Exchange assays using sodium thiocyanate to measure total nuclear content of estrogen receptors in the hypothalamic pituitary axis of mice |
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Authors: | Serge Bélisle Jean-Guy Lehoux Diégo Bellabarba |
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Institution: | From the Departments of Obstetrics-Gynecology, Biochemistry and Medicine, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada, J1H 5N4 |
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Abstract: | We studied the effect of sodium thiocyanate (NaSCN) in the determination of specific nuclear estrogen receptors from the hypothalamic-pituitary axis (HPA) of mice. We compared the results with those of the exchange assay using either suspensions of whole nuclei or KC1-nuclear extracts. Our findings demonstrated that 0.5 M NaSCN was more efficient than 0.5 M KC1 in extracting 3H] E2 nuclear content from HPA (91% vs 79.3%). Nuclear fractions extracted with 0.5 M NaSCN revealed the presence of a single class of low-capacity-high affinity binding sites that sedimented in the 4.0 S area. Nuclear binding was T° dependent and reached maximum levels of 450 ± 156 (S.E.) fmol/mg DNA after an overnight incubation at 4°C. Such levels were comparable to those observed in whole nuclei suspensions after 1 hour incubation at 37°C (618 ± 71 fmol/mg DNA, p > 0.05) but two-fold higher (p <0.01) than the concentration of binding sites measured in KC1-extracted nuclear fractions under similar experimental conditions. We conclude that NaSCN extracted the total content of nuclear estrogen receptors in HPA of mature mice. |
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Keywords: | To whom requests for reprints should be addressed |
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