Characterization of estrogen receptor in estrogen-dependent transplantable rat pituitary tumor MtT/F84

Properties of nuclear and cytosolic estrogen receptors (ERs) were examined in a new transplantable rat pituitary tumor designated as MtT/F84, of which grow this stimulated by estrogen. The optimal incubation conditions of both nuclear and cytosolic exchange were found to be at 37°C for 15 min and at 25°C for 2 hr, respectively. Molybdate increased a specific binding of estradiol (F₂) as determined by [³H]E₂-binding assay. Sucrose density gradient analyses of crude cytosol revealed specific peaks of radioactivity in both 4-5S and 8-10S areas. How ever, only a single 5S peak was present in 0.4M KCl-extractable nuclear ER. Molybdate also enhanced the stability of cytosolic 8-10S receptor in density gradient sedimentation behavior. Scatchard plot analysis for nuclear ER yielded a single class of binding sites with a dissociation constant (Kd) of 0.317 nM and the maximum number of binding sites (NBS(max)) of 25.4 fmol/mg protein. Saturation analysis of [³H]estrogen binding to cytosolic ER also yielded a straight line with a Kd of 0.146 nM and NBS(max) of 58.5 fmol/mg protein. The effect of E₂ administration on the intracellular distribution of ER was also examined. A marked disappearance in the ER binding in cytosol with a concomitant increase in binding in nuclear fraction was found after the administration of the unlabeled E₂ in vivo, whereas the total number of ER did not change. Thus, it is concluded that properties of ER in the MtT/F84 were very similar to those in other target organs such as uterus and pituitary gland.