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 L H/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 de monstrated that histidine residues are involved in hCG-receptor. bindi ng. Fluorescence quenching experiments indicated that DEPC did not cha nge the accessibility of fluorophores for acrylamide. Alterations of q uenching rate generally suggest exposure of tryptophanyl residues. Mod ification of histidyl residues was connected with an alteration of the physical state of ovarian membranes. Membrane lipid rigidity was decr eased after DEPC reaction. Thermal perturbation techniques were used t o monitor structural changes in the receptor due to the action of DEPC on membranes. Heat inactivation of hCG-binding sites demonstrated tha t there was a significant destabilization of the LH/hCG receptor struc ture when the membranes were treated with DEPC. Thermal destabilizatio n produced by 5 mmol/l DEPC caused a decrease in T-50 values by about 12 degrees C. These results suggest that histidine residues are locate d at the binding sites of the receptor, and that they are also involve d in alterations of membrane proteins, tie structural integrity of whi ch secondarily influences the accessibility of the LH/hCG receptor.