Specific binding of 1alpha,25-dihydroxycholecalciferol to nuclear components of chick intestine.
Specific binding of 1alpha,25-dihydroxycholecalciferol to macromolecular components of small intestinal mucosa nuclei is demonstrated in vitamin D-deficient chicks. The nuclear 1alpha,25-dihydroxycholecalciferol-macromolecule complex was isolated on sucrose density gradients and sediments at 3.7 S in the presence of 0.3 M KCl. Agarose gel filtration of the nuclear component indicated an apparent molecular weight of 47,000. The nuclear receptor complexes could not be distinguished from previously described cytoplasmic 1alpha,25-dihydroxycholecalciferol-binding components by the ultracentrifugation and chromatographic procedures employed. The association of the 3-H-sterol with the nuclear component is thermolabile and is destroyed by treatment with pronase, but not by nucleases; the receptor component is therefore presumed to be a protein. The macromolecular-1alpha,25-dihydroxycholecalciferol complex formed in vivo or in vitro at 25 degrees can be extracted from intestinal nuclei by 0.3 M KCl, but not by low salt buffers. Smaller amounts of the 3.7 S binding component can be detected in isolated purified chromatin or after incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin at 0 degrees. Following incubation of the labeled hormone with reconstituted cytosol-chromatin at 0 degrees, 1alpha,25-dihydroxy[3-H]cholecalciferol is primarily associated with the cytoplasmic receptor, After shifting the incubation temperature to 25 degrees, a progressive increase in the concentration of the nuclear receptor complex and a concomitant decrease in the concentration of the cytoplasmic binding component occur. Thus the 1alpha,25-dihydroxycholecalciferol binding molecules appear to exist primarily in the cytoplasm, where they presumably function to transport the hormone into the nucleus. Experiments employing incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin from nontarget tissues indicate a requirement for both intestinal cytosol and chromatin for maximal formation of the nuclear hormone-receptor complex. These results suggest that the nuclear-binding component arises from hormone-dependent transfer of the cytoplasmic 1alpha,25-dihydroxycholecalciferol receptor to intestinal chromatin acceptor sites.
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002843:Chromatin / Q000378:metabolism; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D004100:Dihydroxycholecalciferols / Q000378:metabolism*; D006887:Hydroxycholecalciferols / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D009928:Organ Specificity; D011402:Pronase; D011506:Proteins / Q000378:metabolism; D011956:Receptors, Cell Surface; D014808:Vitamin D Deficiency / Q000378:metabolism
D002843:Chromatin; D004100:Dihydroxycholecalciferols; D006887:Hydroxycholecalciferols; D011506:Proteins; D011956:Receptors, Cell Surface; D011402:Pronase