The stabilizing effect of albumin on structure-functional alteration of LH/hCG receptors was analyzed by thermal perturbation technique. On exposing the membranes to bovine serum albumin (BSA) the heat inactivation profile of hCG-binding sites was shifted to a temperature higher by about 5 degreesC (T50 values). The receptor destabilizing action of arachidonic and oleic acids incorporated into ovarian membranes and reversal of this effect when BSA was used as fatty acid scavenger, may indicate that free fatty acids are responsible for the thermal instability of hCG-binding sites. This presumption was corroborated by digestion of membranes with phospholipase A2 (PLA2). This enzyme exerted effects on the thermal stability of the receptor protein resembling those observed upon insertion of fatty acids. The membrane fluidization induced by arachidonic acid can be reversed by BSA. However, alterations of lipid fluidity in membranes were not found to be a necessary prerequisite for stabilization of the LH/hCG receptor structure. Fluorescence quenching studies indicated that incorporation of oleic acid or digestion of membrane phospholipids with PLA2 elevated the accessibility of fluorophores for acrylamide. BSA scavenging of free fatty acids approached the quenching rate of control membranes. Analysis of fluorescence of membranes bound to monodansylcadaverine probe revealed that the negative surface charge derived from free fatty acids resulted in destabilization of the receptor protein. The effects of free fatty acids on membranes suggest that altered lipid-protein interactions may directly affect the stability of the LH/hCG receptor structure.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.