Vol. 132, No. 1, 1985 October 15, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 7 98-203 EVALUATION OF A PHOTOLABILE DERIVATIVE OF 1,2SDIHYDROXYVITAMIN D3 AS A PHOTOAFFINITY PROBE FOR 1,25-DIHYDROXYVITAMIN-D3 RECEPTOR IN CHICK INTESTINAL CYTOSOL Rahul Ray, Stanley Rose, Sally Ann Holick, and Michael F. Holick USDA/Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111 Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139 Received August 3, 1985 Summary: We evaluated the viability of la,25-dihydroxyvitamin D3-3B-[N-(4-azido-2-nitropheny1)glycinate-J (1,25-(OH)2-D3-ANG), an analog of la,25-dihydroxyvitamin 03 (1,25-(OH)2-D3) as a photoaffinity probe for 1,25-(OH)2-D3 receptor in chick intestinal cytosol. A competitive-binding assay revealed that chick intestinal cytosolic 1,25-(OH)2- 03 receptor bound to 1,25-(OH)2-D3-ANG approximately 20-times less effectively than it did to 1,25-(OH)2-D3. Irradiation of 1,25-(OH)2-D3- ANG in the presence of chick intestinal cytosolic preparation significantly diminished subsequent binding to 3H-1,25-(OH)2-D3, suggesting that the photoaffinity analog was covalently attached to the receptor. 1,25-(OH)2-D Therefore the nitroarylazide derivative of of the 1,25- i may be a valuable photoaffinity probe for the characterization OH)2-D3 receptor. 6 1985 Academic Press, Inc. Introduction: It is established that vitamin D3 undergoes sequential metabolism to 25-hydroxyvitamin D3 (25-OH-D3) in the liver and then to 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) in the kidney (1,2). A receptor-like protein having high affinity and low capacity for 1,25-(OH)2-D3 was identified in the intestinal cytosol and the nuclei (1,2). Photoaffinity labelling of steroid and peptide hormone receptors has been extremely valuable in localizing and isolating membrane-bound, nuclear and cytoplasmic receptors and for structure--function analyses (3). We initiated a program to synthesize a photoaffinity-labelled derivative of 1,25-(OH)2-03. Based on previous structure--activity analyses for the chick intestinal 1,25-(OH)2-D3 receptor (2) that showed that the la-OH and 25-OH are important recognition markers, we derivatized the 3B-OH via an ester linkage with a glycine analog containing a 4-azido-2-nitro-phenyl moiety. We report 0006-291X/85 $1.50 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. 198