Effect of chemical perturbation with NaSCN on receptor-estradiol interaction. A new exchange assay at low temperature

V. Sica, G. A. Puca, A. M. Molinari, F. M. Buonaguro, F. Bresciani

Research output: Contribution to journalArticle

Abstract

When 0.5 M sodium thiocyanate is added to uterine cytosol previously labeled with excess [ 3H]-17β-estradiol, no change can be detected in the steady-state cytosol concentration of [ 3H]estradiol-receptor complex for at least 20 h at 4°C. However, the rate of exchange of bound estradiol in the presence of NaSCN was found to be substantially higher than that in the absence of the chaotropic salt. In the presence of NaSCN, the dissociation rate of the complex increases about 10-fold (K -1(SCN) = 1.10 x 10 -2 min -1 vs. K -1 = 1.07 x 10 -3 min -1) while the rate of association increases about 2-fold (K 1(SCN)=1.2 x 10 7 min -1 M -1 vs. K 1 = 7.4 x 10 6 min -1 M -1). The K(d) changes 6.4-fold (K(d)(SCN) = 9 x 10 -10 M vs. K(d) = 1.4 x 10 -10 M) with no decrease in the number of binding sites as shown by Scatchard plots of saturation experiments. This effect of NaSCN can be exploited to assay preformed estrogen-receptor complex by exchange with [ 3H]estradiol at low temperature. When the sample containing preformed complex is incubated overnight (16h) at 4°C with excess [ 3H]estradiol in the presence of 0.5 M NaSCN, there is a quantitative exchange of nonlabeled for labeled estradiol without loss of binding sites. Hormonal steroids other than estrogens do not interfere, and the exchanged estradiol is bound with high affinity. Precision, accuracy, and linearity of the method are highly satisfactory.

Original languageEnglish
Pages (from-to)83-88
Number of pages6
JournalBiochemistry
Volume19
Issue number1
DOIs
Publication statusPublished - 1980

ASJC Scopus subject areas

  • Biochemistry

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