Identification of the porcine intestinal 1,25-dihydroxyvitamin D3 receptor on sodium dodecyl sulfate/polyacrylamide gels by renaturation and immunoblotting.

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RESUMO

Identification of the porcine 1,25-dihydroxyvitamin D3 receptor protein on NaDodSO4/polyacrylamide slab gels was accomplished by two separate techniques: (i) assay of the specific binding activity of tritiated 1,25-dihydroxyvitamin D3 to protein eluted from NaDodSO4/polyacrylamide gels and renatured and (ii) immunoblotting of the partially purified receptor using two anti-receptor monoclonal antibodies. The porcine receptor preparation used in these studies was isolated from a crude nuclear extract of intestinal mucosa followed by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration HPLC, and DEAE-Sepharose chromatography. These receptor fractions were then electrophoresed on NaDodSO4/polyacrylamide gels. The receptor was eluted from the gel, renatured, and assayed for its ability to bind tritiated 1,25-dihydroxyvitamin D3. The renatured receptor appears as a single peak of specific tritiated 1,25-dihydroxyvitamin D3 binding activity. This binding activity corresponds to a band on a silver-stained gel that correlates with the receptor peak eluted from the DEAE-Sepharose column. It also corresponds to the highest molecular weight species identified on an immunoblot with anti-receptor monoclonal antibodies. The 1,25-dihydroxyvitamin D3 receptor protein has a molecular weight of 55,000 as deduced from its migration on NaDodSO4/polyacrylamide gels.

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