Effect of the Structure-Stabilizing Agent Glycerol on Detergent-Solubilized Follicle-Stimulating Hormone Receptors from Calf Testis*

The nonionic detergent Triton X-1OO is widely used to solubilize integral membrane proteins, such as peptide hormone receptors. Solubilization of the FSH receptor from calf testes membrane with Triton X-100 is accompanied by a complete loss of [125I]iodohuman FSH ([125I]iodo-hFSH)-binding activity after 24 h at 4 C, presumably as a consequence of its structural destabilization. In the presence of Triton X-100 and the structure-stabilizing agent glycerol, however, [125I]iodohFSH-binding activity is significantly preserved. This effect of glycerol is time, temperature, and concentration dependent. FSH receptors extracted from calf testes with 2% Triton X-100 in the presence of 30% glycerol retained 50% of their initial binding activity after 48 h at 4 C and 60% of their initial binding activity after 30 days at -20 C. Association constants (Ka) measured in the presence and absence of glycerol were similar (∼109 M-1). There was a 4-fold increase in recoverable receptor activity in the presence of glycerol, although glycerol itself did not solubilize the receptor. The loss of [125I]iodo-hFSH-binding activity by the receptor with time at 4 C was characterized by a decrease in Ka, with no change in receptor concentration, indicating that this was a consequence of progressive structural or conformational alteration in the receptor rather than receptor loss. A 10-fold dilution of the glycerol-treated, detergent-solubilized receptor with 30% glycerol resulted in a more rapid loss of [125I]iodohFSH- binding activity than occurred with the concentrated receptor. However, even with concentrated receptor, [125I]iodohFSH- binding activity was lost more rapidly at 4 C than at –0 C, indicating that a temperature-related denaturation effect was also operative. Our data are consistent with a model of the FSH receptor as possessing a quaternary structure wherein the loss of [125I]iodo-hFSH-binding activity is a consequence of subunit dissociation, denaturation, or a combination of these phenomena.