Stabilization, partial purification, and characterization of thyrotropin receptors in solubilized guinea pig fat cell membranes.

Abstract

Specimens obtained during the course of efforts to purify the TSH receptor in guinea pig fat cell membranes solubilized with Triton X-100 displayed extensive loss of TSH binding activity, analogous to that seen by others during the purification of the TSH receptor in thyroid membranes. Therefore, as a preliminary to efforts to purify the TSH receptor in solubilized fat cell membranes (SFCM), experiments were undertaken to ascertain the factors responsible for this loss of binding activity and to find means for its prevention. Temperature proved to be the most important variable. SFCM stored at -70 C retained their activity with respect to the binding of bovine TSH (bTSH) for months, but binding activity was rapidly lost during storage of SFCM at 4 C, a loss due to a decrease in receptor number rather than binding affinity. Loss of binding activity during storage of SFCM at 4 C was unaffected by the addition of 1 mM cystine and was only slightly and temporarily retarded by a mixture of three protease inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and leupeptin). These results indicated that loss of TSH receptors during storage at 4 C is not due to reduction of disulfide bonds or to proteolytic degradation. On the other hand, activity of the receptor was largely or entirely preserved during at least a week of storage at 4 C by the formation of a TSH-TSH receptor complex, by the addition of 40-50% glycerol either during solubilization or immediately thereafter, or by lyophilization immediately after solubilization. Receptors preserved by these three measures retained their original affinity for bTSH and their response to the TSH binding inhibitory activity of Graves'-immunoglobulin G. In the light of these results, SFCM were prepared in 40% glycerol and then subjected to TSH-affinity gel chromatography. The resulting materials contained at least 29.3% of the original binding activity, and their specific TSH binding activity was increased 227-fold. Saturation analysis of [125I]bTSH binding to this preparation revealed an association constant (Ka) (2.2 x 10(9) M-1), very similar to that in the original SFCM preparation. Binding of [125I]bTSH was inhibited in a dose-dependent manner by Graves'-immunoglobulin G, and in multiple preparations of the latter, TSH binding inhibitory activity measured in the affinity-purified receptor preparation was closely correlated with that measured in SFCM.(ABSTRACT TRUNCATED AT 400 WORDS)