Tate R L, Schwartz H I, Holmes J M, Kohn L D
J Biol Chem. 1975 Aug 25;250(16):6509-15.
Biologically active bovine 125I-thyrotropin preparations have been prepared, characterized, and used to evaluate the optimal conditions for thyrotropin binding to bovine thyroid plasma membranes in vitro. Binding of 125I-TSH has a pH optimum around 6.0 and is sensitive to the choice and concentration of buffer. Binding is inhibited by salts, especially those containing magnesium and calcium ions; magnesium concentrations optimal for adenylate cyclase assays (2 to 5 mM) result in 85 to 98% inhibition of binding. Binding is temperature sensitive. At 37 degrees binding has its highest initial level; however, instability of the membrane at this temperature causes a rapid loss of binding activity. Binding at 0 degrees is optimal in 30 min and at the same level as initial binding at 37 degrees; since there is no decrease in binding activity, it has been chosen as the optimal temperature. Thyrotropin, luteinizing hormone, the beta subunit of thyrotropin, and the alpha subunit of thyrotropin have relative binding affinities for the thyrotropin receptors of 100, 10, 2, and less than 0.5, respectively. In all of these characteristics, 125I-thyrotropin at 1.5 x 10(-5) M concentrations has the same properties of binding to bovine plasma membranes as do [3H]thyrotropin preparations which have been previously characterized (Amir, S.M., Carraway, T.F., Jr., Kohn, L.D., and Winand, R.V. (1973) J. Biol. Chem. 248, 4092-4100) and used to study binding at 5 x 10(-6) M concentrations. 125I-TSH binding as a function of hormone concentration results in curved Scatchard plots; however, Hill plots of these same binding data are linear and have a slope of 0.65. Taken together, these data suggest that the heterogeneity in thyrotropin binding constants which is evident in the Scatchard plot reflects a negatively cooperative relationship among the thyrotropin receptor sites, i.e. decreased hormonal affinity as hormone concentrations increase. Adenylate cyclase studies yield kinetic plots which also exhibit negative cooperativity; corrections for thyrotropin bound under the adverse binding conditions of the adenylate cyclase assays suggest that Km values for thyrotropin in this enzymatic assay are compatible with binding constants measured by the 125I-thyrotropin preparations. Tryptic digestion destroys binding activity on the thyroid plasma membrane but releases specific thyrotropin receptor activity into the supernatant phase. Chromatography on Sephadex G-100 indicates that this solubilized receptor fragment has a molecular weight between 15,000 and 30,000.
已制备、鉴定了具有生物活性的牛125I-促甲状腺激素制剂，并用于评估促甲状腺激素在体外与牛甲状腺质膜结合的最佳条件。125I-促甲状腺激素（TSH）的结合在pH约6.0时最为适宜，且对缓冲液的选择和浓度敏感。盐类尤其是含镁离子和钙离子的盐类可抑制结合；对于腺苷酸环化酶测定而言，最佳的镁离子浓度（2至5 mM）可导致85%至98%的结合抑制。结合对温度敏感。在37℃时结合的初始水平最高；然而，在此温度下膜的不稳定性会导致结合活性迅速丧失。在0℃时30分钟内结合最为适宜，且与37℃时的初始结合水平相同；由于结合活性没有降低，因此被选为最佳温度。促甲状腺激素、促黄体生成素、促甲状腺激素的β亚基以及促甲状腺激素的α亚基与促甲状腺激素受体的相对结合亲和力分别为100、10、2和小于0.5。在所有这些特性方面，浓度为1.5×10⁻⁵ M的125I-促甲状腺激素与牛质膜结合的性质与先前已鉴定的[³H]促甲状腺激素制剂（Amir, S.M., Carraway, T.F., Jr., Kohn, L.D., and Winand, R.V. (1973) J. Biol. Chem. 248, 4092 - 4100）相同，且后者曾用于研究浓度为5×10⁻⁶ M时的结合情况。作为激素浓度函数的125I-TSH结合产生弯曲的Scatchard图；然而，这些相同结合数据的Hill图是线性的，斜率为0.65。综上所述，这些数据表明Scatchard图中明显的促甲状腺激素结合常数的异质性反映了促甲状腺激素受体位点之间的负协同关系，即随着激素浓度增加激素亲和力降低。腺苷酸环化酶研究产生的动力学图也显示出负协同性；针对腺苷酸环化酶测定不利结合条件下结合的促甲状腺激素进行校正后表明，该酶促测定中促甲状腺激素的Km值与125I-促甲状腺激素制剂测得的结合常数相符。胰蛋白酶消化会破坏甲状腺质膜上的结合活性，但会将特异性促甲状腺激素受体活性释放到上清相中。在Sephadex G - 100上进行色谱分析表明，这种溶解的受体片段的分子量在15,000至30,000之间。
