Effect of protease inhibitors on androgen receptor analysis in rat prostate cytosol.
Prostate androgen receptors are liable to proteolytic digestion during in vitro analysis; thus, various proteolytic enzyme inhibitors were tested for their ability to improve the androgen receptor assay. The serine (phenylmethylsulfonylflouride, aprotinin, p-aminobenzamidine) and thiol-senine (leupeptin, bacitracin) protease inhibitors individually present in the homogenization buffer significantly increased the measurable androgen binding sites by 30-35% in rat prostate cytosol as determined by saturation analysis with [3H]-17 beta-hydroxy-17-methyl- 4,9 11-estratrien-3-one (R-1881) for 20 hr at 4 degrees C. The apparent binding affinity was also increased by these compounds. Various combinations were tried and aprotinin/bacitracin was found to be additive in effect. This combination was also shown to prevent receptor degradation as determined by sucrose density gradient centrifugation. The carboxyl protease inhibitor, pepstatin A, was ineffective in improving the receptor assay. Rabbit bile, an inhibitor of seminin, interfered with receptor binding thus rendering it ineffective for use in saturation analysis. The results show that the use of serine-thiol protease inhibitors significantly improves the cytosol androgen receptor yield and assay sensitivity; therefore, we recommend routine inclusion of these compounds(s) in the homogenization buffer for androgen receptor assays.
D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D001414:Bacitracin / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism*; D008297:Male; D011467:Prostate / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D011944:Receptors, Androgen / Q000187:drug effects*; D011987:Receptors, Steroid / Q000187:drug effects*
D011480:Protease Inhibitors; D011944:Receptors, Androgen; D011987:Receptors, Steroid; D001414:Bacitracin; D007611:Aprotinin