National de la Sante et de la Recherche Medicale, Unite de Recherches sur le Metabolisme Moleculaire et la Physio-Pathologie des Steroides,
Departeinent de Chimie Biologique, Faculte de MWdecine de Bicetre, 94-Bicetre, Francet Communicated by Elwood V. Jensen, March 14, 1977
ABSTRACT In high-salt medium, cytosol from immature rat uteri displays two main high-affinity estradiol-binding peaks after ultracentrifugation in a sucrose gradient. The two components are the estradiol receptor which has a sedimentation coefficient of 5.5 S, and the a-fetoprotein which sediments at 4.5 S. The dissociation rate constants (k-1) of plasma a-fetoprotein-estradiol complexes measured at O0 in the absence or presence of 0.4 M KCI were found to be 7 X 10-5 and 8 X 10-5 sec 1, respectively. The half-time of dissociation of these hormone-plasma protein complexes is 100-200 times more rapid than that of the estradiol-receptor complexes. These data led to the use of two "differential dissociation" methods for the measurement of the hormone-binding protein complexes. In a high-salt cytosol, the charcoal technique measured selectively the receptor binding sites; the hydroxylapatite technique measured the sum of the a-fetoprotein plus receptor binding sites. Under these conditions, binding specificity studies provided evidence that a-fetoprotein is not a subunit of the receptor. This was confirmed by binding specificity studies in high-salt medium of the receptor separated from a-fetoprotein by ultra-
centrifugation. In cytosol from immature rat uteri, two macromolecules that bind estradiol (E2) with high affinity are present concurrently: estradiol receptor, an intracellular soluble protein, and a-fetoprotein, a circulating a-globulin (1). The two proteins can be distinguished by differences of their physicochemical properties. In low-salt medium, the sedimentation coefficients are about 8 S and 4.1-4.5 S for the receptor and the a-fetoprotein, respectively (2-5). Conversely, in a high-salt medium, the two proteins cosediment in the region of 4.5 S (1, 6-8). The apparent equilibrium dissociation constant (KD) is approximately 0.5 X 10-1o M for the E2-receptor complex (9) and 2.5 X 10-8 M for the a-fetoprotein-E2 complex (5, 10-12). The binding specificity of the two proteins is very different. The receptor has similar affinities for E2 and diethylstilbestrol (DES), a synthetic nonsteroidal estrogen, whereas its affinity for estrone (E1), an estradiol metabolite, is lower (13, 14). The affinity of a-fetoprotein for E1 is higher than for E2, and it binds DES weakly (4, 12, 15). Recently, a new model for the structure of the receptor was proposed (16). It implies that a-fetoprotein is a, if not the, hormone-binding subunit of the receptor and can be obtained from the "8S" receptor by salt treatment. This dissociation would result in the observed changes in physicochemical properties of the two macromolecules. This paper reports some properties of the immature rat uterine E2 receptor and a-fetoprotein in high-salt medium. These studies suggest that the receptor and a-fetoprotein are two distinct proteins present simultaneously in immature rat Abbreviations: E2, estradiol; DES, diethylstilbestrol; E1, estrone; TE buffer, 50 mM Tris-HCI, pH 7.4/1.5 mM EDTA.
* Preceding papers published under the name of Truong. t Postal address: Lab Hormones, 94270-Bicetre, France.
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cytosol and that a-fetoprotein is not a hormone-binding subunit of the receptor.
MATERIALS AND METHODS Female Wistar rats 21-25 days old were used in Animals. all experiments. Cytosol. The animals were decapitated, the blood was collected, and the uteri were quickly removed and rinsed in cold 50 mM Tris-HCl, pH 7.4/1.5 mM EDTA (TE buffer). The uteri were homogenized in the TE buffer (three uteri/ml), in a glass-glass homogenizer at 0°, and all subsequent operations were performed at 0-4°. The homogenate was centrifuged at 200,000 X g for 1 hr, and the resulting supernatant was taken as the cytosol. Measurement of Binding Activity. Cytosol was incubated with 7 nM radioactive E2 ([3H]E2, 45 Ci/mmol; CEA France) for at least 2 hr. For competition studies, unlabeled compounds at 70 nM were added to the radioactive E2 prior to incubation. This concentration was chosen in order to be able to distinguish easily between competitors of different affinities. The background binding (nonsaturable) was estimated by isotopic dilution of [3H]E2 in the presence of 3 AM unlabeled E2. High-affinity binding of E2 was measured by "differential dissociation" techniques, either by charcoal/dextran (17) or by hydroxylapatite (18) methods, as previously described (19). Charcoal removes free hormone, including that released during exposure to the adsorbent. The duration of treatment with the adsorbent is critical, considering the rate of dissociation of hormone-protein complexes (17). Hydroxylapatite adsorbs hormone-receptor and hormone-a-fetoprotein complexes, and free hormone is eliminated by an extensive and rapid wash with buffer. Ultracentrifugation through Sucrose Gradient. For analytical studies, samples (0.2-0.3 ml) containing 10 jig of peroxidase, as an internal standard [S = 3.6 (20)], were layered on a 5-20% linear sucrose gradient in TE buffer containing 0.4 M KCI and run for 16-17 hr at 48,000 rpm in an SW 60 rotor (Beckman). Two-drop fractions were collected, and sedimentation coefficients were calculated according to Martin and Ames (21). For preparative studies, samples were layered on a 5-20% linear sucrose gradient in TE buffer and run for 15 hr at 37,000 rpm in an SW 41 rotor. Radioactivity Measurement. Aliquots (0.5 ml) were assayed in 10 ml of Bray's solution and 0.2-ml aliquots were counted in ethanol/toluene (3:10, vol/vol) Omnifluor mixture, with a 3H efficiency of 27% and 30%, respectively.
RESULTS Sedimentation Coefficients of E2-Binding Macromolecules in Cytosol at High Ionic Strength. On the basis of their sedimentation coefficient in low-salt medium, it is easy to dif-
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Biochemistry: Radanyi et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
C',)
0~~~~~~~
g' 5.25]
~~~~~~~~~0
I 5.00
100 5
1
200 Time, min
FIG. 3. Dissociation of the a-fetoprotein-E2 complex as a function of time. B is the concentration of bound E2 expressed in cpm/ml. 0, with TE buffer; 0, with TE/0.4 M KCl buffer.
2.5
5
7.5
S
Fw.. 1. Sucrose gradient ultracentrifugation of [:H]E2-receptor and aY-fetoprotein complexes from immature rat uterine cytosol and [3HJE.-a-fetoprotein complexes from immature rat plasma in highsalt medium. Cytosol (0) (0.3 ml) and plasma (*) (0.2 ml) samples were separately ultracentrifuged at 48,000 rpm for 16 and 17 hr, respectively. In both experiments, S values were calculated according to Martin. and Ames (21).
ferentiate a-fetoprotein from E2 receptor in a cytosol. However, at high ionic strength, the sedimentation coefficients are similar (about 4-5 S). The 4-5S peak can be partially resolved into two peaks, 4.5 and 5.5 S, respectively (Fig. 1).Two sets of data indicated that the 4.5S peak is a-fetoprotein. a-Fetoprotein from plasma of 21- to 24-day old rats has a sedimentation coefficient of 4.5 S (Fig. 1). In presence of E2, a marked decrease in the binding of [3H]E2 was observed in the two peaks, indicating that both binding systems are saturable; however, DES depressed the [3H]E2 binding of the 5.5S peak, more than that of the 4.5S peak (Fig. 2). These ultracentrifugation experiments gave some qualitative indications, but they also illustrated the difficulty in obtaining quantitative results by this method. More precise information may be obtained from binding measurements performed by "differential dissociation" techniques. With the latter, highaffinity ligand macromolecule complexes are selectively
S
FIG. 2. Sucrose gradient ultracentrifugation of cytosol after incubation with [3HJE2 7 nM, alone (0), plus DES 70 nM (0), or plus E2 70 nM (U). Samples (0.3 ml) were layered and the tubes were centrifuged for 16 hr at 48,000 rpm. The small differences in the sedimentation coefficient values observed in the absence or presence of E2 and DES are explainable by the higher amount of free [3H]E2 in the competition experiments.
measured because they dissociate more slowly than the "nonsaturable" and "nonspecific" complexes at 40. Dissociation Rate Constant of the E2-a-Fetoprotein Complexes. In order to work under optimal conditions for measuring binding by differential dissociation techniques, it is necessary to know the dissociation rate constant of the hormone-protein complexes. For a-fetoprotein, the affinity for the hormone is known, but the dissociation rate of the complex is not. This dissociation rate constant (kL1) was measured in the absence or presence of 0.4 M KC1. The measurement of k-1 was performed with plasma of 21-day-old rats, diluted 1:10 in TE or TE/0.4 M KC1 buffer and incubated for 17 hr with 20 nM [3H]E2. At zero time, charcoal/dextran, 1:1 (vol/vol), suspension was added to the incubated plasma. At different times (0-0 min), aliquots were centrifuged at 700 X g for 10 min. The radioactivity of the supernatant represents the a-fetoprotein-E2 complexes. Fig. 3 shows the dissociation of the complexes as a function of time, in the presence or absence of 0.4 M KCL. The half-lives of the complexes at 00 were 160 min with k-1 = 7 X 10-5 sec-1 and 140 min with k.1 = 8 X 10-5 sec-1, respectively. These results are different from those obtained for the E2receptor complex-ti/2 = 10-20 days and k-1 = 7.3 X 10-7 sec-1 (22, 23), and no change in the presence of 0.4 M KCI (24, 25). Comparison of Estrogen Binding to Plasma a-Fetoprotein Measured by Charcoal and Hydroxylapatite Techniques. From the preceding results, one can predict that selective measurement of receptor is possible in a cytosol containing both a-fetoprotein and receptor, if exposure of the extract to the charcoal is performed over a long period of time (17 hr). On the contrary, by a technique such as hydroxylapatite method with a short washing time (30 min), one-measures simultaneously E2 binding to both a-fetoprotein and receptor. It was verified that, with the charcoal technique, the contribution of a-fetoprotein to the high-affinity E2 binding is not measured. Plasma from 21- to 25-day old rats diluted 1:10 with TE/0.4 M KCl buffer was incubated with the hormone and competitors. The highaffinity binding was measured in parallel by the charcoal and the hydroxylapatite techniques. Table 1 shows that no specific binding was detected with the charcoal technique. On the contrary, the hydroxylapatite technique measured the a-fetoprotein-[3H]E2 complex with the expected specificity [already observed with the pure protein (5)] E1 being more potent than E2, and DES not competing. This confirms the prediction based on the difference of the dissociation rate constants for the two protein-E2 complexes. High-Affinity Binding of E2 to Cytosol Proteins. The comparative evaluation of the binding activity in cytosol by the two adsorbent techniques may be a good tool for discriminating between the contribution of the two proteins to total estrogen binding. The charcoal technique gives a measurement of the
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Radanyi et al. Table 1. Binding of estradiol to plasma a-fletoprot~in ithe of competitors: Comparison of hydroxylapatite (HAP) and charcoal methods.
presence
Binding activity, cpm/ml HAP [3H]E2 [:3H]E2 + E2 [3H]E2 + E1 [:PH]E2 + DES
Binding activity, cpm/ml Charcoal* HAP*
%
HAP
0
100
49,700 28,900 26,900 49,300
Table 2. High-affinity binding of estradiol to salt-treated cytosol proteins in the presence of competitors: Comparison of hydroxylapatite (HAP) and charcoal methods.
Residual activity,
Charcoal 0
58
0
54
0
99
binding of the receptor, and the hydroxylapatite technique, the total high-affinity binding. Cytosol containing 0.4 M KCI was incubated with E2 and competitors. Table 2 shows that, as measured by the charcoal technique, the binding affinities with the receptor are in the order E2 DES > El. On the contrary, with the hydroxylapatite technique, the observed order E2 > DES E1 suggests binding of ligands to both receptor and a-fetoprotein. If, as recently published (16), in high-salt medium the receptor dissociates to yield a-fetoprotein, the charcoal measurement would be irtipossible. The dissociated part of the receptor would escape detection after the 17-hr exposure to charcoal. In fact, the binding activity of the cytosol measured by the charcoal technique was found to be the same at low and high ionic strengths, 16,500 and 16,400 cpm/ml, respectively. These results are in agreement with the data previously published, indicating the same dissociation rate constant of the E2-receptor complex from rat at low and high ionic strengths (26). -
Binding Specificity of the Receptor in Absence of a-Fetoprotein at High Ionic Strength. The comparison of the
high-affinity binding specificity obtained by the two techniques in the cytosol at high ionic strength suggests that the receptor does not dissociate to a-fetoprotein in the presence of salt. More direct evidence would be obtained if receptor free of a-fetoprotein and treated by KCI showed the same specificity as the receptor in low-salt medium. To obtain this evidence, lowionic-strength cytosol was run through a sucrose gradient and the fractions corresponding to the top and the heavier side of the "8S" peak were pooled. A tube containing cytosol labeled with radioactive E2 served as a marker for locating the "8S" peak. At low ionic strength, a-fetoprotein migrated in the 4.5S region and the "8S" peak contained the receptor. To the "8S" pool, KCI was added to a final concentration of 0.4 M, and then this was incubated with estrogens. Binding was measured by the hydroxylapatite technique. The data presented in Table 3 show that DES competes strongly with [3H]E2 and that it competes more strongly than E1 (compare also Table 2, charcoal column). These results clearly indicate that a-fetoprotein is not a subunit of the receptor. DISCUSSION
Recently, a new model for the structure of the cytosol estrogen receptor of the immature rat uterus was proposed (16). This model postulated that the "8S" receptor (2), which is dissociated into "4-5S" subunits by salt treatment (0.4 M kCl) (6-8), is mainly if not entirely composed of a-fetoprotein. Evidence for this model was drawn from the following experimental and theoretical considerations. A low-salt-buffered cytosol, containing the "8S" receptor, was treated with an anti-a-fetoprotein immunoadsorbent in order to remove free a-fetoprotein. The nonadsorbed fraction was then exposed to high-salt conditions (0.4 M KCI) in order to dissociate the "8S" receptor into the "4-5S" subunits. The salt-treated cytosol was then divided
2271
Residual activity, %
HAP
Charcoal
100 100 16,400 41,160 [3H]E2 19 13 3,160 [3H]E2+ E2 5,260 74 54 12,200 22,160 [3H]E2 + E1 29 49 [3H]E2 + DES 19,960 4,770 The 29% residual activity observed in presence of DES is due to the relatively low concentration of nonradioactive competitor. Under these conditions, the "nonspecific" binding, higher for DES than for E2, cannot be neglected and explains this value. * The data shown were obtained with two different cytosol preparations.
in two parts. One part was subjected to an anti-IgG immunoadsorbent and served as the control (high-salt control); the concentration of a-fetoprotein left after adsorbtion was 1.84 ,ug/ml. The other part was subjected to an anti-a-fetoprotein immunoadsorbent, and the concentration of a-fetoprotein in the nonadsorbed fraction was 0.14 /ig/ml. The authors were unable to detect any receptor activity in the latter fraction. They concluded that the "4-5S" E2-binding peak obtained by high-salt treatment consisted entirely of a-fetoprotein, and that a-fetoprotein must be present in the "85" receptor peak at low-salt concentrations. They suggested that, under these conditions, a-fetoprotein is not detected by immunological methods because the antigenic determinants of the a-fetoprotein are masked when it is present in an oligomeric form. In fact, the data presented in the above-mentioned paper (16) can be reevaluated as follows. Their "8S" receptor peak (their figure 1, curve A) corresponds to a concentration of binding sites of approximately 0.5 pmol/ml. After incubation with 5 nM [3H]E2, the labeling of this peak is due to the high affinity of the receptor for E2 (KD -10-10 M). Under these conditions, no radioactive a-fetoprotein peak is visible in the "4-5S" region, although, according to the authors, the concentration of afetoprotein in this area is 1.84 ,tg/ml-that is to say, 25 pmol/ ml. From the data of their figure 2, this last result could be due to the competition, by the receptor, with E2 binding to a-fetoprotein. The authors assumed that, after KCI treatment, afetoprotein had been released from the "85" and was present in the "4-5S" peak of their figure 1, curve B. Since "free" afetoprotein has an affinity no more than 5% of that of the "8S" receptor (cited in table 1 of their paper), it is difficult to understand how the transfer of bound [3H1E2 from the "8S" to the "4-5S" region (in which the protein has much lower affinity for the hormone) can give a radioactive peak of approximately the same magnitude. We therefore conclude that the "4-5S" peak of their figure 1 curve B is a-fetoprotein. In consequence, it is not surprising that immunoadsorbtion with anti-a-fetoprotein antibodies resulted in the disappearance of this peak Table 3. Specific binding of radioactive estradiol to 0.4 M KCltreated estradiol receptor obtained from "8S" fraction of immature rat uterine cytosol
[3H]E2 [3H]E2 + E2 [3HJE2 + E1
[3H1E2 + DES
Binding activity,
Residual
cpm/ml
activity, %
6650 810 4100
100 12 62 13
860
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Biochemistry: Radanyi et al.
(curveC). It remains to be explained what happened to the receptor in B and C and the reason for the presence of a "4-5S" a-fetoprotein peak in curve B but not in curve A. We suggest that, for the most part, receptor had not resisted the KCl treatment followed by immunoadsorbtion with anti-IgG as well as with anti-a-fetoprotein because, in both cases, protective proteins can be removed nonspecifically. It is known that estrogen receptor is more fragile in 0.4 M KCI and especially in the absence of E2, the experimental conditions of the reported immunoadsorbtion procedure (16). An important decrease of receptor binding activity would suppress the competitive effect of E2 binding to a-fetoprotein, and therefore it can be understood that the a-fetoprotein peak is absent in curve A but is revealed in curve B. The results presented here show that it is possible to distinguish between a-fetoprotein and receptor present concurrently in immature rat uterine high-salt cytosol by using different approaches. First, this can be obtained on the basis of their sedimentation coefficients, 4.5 and 5.5 S. Both peaks can be saturated with E2. DES competes strongly with E2 for the binding to the 5.5S receptor peak and only slightly for binding to the 4.5S a-fetoprotein peak. Second, precise data concerning the binding specificity of estrogens to the cytosol high-affinity binding proteins can be obtained with differential dissociation methods. Because of the different dissociation rates of the two E2-protein complexes at 00 (receptor-E2, t1/2 = 20 days; afetoprotein-E2, t 1/2 = 140 min), selective measurement of the receptor was achieved by incubating the extract with a charcoal/dextran suspension for 17 hr. The high-affinity binding specificity was E2 - DES > El, which should be expected for receptor specificity. It was observed (26) that salt treatment does not change the concentration of E2 binding sites measured by the charcoal method. Conversely, when receptor and a-fetoprotein were measured simultaneously by the hydroxylapatite technique, the binding specificity was E2 El > DES, suggesting the contribution of both proteins to estrogen binding. Third, when a-fetoprotein is removed by ultracentrifugation, the binding specificity of the receptor, dissociated into subunits -by salt, is typical of the low-salt receptor, with the order E2 DES > E1. The present studies clearly demonstrate that a-fetoprotein is not a component of the receptor. It is difficult to imagine that a-fetoprotein, which binds E2 with high affinity in only two animal species (rat and mouse) and which disappears almost completely in the mature animal, could be a component of the Ea receptor. In any case, attempts to demonstrate binding of a-fetoprotein-E2 complexes to DNA-cellulose (27) or to uterine nuclei (A. M. Kaye, personal communication) were unsuccessful, contrary to corresponding controls with E2-receptor complexes. No a-fetoprotein has been detected in the uterine nuclear fraction (16). It has been observed that the estrogen receptors from somatic cells of all studied species (mammalian, avian,-amphibian) exhibit similar physicochemical properties (28), and they are of major physiological importance in mature animals. A possible role of a-fetoprotein is the protection of the fetus against the circulating steroids coming from the mother. During fetal life in most species, this role may be played by sex-steroid-binding plasma protein (29), and it is just in the rat and in the mouse that this protein has not been found. Note Added in Proof. Unpublished results from B. Attardi and E. Ruoslahti of studies with the mouse uterine cytosol and specific afetoprotein antibody confirm the lack of a-fetoprotein component in KCI-dissociated receptor preparation. The authors thank G. Redeuilh for his help, E. Eigenmann for his active participation in the writing of the manuscript, and F. Trautsch and S. Bellorini for secretarial assistance. This work was partially
Proc. Natl. Acad. Sci. USA 74 (1977) supported by the Centre National de la Recherche Scientifique, the Delegation Generalea la Recherche Scientifique et Technique, and
the Ford Foundation. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1. Michel,G., Jung, I., Baulieu, E. E., Aussel, C. & Uriel, J. (1974) Steroids 24, 437-449. 2. Toft, D., Shyamala, G. & Gorski, J. (1967) Proc. Natl. Acad. Sci. USA 57, 1740-1745. 3. Jensen, E. V., De Sombre, E. R., Hurst, D. J., Kawashima, T. & Jungblut, P. W. (1967) Arch. Anat. Microsc. Morphol. Exp. (Suppl.) 56,547-569. 4. Soloff, M. S., Morrisson, M. J. & Swartz, T. L. (1972) Steroids 20, 597-608. 5. Aussel, C., Uriel, J. & Mercier-Bodard, C. (1973) Biochimie 55, 1431-1437. 6. Jensen, E. V., Suzuki, T., Kawashima, T., Stumpf, W. E., Jungblut, P. W. & De Sombre, E. R. (1968) Proc. Natl. Acad. Sci. USA 59, 632-638. 7. Erdos, T. (1968) Biochem. Biophys. Res. Commun. 32, 338343. 8. Rochefort, H. & Baulieu, E. E. (1968), C. R. Hebd. Seances Acad. Sci. 267, 662-665. 9. Raspe, G., ed. (1971) Advances in the Biosciences (Pergamon Press-Vieweg, Oxford), Vol. 7. 10. Ekka, E. & De Herthog, R. (1976) J. Steroid Biochem. 7,241247. 11. Savu, L., Crepy, O., Guerier, M. A., Nunez, E., Engelmann, F., Benassayag, C. & Jayle, M. F. (1972) FEBS Lett. 22, 113-116. 12. Raynaud, J. P., Mercier-Bodard, C. & Baulieu, E. E. (1971) Steroids 18, 767-787. 13. Baulieu, E. E., Alberga, A. & Jung, I. (1967) C. R. Hebd. Seances Acad. Sci. 265,354-357.
14. Geynet, C., Millet, C., Truong, H. & Baulieu, E. E. (1972) Gy-
necol. Invest. 3,2-29. 15. Nunez, E., Savu, L., Engelmann, F., Benassayag, C., Crepy, 0. & Jayle, M. F. (1971) C. R. Hebd. Seances Acad. Sci. 276, 242-245. 16. Uriel, J., Bouillon, D., Aussel, C. & Dupiers, M. (1976) Proc. Natl. Acad. Sci. USA 73, 1452-1456. 17. Milgrom, E. & Baulieu, E. E. (1969) Biochim. Biophys. Acta 194, 602-605. 18. Erdos, T., Best-Belpomme, M. & Bessada, R. (1970) Anal. Biochem. 37,244-252. 19. Truong, H., Geynet, C., Millet, C., Soulignac, O., Bucourt, R., Vignau, M., Torelli, V. & Baulieu, E. E. (1973) FEBS Lett. 35, 289-294. 20. Shannon, L. M., Kay, E. & Lew, J. Y. (1966) J. Biol. Chem. 241, 2166-2172. 21. Martin, R. G. & Ames, B. N. (1962) J. Biol. Chem. 236, 13721379. 22. Best-Belpomme, M., Fries, J. & Erdos, T. (1970) Eur. J. Biochem. 17,425, 432. 23. Geynet, C., Millet, C., Truong, H. & Baulieu, E. E. (1972) C. R. Acad. Sci. Paris, 275, 1551-1554. 24. Soulignac, 0. (1974) These de doctorat en M6decine, Universite de Paris XI. 25. Salat-Trepat, J. & Reti, E. (1974) Biochim. Biophys. Acta 338, 92-103. 26. Rochefort, H. & Baulieu, E. E. (1971) Biochimie 53,893-907. 27. Fox, T. 0. (1975) Nature 258,441-444. 28. Baulieu, E. E., Atger, M., Best-Belpomme, M., Corvol, P.,
Courvalin, J. C., Mester, J., Milgrom, E., Robel, P., Rochefort, H. & de Catalogne, D. (1975) Vitam. Horm. (NY) 33, 649731. 29. Mercier-Bodard, C., Renoir, J . & Baulieu, E. E. (1976) Symposium au V Congre's International d'Endocrinologie, Ham-
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