Abstract
The oligomeric glycoprotein hormone, ovine lutropin was treated with anhydrous HF at 0 degrees C for 30, 60 and 180 min and at 23 degrees C for 60 and 180 min. The products, designated deglycosylated lutropin 1 (DGLH-1) to deglycosylated lutropin 5 (DGLH-5) respectively, were characterized by gel filtration, concanavalin A-Sepharose binding, disc electrophoresis, amino acid analysis, carbohydrate composition and spectral properties. The preparations were also evaluated for receptor binding activity and immunological activity and bioassayed in vitro in collagenase-dispersed rat interstitial cells. In DGLH-1, fucose and galactosamine were removed completely, and there was a 94% decrease in hexoses and 39% decrease in N-acetylglucosamine. Reaction with HF at 0 degrees C for 1 or 3h led to removal of all hexoses and additional loss of hexosamines. Reactions at 23 degrees C for either 1 or 3h were not of additional value in deglycosylation and none of the reaction conditions yielded the apohormone. All the five deglycosylated hormone preparations were not retained on immobilized-concanavalin A columns and on Sephadex G-100 they were eluted with an increased V(e)/V(0) ratio consistent with the loss of carbohydrate residues. Loss of all but the last of the N-acetylglucosamine residues decreased the abnormality of lutropin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but did not eliminate it. Receptor binding activities of DGLH-1 and DGLH-2 were not different from that of the native hormone, but that of DGLH-3 was slightly decreased and the products obtained at 23 degrees C (DGLH-4 and DGLH-5) had lower activity. Immunoreactivities followed a similar pattern. None of the derivatives had activity in the bioassay in vitro. All of the five derivatives inhibited the action of the native hormone in the bioassay in vitro. Their hormonal antagonistic activity was consistent with the receptor binding activity, with DGLH-5 being the least potent in this respect. The DGLH-4 and DGLH-5 preparations had undergone conformational changes as revealed by 8-anilinonaphthalene-1-sulphonate fluorescence, but this did not result in loss of quaternary structure.