Prostate tissue was homogenized on ice in low-salt TEGD buffer (1.5 mM disodium ethelynediaminetetraacetate, 1.0 mM phenyl-methyl sulfonyl fluoride, 1.0 mM sodium molybdate, 1.0 mM dithiothre-itol, 10 mM Tris, and 10% glycerol) at 10 ml per gram of tissue. Homog-enates were centrifuged (30 000 X g) and supernatants were pooled.
PZ was used at concentrations of 1, 3, 10, 30, 100, and 300 |xM; and 1 and 3 mM in duplicate during three separate runs. The assay mixture including PZ was incubated on a rotary mixer for 24 h at 4°C in 12 X 75 mm glass tubes with [3H] R1881 (83.5 Ci/mmol) and 10 |xM triamcinolone acetate (to fully bind progesterone and glucocorticoid receptors). Incubations with a 100-fold molar excess of inert R1881 were used to estimate nonspecific binding. All incubations were performed using 300 |xl of pooled prostate homogenate. Ligand-bound receptor was separated from unbound ligand using 500 |xl of 60% hydroxyapatite slurry in 50 mM Tris buffer. Samples were washed three times in 50 mM Tris (with 600 times; g centrifugation between washes) to completely remove unbound ligand.