Comparison of the binding of [2,4,6,7-3H] estradiol-17β [E23H] to the immature rat uterus under in vivo, in vitro and “cell-free” conditions—I. Some characteristics of the cytoplasmic estradiol-receptor complex

Abstract A comparative study was done on the cytoplasmic estradiol-receptor complex obtained after in vivo infusion of E 2 3 H or after whole tissue incubation in the presence of E 2 3 H or after “cell-free” incubation with E 2 3 H of cytosol prepared in buffers of varying ionic strengths. Sucrose density gradients containing 0.3 M KCl showed that cytosol after in vivo infusion sedimented as a distinct peak in the “4S” region whereas after either “whole-tissue” or “cell-free” incubations in the presence of E 2 3 H both “4S” and “5S” peaks were present simultaneously, provided the incubation was done at higher temperatures. Under “cell-free” conditions, a comparison of the dissociation kinetics of cytosol estradiol-receptor complexes showed two first-order components irrespective of the buffer utilized (k 1 D:1 to 2 × 10 −4 s −1 and k 2 D:1 to 2 × 10 −5 s −1 . However, the quantitative importance of the two components was different depending on the presence of NaCl. The rapidly dissociating component was 3 or 4 times more important than the slowly dissociating component in the absence of salt, whereas in the presence of salt, the slowly dissociating component was twice the rapidly dissociating component. Similarly, the apparent dissociation constants were the same in Tris-EDTA and Tris-NaCl buffers (1.5 × 10 −10 M). Sucrose density gradients revealed the presence of aggregates in the presence of NaCl. These observations tend to show that experimental procedures may have a preponderant influence on some characteristics of the cytosol receptor, when analyzed in a “cell-free” system, and which appear to be more complex as compared to in vivo conditions.