The Role of the Amino Group in Subunit Association and Receptor Site Interaction for Ovine Luteinizing Hormone as Studied by Acylation

Abstract

Abstract The positively charged amino groups (in physiological pH range) of ovine luteinizing hormone (oLH) and its subunits (oLHα and oLHβ) were acylated to produce negatively charged (maleylated) or neutral groups (carbamylated) in their place. In both reactions, the number of modified e-lysine amino groups in luteinizing hormone (LH), LHα, and LHβ was 10, 9, and 2, respectively. The NH2 termini were also completely acylated in both reactions. Maleylation or carbamylation did not alter the hydrodynamic volume of oLH or oLHβ significantly, although a greater Stokes radius was obtained with the oLHα derivatives compared to native oLHα as judged by Kav values from molecular exclusion chromatography. Maleylated LHβ combined with native LHα as well as the native subunits recombine, but maleylated LHα recombined with native LHβ less effectively. Efficiency of recombination was judged by the quantity of material with a hydrodynamic volume similar to LH under specified recombination conditions. Maleylated LHα and maleylated LHβ recombined very poorly. The series of recombination products with carbamylated subunits were all obtained in yields comparable to those observed with the recombination of the native subunits. Biological activities of the various recombined products were examined by both the in vivo ovarian ascorbic acid depletion assay and the in vitro radioligand receptor assay and the results agreed. Intact oLH which had been carbamylated or maleylated, as well as the recombined subunit derivatives from these reactions were completely inactive. Either the carbamylated or maleylated oLHα plus native oLHβ, and native oLHα plus maleylated oLHβ gave a low biological activity with response characteristics different from that of the native hormone. The recombined native subunits, or native oLHα plus carbamylated oLHβ, gave products that were slightly less potent than native oLH, with response characteristics that paralleled those of the native hormone. These results suggest that the charges on lysine e-amino groups in the oLHα play a role in the recombination with oLHβ, but that the charge on the oLHβ amino groups is not crucial for subunit-subunit interaction. It may also be concluded that the charge on the free amino groups of oLHβ must be positive or neutral for receptor site interaction. At least a portion of the amino groups on the oLHα must carry a positive charge for maximum hormone-receptor site interaction.