SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF THYROID MEMBRANE TSH BINDING PROTEINS

Abstract

Crude plasma membranes obtained from bovine thyroids possess 1 class of high affinity, low capacity binding sites for TSH [thyroid stimulating hormone] with average association constant (Ka) of 1.301 .times. 109 M-1 and maximal capacity 8.76 .times. 10-10 M/mg of protein. Treatment of crude membranes fraction with 0.1% Triton X-100 and the subsequent sonication in ultrasonic disintegrator resulted in solubilization of membranes proteins with mean recovery of 40.0 .+-. 6.2%. Soluble proteins retained the property to bind [125I]TSH, but the binding of the hormone was decreased. The removal of the detergent from the solubilizate by gel filtration on Sephadex LH-20 increased the binding of TSH well above that demonstrated for crude thyroid membranes. The chromatography of soluble proteins on Ultrogel AcA-44 revealed the presence of 2 TSH binding proteins, one with the MW above 130,000 daltons and the other with the MW approximately 30,000 daltons. The electrofocusing of solubilizate on Ampholine resulted in 2 protein peaks, one at pH 4.0-4.1 and the other at pH 4.4-4.6. The latter peak bound [125I]TSH specifically. The present results have confirmed the heterogeneous character of solubilized TSH receptor preparation. The hormone binding sites belong to acid proteins.