Specific binding of 1α,25 dihydroxycholecalciferol to nuclear components of chick intestine

P. F. Brumbaugh, Mark R Haussler

Research output: Contribution to journalArticle

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Abstract

Specific binding of 1α,25 dihydroxycholecalciferol to macromolecular components of small intestinal mucosa nuclei is demonstrated in vitamin D deficient chicks. The nuclear 1α,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 1α,25 dihydroxycholecalciferol binding components by the ultracentrifugation and chromatographic procedures employed. The association of the 3H 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 1α,25 dihydroxycholecalciferol complex formed in vivo or in vitro at 25° 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 1α,25 dihydroxy[3H]cholecalciferol with reconstituted cytosol chromatin at 0°. Following incubation of the labeled hormone with reconstituted cytosol chromatin at 0°, 1α,25 dihydroxy[3H]cholecalciferol is primarily associated with the cytoplasmic receptor. After shifting the incubation temperature to 25°, 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 1α,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 1α,25 dihydroxy[3H]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 1α,25 dihydroxycholecalciferol receptor to intestinal chromatin acceptor sites.

Original languageEnglish (US)
Pages (from-to)1588-1594
Number of pages7
JournalJournal of Biological Chemistry
Volume250
Issue number4
StatePublished - 1975

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Calcitriol
Chromatin
Intestines
Cytoplasmic and Nuclear Receptors
Cytosol
Cholecalciferol
Hormones
Pronase
Calcitriol Receptors
Ultracentrifugation
Sterols
Intestinal Mucosa
Macromolecules
Vitamin D
Sepharose
Gel Chromatography
Sucrose
Buffers
Sediments
Cytoplasm

ASJC Scopus subject areas

  • Biochemistry

Cite this

Specific binding of 1α,25 dihydroxycholecalciferol to nuclear components of chick intestine. / Brumbaugh, P. F.; Haussler, Mark R.

In: Journal of Biological Chemistry, Vol. 250, No. 4, 1975, p. 1588-1594.

Research output: Contribution to journalArticle

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abstract = "Specific binding of 1α,25 dihydroxycholecalciferol to macromolecular components of small intestinal mucosa nuclei is demonstrated in vitamin D deficient chicks. The nuclear 1α,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 1α,25 dihydroxycholecalciferol binding components by the ultracentrifugation and chromatographic procedures employed. The association of the 3H 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 1α,25 dihydroxycholecalciferol complex formed in vivo or in vitro at 25° 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 1α,25 dihydroxy[3H]cholecalciferol with reconstituted cytosol chromatin at 0°. Following incubation of the labeled hormone with reconstituted cytosol chromatin at 0°, 1α,25 dihydroxy[3H]cholecalciferol is primarily associated with the cytoplasmic receptor. After shifting the incubation temperature to 25°, 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 1α,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 1α,25 dihydroxy[3H]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 1α,25 dihydroxycholecalciferol receptor to intestinal chromatin acceptor sites.",
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AB - Specific binding of 1α,25 dihydroxycholecalciferol to macromolecular components of small intestinal mucosa nuclei is demonstrated in vitamin D deficient chicks. The nuclear 1α,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 1α,25 dihydroxycholecalciferol binding components by the ultracentrifugation and chromatographic procedures employed. The association of the 3H 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 1α,25 dihydroxycholecalciferol complex formed in vivo or in vitro at 25° 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 1α,25 dihydroxy[3H]cholecalciferol with reconstituted cytosol chromatin at 0°. Following incubation of the labeled hormone with reconstituted cytosol chromatin at 0°, 1α,25 dihydroxy[3H]cholecalciferol is primarily associated with the cytoplasmic receptor. After shifting the incubation temperature to 25°, 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 1α,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 1α,25 dihydroxy[3H]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 1α,25 dihydroxycholecalciferol receptor to intestinal chromatin acceptor sites.

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