Treatment of the rat ovarian membrane-bound and Triton X-100 solubilized LH/hCG receptor with the tryptophan-specific reagents N-bromosuccinimide (NBS) and 2-hydroxy-5-nitrobenzyl bromide (HNB-Br) resulted in inactivation of the receptor to bind hCG. Fluorescence quenching studies indicated that oxidation of tryptophan residues by NBS decreased the accessibility of fluorophores for acrylamide. Preceding binding of hCG to receptor sites was found to protect fluorophores from NBS action. Modification of tryptophan residues was associated with alteration in the rigidity of ovarian membranes and with destabilization of the LH/hCG receptor structure. The results suggest that tryptophan residue is essential for hCG binding to the receptor.
The role of the physical state of ovarian membranes was studied in the process of the early desensitization of the LH/hCG receptor. Thirty min after injection of a desensitizing dose of hCG to rats, the hCG-responsive adenylylcyclase activity was reduced, whereas hCG binding to ovarian membranes was still normal. Early desensitization decreased rigidity of membrane lipids, determined by fluorescence polarization of DPH. Possible structure-functional properties of the LH/hCG receptor were analyzed by thermal perturbation technique. Desensitization decreased thermal stability of the LH/hCG receptor in membranes and in proteoliposomes. Desensitization modified the quenching of protein fluorescence and intrinsic fluorescence spectral properties of membranes. The Stern-Volmer constants for control and desensitized membranes were found to be 4.3 and 5.5, respectively, indicating that desensitization elevated the accessibility of fluorophores for acrylamide. The changes of the physical properties of membranes resulting from desensitization was exhibited solely in the treatment with hCG in vivo, but not in vitro. The results suggest that the hCG-induced alteration of the physical state of luteal membranes may be a requirement for the induction of changes that lead to desensitization.
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