Δ9-[16α-125I]Iodo-19-Nortestosterone: A Gamma-Emitting Photoaffinity Label for the Progesterone Receptor*

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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.