Ultraviolet irradiation of the estrogen binding protein in rat uterine cytosol results in a progressive photoinactivation which is rapid at 254 nm and slower at greater than 315 nm. Both unfilled and estradiol-filled sites are inactivated at approimately the same rates at 254 nm (t 1/2 equals 8 min and 11 min, respectively), but at 315 nm, empty sites are consumed much more rapidly (t 1/2 equals 3.4 hr) than filled ones (t 1/2 equals 24 hr). The protective effect of the estrogen ligand at this wavelength appears to depend on its binding to the estrogen-specific binding site, as inactivation rate studies at different concentrations of estrone, estradiol, and estriol show a good correlation between the extent of protection and the fractional saturation of the high affinity estrogen binding sites. Scatchard analysis indicates that inactivation is the result of a loss of binding sites and not a decrease in their affinity, and sedimentation analysis shows that increased heterogeneity and aggregation of the estrogen binding species accompanies the photoinactivation process. Photoinactivation appears to be the result of direct irradiative damage of the animo acid residues, as the inactivation rate is the same under air and nitrogen atmospheres, and is unaffected by nucleophiles, reductants, and radical scavengers. When photoinactivation is measureed by irradiation of cytosol containing [3-H]estradiol, a concurrent photocovalent attachment process is noted; the steroid becomes linked to protein in a solvent-extractable manner (boiling ethanol inextractable). This attachment, however, does not appear to be related to the steroid binding at the estrogen binding site. Its rate is affected by reductants and scavengers. A similar photocovalent attachment reaction occurs when bovine serum albumin or ovalbumin is irradiated in the presence of [3-H]estradiol or [3-H]diethylstilbestrol. The detailed reactions involved in this photocovalent attachment process have not been defined at present.

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Experiments were performed to determine the in vivo effect of various estrogens and anti-estrogens on the nuclear accumulation and retention of estrogen receptors, the cytoplasmic levels of estrogen receptors, and the formation of salt-resistant and salt-extractable forms of the nuclear estrogen receptor in immature rat uteri. A 5 mug injection of estradiol-17beta (E2) or diethylstilbestrol (DES) resulted in a maximal nuclear translocation of the receptor complexes by 1 h with a subsequent rapid decrease of both the estradiol receptor complex (ERC) and diethylstilbestrol receptor complex (DRC) to levels found in uteri of saline-injected rats by 12 h. However, the antiestrogens U-11,100A, zuclomiphene and enclomphene (100 mug/injection) resulted in a slower nuclear accumulation of receptor complex which continued to increase through 24 h. The cytosol receptor levels with E2 and DES were depleted to 10--20% of control levels within 1 h, but then were replenished so that they were above control levels by 24 h. The clomiphene-type compounds also showed an initial depletion of cytosol estrogen receptor, but the antiestrogens were almost ineffective in receptor replenishment. The estrogen receptor translocated to the nuclear fraction by estrogens demonstrated both salt-extractable (0.3M KCl) and salt-resistant forms at 1--6 h, whereas the clomiphene-type compounds resulted in the formation of only a salt-extractable form of the estrogen receptor at all times. By 12--24 h after injection, the salt-resistant forms of the ERC and DRC were no longer present. The effect of varying the dosage of injected E2 (0.05 mug-5 mug) resulted in the formation of an identical amount of salt-resistant ERC at 1--2 h, whereas the total amount of nuclear ERC (salt-resistant and salt-extractable) varied with the injected dose of E2. However, at 6 h, the amount of salt resistant ERC varied with the injected dose of E2 (0.005-5 mug). These results suggest that the nuclear salt-resistant form (formed by estrogens only) of the estrogen receptor is required for true uterine growth, whereas the nuclear salt-extractable form may be only sufficient for short term estrogenic responses.

Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.

Various aspects of the interaction of oestrogen-receptor complexes with calf uterine chromatin covalently coupled to cellulose were analysed. Partially purified [3Hloestradiol-receptor complexes were bound to intact, or partially deproteinized, chromatin resins. Proteins were removed from the chromatin-cellulose resins by extraction with high molarities of salt, including NaCl/urea, guanidine hydrochloride and guanidine thiocyanate. After extensive washing to remove the salt, [3Hloestradiol-receptor-complex solutions were added to the resins and the degree of binding was determined. The extent of [3H]oestradiol-receptor-complex binding to chromatin was enhanced by extraction of chromosomal proteins. By varying the molarity of the salt, and consequently the extent of protein removal, it was possible to resolve [3Hloestradiol-receptor-complex binding to guanidine thiocyanate-extracted chromatin into two components. Similarly, [3Hloestradiol-receptor-complex binding to guanidine hydrochloride-treated chromatin included three regions of enhanced binding capacity. The [3Hloestradiol-receptor-chromatin interaction was saturable with respect to both intact and salt-extracted resins. Thus uterine chromatin may contain three or more specific classes of acceptors for the oestrogen-receptor complex.For many years the hypothesis that steroidhormone receptors interact specifically with a component of the nucleus has been included in the schematic representation of steroid-hormone action (Liao & Fang, 1969;O'Malley & Means, 1974;Gorski & Gannon, 1976). Early data favoured this general concept; however, the precise nature of the acceptor region has been difficult to determine. More recent efforts probing the nuclear site of action of steroid hormones have suggested the acidic-protein fraction of chromatin as being important in steroidhormone-receptor interactions (Spelsberg et al., 1979).The initial binding by oestrogen to its receptor is characterized by specificity, high affinity and saturability; presumably binding to specific regions in the nucleus is regulated by similar parameters. Thus the nucleus represents a second level of specificity.O'Malley et al. (1972) reported that progesteronereceptor complexes interact specifically with an acidic protein fraction of chick oviduct chromatin, suggesting that non-histone proteins constitute the nuclear site of action of steroid-hormone-receptor complexes. Subsequently, the binding activity of thisAbbreviations used: GuHCl, guanidine hydrochloride; GuSCN, guanidine thiocyanate.Vol. 200 non-histone protein fraction was dissociated from isolated chromatin by 7 M-GuHCI (Thrall et al., 1978). When these chromosomal proteins were separated by isoelectrofocusing and selectively reannealed to DNA, the ability of the progesterone receptor to bind was not limited to a single protein.Rather, three distinct sets of acidic protein-DNA complexes exhibited acceptor activity. These reports from cell-free systems are consistent with other studies which suggest the presence of at least two disti...