Photoaffinity labeling of the 1,25-dihydroxyvitamin D-3 receptor.
Underivatized 1,25-dihydroxy[26,27-3H]vitamin D-3 was successfully used to photoaffinity label the 1,25-dihydroxyvitamin D-3 receptor. The covalent incorporation of tritium into the receptor protein was induced by ultraviolet irradiation of the receptor-1,25-dihydroxy[26,27-3H]vitamin D-3 complex in crude pig intestinal nuclear extract. The amount of incorporated label increased with increasing time of irradiation and was dependent on light of wavelengths 220-280 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography were used to demonstrate that label was incorporated primarily into the 1,25-dihydroxyvitamin D-3 receptor. In addition, the label incorporation was eliminated by competition with a 100-fold excess nonradioactive 1,25-dihydroxyvitamin D-3, indicating that the label was specific for the steroid binding site. Since 1,25-(OH)2[26,27-3H]vitamin D-3 is readily available and requires no special precautions for its preparation and handling, it should be a useful photoaffinity label for future studies of the receptor.
Brown
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Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin, Madison 53706.
DeLuca
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D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002117:Calcitriol / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D007422:Intestines / Q000648:ultrastructure; D008970:Molecular Weight; D010782:Photolysis; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000378:metabolism* / Q000528:radiation effects; D013552:Swine; D014316:Tritium; D014466:Ultraviolet Rays
D000345:Affinity Labels; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D014316:Tritium; D002117:Calcitriol