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Fig. 6. A typical example of separation of iodinated E-6-BSA (E-1251-BSA) from free 125I by a PD-10 column. Five hundred ml fractions were collected and 5 ¡¡L from each fraction were taken and counted in a gamma counter. The first radioactive peak corresponded to bound 125I and the second radioactive peak corresponded to free 125I.

homogenates. For example, from five rats, the total wet weight of the brain was 7.3 g. In the Pj, mP2, and P3 fractions the protein recovered were 3.9, 0.62, and 0.14%, respectively, of the total protein in the homogenate (about 730 mg protein). The X ± SE values for the P3 fraction in four experiments were 0.19 ± 0.038% corresponding to 3.2 ± 0.85 mg of protein. Thus, the plasmalemma-microsomal fraction contains the least amount of proteins and the largest amount of cell membranes compared to the other two fractions.

Consistent with the ultrastructural differences between the mitochondrial (mP2) and plasmalemma-microsomal (P3) enriched fractions, the radioiodinated ligand E-6-125I-BSA (using the iodogen procedure to label the conjugates shown in Fig. 6) differentially binds to these two fractions, as depicted in Fig. 7. Interestingly, the highest affinity (Kd = 0.14 ± 0.08 nM, n = 3) and the greater number of binding sites (Bmax = 31.3 ± 11.8 pmol/mg protein) correspond to the P3 fraction with a Kd of at least 10 times lower than the one determined for the mP2 fraction. This difference indicates that the sought-out mER is probably an estrogen protein binder localized in the cell membrane of neurons or glial cells with high affinity for E, as one would expect because of the low circulatory levels of this hormone in blood (3). Besides the high affinity and abundance of sites, the binding sites are also selective for E, because P-3-BSA, a progesterone-BSA conjugate, shows very little specific binding when low doses of protein of the P3 fraction are used in the radioreceptor assay (Fig. 8). In addition, unlabeled P-3-BSA is a poor competitor of the ligand binding since the IC50 is greater than 1000 nM (Fig. 9).

If one considers what are thought to be classical features of the estradiol structure for binding—that is, "the molecule should be flat with hydrophilic groups (OH groups) at either end of the molecule, that the middle portion of the molecule be hydrophobic and

Fig. 7. Homologous competitions curves of E-6-125I-BSA binding to subcellular fractions (mP2 and P3) from the whole female rat brain. The data are analyzed by LIGAND program and the Kds are shown in the figure. The concentrations of specific binding sites for mP2 and P3 are 10.2 ± 4.3 and 31.3 ± 11.8 pmol/mg proteins, respectively. The nonspecific binding was obtained in the presence of 1 ^M unlabeled E-6-BSA. Data are expressed as X ± SD. "No" indicates the absence of competitor.

Fig. 7. Homologous competitions curves of E-6-125I-BSA binding to subcellular fractions (mP2 and P3) from the whole female rat brain. The data are analyzed by LIGAND program and the Kds are shown in the figure. The concentrations of specific binding sites for mP2 and P3 are 10.2 ± 4.3 and 31.3 ± 11.8 pmol/mg proteins, respectively. The nonspecific binding was obtained in the presence of 1 ^M unlabeled E-6-BSA. Data are expressed as X ± SD. "No" indicates the absence of competitor.

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