The thermolability of rat uterine cytosol estrogen receptors: Basis for a novel cytosol exchange-denaturation assay

Abstract

Thermal denaturation of the rat uterine cytosol estrogen receptor obeys simple first-order reaction kinetics in the temperature range from 0–48°C, whether 17β-estradiol is present or not. The rate of inactivation is five times faster in the absence of 17β-estradiol than in its presence, and was shown to be independent of protein concentration over a wide range. Analysis of denaturation data as a function of variable temperature by Arrhenius plots showed that: (1) the stability of the receptor to heat effects is enhanced dramatically by the presence of 17β-estradiol; (2) 5α-dihydrotestosterone exerts a protective effect on the receptor, whereas progesterone does not; and (3) inactivation of binding at temperatures below 30°C is much more rapid than would be predicted from its behavior above 30°C. A cytosol receptor assay has been developed which exploits the fact that denaturation of the estrogen receptor does occur during thermally-accelerated isotope exchange procedures. When receptor site exchange is performed at 40°C, bound radio-labeled steroid achieves a maximal level within 3 min, at which point bound and unbound pools of steroid are at equilibrium, with the same specific activity. For the remainder of a 10-min incubation period at 40°C, binding decreases linearly as the receptor undergoes denaturation. By extrapolation of the receptor denaturation curve to zero time and correction for dilution of specific activity, an accurate determination of total specific estrogen receptor binding site concentration is realized.