The role of estradiol in the equilibrium binding of estrogen receptor to rabbit uterine chromatin

480

Biochimica et Biophysica Acta, 343 (1974) 480--491

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27406 THE ROLE OF E S T R A D I O L IN THE EQUILIBRIUM BINDING OF E S T R O G E N R E C E P T O R TO RABBIT UTERINE CHROMATIN

MARVIN L. CHATKOFF, J.A. JULIAN, JOSEPH E. MARTIN and ROBERT J. YOUNG Department o f Obstetrics and Gynecology, Biochemistry and Laboratory Animal Medicine, The University o f Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284 (U.S.A.)

(Received December 7th, 1973)

Summary Estradiol complexed with receptor stimulates transcription in target organs. The precise function of the steroid molecule in the mechanism is unknown. Among its many suggested functions, is that estradiol participates as an active c o m p o n e n t in the binding of receptor to a chromatin fraction. In vitro evidence is presented that estradiol is neither necessary for binding of receptor to chromatin nor does it significantly influence the equilibrium parameters which describe the extent of binding. It is further shown that binding of estradiol to receptor previously bound to chromatin has similar equilibrium parameters to that for the binding reaction of estradiol to free receptor. These results suggest that the receptor may have an estradiol binding site disjoint from the chromatin binding site which is not functionally hindered by the receptor being b o u n d to chromatin.

Introduction Estrogen--receptor complexes have been shown to bind to target tissue [1] and perhaps to non-target tissue chromatins [2]. Previously we have described a method of estimating from estrogen--receptor complex to chromatin equilibrium binding assays, the equilibrium constant and the number of available chromatin acceptor sites [3] with the assumption that binding approximates a second order to first order reaction. The influence of estradiol on the equilibrium binding parameters may be determined by means of competition between e m p t y receptors and estrogen--receptor complexes for chromatin binding sites. Binding studies of estradiol alone [4--5 ], e m p t y receptor [6] and estrogen--receptor complex [7--8] to DNA and RNA [9] suggest that the estradiol molecule either directly participates in receptor binding or modifies the extent of binding by influencing binding kinetics. On the other

481 hand, estrogen--receptor to chromatin binding which apparently involves acidic nuclear proteins [10] may be independent of the presence of the steroid. This study evaluates the equilibrium parameters of the estrogen--receptor complex to chromatin reaction in the presence of varying concentrations of empty receptor and estrogen--receptor complex. The equilibrium parameters for the estrogen--receptor complex to chromatin and the empty receptor to chromatin reactions were determined. In addition, an approximate number of total chromatin acceptor sites per #g DNA was determined for 1 week castrate rabbit uterine chromatin under the in vitro experimental conditions and calculational assumptions described. The present concept of the mechanism of steroid action involves as the first step in the sequence, the binding of the steroid to its specific cytoplasmic receptor. The complex enters the nucleus and binds to chromatin as demonstrated in vivo and in vitro. Under equilibrium conditions a very small amount of empty receptor necessarily must dissociate within the nucleus provided that both the estrogen to empty receptor and the estrogen--receptor complex to chromatin reactions are reversible, that is, have non zero dissociation constants even though only estrogen--receptor complexes apparently enter the nucleus. However, these quantities of free receptor would be so small that the presence of empty receptors in nuclear extracts would be difficult to demonstrate. If empty receptors will bind to chromatin in vitro, then through this artificial procedure the equilibrium parameters of estradiol binding to empty receptors attached to chromatin can be compared with that of estradiol binding to free receptor as normally occurs in the cytoplasm. Differences in these parameters under the two conditions reflect the perturbing influences of chromatin on the estradiol binding site of the receptor. Methods

Preparation of chromatin Uterine nuclei were prepared from ovariectomized mature female New Zealand white rabbits one week post-castration following the method of Steggles et al. [11]. Chromatin was purified by the method of Spelsberg and Hnilica [12]. Chromatin preparations were stored in liquid nitrogen in 1.5 mM NaC1--0.15 mM sodium citrate pH 7.0. The ratio of basic proteins to DNA for the chromatin prepared by this method averaged 1.43 -+ 0.05 mg/mg and the ratio of acid proteins to DNA was 0.45 + 0.003 mg/mg [3].

Preparation of estradiol--receptor complex [6,7 -3 H] Estradiol-17~ (spec. act. 48 000Ci/mole) was obtained from New England Nuclear and purified before use by column chromatography using Sephadex LH-20. Uteri were removed from rabbits two weeks post-ovariectomy and immediately homogenized in two volumes 0.01 M Tris--HC1--0.001 M EDTA, pH 7.4 with a Polytron PT-10ST for four 5-s intervals at a rheostat setting of 5. The homogenate was centrifuged at 1 0 5 0 0 0 × g for one hour. The 1 0 5 0 0 0 X g supernate (cytosol) was diluted to 10 mg protein per ml with 0.01 M Tris-HC1--0.001 M EDTA buffer. Cytosols from non-target tissue were prepared in the same manner.

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Fraction No. Fig. 1. Sucrose g r a d i e n t s e d i m e n t a t i o n . U t e r i n e c y t o s o l c o n t a i n i n g e s t r o g e n - - r e c e p t o r c o m p l e x was prep a r e d as d e s c r i b e d in M e t h o d s . 2 0 0 pl a i i q u o t s w e r e a p p l i e d to s u c r o s e g r a d i e n t s (5 to 20% s u c r o s e in 0.01 M T r i s - - H C l - - O . 0 0 1 M E D T A b u f f e r , p H 7.4) a n d c e n t r i f u g e d 16 h in an SW 6 5 r o t o r a t 45~)~)~ r e v . / m i n a t 4 ° C.

[6,7 -3 H] Estradiol-17~ was added to the cytosol at the desired concentration and incubated 30 min at 4°C. Free estradiol was removed by incubating the cytosol-[ 3 H] estradiol mixture with a resuspended pellet of dextran-coated charcoal for 15 min at 4°C. The charcoal was removed by centrifuging 10 min at 1200 × g at 4°C. Sucrose gradient sedimentation analyses of the state of the initial estradial-receptor complexes, formed by the above procedures, consistently had a single peak at approx. 8 S (Fig. 1) insuring that a homogeneous form of receptor complex was added to the incubation medium. Assays

Basic receptor--chromatin binding assay (constant empty receptor to estrogen-receptor complex) Increasing amounts of [3 H] estradiol--receptor complex were incubated for one hour at 4°C with 30--50 pg chromatin. The assay procedure followed that of Steggies et al. [1] except that the chromatin pellets were rinsed twice with 1 ml 0.15 M NaC1 before resuspending in 1 ml 0.15 M NaC1--0.01 M MgC12 for filtrations. Reverse binding assay Aliquots containing 30--50/~g chromatin were incubated 1 h at 4°C with a single concentration of empty receptors, sufficient to saturate the chromatin acceptor sites when complexed with estradiol, or non-target tissue cytosol in 0.5 ml total volume with 0.15 M NaC1. 0.5 ml 0.15 M NaC1 was added to each

483 sample and the solutions were centrifuged 10 min at 1200 × g to sediment the chromatin. The pellets were resuspended in 1 ml cold 0.01 M Tris--HC1--0.001 M EDTA buffer and recentrifuged. The pellets were resuspended in 100 pl 0.01 M Tris--HC1--0.001 M EDTA buffer containing increasing concentrations of [3 H] estradiol and incubated 30 min at 4°C. 1.0 ml cold 0.15 M NaC1 was added and the samples were centrifuged 10 min at 1200 × g. The samples were rinsed and filtered as in the basic assay procedure. Controls contained only buffer and chromatin in 0.15 M NaC1 during the initial incubation.

Inhibition assay (constant estrogen--receptor complex) Increasing amounts of e m p t y receptor and a single concentration of estrog e n - r e c e p t o r complex were incubated with 30--50 pg DNA for 1 h at 4°C. The samples were rinsed and filtered as in the basic assay procedure. For those assays where the estrogen--receptor complex was incubated with chromatin, appropriate controls containing the complex but not chromatin were included. These were subtracted as background. The filters were dried at room temperature and counted in a standard toluene--PPO-POPOP mixture. Filters were then dried at r o o m temperature and the DNA extracted by incubating the filters in 0.5 M HC104 for 30 min at 90°C. The extracts were assayed for DNA by the m e t h o d of Burton [ 1 3 ] . Protein concentrations of the cytosols were determined by the method of Lowry et al. [14] using bovine serum albumin as a standard. In assays in which it was necessary to determine the a m o u n t of e m p t y receptor remaining after incubation with [3 HI estradiol {empty receptor to estrogen--receptor complex ratios) it was assumed that emp ~y receptor was the difference between total receptor sites as determined by dextran-coated charcoal assay [ 15] and estrogen--receptor complex concentrations determined by the amount of [3 H] estradiol b o u n d after charcoal treatment. In calculating Lineweaver--Burk plots, the estrogen--receptor complex b o u n d to chromatin on the filters was adjusted to pmoles/pg DNA. The estrog e n - r e c e p t o r complex added was adjusted to pmoles/pg DNA by assaying an aliquot of chromatin added in the assay for DNA. Results Three Lineweaver--Burk lines were constructed from estrogen--receptor c o m p l e x to chromatin binding assays in which three different relative concentrations of e m p t y receptor to estrogen--receptor complex (Fig. 2) were incubated with aliquots of a uterine chromatin preparation (purified chromatin pooled from 15 rabbits). The receptor aliquots were from a single preparation of uterine cytosol (uteri pooled from 5 rabbits). The total receptor concentration, e m p t y receptors plus estrogen--receptor complexes, was equal for all assays. The extrapolated vertical and horizontal intercepts are sets of apparent chromatin acceptor sites and dissociation constants, K D which vary with the percentage of e m p t y receptors present and are smaller than the true values. If e m p t y receptors did n o t compete with estrogen--receptor complex for binding sites or kinetically alter the equilibrium constant then the three assays would have identical intercepts. The parallelism of the lines indicates that the appar-

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Fig. 2. L i n e w e a v e r - - B u r k p l o t s o f e s t r o g e n - - r e c e p t o r c o m p l e x to c h r o m a t i n binding at various initial e m p t y r e c e p t o r to e s t r o g e n - - r e c e p t o r c o m p l e x ratios. A, e m p t y r e c e p t o r / e s t r o g e n - - r e c e p t o r c o m p l e x = 1 . 3 2 2 ; B, e m p t y r e c e p t o r / e s t r o g e n - - r e c e p t o r c o m p l e x = 0 . 3 3 ; C, e m p t y r e c e p t o r / e s t r o g e n - - r e c e p t o r c o m p l e x = 0 . 0 6 2 . A single c y t o s o l w a s divided and i n c u b a t e d w i t h t h r e e c o n c e n t r a t i o n s o f [ 3 H ] estradiol (1.2, 2.5, a n d 6.3 • l ( r 8 M ) in the p r e p a r a t i o n o f e s t r a d i o l - - r e c e p t o r c o m p l e x to o b t a i n t h e three e m p t y recept o r / e s t r o g e n - r e c e p t o r c o m p l e x ratios. T h e assays w e r e p e r f o r m e d s i m u l t a n e o u s l y a c c o r d i n g to the p r o c e dure for t h e basic r e c e p t o r - - c h r o m a t i n binding assay. Fig. 3. E x t r a p o l a t i o n o f lIB values. 1/B values for t h e three e m p t y r e c e p t o r / e s t r o g e n - - r e c e p t o r c o m p l e x ratios w e r e t a k e n f r o m t h e assays d e s c r i b e d in Fig. 2. T h e labeled v o l u m e for each line i n d i c a t e s the a m o u n t o f c y t o s o l a d d e d to the c h r o m a t i n aliquot. The line labeled CAS w a s derived f r o m the vertical i n t e r c e p t s in Fig. 2.

ent chromatin acceptor sites vary proportionally with changes in the ratio of empty receptors to estrogen--receptor complex and therefore the constant slope of the lines is a characteristic of the particular chromatin. Three repeat assays, each at two levels of empty receptors to estrogen--receptor complex, were performed. The average slope for the four assays was 9.32 + 1.45 and agrees with our previously reported value of 9.0 -+ 1.1 for one week castrate rabbit uterine chromatin [3]. The real value of chromatin acceptor sites for the particular chromatin sample is obtained by graphical extrapolation of the assays to the saturated condition (Fig. 3) as described in Appendix 1. Reciprocal bound values ( l / B ) from an individual Lineweaver--Burk graph transform into a vertical series of points at a constant ratio of empty receptors to estrogen--receptor complex. A line through the points of equal total receptor concentration intersects the vertical axis at a value of lIB which would be expected if estrogen-receptor complex were equal to the total receptor concentration. The series of vertical intercepts defines an ideal binding curve. The vertical intercept of a line through apparent chromatin acceptor site points is the real value for the particular chromatin assayed (pmoles/460 pg DNA for this one week castrate chromatin preparation). Theoretically all lines should intersect the horizontal axis at a common point. This point defines the ratio of g D values for empty receptors and estrogen--receptor complex binding respectively KD R/KD R E. In this representative example the ratio is 1.04. The mean value

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Fig. 4. I n h i b i t i o n assay. Assay p r o c e d u r e d e s c r i b e d u n d e r M e t h o d s . 0, 25, 1 0 0 , 1 5 0 , a n d 2 0 0 #1 of a e y t o s o l c o n t a i n i n g e m p t y r e c e p t o r o n l y a n d 50 #1 of e y t o s o l c o n t a i n i n g e s t r o g e n - - r e c e p t o r c o m p l e x plus empty receptor (empty receptor/estrogen---receptor complex = 0.0865, estrogen--receptor complex = 0 . 9 8 2 4 p m o l e s / 5 0 /~1) w e r e a d d e d to c h r o m a t i n aliquots, A. E x p e r i m e n t a l i n h i b i t i o n . T h e v a l u e f o r B w h e r e e m p t y r e c e p t o r = O was o b t a i n e d b y c o n s t r u c t i o n of t h e ideal b i n d i n g c u r v e f o r this c y t o s o l a n d c h r o m a t i n c o m b i n a t i o n . T h e o t h e r six p o i n t s c o r r e s p o n d t o the a m o u n t s o f a d d i t i o n a l c y t o s o l c o n t a i n i n g only e m p t y r e c e p t o r d e s c r i b e d a b o v e . B. T h e t h e o r e t i c a l c o m p e t i t i o n c a l c u l a t e d u n d e r t h e a s s u m p t i o n t h a t K D R = K D R E is i n d i c a t e d b y t h e solid line. E x p e r i m e n t a l values are i n d i c a t e d b y i n d i v i d u a l points. B O = B w h e r e e m p t y r e c e p t o r = O for a n y t o t a l r e c e p t o r c o n c e n t r a t i o n ( t o t a l r e c e p t o r c o n c e n t r a t i o n = e s t r o g e n - - r e c e p t o r c o m p l e x ) ; B = B w h e r e e m p t y r e c e p t o r ¢ O for t h e s a m e t o t a l r e c e p t o r c o n c e n t r a t i o n = empty receptor + estrogen--receptor complex).

for four assays is 0.98 + 0.05. The horizontal intercepts is sensitive to small errors in estimating the ratio of e m p t y to full receptor levels in the cytosol, nevertheless, the results indicate that an e m p t y receptor has approximately the same dissociation constant as the estrogen--receptor complex. The inhibition assay is a further demonstration of the competitive binding of estrogen--receptor complexes and e m p t y receptors. In this assay the amount of estrogen--receptor complex added was held constant while e m p t y receptor were increased, resulting in increasing total receptor concentration and e m p t y receptor to estrogen--receptor complex ratios. Under these conditions the amount of estrogen--receptor complex b o u n d per pg DNA should have decreased as the number of e m p t y receptors increased (Fig. 4A). To confirm that the amount of competitive inhibition by the e m p t y receptors was quantitatively correct, the data in Fig. 4A was compared to theoretical values as follows (Fig. 4B). Since binding of the estrogen--receptor complex is a function of both the cytosol and chromatin, a binding assay was performed simultaneously with the inhibition assay. Utilizing the K D R/KDR E value of 1.0 an ideal binding curve was extrapolated from the experimental binding curve. From the ideal binding curve the a m o u n t of estrogen--receptor complex which would have b o u n d in the absence of e m p t y receptors (i.e. estrogen--receptor complex = total receptor concentration) was derived for any total receptor concentration. The a m o u n t of estrogen--receptor complex which would have b o u n d (lIB value) at an e m p t y receptor to estrogen--receptor complex ratio (where total receptor concentration 4= estrogen--receptor complex) for the same total receptor concentration should have fallen on the line from 1.0 through the 1 / b o u n d

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Fig. 5. C o m p a r i s o n b e t w e e n target tissue and n o n - t a r g e t tissue c y t o s o l s in an i n h i b i t i o n assay. A s s a y p r o c e d u r e d e s c r i b e d u n d e r Methods. 50 #1 o f a u t e r i n e c y t o s o l c o n t a i n i n g e s t r o g e n - - r e c e p t o r c o m p l e x and empty receptor (empty receptor/estrogen~receptor complex = 0.046, estrogen--receptor complex = 0 . 8 7 1 5 p m o l e s / 5 0 ~1) plus either O ( c o n t r o l ) , 25, 50, 1 0 0 , 1 5 0 or 2 0 0 #1 o f uterine c y t o s o l c o n t a i n i n g o n l y e m p t y r e c e p t o r ( e ) or similar a l i q u o t s o f a m u s c l e c y t o s o l ( o ) w e r e added to standard c h r o m a t i n a l i q u o t s . Bovine s e r u m a l b u m i n in 0 . 0 1 M Tris-HC1-0.001 M E D T A b u f f e r w a s a d d e d t o m a i n t a i n c o n s t a n t p r o t e i n c o n c e n t r a t i o n . Per c e n t c o n t r o l c a l c u l a t i o n s based o n p m o l e s / p g D N A b o u n d . Fig. 6. R e v e r s e binding a s s a y - - u p t a k e curve. Assay p r o c e d u r e d e s c r i b e d u n d e r M e t h o d s . 2 0 0 #l c y t o s o l ( 1 0 m g ] m l ) w e r e a d d e d t o c h r o m a t i n a l i q u o t s ( 4 0 g g D N A ) for t h e initial i n c u b a t i o n , A f t e r rinsing, the c h r o m a t i n p e l l e t s w e r e r e s u s p e n d e d in 1 0 0 pl 0 . 0 1 M T r i s - - H C 1 - - 0 . 0 { ) l M E D T A b u f f e r c o n t a i n i n g [ 3 H ] e s t r a d i o l in c o n c e n t r a t i o n s ranging f r o m 0 . 0 3 0 6 to 0 , 2 3 7 p m o l e s / 1 0 0 #1. A, u t e r i n e c y t o s o l ; B, skeletal m u s c l e c y t o s o L

value on the vertical axis for that total receptor concentration. The percent difference between the estrogen--receptor complex bound value where total receptor concentration ¢ estrogen--receptor complex and the ideal estrogen-receptor complex bound value where total receptor concentration = estrogen-receptor complex is the theoretical line of Fig. 4B. The percent difference between experimental values of estrogen--receptor complex bound and the ideal values (Fig. 4B) were calculated in the same manner (difference between experimental values where total receptor concentration ¢ estrogen--receptor complex and ideal estrogen--receptor complex bound where total receptor concentration = estrogen--receptor complex). Simultaneous inhibition assays performed with aliquots from a c o m m o n chromatin preparation and a c o m m o n preparation of estrogen--receptor complex show that muscle cytosol is unable to inhibit estrogen--receptor complex binding to chromatin (Fig. 5). Inhibition is therefore caused only by specific components of uterine cytosoh The reverse binding equilibrium uptake curve demonstrates the saturable binding of estradiol to empty receptors preincubated with chromatin (Fig. 6). Low binding levels (approximately 5% of the value for uterine cytosol) were obtained for the same chromatin preincubated with skeletal muscle cytosol. The Lineweaver--Burk plot indicates that the number of receptors bound per pg D N A is of the same order as the number of estrogen--receptor complexes which would bind for a similar cytosol concentration (Fig. 7A). No binding was obtained in controls in which the cytosol incubation was replaced by buffer (3

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to 10 cpm above instrument background from the lowest to highest concentration). Lineweaver--Burk estimates of the dissociation constant (per unit of volume rather than per pg DNA (Fig. 7B)) of 1.11 • 10 -9 M agrees with the mean dissociation constant determined from two-week castrate rabbit uterine cytosol charcoal assays in our laboratories (1.0 • 10 -9 M). Discussion The reverse binding assay is direct in vitro evidence that estradiol will bind specifically to e m p t y receptor which was prebound to chromatin. This is shown by the inability of estradiol to bind when the chromatin is preincubated with skeletal muscle cytosol. This result eliminates as potential binding factors cytosol components c o m m o n to both target tissue and non-target tissues and implies that the target tissue estradiol receptor is involved. The extremely low number of chromatin b o u n d [3 H] estradiol counts in the absence of receptor appears to conflict with reported findings of estradiol-17-~ binding to purified DNA [5]. The difference may be due to the presence of the nuclear proteins, or DNA binding of estradiol may be a nonspecific artifact. Furthermore, the dissociation constant for the reaction of estradiol with previously b o u n d e m p t y receptor determined from Lineweaver--Burk extrapolation is similar in value to that found in dextran-coated charcoal assays of estradiol binding to cytosol receptor, implying that the receptor binding site for estradiol is n o t influenced by r e c e p t o r - c h r o m a t i n binding. This evidence indicates t w o disjoint binding sites on the receptor, one for binding to a chromatin site and the other for binding estradiol. Although the reverse binding assay indicates that e m p t y receptor will bind to chromatin in vitro, neither the specificity of the binding nor the equilibrium

488 constant can be derived from this type of assay. The equilibrium assays {constant empty receptor to estrogen--receptor complexes) which provide quantitative evidence also suggest that the estradiol molecule does n o t participate or at most minimally interacts in the binding. The estimated equal dissociation constants for estrogen--receptor complexes and empty receptors are predicated on the assumption of second order, competitive kinetic mechanisms for initial receptor binding to chromatin. Among the supportive evidence for equal dissociation constants is the linearity and parallelism of the Lineweaver--Burk lines independent of empty receptor to estrogen--receptor complex levels as required by Eqn 5, Appendix 1. Also, the existence of a c o m m o n extrapolated intercept at the value of 1.04 defined by the intersection of lines drawn through equal concentration values gives further support to this conclusion. There may be small steroid effects on receptor binding which were n o t determinable within the limits of the assay method. The results of the inhibition assay are further support for the equality of K D R and K D R E. For any other ratio of K D R t o K D R E, percent difference curves above or below the indicated theoretical line would have been obtained as a function of empty receptors/ total receptor concentration. Curves which would fall above the theoretical line would correspond to K D R / g D R E values less than 1, curves below to values greater than 1. The diminishing amounts of estrogen--receptor complex bound for a constant a m o u n t of estrogen--receptor complex added with increasing amounts of e m p t y receptor is evidence that empty receptors compete with estrogen--receptor complexes (Fig. 4A), independently of any theoretical model. The basic binding assay performed at different ratios of empty receptor to estrogen--receptor complex proves that e m p t y receptors compete with estrog e n - r e c e p t o r complexes because in these assays the total receptor concentration (in the same volume of the same cytosol) and therefore all other components were held constant and only the ratio empty receptor/estrogen--receptor complex was varied. However, this result does n o t eliminate the possibility of the existence of additional non-receptor inhibitors. The lack of inhibition by muscle cytosol lends further support to the view that the empty receptor is the only inhibitor. Because this interpretation is based upon the assumption of chemical equilibrium conditions, the basic assay binding was performed at four time periods which established that equilibrium had been essentially reached at 1 h. A conclusion that 20 min was sufficient to reach equilibrium has been reported [15] for progesterone receptor binding to chromatin. In binding rat uterine receptor to R3230 AC m a m m a r y t u m o r chromatin at 4 ° C, it has been found that equilibrium could n o t be achieved even at 12 h [16]. Whether the heterogeneity of the system, species difference or some property of t u m o r chromatin makes this result differ from ours is n o t clear. Our conclusion that estradiol is n o t a dynamic factor in the binding of receptor to chromatin appears at variance with DNA--receptor binding studies which show that receptor binding is enhanced in the presence of estradiol [6]. These studies based upon sucrose gradient sedimentation of complexes do n o t yield kinetic parameters so that a direct comparison of binding parameters is not possible.

489 In conclusion, it appears that the presence of the estradiol molecule does n o t significantly change the equilibrium parameters of the reaction. The fact that estradiol can bind to receptor chromatin complex with the same equilibrium parameters as that for estradiol binding to receptor alone indicates that the estradiol binding site is independent of the chromatin binding site. Estradiol function may be limited to transport of the receptor into the nucleus and/or transcriptional mechanisms activated by estradiol--receptor complexes. Appendix 1

Competitive inhibition One possible mechanism for competitive reactions between empty receptor (R) and estradiol bound receptor (ER) for e m p t y chromatin sites (C) (at constant temperature in a non-reactive medium) is defined by kl ER + C ~ ' ER--C k_l

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(2)

where R--C are receptor-chromatin acceptor complexes and ER--C are estrogen--receptor--chromatin acceptor ternary complexes. The rate constants for the two reactions may differ. The initial concentrations ERI, RI, and CAS (estrogen receptor, empty receptor and chromatin acceptor sites) are arbitrary. Because of the small dissociation constant for estrogen-receptor complex, the free estradiol is assumed to be negligible throughout the reaction, and the kinetics of estrogen with its receptor is ignored. The Lineweaver-Burke equation modified to include competitive inhibition (expressed solely in terms of the measurable equilibrium values F and B corresponding to ER and ER-C respectively) is, CAS

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where the right-handmost term is the non-linear perturbation due to the presence of empty receptors. The initial number of empty receptors RI is estimated from dextran-coated charcoal analysis of the cytosol. In the absence of empty receptor (RI equal to zero) Eqn 3 reduces to the ordinary Lineweaver--Burk equation. The presence of the variable F in the right handmost term makes Eqn 3 non-linear in the variables lIB and 1/F except for the special condition of equal dissociation constants (KDRZ = KDR ), in which case, the right-hand term is the constant initial ratio of empty to estradiol bound receptors. For sufficiently large concentrations of estrogen--receptor added (ERI), Eqn 3 is approximately linear. In going to the limit of infinite receptor added in Eqn 3, ER/ERI approaches unity while the ratio RI/ERI is constant. This is

490 accomplished experimentally by adding increasing amounts of solution containing e m p t y receptor and estradiol-receptor complex maintained in a fixed ratio. In the limit of infinite concentrations of free estrogen-receptor, the vertical intercept is n o t the usual true number of chromatin acceptor sites, but rather an apparent number CAS given by the reciprocal expression

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(4)

KDR

The apparent number of acceptor sites is diminished in the presence of e m p t y receptor and is a function of ratio of the initial concentrations and of the ratio of the dissociation constants. If empty receptors do n o t bind to acceptor sites then K D R is infinite and the apparent number of sites are equal to the true number. Similarly in the absence of initially u n b o u n d receptor R~ the true and apparent values are identical. Eqn 4 may be used to estimate the ratio of the dissociation constants and the absolute number of acceptor sites from the apparent chromatin acceptor sites for a series of known initial conditions. A plot of Eqn 4 which is linear in the inverse variable 1/CAS and the second variable R~/ERI intercepts the horizontal axis at the ratio K D R E lED R. This construction is similar to the horizontal intercept in a Lineweaver--Burk reciprocal plot which yields the single value K D . For the special condition of equal dissociation constants

ERI CAS = CAS - T ERI KD=

(5)

K D - -

T

where T is the total initial receptor i.e. full plus empty and KD is the apparent dissociation constant. Both CAS and KD differ from the true values by the same factor. Therefore under the condition of equal dissociation constants for empty and full receptors, Lineweaver--Burk lines should be parallel for all initial ratios of e m p t y to estradiol-bound receptor. Appendix 2

Competitive inhibition of estrogen--receptor complex binding by variable amounts of empty receptor The Lineweaver--Burk form (Eqn 3, Appendix 1) for the a m o u n t of bound estrogen receptor complex (B) for the condition g D R = KD R E K D is ----

CAS

KD

B

F

+ I + RdERI •

(6)

In ideal binding in which R~ = 0, the ideal a m o u n t bound (B0") is given by CAS

KD

Bo

Fo

+ 1.

(7)

491 Because for equal dissociation c o n s t a n t , F Fo

ERI -

(8)

T

it f o l l o w s that ER~ B = ~ - - Bo •

(9)

Therefore, the fractional difference b e t w e e n the ideal a m o u n t b o u n d B0 and the actual a m o u n t B is B o -- B B0

-

T - - ERI T

-

RI T

(10)

Eqn 10 w h i c h is linear in RI/T is t h e theoretical line o f Fig. 4A. References 1 Steggles, A.W., Spelsberg, T.C., Glassner, S.R. and O'Malley, B.W. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1 4 7 9 - - 1 4 8 2 2 Chamness, G.E., Jennings, A.W. and McGuire, W.L. (1973) Nature 2 4 1 , 4 5 8 - - 4 6 0 3 Chatkoff, M.L. and Julian, J.A. (1973) Biochem. Biophys. Res. Commun. 51, 1 0 1 5 - - 1 0 2 2 4 Goldherg, M.L. and Atchley, W.A. (1966) Proc. Natl. Acad. Sci. U.S. 55, 989--996 5 T'so, P.O.P., and Lu, P., (1964) Proc. Natl. Acad. Sci. U.S. 51, 17--23 6 Andre, J. and Rochefort, H. (1973) FEBS L e t t . 29, 135--140 7 Musliner, T.A. and Chader, G.J. (1972) Biochim. Biophys. Aeta 262, 256--263 8 Clemens, L.E. and Kleinsmith, L.J. (1972) Nat. New Biol. 237, 204--206 9 Liao, Shutsung, Liang, Tehming, and T y m o c z k o , J.L. (1973) Nat. New. Biol. 2 4 1 , 2 1 1 - - 2 1 3 10 Spelsberg, T.C., Steggles, A.W., Chytfl, F. and O'Malley, B.W. (1972) J. Biol. Chem. 247, 1 3 6 8 - - 1 3 7 4 11 Steggles, A.W., Spelsberg, T.C. and O'Malley, B.W. (1971) Biochem. Biophys. Res. Commun. 43, 20--27 12 Spelsberg, T.C. and Hnilica, L.S. (1971) Biochim. Biophys. Acta 228, 202--211 13 Burton, K. (1956) Biochem. J. 62, 315--323 14 Lowry, O.H., Rosebrough, N.J., F a ~ , A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 15 Spelsberg, T.C., Steggles, A.W. and O'Malley, B.W. (1971) J. Biol. Chem. 246, 4 1 8 8 - - 4 1 9 7 16 McGuire, W.L., Huff, K. and Charnness, G.C. (1972) Biochemistry 11, 4 5 6 2 - - 4 5 6 5