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Endocrinology, Vol 122, 1923-1932, Copyright © 1988 by Endocrine Society

ARTICLES

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

DJ Lamb, DW Bullock, RM Hoyte and RB Hochberg
Scott Department of Urology, Baylor College of Medicine, Houston, Texas 77030.

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.




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Copyright © 1988 by The Endocrine Society