Diethylpyrocarbonate, a histidine selective reagent, causes structural alteration of rat ovarian LH/hCG receptor

Treatment of rat ovarian membrane-bound and Triton X-100 solubilized LH/hCG receptor with a histidine-specific reagent diethylpyrocarbonate (DEPC) resulted in inactivation of the ability of the receptor to bind hCG. The partial reversibility of this inhibition by hydroxylamine demonstrated that histidine residues are involved in hCG-receptor binding. Fluorescence quenching experiments indicated that DEPC did not change the accessibility of fluorophores for acrylamide. Alterations of quenching rate generally suggest exposure of tryptophanyl residues. Modification of histidyl residues was connected with an alteration of the physical state of ovarian membranes. Membrane lipid rigidity was decreased after DEPC reaction. Thermal perturbation techniques were used to monitor structural changes in the receptor due to the action of DEPC on membranes. Heat inactivation of hCG-binding sites demonstrated that there was a significant destabilization of the LH/hCG receptor structure when the membranes were treated with DEPC. Thermal destabilization produced by 5 mmol/l DEPC caused a decrease in T50 values by about 12 degrees C. These results suggest that histidine residues are located at the binding sites of the receptor, and that they are also involved in alterations of membrane proteins, the structural integrity of which secondarily influences the accessibility of the LH/hCG receptor.