THE JOURNAL OF BIOLOQICAL CHE~~WTRY Vol.249, No. 5, Issue of March IO, pp. 1815-1636, 1974

Printed in U.S.A.

The Binding of Estradiol-17@ to the Bovine Endometrial

Nuclear Membrane*

(Received for publication, November 27, 1972, and in revised form, June 18, 1973)

VAUGHN JACKSON AND ROGER CHALKLEY~

From the Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242

SUMMARY

The interaction of estradiol-1’7/3 with mature bovine endo- metrial tissue, and with isolated nuclei has been studied. The hormone binds to an insoluble nuclear fraction. This fraction contains membrane and evidence is presented to show that estradiol is bound to the nuclear membrane. Incu- bation of isolated nuclei and microsome fractions with estra- dial-17@ shows that the hormone binds essentially instan- taneously to microsomes and nuclei. Such binding is non- saturable up to estradiol-170 concentrations as great as 2.5 x 10m6 moles per mg of membrane protein and it is likely that this interaction is not biologically significant. A second form of binding is observed in nuclei which is of higher affinity and saturable, with 507 •t 47 sites per nucleus. This class of binding sites is found to be blocked in cattle that are main- tained in artificially induced estrus by feeding with diethyl- stilbesterol.

The high affinity binding site is present only in the mature endometrium and is absent in nuclei from the mature myo- metrium (the muscular tissue surrounding the endometrium in the uterus) and the immature uterus. Potent estrogenic agents compete effectively with estradiol-170 for binding to this site, whereas weak estrogenic steroids (such as proges- terone and testosterone) are inefficient competitors. The sensitivity of the high affinity site to pH and hydrolytic en- zymes has been studied and compared with the effect of such agents on the low affinity, nonspecific, membrane site. Such a comparison underscores the inherent differences between these two sites of interaction. The thermodynamic param- eters of the high affinity binding site have been measured and compared to reported values for the specific cytoplasmic receptor binding site. The nuclear high affinity binding site possesses a favorable free energy of interaction as a result of entropic forces (AS0 = +36.4 cal deg-l mole-‘, AH0 = - 0.66 Cal mole-‘). The number of high affinity nuclear sites varies dramatically during the estrous cycle in a bicyclic fashion. Sites are available during estrus and diestrus, whereas they are blocked in later metestrus and proestrus.

* This work was supported by United States Public Health Service Grant CA-10871 and by the American Cancer Society (Iowa Division) Grant P451.

$ Research Career Development Awardee of the National Insti- tutes of Health, Grant GM-48410.

Estradiol-17/3 causes cell proliferation in endometrial tissue approximately 48 hours after blood levels of the steroid rise during the estrous cycle (1). Prior to cell division there is an initial increase in ribosomal RNA synthesis (2) and 24 hours later an increase in DNA synthesis (3). It has been proposed that the hormone itself mediates at least part of this response by interacting directly with the genome (4-6). Following injection of the hormone in the rat, or direct incubation of the uterus with the hormone, a portion of the estradiol becomes associated with the uterine nuclear fraction in a specific manner (7-9). Although the nuclear fraction has not been analyzed in great detail, some evidence has been presented that estradiol binds to the chromatin fraction (7). We will present data which support the proposal that in vivo, estradiol binds to the nuclear membrane, which ap- pears to be an integral part of chromatin as isolated. During in vitro incubation using isolated nuclei, the hormone binds nuclei both at a nonsaturable low affinity site (in a rapid reaction, which resembles its binding to isolated microsomal material) and also at a saturable high affinity site (with a measurable rate) which we propose is the same site as that involved in incubations with in- tact tissue.

We also report on the thermodynamic parameters of hormone binding and on the sensitivity of the high affinity site to various hydrolytic enzymes and to pH. In several of these properties, the high affinity sites on the nucleus are clearly different from the cytoplasmic receptors and we will argue that the high affinity nuclear binding site which is present in endometrial tissue is a discrete entity in itself and is distinct and separate from the cytoplasmic receptor site.

Finally, we have found that the availability of the nuclear high affinity sites varies during the estrous cycle, though this variation is not reflected in the concentration of cytoplasmic receptor in the cell, which remains relatively constant.

MATERIALS AND METHODS

Animals used--Young Hereford heifers (18 to 22 months old) were used in thcsc studies. C arc wa.s taken to ensure that these animals had not been fed fattening agents and that their estrous cycle was normal as monitored by vaginal smears. On occasion heifers which were fed grain containing diethylstilbesterol (5 mg per pound of protein supplement per day) were utilized.

Isolation oj Nuclei and Preparation of Nucleohistonel-Endo-

1 The nomenclature used in this paper is that common in the chromosomal nucleoprotein field. Chromatin refers to the gel- like high molecular weight product obtained by lysis of nuclei and subsequent removal of divalent cations. Nucleohistone is

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metrial tissue was removed from the uterus at 4” and incubated in Eagle’s medium (10) (minus vitamins and amino acids) under constant aeration with 95y0 02-5% COZ at 37” for 45 min. L3H]Es- tradiol-17/3 (40 Ci per mmole, New England Nuclear) was present at a concentration of 1.5 x 10-E g/100 ml. The tissue (~20 g) was then removed and washed five times with Eagle’s medium, twice with distilled water, and blended in 20 ml of 0.25 M sucrose- Buffer A (0.1 M NaCl, 5 m&l KCl, 3 rnbr MgClp, 1.5 mM CaCl,, 10 mM Tris, pH 7.0) using a Waring Blendor. The solution was filtered successively through double layers of cheesecloth and Miracloth (Chicopee Mills, Inc.) and centrifuged at 480 x g for 10 min. The nuclear pellet was washed twice in Buffer A by dispersing with a Dounce homogenizer (10 strokes) and centri- fuged at 480 x g for 10 min. The nuclear pellet was rcsus- pended in 2.4 M sucrose-Buffer A, layered on 2.4 M sucrose-Buffer A, and centrifuged at 75,000 x g for 3 hours at 4”. The resulting nuclear pellet was homogenized in Buffer A for nuclear incuba- tions. To make nucleohistone the nuclear pellet was homog- enized in 5 mM EDTA-10 mM Tris-Cl, pH 7.4 and centrifuged at 12,000 x g for 10 min. The pellet was washed twice by homog- enization in distilled water and centrifuged at 27,000 x g for 20 min. The viscous chromatin gel was sheared violently in 10-s bursts at l-mm intervals over a lo-min period in a VirTis model 45 (70 volts) at 2”. When nuclei are isolated for incubation with [3H]estradiol, the tissue should be frozen prior to homogenization as this greatly minimizes the extent of nuclear contamination by microsomes.

Sucrose Gradient Analysis of Nucleohistone-The nucleohistone solution was adjusted to 10 mM Tris, pH 7.4 and layered (0.2 ml) on a sucrose gradient (10 to 30%) in 5 mM Tris-Cl, and centri- fuged at 140,000 x g for 3 hours at 4” in a Beckman SW 50 rotor. Fractions were collected (0.1 ml), diluted, and assayed for nu- cleohistone by absorbance at 260 nm and for the presence of [3H]estradiol-17fi by counting in Bray’s solution (11) using a Nuclear Chicago Unilux III scintillation counter. Efficiency was 22%.

Isolation oj 2lRembrane Fractions for Enzymatic Assays and Determinafion of Estradiol-I 70 Binding-Cell membranes were isolated and purified using a modification of the procedure de- scribed by Moore et al. (12) and Berman et al. (13), employing discontinuous sucrose gradients. The membrane fractions were suspended in Buffer A for the enzymatic assays and determina- tion of [aH]estradiol-17fi binding. The marker enzymes used to monitor the extent of cross contamination between membrane fractions were: glucose-6-phosphatase (14) and DPNH cyto- chrome c reductase (15) for microsomes; cytochrome c oxidase (16) for mitochondria; and 5’-mononucleotidase (17) for microso- ma1 and plasma membrane contamination. Membrane systems were also identified by electron microscopy.

Chemical Analysis-Membrane was measured for protein content using the method of Lowry (18) with bovine serum al- bumin as standard. DNA was measured by the Burton di- phenylamine method (19) using calf thymus DNA as standard. Lipid content was estimated by extraction of nuclear membrane fractions with chloroform-methanol (2: 1) according to the method of Bligh and Dyer (20), evaporating the solution to dryness and weighing the residue. Phosphate determinations utilized the procedure of Rouser (21). Thin layer chromatog-

obtained by vigorously shearing chromatin at low ionic strength and clarifying by centrifugation. The supernatant nucleohistone is rich in DNA and histone (DNA-histone-nonhistone protein, 1:l: -0.2). The content of nonhistone protein is highly variable and depends upon tissue of origin.

raphy of polar lipids used the solvent system chloroform-metha- nol-ammonium hydroxide-water (75 : 30 :4 : 0.5) with Silica Gel G as the stationary phase.

Preparation of Cytosol Fraction and Charcoal-dextran Assay of [3H]Estradio2-17’@ Binding-Cytosol fractions were prepared by centrifuging the homogenate of endometrial &sue at 130,000 x g for 1 hour at 4’. The extent of hormone binding was deter- mined by the procedure described by Baulieu (22).

Assay of Endometrial Nuclei and Microsome Binding of [3H]- Esiradiol-1 ?‘+Microsomes were prepared from the supernatant after centrifugation of the tissue homogenate at 7700 x g for 10 min. Microsomes were then collect.ed by centrifugation at 27,000 x g for 20 min. This peliet was washed once in Buffer A prior to use.

Nuclei and microsomes were suspended in Buffer A at a con- centration of 3.0 mg of DNA per ml for nuclei and 5.0 mg of membrane protein per ml for microsomes. Aliquots (0.6 ml) were incubated in a tube containing the solid steroid which had been added previously in ethanol. The ethanol was removed under vacuum. When competing steroids were present, their concentrations were determined gravimctrically. In the com- petitive binding studies both [%]estradiol and the competing steroid were added to the test tube prior to addition of the nuclei or microsomal solution. At the concentration used, the steroids go into solution rapidly. After incubation at 4” for specified times an aliquot (0.2 ml) was taken both before and after centrifugation at 10,000 X g for 1 min to isolate nuclei and at 27,000 X g for 5 min to yield the microsomal fraction. The difference in the radioactivity of the two samples is a direct measure of the amount of hormone bound which is independent of adventitious binding of hormone to the centrifuge tube. All samples were counted in Bray’s solution.

Electron Microscopy of Nuclei and Nuclear Membrane-The nuclei and nuclear membrane preparations were fixed as a pellet in 1.0% osmic acid-Buffer A for 5 hours at 4”. Dehydration was with acetone and embedding with Vestopal W (Polysciences, Inc.). The fractions were examined in an Hitachi HU-125E electron microscope. Staining was with uranyl acetate and lead citrate (23).

Sensitivity of High Afinity Nuclear Binding Site to pH and Hydrolytic Enzymes-Nuclei and microsomes were incubated with [3H]estradiol-17/3 at 4” for 6 hours and then centrifuged at 480 x g for 10 min to isolate nuclei and at 27,000 x g for 10 min for microsomes. The pellets were suspended at 4” in the appropriate environment under investigation, and incubated at 4” for 2 hours. The solutions were centrifuged at 27,000 x g for 5 min and 0.2-ml aliquots taken from the supernatants for determination of the amount of hormone bound.

Studies of Number of Nuclear Binding Sites in Uterus during Estrous Cycle-The binding analysis was done as described above. The determination of the stage of the uterus in the reproductive cycle was based upon physiological and histological criteria (24, 25). The cow reproductive system was analyzed in the following ways: (a) the condition of the ovaries was examined for follicular development and (after estrus) for development of the corpus luteum; (b) the endometrial tissue was examined for hyperemia, heavy vasculariza,tion, and subsequent proliferation to define the beginning of proestrus and entrance into estrus; (c) the stages of mucous development were recorded. The mucous changes from a slightly viscous, transparent fluid in estrus to a heavily viscous, orange-colored fluid in late metestrus; (d) vaginal smears were also collected to assay for the charac- teristic cycling of lymphocytes, epithelial cells and keratinization.

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RESULTS TABLE II

Estradiol Binding to Endowletrial Tissue

Endometrial tissue was incubated at 37” for 45 min in the presence of [3H]estradiol-17P and chilled to 4”. After homoge- nization in Buffer A it was fract.ionatcd by differential centrifuga- tion. As shown in Table I, -357, of the t,otal estradiol in the tissue was associated with the nuclear material. The amount of free hormone in the tissue is quite large and an assessment of free hormone both within the cell itself and Gthin the extra- cellular matrix is described in a later paper (26).

Various cellular membrane fractions were isolated from endo- metrial tissue labeled with [3H]estradiol and the specific activity of binding (moles of cstradiol bound per mg of membrane protein) determined for each fraction as shown in Table II. The specific activity of binding for the nuclear membrane is 7-fold higher than that observed for the other cell membranes. Extraction with organic solvents and a subsequent analysis by thin layer chromatography indicated that the hormone bound to all mem- brane fractions (including the nuclear membrane) was not metabolized (data not shown) in agreement with previous reports (27).

Binding of estradiol-l7p to cellular membranes The membrane fractions were isolated from 20 g of endometrial

tissue previously incubated with 5.5 X lo-l0 M [3H]estradiol-17p at 37” for 60 min. Fractionation of the cellular membranes was as described under “Materials and Methods.”

Plasma membrane. ................... Mitochondria. .......... ........ Light microsomes. ...................... Heavy microsomes. ..................... Rough endoplasmic reticulum. ........... Nuclear membrane. .....................

KC1 Extractability of Estradiol Bound to Nuclei from Mature Endometrium

BSA

I

Several previous studies, particularly those concerning the binding between the nucleus and the cytoplasmic estradiol- receptor complex in vitro, have utilized the observation that the resulting complex can be disrupted with 0.4 M KC1 with an efficiency of -50% (5). We therefore tested the estradiol- labeled nuclei isolated from a whole tissue incubation by ex- tracting with KCI. Very little estradiol (-6y0) is released by this treatment, though if the cytoplasmic estradiol-receptor- nuclear complex is constructed in u&o, we find that we can indeed release 50y0 of the bound estradiol by extraction with KC1 solutions (Fig. 1).

TOP 20 10 BOTTOM

FRACTION NUMBER

Identification oj Nuclear Binding Site in Mature Endometrium

Isolation of Subnuclear Fraction which Binds Estradiol- Chromatin was prepared from labeled tissue as described under “Materials and Methods.” It was disrupted by shearing to

FIG. 1. Extraction of [3H]estradiol-nuclear complex from ma- ture endometrium with KCl. .Nuclei from an incubation of tissue in the presence of [3H]estradiol were prepared and extracted with 0.4 M KC1 as described under “Materials and Methods.” The extract was analyzed on a 5 to 20yc sucrose density gradient (0-O). The estradiol-receptor-nuclear complex was pro- duced in vitro as described in a later paper (26), extracted in 0.4 M KCl, and analyzed on sucrose density gradients (O--O ).

TABLE I Subcellular fractionation of endometrial

tissue labeled with [SH]estradiol Twenty grams of endometrial tissue was incubated with 5.5 X

lO-‘o~ [3H]estradiol at 37” for 45 min. The tissnewas homogenized in 20 ml of Buffer A, and a subcellular fractionation perforined by differential eentrifugation. Centrifugation was at 480 X g/10 min (nuclei) ; 7700 X g/10 min (fragmented chromosomal material); 27,000 X g/20 min (mitochondria and heavy microsomes); and 130,000 X g/60 min (light microsomes).

yield nucleohistone and subsequently analyzed on a sucrose gradient as shown in Fig. 2. The hormone is almost entirely associated with an insoluble pellet. Estradiol is not bound to nucleohistone, the major portion (95%) of which is located within the gradient. Throughout all these operations the temperature was maintained at 2”, and it is reasonable to expect that the hormone would presumably still bc associated to the same binding site as it was prior to the &taring process. This is supported by the observation that the dissociation rate at 4” of the hormone from the nuclear binding site is negligible.

Per cent bound

x 104

Total homogenate. 603 Intact nuclei........... 158 26.2 Fragmented chromosomal material. 66 11.0 Mitochondria and heavy microsomes. 55 9.1 Lightmicrosomes......... 68 11.3 Hormone bound to cytoplasmic receptor. 116 19.3 Free hormone.............................. 140 23.1

Chemical Composition of Subnuclear Fractions-Chemical analyses of the pelleted fraction and the nucleohistone from within the gradient are shown in Table III. The nucleohistone has a T, N 75” in 5 X 1OW M Tris, pH 7.5 and possesses the normal complement of histone. There is no indication that significant amounts of any histone fraction had become separated from the nucleohistone. The pellet fraction associated with estradiol contains approximately equal weights of lipid and protein and only a small fraction of the input DNA applied to the gradient (-57,). The nature of the polar lipids is shown in Table IV along with the lipid composition of other cellular membranes. It is immediately apparent that its lipid content is characteristic of membrane in general. There is a strong

Moles of [W&radio1 per mg of membrane protein

x 10-1s

1.87 4.27 1.48 1.83 1.86

14.0

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20 10 TOP BOTTOM

FRACTION NUMBER

FIG. 2. The fractionation of sheared [3H]estradiolkhromatin. Chromatin containing bound estradiol was sheared and applied to a 10 to 30yc sucrose gradient in 5 mM Tris, pH 7.4 and centri- fuged 140,000 X g for 3 hours.

TABLK

Chemical composition of nucleohisto?~e

and membrane fraction

Endometrial chromatin was separated into nucleohistone and membrane fractions on sucrose gradients and then assayed for lipid, protein, and DNA content as described under “Materials and Methods.”

Nuclear membrane. 0.04 0.03

Nucleohistone

mg 0.05 0.10

I I 0.03

1.0 1 0.12 0.95

TABLE IV

Phospholipid content of several cell membrane systems a?kd calf endometrial

nuclear membrane The thin layer chromat.ography and assay for phosphate are

described under “Materials and Methods.” The data are ex- pressed in terms of mole per cent phosphate.

Sphingomyelin. Phosphatidylcholine

Phosphatidylethanola- mine.

Phosphatidylserine Phosphatidylinositol Phosphatidic acid. Lysophosphatidylcholine Lysophosphatidyl-

ethanolamine Unidentified. Lysoinositol Cardiolipin

u N.D., not detected.

I- /(

--

Mole per cent phosphate

Mito- :hondria

(28)

40.0

28.4

7.0

2.3

22.5

Plasma W)

23.1

30.6

11.4 4.2 5.4

11.1 5.6

1.9

Endo- 3lasmic :ticulun

(30)

9.8 44.6

15.2 7.8 8.0 1.6

3.8

1

_-

-

Endo- metrial nuclear mem- brane (calf)

2.4 5.0 58.2 54.7

25.9 16.6 4.4 N.D. 8.9 8.2 1.0 N.D. 1.0 13.3

N.D.a

N.D. N.D.

N.D. 2.2

N.D. N.D.

similarity to the lipid composition of pig nuclear membrane (31) reflected in the high content of phosphatidyl choline, but nonetheless significant differences are observed in phosphatidyl serine and lysophosphatidyl choline. The nuclear membrane fragments have essentially the same phospholipid content as that recently reported for rat liver nuclei (32).

Microscopic Study of Nuclear Fraction-The estradiol-binding fraction was examined under the electron microscope. A trilaminar structure characteristic of membrane can be seen as shown in Fig. 30. When the nuclei are examined under the eiectron microscope prior to the shearing and sucrose gradient centrifugation, both the inner and outer nuclear membrane are intact as shown in Fig. 3, A and B. The nuclei are quite pure, but not totally free of cytoplasmic membraneous material.

Extent of Contamination with Other Cellular Membranes-The membrane fraction obtained as described above was assayed for contamination with other cellular membrane fractions using assays for enzymes generally thought to be characteristic of a given membrane system. The data in Table V indicate that while there is no detectable contamination with mitochondria, there is indeed a low level (-1501,) contamination of the nuclear membrane material with other membrane fractions which is primarily microsomal in nature.

In Vitro Studies of Estradiol-17fi Binding to Nuclei and Microsomes from Mature Endometrium

Kinetics of Binding-Nuclei and microsomes were isolated as described under ‘%aterials and Methods” and then incubated with free [3H]estradiol-17fl to determine the rate of hormone binding at 4”. The data are shown in Fig. 4. The single association rate for microsomal binding is so rapid that it is not measurable by these techniques; on the other hand, the nuclear binding shows two distinct association rates, one which is instantaneous and a second which is much slower and meas- urable. The second-order rate constant for the slower binding is 1.94 f 0.23 X lo5 SC M-I if we assume it is independent of the faster binding and that the contribution of the latter to the total binding can therefore be subtracted.

III order to gain an insight into the physiology of the two observed rates we utilized endometrial tissue in which we ex- pected to find the bulk of the active sites blocked, namely in cattle fed a diet containing a continuous supply of diethylstil- besterol which is used as a fattening agent. The endometrial tissue from these animals was in the semiestrus state, charac- teristic of a uterus heavily treated with estrogen. The nuclei were isolated from this tissue and the rate of association of estradiol-17/3 studied at 4” with the results shown in Fig. 4. Only the instantaneous binding mode was observed in t.he nuclei from such treated cattle. The microsomes from this tissue also bound estradiol-17/3 instantaneously as before and to the same extent.

Equilibrium Studies-Nuclei from endometrial tissue treated with diethylstilbesterol in vivo bind the [3H]estradiol in a similar mamler to normal microsomes (not diethylstilbesterol treated) though to a different extent as is shown in Fig. 5. In both cases the binding is nonsaturable and linear up to estradiol coucentrations of 2.5 x low6 moles per mg of protein and there- fore an equilibrium constant could not be calculated. The final estradiol concentration used is 10’ times greater than that ob- served physiologically and we suspect we are simply observing a nonspecific binding of estradiol to membranous material. On the other hand, nuclei from endometrial tissue in diestrus show two distinct forms of binding. One binding form is nonsaturable

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TMLE V

Enzymatic assay jor nuclear contamination by cell membranes

The methods for isolation of the membrane fractions and for enzymatic activity are as described under “Materials and Methods.”

Glucose 6.phosphatase (microsomal)

DPNH cytochrome c-reductase

(microsomal)

5’.Nucleotidase (plasma membrane and microsomal)

Cytochrome oxidase (mitochondrial)

Plasma membrane. Mitochondria.

Light microsomes . Heavy microsomes.. Rough endoplasmic reticulum..

Nuclear membrane. . . . .

pmoles PCk/hr/mg pmoles reduced/min/mg x 10-2 pm&s POa/hr/mg pm&s ozidized/min/mg x 10-n

84 7.2 39.2 1.f.x

50 14.3 14.5 7.03

388 9.5 102.0 0.73

83 22.2 80.5 4.08

32 8.5 14.7 3.13

15 4.1 3.0 N.1j.o

a ND., not detected.

I J 2 4 6 8 10

TIME (HOURS)

1 2 3

TIME (HOURS)

FIG. 4. The rate of [%]estradiol binding to endometrial nu- clei. A, binding as a function of time was studied using nuclei (O-O), endometrial microsomes (O--D), and nuclei from

E+ log sR = K,t 0 0

endometrial tissue taken from cattle fed diethylstilbesterol where E refers to [3H]estradiol concentration, R to the concen- (O-O). B, the second order rate constant for the binding of tration of high affinity sites as determined from the Scatchard [3H]estradiol to the high affinity site of nuclei was determined plot (34) of Fig. 5C. The concentration of sites was 1.05 nM and from the following equation (33): the estradiol concentration was 2.53 nM.

TOTAL DPM/MG MEMB. PROT. b1D4 ) CORRECTED TOTAL DPMIMG MEMB. PROT.

(x1$)

FIG. 5. The in vitro binding of [3H]estradiol to microsomes and nuclei from mature endometrium. A, binding was with micro- somes from tissue in diestrus (O-O), nuclei from tissue in diestrus (O-O ), nuclei from tissue of cattle fed diethylstilbes- terol (O-O), and nuclei from tissue in diestrus and treated with lOOO-fold excess diethylstilbesterol at 4O for 10 hours prior to addi- tion of [3H]estradiol (+m). B, saturation curve for nuclei from endometrial tissue in diestrus. The curve is generated by

BOUND ( x IOe’oMO~~~ )

correcting for the nonsaturable binding to nuclei which is given by the linear equation, y = 0.15 x as calculated from A using nuclei treated with lOOO-fold excess diethylstilbesterol prior to [YH]estra- diol addition. The amount bound nonspecifically to diethylstil- besterol-treated nuclei at any given concentration is subtracted from total bound hormone. C, the Scatchard plot (34) is gen- erated from the saturation curve of H in order to determine the equilibrium constant and number of binding sites.

and of low affinity and the other is of high affinity and fully level observed in diestrus nuclei. The saturation curve so saturable. It is possible to estimate the equilibrium constant obtained is shown in Fig. 5B. The dissociation constant (Fig. for the high affinity sites by subtracting the level of e&radio1 5C) is 1.33 •t 0.26 x 1OP M and the level of maximum binding bound to the nonspecific weaker binding site (as measured in is 10.5 f 0.9 x lo+ moles per mg of membrane protein. Since nuclei pretreated with excess diethylstilbesterol) from the total we find that there are 0.13 =t 0.02 mg of nuclear membrane

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protein per mg of DNA and by assuming 6.0 x 1OP2 g of DKA per nucleus (35), we calculate that there are 507 f 45 sites for estradiol-17fi binding per nucleus.

The low affinity sites are those with the rapid kinetics of binding, as is shown in Fig. 6 in which we see that a graph of the zero time binding as a function of input estradiol concentration is linear and evidences no saturation. The development of the characteristic saturation curve for estradiol binding to diestrus nuclei requires sufficient time at each point to permit the higher affinity interaction to reach equilibrium. The zero time binding curve for normal nuclei closely parallels the binding curve to diethylstilbesterol nuclei and we conclude (a) that binding to the low affinity occurs essentially instantaneously; (6) that binding to the high affinity site occurs at a finite and measurable

20 40 64 so 00

TOTAL DPM/MG MEMB. PROT. (x,04)

FIG. 6. The low affinity binding of mature endometrial nuclei in diestrus. The nuclear suspension was incubated with [3H]- estradiol for 2 min with 0.2 ml taken before and after centrifuga- tion. Nuclei from diestrus tissue (0-O ) and from tissue taken from cattle fed on diethylstilbesterol (0-O).

A Ail G 2 100

!J n 5 80

u 0 5 60

R 5 40

x Q F z 20

ZE a9

-15 -13 -11 -9 -7

1621

rate; and (c) that the use of nuclei from diethylstilbesterol treated cows provides a valid measure of the cxtcnt of low affinity binding needed for the development of saturation curves of the type shown in Fig. 5B.

SpeciJicity of High Afinity Nuclear Binding Site

Competitive Inhibition of Binding by Other Steroids-Competi- tion for the high affinity binding site of isolated nuclei was studied using steroids of differing estrogenic potency. The extent of competition was assayed by measuring the decrease in the

amount of [aH]estradiol bound to the high affinity site in the presence of varying amounts of the competitor. The results of control studies shown in Fig. 7C indicate that the competitors do not interfere with the low affinity binding of estradiol-17@ in any way. This result is consistent with the previous observa- tion that the nonspecific binding appears to be nonsaturable.

Nuclei from mature endometrial tissue in diestrus were incu- bated with [3H]estradiol-17/3 at a concentration sufficient to reach approximately 50% saturation. Increasing concentrations of competitor steroids were also added at the same time as the labeled estradioL17P and the subsequent effect of the competitor on the amount of estradiol bound to the high affinity site was measured directly. The results are shown in Fig. 7, A and B.

The potent estrogcnic compounds, diethylstilbesterol and estradiol-17fi at low concentrations are efficient competitors for the high affinity site. Estradiol-17Lu, estrone, and estriol must be present at a lo-fold higher concentration to cause 50% inhibition. 1 lol-Hydroxyprogesterone and hydrocortisone show no significant, inhibition of binding of estradiol-178 up to a con- centration which is four orders of magnitude greater than that of the labeled hormone.

Inhibition was studied at different concentrations of com- pctitor and [aH]estradiol-17P so that the equilibrium constants between the competitors and the high affinity binding site could be determined. Double reciprocal plots of such binding studies

C

log&OLES STEROIDIMG MEMB. PROT. i loglOiMOLES STEROID/MG MEMB. PROT) log&OLES STEROID/MG MEMB.

FIG. 7. Studies on the inhibition by estrogenic and nonestro- petitors were progesterone (A-A), lla-hydroxyprogesterone genie compounds of [3H]estradiol-17fl binding to the nuclear high (O---U), hydrocortisone (O-O), and testosterone (0-O). affinity site. A, competition studies between [3H]estrsdiol-170 C, competitive studies between [3H]estradiol-17p and estrogcnic and estrogenic compounds using nuclei isolated from endometrial compounds using nuclei from endometrial tissue taken from cattle tissue in diestrus. Competitors were: unlabeled estradiolL17fl fed on diethylstilbesterol (high affinity site either absent or (O-O ), diethylstilbesterol (A---A), estradiol-17ol (*--*), blocked). Competitors were: unlabeled estradiol-170 (O--O ), estrone (O-O), and estriol (0-U). B, competitive studies diethylstilbesterol (A---A), estradiol-17a (*-*), estrone between [3H]estradiol-17p and nonestrogenic steroids. Com- (O--O), and estriol (O----O).

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TABLE VI

Competitive itlhibition of estradiol-1’7p bixdi,lg to nuclear high ufinity site

Inhibitor constants (K,) for estrogenic competitors were deter- mined from the double reciprocal plots of Fig. 8. The data of Fig. 8 were analyzed in a Lint 8 computer programmed to calculate Kf assuming competitive inhibition and also to obtain t.he stand- ard estimated error. The mathematical program was that of Cleland (36). The higher concentration of diethylstilbesterol used for inhibition (8.62 X lo-l2 moles per mg) which does not intercept on the ordinate with the other lines was not included in the calculation for diet.hylstilbesterol inhibition. Effectiveness as inhibit.or is calculated from the following formula:

1.0 2.0 3.0 4.0

KI (Estradiol) Kr (Inhibitor)

X 100 = y0 Effectiveness (xl;’ MOLESIMG.) ,A,,?;%

-

[~HlEstradiol- 17i3

Standard estimated

errm

Effectiveness as inhibitor 1 Inhibitor

KI (X 10-S M)

5.72 25.9

4.74 67.9 72.5

Kep (x 10-s M) X10-9.W

4.18 0.0255 3.91 0.0231

7.09 4.29 4.54

0.0259 0.0238 0.0254

Estradiol-17p lSstradiol-17ol. Diethylstilbes-

terol Estrone J&trio1

-

8.0 - /'

2 4 6 8 10 12

P”

FIG. 9. The sensitivity of estradiol binding sites to pH. Nuclei (O--O) and microsomes (O-O) were labeled wit.h [3H]- estradiol, centrifuged and resuspended at the appropriate pH, and then incubated at 4” for 2 hours. For the pH range 3 to 7, 0.02 M sodium phosphate-O.01 M sodium citrate-Buffer A was used. For the ~1% range 7 to 11, Buffer A was employed. The ordinate is expressed as the per cent of the hormone found in the snper- natant after the nuclei and microsomes were removed by sedi- mentation. The dotted line (- - -) is the binding observed by Puca and Bresciani (37) for the calf endometrium cytoplasmic receptor.

are shown in Fig. 8. The curves in all cases are made to intercept on the ordinate, thereby assuming t,hat the inhibition of estradiol binding is competitive. The calculated dissociation constants along with standard errors are presented in Table VI. Since the standard estimated error is very low, it would appear t,hat the assumption that competitive inhibition is occurring is a valid one.

Sensitivity oj High Afinity Nuclear Sites to Chemical Environ- ment-The high affiility nuclear site is critically affected by changes in pH as is documented in Fig. 9, in cont.rast to the microsomal binding which is much less sensitive to extremes of

1/( DPM/MG. MEMB. PROT.) (~16~)

FIG. 8. Double reciprocal plots of the competitive inhibition of binding between [3H]estradiol-170 and estrogenic compounds. The binding of increasing concentrations of [3H]estradiol to nuclei was measured in the presence of different amounts of competitor. Each line is a measure of [“Hlestradiol binding as a function of concentration at a specific constant concentration of inhibitor. The concentration of inhibitor is marked directly on the curves and can be comput,ed by multiplying the number on the line by the constant factor written immediately under the name of the inhibitor.

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PI-I. A clear distinction between the nuclear and cytoplasmic receptors emerges when comparing these data with the pH sensitivity profile for cytoplasmic receptor binding as determined by Puca and Bresciani (37) as is seen in Fig. 9.

Neither the microsomal nor the nuclear binding is affected by extensive incubation with DNase or RNase (data not shown). However, there is a considerable difference ill their sensitivity to proteolytic digestion as shown in Fig. 10. Essentially all the estrogen is rapidly released from the nuclear high affinity

site, whereas less than 20y0 of the hormone is released from the

microsomal material, even upon prolonged incubation.

Thermodynamic Characterization of Nuclear High Afinity Binding Site-The low affinity binding sites (both microsomal and nuclear) were assayed for changes in binding capacity as a function of temperature. No change was observed, and we

conclude that any variation in binding as a function of tem- perature is due to changes in the high affinity site. Nuclei were

labclcd with estradiol-17/3 at 36” for 17 min; 23” for 60 min;

12” for 3 hours; and 4” for 6 hours. The time at these tempera-

30 60 90 1920

~IME(MINUTES)

FIG. 10. The release of estradiol from its binding sites as a result of pronase treatment. Nuclei (O--O) and microsomes (O--O ) were labeled with [3H]estradiol, centrifuged, and re- suspended in fresh Buffer A containing pronase at a concentration of 40 pg per mg of DNA for nuclei or 40 pg per mg of protein for microsomes.

TABLE VII

Thermodynamic parameters of estradiol-17p binding

The binding studies were performed as described under “Ma- terials and Methods.” The enthalpy was calculated from the

Van’t Hoff plot as described in the text. The free energy calcula- tion assumes one receptor site binds one estradiol molecule.

Temperature Dissociation constant No. of binding sites

I x 10-o M x lo-‘2 moles/mg membrane $rotein

4” 3.09 zt 0.40 1.02 & 0.05 12 3.58 zk 0.41 0.98 ~6 0.06 23 3.51 zt 0.88 0.92 zt 0.11

36 3.40 It 1.50 0.70 + 0.20

Comparalise aala

Thermodynamic parameters Nuclei high affinity binding site

Cytosol receptor binding site (38)

tinthalpy (AH”)

Free (AG’& energy Entropy (As”)

-0.66 f 0.19 -18

-11.33 dz 0.14 -14

+36.4 -12

tures is sufficient to permit the reaction to reach equilibrium. Incubation was not continued for extended time periods at the higher temperatures to minimize degradation. The equilibrium constant at each temperature was calculated and plotted against the reciprocal of temperature.

AH0 was calculated from the slope of this line and AS0 com- puted from the Gibbs-Helmholtz equation. The various thermo- dynamic parameters are presented in Table VII. The thermo- dynamic parameters for the cytoplasmic receptor calculated by Sanborn and Korenman (38) are included in Table VII. A major difference is seen in the entropy of the binding. The entropy is negative for the binding of e&radio1 to the cytoplasmic receptor and the large enthalpic contribution is the factor which overcomes the unfavorable entropy and facilitates binding. In the case of the nuclear receptor the major contribution to estra- diol binding is due to the large positive entropy.

Variation in High Afinity Nuclear Binding Sites as Function of Age and Ovarian Cycle

Immature uterine tissue was obtained from 5 to 10 day old heifers. Nuclei were isolated from both whole uterus and from the dissected endometrium and assayed for in vitro estradiol binding (Fig. 11). The binding curves obtained in this way show no indication of saturation and resemble those obtained for adult endometrial tissue which has been preincubated with high levels of estradiol-17P or diethylstilbesterol. The same

20 40 60 80 100

TOTAL DPM/MG. MEMB. PROT. b1D4)

FIG. 11. The variation in in vitro binding of [sHIestradio to uterine nuclei as a function of animal maturity. The binding of [3H]estradiol to nuclei from mature endometrium in diestrus (O--O ), immature calf uteri (O-O), immature calf endo- metrium (O--O ), and mature myometrium in diestrus (m-m) was studied as described under “Materials and Methods.”

TABLE VIII

Binding properties for mature and immature uterine cytoplasmic receptor

binding to estradiol

The dissociation constant and number of binding sites were

calculated from Scatchard Dlots.

Tissue (calf) Dissociation

constant No. of binding sites

Myometrium (mature).

Endometrium (mature). Endometrium (immature).

Total immature uterus.

x 10-g M x lo-” moles/mg protein

1.35 20.0 1.20 23.0

0.52 51.0

0.46 38.0

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FIG. 12. The high affinity nuclear

300

binding sites in mature bovine endo- metrium and myometrium during t,he estrous cycle. Endometrium ( and Myometrium ( ). Determi- nation of number of binding sites involves treatment of nuclei with [3H]estradiol at 4” for 6 hours, and constructing a Scatchard plot as described in Fig. 5C. Identification -t-r.--- I- 11 -I 1 I~ L..- VI stages in me eswous cycle nas men described under “Materials and Methods.”

0

t-

I I I I I I I I

LATE I

METESTRUS ; DIESTRUS ; I

I

I- (PRoESTRUS)

2

result was also observed for mature myometrium (the muscular tissue surrounding the endometrium). There are no specific nuclear binding sites in immature tissue, neither in the whole uterine tissue nor in the endometrial tissue which of course is the target organ for estrogen in the adult and as such shows t.he specific high afliuity sites described above. Despite the absence of specific nuclear sites in all of these uterine tissues except the mature endometrium, cytosol estradiol receptor protrius are present in all the tisues as shown in ‘l’able VIII.

Uterine tissue was collected from adult heifers and grouped ‘into the various phases of the estrous cycle as described under “Materials and 3Tethods.” The tissue was dissected into the endomctrial aud myometrial fractions. Nuclei were isolated and assayed for estradiol bin&g iu the usual manner. The data are presented in Fig. 12. The lack of sites in late metestrus proved to be highly reproducible as did the extensive availability of sites in estrus itself. Essentially no high affinity nuclear binding sites were observed in nuclei isolated from the myo- metrial layer of the uterus at any stage of the estrus cycle.

lxscussIoY A

Three maiu points have emerged from these studies. (a) Following incubation of mature endometrial tissue with [“Ii]- estradiol, the lab&d material associated with thq nucleus is bouud largely to the nuclear envelope. Essentially 110 hormone is found iu the uuclcohistone fraction (though a small amount Of nucleohistone is invariably found closely associated with the nuclear membrane fraction). (b) The hormone cat1 interact with the same sites upon incubation with isolated nuclei though in addition a less specific interaction is also observed under these conditions. (c) Alt.hough the binding described is highly sterco- specific, it differs somewhat from previous, simiIar studies in that the hormouc is not dissociated from its nuclear site by 0.4 h3 KC1 solut.ions.

Biuding curves for the interaction between estradiol and isolated endomet.rial nuclei have been presented. If the nuclei were isolated from tissue which had been exposed to diethylstil- bcsterol or to estradiol-17P, we observe a linear relatiorlship between hormone input into the binding reaction aud the amount

4 6 8 10 12 14 16 18 20 22

TIME (DAYS)

bound to the nuclei. The biuding curve shows uo sign of satura- tiou even in the presence of vastly hyperphysiological concentra- tions of hormone. However, if nuclei are isolated without pre- treatment with estradiol, from the tissue of heifers in diestrus, the binding curve is a reflection of two types of binding: a low affinity nonsaturable compouent discussed above, together with a saturable component of much higher affinity. By subtracting the coutributiou of the lower affinity interaction, the binding curve of estradiol to the high affinity sites becomes apparent. We compute that the binding constant has a value of (1.33 f 0.26) x 10eg M and that there are 507 f 47 binding sites per nucleus. This number of binding sites is somewhat lower than those observed by Gorski (9) in immature rats and Maurer and Chalkley (7) in young calves. That this is consistent with t.he behavior of such immature uteri will be discussed in a later paper (39). The high affinity site binds hormone with meas- urable kinetics, whereas the low affinity site binds very rapidly.

l’he subnuclear site of e&radio1 binding in incubations of both tissue and nuclei, is an insoluble material composed of about equal parts of lipid and hydrophobic proteins (40), which WC propose is the nuclear membrane for the following reasons. (a) Electron microscopy of the lluclci to which the hormone is bitldiug indicates relatively little contamiuatiou with other cellular membranes and microscopy of the insoluble subnuclcar fraction reveals a membrane-like structure even though it was sheared during preparation. (b) Analysis for cnzymcs charac- t&tic of other membrane systems indicates that the level of cross coutamiiiatioii is low; the maiu coiitamiilaiit is microsomal in nature, and microsomes do not bind estradiol with high affinity. (c) The lipid composition of the membrane fraction strongly resembles that described for other nuclear membrane prepara- tions by other workers who used different methods of preparation.

The following observations support the idea that the high affinity nuclear binding site is highly stereospecific and quite distinct from the low affinity nuclear site or the microsomal binding site. (a) A variety of competitor molecules can inhibit estradiol binding to the high affinity nuclear site in a manner which directly reflects their known estrogenic potency in viuo. No competition was observed for the low affinity, nonspecific

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nuclear binding sites. (b) The high affinity interaction is absent in tissues which do not show the cellular proliferation response characteristic of the endometrial tissue (41). Interestingly, this observation holds true for the mature myometrial tissue. The myometrium is the muscular support tissue upon which the cndometrium rests. It is known to respond to estradiol in terms of increased muscular tone and hypertrophy, but this tissue does not have a hyperplastic response to estrogens in the estrous cycle (42, 43). The uterine tissue from immature rats has fre- quently been used in studies of hormone action and yet a con- siderable part of this tissue (-90%) is myometrial and as such may be responding in a very different fashion to the pure endo- metrial tissue used in experiments described above. (c) The high affinity nuclear site shows significant differences from the microsomal site in its response to va,riations in pH and in sensi- tivity to pronase treatment. Incubation with pronase com- pletely destroys the high affinity sites, whereas the low affinity sites are unaffected.

We have investigated the possibility that the nuclear binding site is a result of a cytoplasmic receptor becoming associated with the nucleus and thus providing the element of specificity to the interaction. Certainly the interaction of estradiol and the cytoplasmic receptor is of a specificity analogous to that de- scribed for the nuclear high affinity site. This possibility is rendered unlikely, however, by several lines of cvidcnce. (a) The thermodynamic parameters of the estradiol-nuclear mem- brane interaction arc dramatically different from those reported for the cytoplasmic receptor (38). (b) The variation in the number of nuclear sites available during the estrous cycle is not correlated with the relatively constant amount of cytoplasmic receptor per cell at all stages of the cycle (41). (c) No specific nuclear sites are available for estrogen binding in immature calf endometrium, though the level of the cytoplasmic receptor in this tissue is 2-fold higher than in the adult. (d) No specific nuclear sites are seen in myometrial nuclei even though the cytoplasmic receptor concentration is the same as that found in the endometrium. (e) The nuclear site has a radically different response to p1-I compared to the cytoplasmic receptor. (.f) The nuclear second-order rate constant of binding is significantly different from the second-order rate constant for the cytoplasmic receptors (1.94 & 0.23 X lo5 s-l M-~ compared to 3.8 + 0.2 X

lo5 s-l M-l (38)).

10. 11. 12.

13.

14.

15. MACI~LER, B. (1967) Methods Enzymol. 10, 551 16. WHARTON. 11. C.. AND TZAGOLOFF. A. (1967) Methods Enzvmol.

17.

18.

19. 20.

Biophys.‘lll, 559’ ,

EAGLE, H. (1959) Science 130,432 BRAY, G. A. (1960) Anal. Biochem. 1, 279 MOORE, D. J., HAMILTON, R. H., MOLLENHAUER, H. H., MAH-

LEN, R. W., CUNNING&AM, W. P., CHEETHA~, R. d., AND LISQUIRE. V. S. (1970) J. Cell Biol. 44.484

BERMAN, fi. M., ERA& W., AND SPIR~ES, M. A. (1969) Bio- chim. Biophys. Acta 183, 10

NORDIE, R. C., AND ARION, W. J. (1957) Methods Enzymol. 9, 619

10, 245 ’ ’ I \ I

CENTER, M. S., AND BEHAL, F. J. (1966) Arch. Biochem. Bio- phys. 114, 414

Finally, we note that the in vitro high affinity site is absent in immature calves aud also in mature heifers during the late metestrus and the proestrus stages of the estrous cycle. During diestrus the blood level of estradiol is low (42) and the tissue is prepared to respond, and thus the availability of open sites 011

the nucleus seems reasonable. During procstrus the blood level of estradiol is rising rapidly, the tissue is visibly responding and presumably the sites are unavailable because they arc blocked by the bound endogenous hormone. As expected the number of sites increases through estrus where the hormone has com- pleted its stimulation of endometrial proliferation, and the blood level of estrogen is declining. However, during early metestrus and late metestrus when the blood level of estradiol is quite low, the number of nuclear sites become vanishingly small, even though it is unlikely that at this stage of the cycle that the sites would be blocked by endogenous hormone. However, it is known that animals iu late metestrus cannot reinitiate an appro-

21.

LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193,265

BURTON, K. (1968) Methods Enzymol. 12, 163 BLIGH, E. G., AND DYER, W. J. (1959) Can. J. Biochem. Phys-

iol. 37, 911 ROUSER, B., SIAKOTOS, A. N., AND FLEISCHER, S. (1966) Lipids

1,85 22.

23. 24.

MESTER, J., AND BAULIEU, E. E. (1972) Biochim. Biophys. Acta 261, 236

WOOD, It. L., AND LUFT, J. H. (1965) J. Ullrastrucl. Res. 12,22 HAMMOND, J. (l(327) in The Physiology of Reproduction in the

25. Cow, Cambridge University Press, London

PARKES. A. S. (19651 in Marshall’s Phusiolosv of Reproduction.

26.

27.

28.

Vol. 1; 3rd Eb, Ldngmans, Green aid Co:,‘L&ddn JACKSON, V., AND CHALKLEY, R. (1974) J. Biol. Ckem. 249,

1627-1636 ABE, T., HAGERMAN, D. D., AND VILLEE, C. A. (1964) J.

Biol. Chem. 239, 414 QUGLIARIELLO, F., PAPA, S., SLATER, E. C., AND TAQER, J.

M. (1967) in Mitockondrial Structure and Compartmentation, p, 59, Adriatica Edetrice, Bari

29. PFLEGER. R. C.. ANDERSON. N. G.. AND SNYDER. F. (1968)

30.

-_

Biochekistry 7: 2826 ’ ’ ,

DULLNER, B., SIEKEVITZ, P., AND PALLADE, G. E. (1966) J. Cell Biol. 30, 73 (1966)

priate response to estradiol until they have passed into the 31. KLEINIG, H. (1970) J. Cell. Biol. 46, 396

diestrus state (44). Perhaps a second device is available to 32. TATA, J. R., HAMILTON, M. J., AND COLE, R. D. (1972) J. Mol.

block the specific hormone sites against an unprogrammed rise Biol. 67, 231

33. FROST, A. A., AND PEARSON, R. B. (1961) in Kinetics andMeck-

We have demonstrated that the high affinity nuclear binding site is specific for estrogenic compounds. This site is observed it1 mature endometrial tissue and not in thymus, mature myo- metrium, or immature calf uterus. This binding site would appear to be an extremely pa-sensitive protein that interacts hydrophobically with estradiol-17/3. The hormone is not metabolized in the high affinity site suggesting that the binding is not directly related to enzymatic activity (41). The actual function of the binding protein remains a mystery, but its location is at least consistent with the idea that it might be in- volved in nuclear control.

REFERENCES

1. WYNN, R. M. (1967) in Cellular Biology qf the Uterus. D. 138. Appleton-Century-Croft, New York- .

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2. BILLING, R. J., BARBIROLI, B., AND SMELLIE, R. M. S. (1969) Biockim. Biopkys. Acta 190, 60

3. JEENER, It. (1948) Biochim. Biophys. Acta 2, 439 4. JENSEN, IX. V., AND JACOBSON, J. I. (1962) Recent Prog. Hor-

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JUNGULUT, P. W., AND I>I~SOMURE, E. It. (1968) Proc. Nat. Acad. Sci. U. S. A. 69, 632

6. STKGGLICS, A. W., SPELSBERG, T. C., AND O’MALLEY, B. W. (1971) Biochem. Biophys. Res. Commun. 43, 20

7. MAURKIZ, H. It., AND CHALKLEY, G. It. (1967) J. Mol. Biol. 27, 431

8. SHYAMALA, G., AND GORSKI, J. (1969) J. Biol. Chem. 244.1097 9. NOTID~OOM, W. I).. AND GORSKI. J. (1965) Arch. Biochem.

in the blood estradiol levels. anisms, Ed. 2, p. 15, Wiley, New York

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34. SCATCHARD, (:. (1949) Ann. N. Y. Acad. Sci. 61, MO 39. J.\CKSON, V., AND CHALKLIIY, Ii. Biochemistry, in press 35. VONDRKLY, lt.. (1955) Nucleic Acids, Vol. 2, p. 155, Academic 40. J.\ctisox, v. E., l':ARNII.U<DT, J., AND CRILKL14:Y, It. (1968)

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Wiley, New York 42. WYNN, 11. hf. (lM7) in Cellular I3iology (?f the Uterus, p, 363, 37. Puca, G. A., NOLA, I<., SICA, V., AND ~I~I:SCIANI, F. (1071) Applet.on-Century-Croft, New York

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Biochemislry 10, 4955 44. GOOD, 11. G., AND MOYER, L). L. (1968) Fertil. Sleril. 19, 37

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Vaughn Jackson and Roger Chalkley to the Bovine Endometrial Nuclear MembraneβThe Binding of Estradiol-17

1974, 249:1615-1626.J. Biol. Chem. 

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