Dye-ligand interactions with 1,25-dihydroxyvitamin D3-receptor complexes from chicken intestine

WS Mellon

A number of dye-ligand adsorbents have been examined for purifying and characterizing 1,25-dihydroxyvitamin D3-receptor complexes from intestines of vitamin D3-deficient chickens. In particular, several triazinyl dyes--Cibacron blue F3GA, Procion red HE3B, and Green A dye, immobilized to agarose via an ether linkage--retain specifically bound 1,25-dihydroxyvitamin D3-receptor complexes formed at 0-4 degrees which are eluted at high salt concentrations. Moreover, receptor binding to these dye-ligand matrices occurs in the presence and absence of sterol. At least for Cibacron blue, the strength of receptor binding depends critically on the method of dye coupling to matrix. The concentration of KCl required for elution of receptor from the triazine ether-linked matrix is greater than coupling through the amine of the anthraquinone via a 10-atom spacer arm approximately equal to coupling through the amine of the anthraquinone via an isourea bond greater than Cibacron blue dextran. Data are presented which demonstrate that sterol-receptor complexes formed at 25 degrees have reduced affinity for dye-ligands when compared with sterol-receptor complexes formed at 0-4 degrees. It is suggested that this finding is related to proteolytic alterations of the receptor, since limited digestion with trypsin can mimic this phenomenon and several protease inhibitors can reduce the thermal- induced alterations. Biospecific elution of receptor is demonstrated using synthetic polyribonucleotides. Preference for polyguanylic and polyinosinic acid is observed over several other polyribonucleotides and mononucleotides. The data in this study, viewed collectively, suggest that there is a specific interaction between the polynucleotide domain of the 1,25-dihydroxyvitamin D3-receptor and several triazinyl dye-ligands. It is concluded that these dye-ligands should prove to be of considerable interest for facile chromatography to purify and characterize this receptor.

Volume 25, Issue 1, pp. 79-85, 01/01/1984
Copyright © 1984 by American Society for Pharmacology and Experimental Therapeutics