title: Characterization and stabilization of progesterone receptors in human benign prostatic hypertrophy.
abstract: Experimental conditions for the optimum detection and measurement of the cytosolic progestogen receptor in human prostatic hyperplasia and neoplasia are described. In presence of 20 mM molybdate ion and a reaction time of 0.5-2 h at 0-2 degrees C, we are able to detect the appearance of a [6.7-3H]-17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione ([3H]-R5020) and [3H]-progesterone binding moiety in human prostatic cytosol. The [3H]-R5020 binding protein sediments at approximately 8-11S in a glycerol density gradient centrifuged in a Sorvall TV865 rotor (vertical rotor). Maximal binding of [3H]-R5020 occurs at 30 min at 25 degrees C and 1 h at 0 degree C. Considerable overall improvement in receptor detection and measurement was made when gradient centrifugation was carried out in a vertical rotor instead of swing bucket rotor. The specifically bound [3H]-R5020 is displaced by progesterone, triamcinolone acetonide and R5020, but not by cortisol, dihydrotestosterone, 17 beta-estradiol, or diethylstilbestrol. High affinity was demonstrated by Scatchard analysis on binding of [3H]-5020 to receptors from cytosol of benign prostatic hypertrophy (BPH) giving a KD of 2.4 X 10(-10)M and a binding site concentration of 50 fmol/mg protein. Molybdate ion maintains the hormone receptor complex in 8-11S form which is totally converted to the 4-5S form in the presence of 0.6 M K Cl. The optimum concentration of fluoride ion for enhancing the specific binding of [3H]-R5020 was determined to be between 20 and 50 mM. Cytosol from human BPH in the presence of 25 mM sodium fluoride contains [3H]-R5020 receptors which sedimented mostly in the 4-5S region of glycerol density gradient. Sodium-molybdate and sodium fluoride stabilize and stimulate the specific [3H]-R5020 receptor complex at 25 and 0 degree C. The loss of specific receptor hormone binding capacity can be reversed by fluoride and, to a lesser extent, by molybdate.
mesh_terms: D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D008297:Male; D008982:Molybdenum / Q000494:pharmacology; D011374:Progesterone / Q000378:metabolism; D011397:Promegestone / Q000378:metabolism; D011467:Prostate / Q000378:metabolism*; D011470:Prostatic Hyperplasia / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000378:metabolism; D011980:Receptors, Progesterone / Q000187:drug effects / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology
chemical_list: D011980:Receptors, Progesterone; D011374:Progesterone; D008982:Molybdenum; D012969:Sodium Fluoride; C024687:sodium molybdate(VI); D011397:Promegestone