Morris J C, McCormick D J, Ryan R J
Division of Endocrinology and Metabolism, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
J Biol Chem. 1990 Feb 5;265(4):1881-4.
In order to study the structure and function relationships of the thyrotropin (TSH)-specific beta-subunit, we produced 11 synthetic overlapping peptides containing the entire 112-amino acid sequence of human beta TSH and tested them for activity in TSH radioreceptor assay using both human and porcine thyroid membranes. Synthetic peptides representing four regions of the beta-subunit demonstrated the ability to inhibit binding of 125I-bovine TSH to crude thyroid membranes. The peptide representing the -COOH terminus of the subunit (beta 101-112) possessed highest binding activity, inhibiting binding of labeled TSH with an EC50 of 80 microM. The remaining active peptides were: beta 71-85 (104 microM), beta 31-45 (186 microM), beta 41-55 (242 microM), and beta 1-15 (331 microM). Specificity of the binding activity was shown by the inability of the peptides representing the remainder of the subunit to inhibit binding of label and by the inability of any of the peptides to inhibit binding of 125I-epidermal growth factor to the same thyroid membranes. The low affinity of the peptides as compared with native hormone is in agreement with previous studies of synthetic alpha-subunit peptides and, further, suggests that the interaction of beta TSH with receptor is multifaceted, requiring cooperative binding of these sites for the observed high affinity of the whole hormone. These studies are in agreement with previous predictions of active regions by chemical modification but add two regions to the list, showing the utility of the synthetic peptide strategy in the study of peptide hormone structure-activity relationships.
为了研究促甲状腺激素（TSH）特异性β亚基的结构与功能关系，我们合成了11个重叠肽段，它们包含人β-TSH完整的112个氨基酸序列，并使用人及猪的甲状腺膜，通过TSH放射受体分析检测了这些肽段的活性。代表β亚基四个区域的合成肽段显示出能够抑制125I-牛TSH与粗制甲状腺膜的结合。代表该亚基羧基末端的肽段（β101-112）具有最高的结合活性，以80μM的半数有效浓度（EC50）抑制标记TSH的结合。其余具有活性的肽段为：β71-85（104μM）、β31-45（186μM）、β41-55（242μM）和β1-15（331μM）。代表该亚基其余部分的肽段无法抑制标记物的结合，且任何肽段均不能抑制125I-表皮生长因子与相同甲状腺膜的结合，这表明了结合活性的特异性。与天然激素相比，这些肽段的亲和力较低，这与之前对合成α亚基肽段的研究结果一致，并且进一步表明β-TSH与受体的相互作用是多方面的，整个激素要表现出高亲和力需要这些位点的协同结合才能实现。这些研究与之前通过化学修饰对活性区域的预测结果一致，但在活性区域列表中新增了两个区域，显示了合成肽策略在研究肽类激素结构-活性关系中的实用性。
