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The properties of a nuclear acidic protein fraction that binds [6,7-3H]oestradiol-17beta.

ABSTRACT: 1. Additional evidence was obtained that the nuclear oestradiol-17beta receptor is an acidic protein. Partial purification of the receptor protein was obtained by chromatography on hydroxyapatite and it contains protein-bound phosphate. 2. The nuclear ;5s' and cytoplasmic ;9.5s' and ;5s' receptors from uterus, dimethylbenzanthracene-induced mammary adenocarcinoma and kidney are precipitated together with bound oestradiol-17beta by protamine sulphate. This common property suggests that the nuclear and cytoplasmic receptors are related to each other. 3. The properties of two acidic protein fractions from both liver and dimethylbenzanthracene-induced mammary adenocarcinoma are described. Fraction 1 contains two major components and fraction 2 contains one component, as judged from polyacrylamide-gel electrophoresis. Fraction 2 contains RNA and both fractions contain protein-bound phosphate. 4. These fractions form insoluble complexes with calf thymus histone, protamine sulphate and poly-l-lysine. The formation of these complexes is markedly affected by ionic strength and pH. Ionization of both the in-amino group of lysine and carboxyl group are involved. RNA and DNA do not appear to be involved. The interaction is not affected by EDTA or 1mm-Na(+), -K(+), -Ca(2+), -Mg(2+) or -Mn(2+). Per unit weight, whole histone has 4-5 times as many binding sites for the acidic proteins as the latter have for the former. 5. No convincing evidence was obtained for DNA-acidic protein interaction, but, as judged from precipitation experiments, there was competition between DNA and acidic protein for histone. 6. Relatively large amounts of acidic protein partly relieved the histone inhibition of the template activity of DNA for Escherichia coli RNA polymerase (EC 2.7.7.6).

SUBMITTER: King RJ

PROVIDER: S-EPMC1184940 | biostudies-other | 1969 Sep

REPOSITORIES: biostudies-other

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Project description: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.

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The properties of a nuclear acidic protein fraction that binds [6,7-3H]oestradiol-17beta.

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