Photoaffinity labeling of the 1,25-dihydroxyvitamin D-3 receptor

• Published in: Biochimica et biophysica acta Vol. 1073; no. 2; pp. 324 - 328
• Main Authors: Brown, Thomas A., DeLuca, Hector F.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 04.03.1991; Elsevier
• Subjects: Affinity Labels - metabolism; Animals; Binding, Competitive; Biological and medical sciences; Calcitriol - metabolism; Calcium; Cell Nucleus - chemistry; Cell receptors; Cell structures and functions; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Immunosorbent Techniques; Intestinal nuclei; Intestines - ultrastructure; Molecular and cellular biology; Molecular Weight; Photolysis; Pig intestine; Receptors, Calcitriol; Receptors, Steroid - metabolism; Receptors, Steroid - radiation effects; Steroid hormone receptor; Swine; Tritium; Ultraviolet Rays; Vitamin D; Vitamin D metabolite; Steroid hormone receptor; Vitamin D metabolite; Vitamin D; Intestinal nuclei; Calcium; Pig intestine; Vertebrata; Mammalia; Ultraviolet irradiation; Cholecalciferol(1,25-dihydroxy); Gut; Artiodactyla; Photoaffinity labelling; Ungulata; Pig; Biological receptor
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(91)90138-7

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.