ヒトTSH受容体の可溶化および部分純化

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To elucidate the role of TSH recptor antibodies in the pathogenesis of Graves' disease, human thyroid membrane fractions were solubilized in Triton X-100 and its properties were characterized. Partial purification of soluble protein fractions were also performed. The results were as follows. 1) Treatment of thyroid membranes with 0.5% Triton X-100 resulted in solubilization of membrane proteins which retained the property to bind <125>^I-TSH. Approximately 40% of the total membrane protein was extracted by this procedure. 2) Binding of <125>^I-TSH to the soluble fractions was markedly inhibited by increasing concentrations of Triton X-100, was dependent on pH, time and temperature. 3) Binding of <125>^I-TSH to the soluble fractions was inhibited by unlabeled TSH in the concentration range from 25μU to 10mU/tube in a dose dependent manner. A Scatchard plot analysis demonstrated the presence of high affinity binding site with Ka of 1.12 × 10^9M-. 4) Binding of <125>^I-TSH to the fractions was markedly inhibited by Graves' IgG with high LATS/HTACS activity. Using this soluble TSH・RRA, TBIIwas detected in about 70% of patients with Graves' disease. 5) The chromatography of the soluble fractions on Sepharose CL-6B revealed the presence of two TSH binding proteins, with apparent molecular weight 280,000 and 120,000daltons, respectively. 6) Soluble fractions were partially purified with DEAE Sepharose CL-6B chromatography and chromatofocusing. The fractions obtained with these two step purification procedures exhibited a 40- fold increase in its TSH binding activity as compared with that of original soluble fractions. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, there appeared a major band with about 70,000 daltons. These partially purified TSH receptors may provide a useful preparations for producing receptor antibodies which could be involved in a pathogenesis of Graves' disease.
神戸大学の論文

ヒトTSH受容体の可溶化および部分純化

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