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Fig. 8. Differential binding of E-6-125I-BSA and P-3- 125I-BSA to CS-P3 fractions (0.5 ^g protein). A single experiment in duplicate for each case.

UNLABELED LIGAND (MOLAR)

Fig. 8. Differential binding of E-6-125I-BSA and P-3- 125I-BSA to CS-P3 fractions (0.5 ^g protein). A single experiment in duplicate for each case.

NO -11 -10 -9 -8 -7 -6 -5 UNLABELED COMPETITORS (MOLAR)

Fig. 9. Competition curve of several steroid-BSA conjugates of ligand binding E-6-1251-BSA to CS-P3 fractions (0.5 ^g protein). One experiment in duplicate for each case.

NO -11 -10 -9 -8 -7 -6 -5 UNLABELED COMPETITORS (MOLAR)

Fig. 9. Competition curve of several steroid-BSA conjugates of ligand binding E-6-1251-BSA to CS-P3 fractions (0.5 ^g protein). One experiment in duplicate for each case.

Fig. 10. Specific binding of E-6-125I-BSA (45,000 cpm/0.5 mL, 0.14 nM) to CS-P3 (0.5 ^g) in duplicates displaced by unlabeled E-6-BSA and 17a-E-6-BSA. The total binding without competitor (about 4000 cpm) is considered as 100%. "NO" indicates the absence of competitor.

Fig. 10. Specific binding of E-6-125I-BSA (45,000 cpm/0.5 mL, 0.14 nM) to CS-P3 (0.5 ^g) in duplicates displaced by unlabeled E-6-BSA and 17a-E-6-BSA. The total binding without competitor (about 4000 cpm) is considered as 100%. "NO" indicates the absence of competitor.

that the distance between terminal hydroxyls (1.09 nm) be highly specific" (32,33), it is intriguing that another conjugate testosterone-BSA (T-3-BSA) with the OH (C-3) blocked by the BSA has a comparable high affinity for the E binding sites in these membranes (Fig. 9), indicating that the OH (C-17) is important for the binding. Moreover, if the BSA blocks the OH (C-17) and leaves the OH (C-3) available for binding, higher affinity is observed. In addition, the binding of E-6-BSA is highly sterospecific because its a isomer is a poor competitor (Fig. 10). Therefore, it seems that both hydroxyl groups are required

Fig. 11. Specific binding curves for B-mP2 and B-P3 using several doses of proteins. Each point represents three duplicate experiments and the values are expressed as X ± SD.

for binding, and they must be oriented in the p position. It would be of interest to know if E-17-BSA and T-3-BSA are also active in releasing DA from striatal fragments. The specific binding of E-6-125I-BSA represents over 90% of total binding and increases linearly as a function of low doses of protein either in P3 fractions derived from brain or CS tissues (Figs. 11 and 12, respectively). Note that at equivalent concentration of proteins much higher specific binding is detected in the P3 than in the mP2 fractions from brain origin (Fig. 11) This is consistent with the finding that there are greater number of binding sites in the cell membranes than in the mitochondrial fraction, as previously shown.

Further experiments were performed to determine the binding kinetics of E-6-125I-BSA to CS-P3 from female rat brains. Figure 13A shows the association curve of E-6-125I-BSA to CS-P3 membranes over time. The binding was rapid and reached equilibrium by about 20 min at 4°C. This association kinetics is more rapid (k1 = 0.33 nM-1 min1) than that of P2 from brain, which reached equilibrium only after about 30 min (29). Figure 13B shows the dissociation curve of the labeled ligands from their binding sites in CS-P3. This dissociation constant is estimated to have a k2=0.075 min1. The Kd calculated from these data revealed a Kd value of 0.23 nM close to the one obtained by competition binding, demonstrating the validity of the assay.

Though we have reported earlier that free estradiol (but not 17a-E) is a partial competitor (IC50 about 1000 nM) of the ligand binding to P2 brain fractions (11) and that 125I-BSA does not bind this fraction nor the unlabeled BSA competes, it seems of interest to compare how these estradiol conjugates will behave in a classical uterine cytosolic preparation in which the nuclear estradiol receptor (nER) is labeled with 3H-estradiol. Uterine

Fig. 12. Similar to Fig. 11, but using CS-P3 fractions.

cytosols (rich in nERs) were prepared from immature female rats in TE buffer (10 mM Tris, 0.2 mM ethylenediaminetetraacetic acid [EDTA], pH 7.5) according to a modified procedure of Bruns et al. (34). Briefly, immature female rats (30 d old) were sacrificed by decapitation and the uteri were removed quickly into cold TE buffer. The uteri were minced and homogenized in the same Teflon glass homogenizer at 1 uterus/mL TE for 2 min. The homogenate was then ultracentrifuged at 125,000g for 60 min; the supernatant (S3) obtained was considered the uterine cytosol. The protein concentration was estimated by the method of Bradford (35) using BSA as standard, and stored in aliquots at -70°C. The saturation and competition assay of the binding of [3H]- estradiol (85 Ci-mmol, NEN) to intracellular estradiol receptors (nER) in uterine cytosol of immature rats were performed at 4°C in TE buffer or TE plus 0.08% BSA (TEB). [3H]-estradiol (2 or 10 nM for competition assay and 0.1-10 nM for saturation assay) was incubated for 1.53 h with uterine cytosol (80-440 mg proteins) and competing compounds in 500 mL or 250 mL volume. The ethanol used to dissolve the ligand and steroids was in the range of 0.004-0.4%. 200-500-fold E or DES was used to determine the nonspecific binding. The bound [3H]- estradiol was separated from free using the following two methods: 1. Rapid filtration by polyethylenimine (PEI)-treated filters: The rapid filtration using PEI-treated Whatman GF/B glass filters for separation of bound from ligand was based on the method of Bruns et al. (34) Briefly, the glass filters were soaked in 0.3% PEI (v/v) for 1-3 h at room temperature and placed on a 1225 sampling manifold. The manifold was then transferred to the cold room (4°C) for about 1 h before use. After the incubation, the reaction solution was poured onto PEI-treated filters in the manifolds under reduced pressure. The tubes and filters were washed with two 5 mL P2-Tris incubation buffer (TE or TEB). The radioactivity retained by the filters was counted using aqueous scintillation

Association

o -■-1-■-1-■-10 10 20 30 Time (min)

Dissociation

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