Isolation of luteinizing hormone receptor binding inhibitor from ovine corpora lutea

Procedures for the isolation of luteinizing hormone receptor binding inhibitor (LHRBI) from crude extracts of ovine corpora lutea are described. Microsomal pellets recovered by differential centrifugation of homogenates of fresh corpora lutea were stored at -20 degrees C for 3 weeks and then extracted with Tris-HCl buffer (pH 7.4). On sequential filtration of the extract on Amicon UM-20, PM-10 and UM-2R filters, the corresponding retentates (UM-20R, PM-10R and UM-2R) demonstrated inhibition of radio-labeled human chorionic gonadotrophin (hCG) to ovine luteal cells. The retentate of UM-2 filter (UM-2R) was further fractionated by reverse phase high pressure liquid chromatography. The active fractions- III, IX, X and XIII, thus obtained, calculated by their ability to inhibit 125I-hCG binding in dose dependent manner, were purified 150 to 1000-fold.