Exchange assays using sodium thiocyanate to measure total nuclear content of estrogen receptors in the hypothalamic pituitary axis of mice.
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 degrees dependent and reached maximum levels of 450 +/- 156 (S.E.) fmol/mg DNA after an overnight incubation at 4 degrees C. Such levels were comparable to those observed in whole nuclei suspensions after 1 hour incubation at 37 degrees C (618 +/- 71 fmol/mg DNA, p greater than 0.05) but two-fold higher (p less than 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.
D000818:Animals; D002467:Cell Nucleus / Q000032:analysis; D005260:Female; D007030:Hypothalamo-Hypophyseal System / Q000032:analysis*; D008722:Methods; D051379:Mice; D008810:Mice, Inbred C57BL; D011960:Receptors, Estrogen / Q000032:analysis*; D013861:Thiocyanates
D011960:Receptors, Estrogen; D013861:Thiocyanates; C024553:sodium thiocyanate