delta 9-[16 alpha-125I]iodo-19-nortestosterone: a gamma-emitting photoaffinity label for the progesterone receptor

Abstract

We have synthesized 16 alpha-iodo-4,9-estradien-17 beta-ol-3-one [delta 9-16 alpha-iodo-19-nortestosterone (delta 9-INT)] labeled with 125I (delta 9-[16 alpha-125I]INT) to provide a new gamma-emitting photoaffinity ligand for the progesterone receptor that has many advantages over the currently available [3H]R5020. We have characterized the interaction of delta 9-[16 alpha-125I]INT with the rabbit uterine progesterone receptor and have demonstrated the usefulness of this compound for studies of receptor structure. The binding of 2 nM [3H]progesterone to receptor in rabbit uterine cytosol was specifically competed for by 19-nortestosterone, 16 alpha-iodo-19-nortestosterone, and delta 9-INT. Scatchard analysis demonstrated that delta 9-[16 alpha-125I]INT and [3H]progesterone estimated the same number of binding sites in rabbit uterine cytosol, with a Kd for delta 9-[16 alpha-125I]INT of about 2.7 nM. The binding of delta 9-[16 alpha-125I]INT was inhibited by both progesterone and R5020, whereas testosterone, estradiol, and 5 alpha-dihydrotestosterone were ineffective. In cytosol, delta 9-[16 alpha-125I]INT covalently labeled the same mol wt receptor forms as [3H]R5020. Although the efficiency of cross-linking was similar for [3H]R5020 (3%) and delta 9-[16 alpha-125I]INT (4%), the radioactivity was 10-fold greater due to the higher specific activity of delta 9-[16 alpha-125I]INT and the lack of sample quench. The use of delta 9-[16 alpha-125I]INT greatly increases the sensitivity and efficiency of the photoaffinity labeling technique; it will provide a valuable tool for further studies of the progesterone receptor, allowing the detection of receptor in dilute cytosol after gel electrophoresis under denaturing conditions.