Deglycosylated ovine lutropin: preparation and characterization by in vitro binding and steroidogenesis.
Ovine lutropin (oLH) subunits were deglycosylated in anhydrous hydrofluoric acid. All possible combinations of native and deglycosylated alpha- and beta-subunits were prepared and characterized. The two native plus deglycosylated counterpart preparations showed properties intermediate between the native only and the deglycosylated subunit hybrid. For brevity, only the properties of the latter hybrid were described in detail. Deglycosylation did not alter subunit-subunit recombination, protein conformation (circular dichroism) or the peptide chains (N-termini). Deglycosylation enhanced receptor-binding activity, but the carbohydrate moieties are important for maximal steroidogenesis (Leydig cell testosterone production). The deglycosylated hybrid molecule acts as an antagonist or agonist to native hormone action depending on the test system and conditions. The deglycosylated hormone binds to the receptor much more rapidly than the native hormone and binding plateaued at a higher level in the case of the deglycosylated hormone, although cross-labeling of tracers (either native or deglycosylated) indicated that competition was for the same type of receptors. To eliminate internalization of the hormone as a factor in the binding studies we used broken cell (membrane) preparations of Leydig cells. Differences in the preparations are thus attributable to a greater 'on-rate' for the deglycosylated hormone. The 'off-rate' from the receptor was essentially equal for both native and deglycosylated hormones. For this reason the deglycosylated hormone has only limited antagonistic action for native hormone activity.
D000818:Animals; D001681:Biological Assay; D002241:Carbohydrates / Q000502:physiology*; D006858:Hydrofluoric Acid / Q000494:pharmacology; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism; D007986:Luteinizing Hormone / Q000494:pharmacology / Q000502:physiology*; D046911:Macromolecular Substances; D008297:Male; D008968:Molecular Conformation; D055503:Protein Multimerization; D011869:Radioligand Assay; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D011974:Receptors, LH; D013329:Structure-Activity Relationship; D013737:Testis / Q000378:metabolism; D013739:Testosterone / Q000096:biosynthesis
D002241:Carbohydrates; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D011974:Receptors, LH; D013739:Testosterone; D007986:Luteinizing Hormone; D006858:Hydrofluoric Acid