The use of protamine to study [6,7-3H]-oestradiol-17β binding in rat uterus

Abstract

1. The binding of [6,7-(3)H]oestradiol-17beta to uteri has been studied by using sucrose-gradient analysis and also the property of oestradiol receptors to form insoluble complexes with protamine. 2. Protamine precipitates the 8S and part of the 4S oestradiol-binding proteins in uterine cytoplasm from mature rats. It does not precipitate the oestradiol-17beta-binding proteins present in cytoplasm from non-target tissues or serum. No tritium-labelled material was precipitated by protamine after equilibration of [6,7-(3)H]oestradiol-17beta with either serum albumin or phosvitin. 3. Protamine precipitated a small amount of progesterone but not testosterone or cortisol that had been equilibrated with uterine cytoplasm. It did not precipitate any tritium radioactivity from muscle cytoplasm that had been equilibrated with either [1,2-(3)H]testosterone sulphate or [1,2-(3)H]dehydroepiandrosterone. 4. A simple method has been devised for measuring binding constants of tissue extracts for [6,7-(3)H]oestradiol-17beta, based on precipitation with protamine. Reasonable agreement was obtained between the values obtained by this method and those obtained by sucrose-gradient analysis. 5. This method has been used to study the effect of maturity, ovariectomy, adrenalectomy and hypophysectomy on the cytoplasmic binding of [6,7-(3)H]oestradiol-17beta. None of these procedures affected the dissociation constant K(d) or the number of binding sites/mg of cytoplasmic protein. When measured per uterus or per mg of DNA, ovariectomy and hypophysectomy decreased the number of binding sites. Adrenalectomy had no effect. 6. The properties of the 4S oestradiol-binding protein present in cytoplasm from mature uteri have been studied. It is not present in uteri from immature, ovariectomized, or hypophysectomized rats and it does not bind testosterone or cortisol. Unlabelled oestradiol-17beta, U-11,100A, N-ethylmaleimide and N-bromosuccinimide all decrease the binding of [6,7-(3)H]oestradiol-17beta to both 8S and 4S receptors. Binding to both 8S and 4S receptors decreases when oestradiol is transported to the nucleus. The 4S receptor is not the same as the 4S binding component formed by salt dissociation of the 8S receptor.

Identification of a High Affinity Nuclear Acceptor Site for Estrogen Receptor of Calf Uterus

“…In all reports except one (38), however, the results show that a very limited number of acceptor sites exist on DNA (37). These estimations of the number of sites are in conflict with certain indirect in vivo (27) (40), or to form nonspecific complexes with various basic proteins (41) or polyamines (42). Such mediated interactions are reduced to a minimum, and even the most purified preparations of estrogen receptors bind to the specific agarose derivatives when experimental conditions reported here, are used.…”

Estrogen Receptors in Human Breast Cancer

“…This may well turn out to be true but at present the gradient centrifugation is still necessary to evaluate 4-5S binding. Steggles and King reported that a 4-5S EBP distinct from the usual 8-1OS or salt-derived 4-5S form was present in uteri of mature rats (18). Furthermore, this 4-5S form disappeared after ovariectomy or hypophysectomy and thus could be very important in breast tumor response to endocrine ablation.…”

Soluble Complexes between Steroid Hormones and Target-Tissue Receptors Bind Specifically to Target-Tissue Chromatin

“…Evidence is available showing that there is specific binding in the 4S region (10) as well as in the 8S (5), but not all investigators have confirmed this result. Under the conditions used in our experiments, only 8S estradiol receptors have been found in the cytosols from uteri of ovariectomized rats (27).…”