An exchange assay for quantitating 1,25-dihydroxyvitamin D3 receptors.
Steroid hormone receptors, including those for vitamin D3, contain reactive sulfhydryl group(s) essential for hormone binding. On this basis, several compounds that are capable of interacting with sulfhydryl groups were tested for their ability to dissociate 1,25-dihydroxyvitamin D3 from chicken intestinal receptors. At concentrations resulting in 50% displacement of specifically bound cytoplasmic 1,25-dihydroxyvitamin D3, the order of potency for these displacing reagents is mersalyl acid greater than sodium thiocyanate approximately equal to p-hydroxymercuribenzoate approximately equal to mercuric chloride much greater than 5,5'-dithiobis-(2-nitrobenzoic acid). Hormone displacement by mersalyl acid and p-hydroxymercuribenzoate was completely reversible upon dithiothreitol addition. In contrast limited rebinding of 1,25-dihydroxyvitamin D3 occurred after, 5,5-dithiobis-(2-nitrobenzoic acid) and mercuric chloride treatment. Furthermore, at least for mersalyl acid treatment, hormone displacement and subsequent regeneration of sterol binding did not seem to alter the integrity of the receptor as evidenced by sucrose gradient analysis, DNA-cellulose and phosphocellulose chromatography. Additionally, treatment of the nuclear 1,25-dihydroxyvitamin D3 receptor did not significantly affect the apparent equilibrium dissociation constant for hormone binding (Kd = 2.7 X 10(-10) M; Kd = 1.7 X 10(-10) M, for control and mersalyl acid treated receptor, respectively). Finally, a method has been developed for measurement of occupied 1,25-dihydroxyvitamin D3 receptors. The unfilled receptors were quantitated in the cytoplasm or chromatin extracted fraction by incubation with radioactive 1,25-dihydroxyvitamin D3 at 0-4 degrees C for 3-6 h without interference from previously filled sites. The total receptor measurement is carried out by incubation of cytosol or nuclear extract with 0.5-1.0 mM mersalyl acid for 1.0 h. Exchange of radioactive sterol for the bound nonradioactive sterol is accomplished by incubation with 1-2 mM dithiothreitol and 1.0 nM radioactive 1,25-dihydroxyvitamin D3 at 0-4 degrees C for 16 h. Subtracting the unfilled sites from total sites results in a measurement of filled sites. With this exchange assay, estimates of total receptor in untreated and 1,25-dihydroxyvitamin D3 treated chicks (estimated 2 h after injection of doses 0.3-300 nmol) were not significantly different (66.2 +/- 4.5 versus 64.4 +/- 10.4 to 69.0 +/- 5.9 pg/mg protein, respectively). Furthermore, quantitation of total receptor by direct or exchange assay obtained from in vitro incubations of intestinal slices with radioactive and nonradioactive 1,25-dihydroxyvitamin D3 were not[1]References
- An exchange assay for quantitating 1,25-dihydroxyvitamin D3 receptors. Radparvar, S., Mellon, W.S. J. Steroid Biochem. (1984) [Pubmed]
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