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, Mark 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 - 1987

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Calcitriol Receptors
Electrophoresis
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
Chickens
Proteolysis
Liver Extracts
Collodion
Intestinal Mucosa
Chromatography
Purification
Proteins
Molecular Weight
Gels
Molecular weight
Monoclonal Antibodies
Tissue
Staining and Labeling
Peptides
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chicken intestinal receptor for 1,25-dihydroxyvitamin D3. Immunologic characterization and homogeneous isolation of a 60,000-dalton protein. / Pike, J. W.; Sleator, N. M.; Haussler, Mark R.

In: Journal of Biological Chemistry, Vol. 262, No. 3, 1987, p. 1305-1311.

Research output: Contribution to journalArticle

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