Chicken intestinal receptor for 1,25-dihydroxyvitamin D3. Immunologic characterization and homogeneous isolation of a 60,000-dalton protein

J. W. Pike, N. M. Sleator, M. R. Haussler

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Abstract

The chick 1,25-dihydroxyvitamin D3 receptor has been identified via immunoblot analysis and isolated to homogeneity via positive immunoselection and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cytosolic extracts of intestinal mucosa, as well as purified samples highly enriched for receptor by nonimmunologic methodology were electrophoresed on denaturing gels, transferred to nitrocellulose, and probed utilizing a purified monoclonal antibody against the chick receptor. Two protein signals were detected by this approach, a major species of 60,300 daltons and a minor form at 58,000 daltons. Both immunologically identified species were present in receptor-positive tissues but were absent in receptor-negative liver extracts. The two immunoreactive cytosolic proteins comigrated with two polypeptides detected via Coomassie Blue staining as well as by immunoblot analysis after enrichment utilizing DNA-cellulose, blue dextran-Sepharose, and other chromatographic separation techniques. Increasing concentrations of the minor form during purification suggest it arises from the larger molecular weight species via proteolysis. Finally, both forms of the receptor were isolated to near homogeneity employing positive immunoselection and each individually purified to homogeneity employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These experiments show that the chick receptor exists as a major species of 60,300 as well as a minor form of 58,600 and that both forms can be purified to homogeneity via immunoaffinity chromatography.

Original languageEnglish (US)
Pages (from-to)1305-1311
Number of pages7
JournalJournal of Biological Chemistry
Volume262
Issue number3
StatePublished - Jan 1 1987

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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