The effect of leupeptin on progestin receptors of human uterine tissues

In preparation for studies of progestin receptor dynamics in human uterine tissues, we have investigated the effect of tissue handling and the protease inhibitor leupeptin on the molecular forms of progestin receptors in human endometrium, myometrium, and leiomyoma. Tritiated R5020 [17,21-dimethyl-19-nor-4,9-pregnadiene-3.20-dione) (promegestone)] was the labeled ligand, and sucrose density gradient ultracentrifugation was used to determine the sedimentation coefficients of the receptors. Scatchard plots of saturation analyses were used to determine the number of binding sites and dissociation constants. We found that rapid chilling of tissue at surgery was necessary to maintain specific progestin binding in myometrium, that leupeptin permitted demonstration of 8S receptors in myometrium and leiomyoma, and that 8S receptors were present in endometrium, with or without leupeptin. With careful handling of tissue and minimal homogenization, leupeptin may not be necessary to preserve 8S receptors in myometrium and leiomyoma cytosols; however, the use of leupeptin gives greater assurance that 8S receptors will be preserved. The number of binding sites varied from 0.6-2.0 pmol/mg cytosol protein, and the Kd values observed were between 1.7-6.9 X 10(-10) M.