Characterization and stabilization of progesterone receptors in human benign prostatic hypertrophy

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.