Experimental conditions for the optimal measurement of estrogen (ER) and progesterone (PR) receptors in normal vervet monkey (Cercopithecus aethiops pygerythrus) uteri are described. The uteri of this primate were found to contain relatively high concentrations of both ER and PR. Levels of ER ranged from 151 to 822 femtomoles per mg protein (mean for group assayed is 327 +/- 165 femtomoles per mg protein). PR assays were performed on the same cytosols and the levels ranged from 444 to 2267 femtomoles per mg protein (mean of 1285 +/- 511 femtomoles per mg protein). Mean Kd values for the ER- and PR-ligand complexes were found to be 3.15 +/- 1.4 X 10(-10)M and 2.38 +/- 0.2 X 10(-9)M respectively, within the group analysed (n = 21). The ratio of PR to ER varied between 1.1 and 13.1 with a mean of 4.5 +/- 2.4. Ligand specificity studies revealed that [3H]-17 beta-estradiol binding to the ER could only be inhibited by estrogens or estrogen analogues. The PR however exhibited an affinity for a wider range of ligand types. In low ionic strength buffers both ER and PR sedimented as approximately 8S type molecules in the presence or absence of 10mM sodium molybdate. Both receptors dissociated into smaller components, following a short exposure to 0.4 M KCl and subsequent centrifugation in a gradient containing 0.4 M KCl.
Sodium molybdate affected the stability of vervet monkey (Cercopithecus aethiops pygerythrus) uterine estrogen (ER) and progesterone (PR) receptors. Yields of receptors were invariably higher (20-40%) when cytosols were prepared in the presence of 10mM sodium molybdate. No changes were observed in the binding affinities for the natural ligands as reflected in dissociation constants. Receptor-ligand association at 0 degrees C and 20 degrees C was not affected in the presence or absence of molybdate. Stability studies at 37 degrees C indicated both receptors to be more resistant to inactivation in the presence of molybdate. Dissociation of ER and PR was biphasic, indicating the existence of slow (SDC), as well as fast dissociating (FDC) complexes. Rate constants of dissociation were significantly affected by the presence of sodium molybdate. Although no significant changes in the sedimentation coefficients were observed, marked differences in the actual gradient profiles could be illustrated in the presence or absence of sodium molybdate. Observed effects could only be partially reversed in sedimentation dialysis experiments. Proteolytic inhibitors phenylmethylsulfonylfluoride (PMSF) and leupeptin had no inhibitive effect on the molybdate stabilization of ER and PR.
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