Characterization of 7-8S progestin binding protein in human prostate using vertical tube rotor.
The specific binding of the synthetic progestin, 17 alpha-methyl[3H]promegestone (R5020), to the cytosol of human benign hyperplastic prostate has been studied in sucrose density gradients using a vertical tube rotor. The cytosol of human prostate was shown to contain substantial amounts of a 7-8S macromolecule with a high affinity (Kd = 0.5-1 nM) for R5020 which is saturated at low concentrations (10 nM). The conventional technique of sucrose density gradient analysis in a swinging bucket rotor was not suitable for reproducible optimal analysis of a 7-8S high affinity complex. The use of the salt, Na2MoO4, had a stabilizing effect on the complex. Comparison of saturation analysis assays using dextran charcoal assay and vertical tube rotor assay showed that the charcoal assay can give an over-estimation of the 7-8S saturable binding. Progestational steroids competed with R5020 for binding to 7-8S, whereas androgenic steroids, with the exception of 19-nor-testosterone, did not compete. Incubation of cytosol at elevated temperatures in the presence of DNA-cellulose resulted in the binding of the hormone-protein complex to DNA-cellulose. High ionic strength buffer was required to extract the complex which sedimented at 4.5S in sucrose gradients prepared in 0.4 M KCl. Based on the data presented, progestin binding in human prostate is clearly similar in physical chemical properties to progesterone receptors in "classical" target tissues. However, rapid sucrose gradient analysis with a vertical tube rotor is preferred over conventional techniques to evaluate progestin receptor binding in human prostate.
D002482:Cellulose / Q000031:analogs & derivatives; D002499:Centrifugation, Density Gradient / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000031:analogs & derivatives; D006801:Humans; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D011397:Promegestone / Q000378:metabolism; D011467:Prostate / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000302:isolation & purification / Q000378:metabolism*
C033190:DNA-cellulose; D011980:Receptors, Progesterone; D002482:Cellulose; D004247:DNA; D011397:Promegestone