The measurement of oestrogen receptors in the nuclei of cells of human breast cancer is becoming increasingly important for patient management. However, the steroid-binding properties of the oestrogen nuclear receptor of human cells under different conditions of temperature and ionic strength have received little attention despite the relevance of the receptor for interpretation of assay data. This paper reports a study on the influence of temperature and ionic strength on the exchange rate of [3H]oestradiol from human breast and endometrial nuclear receptor.
When the oestrogen–nuclear receptor complex was bound to intact or sheared nuclei, the displacement of bound [3H]oestradiol into buffer containing excess unlabelled oestradiol increased with temperature but was significant over 24 h even at 4 °C for nuclei from both breast and endometrium. The use of protease inhibitors combined with relabelling of nuclear receptor after incubation confirmed that the observations at 4 °C represented exchange of hormone rather than degradation of the hormone–receptor complex. Degradation was seen at higher temperatures.
Measurement of the on-rate of [3H]oestradiol onto nuclear receptor prefilled with unlabelled oestradiol showed that on-rate was also significant over 24 h at 4 °C The displacement of oestradiol from salt-extracted, hydroxylapatite-precipitated receptor also increased with temperature although, in this case, no displacement could be detected until temperatures above 4 °C were reached.
Only 40% of total oestrogen receptor detectable in intact human nuclei was solubilized by the standard method of salt extraction in 0·6 mol KC1/1. As the salt concentration was raised (0–0·6 mol KC1/1), an increase in the stripping of oestradiol from the hormone–receptor complex was observed. Such stripping of nuclear receptor during salt extraction would lead to a false impression of the proportion of 'empty' nuclear receptors.
The results show that filled oestrogen nuclear receptor from human tumour tissue may be assayed by exchange at 4 °C over 24 h. This eliminates the protease degradation of receptor which occurs at higher assay temperatures.