Resolution of radioiodinated thyrotropin into receptor active and inactive components by column chromatography.
Resolution of radioiodinated thyrotropin into receptor active and inactive components by column chromatography.
复制标题
通过柱色谱法将放射性碘化促甲状腺素拆分成受体活性和非活性成分。
DOI：
10.1016/0303-7207(82)90126-5
复制
发表时间：
1982
影响因子：
4.1
通讯作者：
Nayfeh,SN
中科院分区：
文献类型：
--
作者：
Powell-Jones,CH;McQuade,RD;ThomasJr,CG;Nayfeh,SN
经乳过氧化物酶标记和Sephadex G100纯化后，发现[~(125)I]TSH具有不同程度的促甲状腺激素受体结合活性。为了进一步纯化放射性标记的激素，[125I]TSH制剂在Sepharose6B上进行了层析。恢复了放射性物质的两个峰(峰I和峰II)，大约包含。60%的应用放射性。在用镁离子洗脱后，放射性标记物质的其余部分被恢复为单峰(峰III)。放射免疫分析表明这3个峰均具有免疫活性，但峰I和峰III的免疫活性是峰II的3-4倍。放射受体分析表明峰III的受体结合量增加(与Sephadex G100单独纯化的[~(125)I]TSH制剂相比)，而峰I和峰II的结合活性显著降低。相反，人促甲状腺激素(NIH)尽管具有完全的免疫活性，但主要作为受体非活性峰出现。Scatchard分析从峰III与牛的[125I]TSH受体结合，得到了一条曲线，其亲和力类似于我们之前报道的用Sephadex G100层析纯化的[125I]TSH。然而，结合位点的总数随着[125I]TSH制剂的活性部分的增加而成比例增加。由于结合激素的质量是从总放射性的百分数范围计算出来的，而且只有一小部分测量的总激素参与了结合，因此在计算总受体数量时，有必要对无效部分进行校正。
Following radioiodination by the lactoperoxidase method and subsequent purification on Sephadex G100, it was found that [125I]TSH exhibited varying degrees of binding activities to the thyrotropin receptor. In order to further purify the radiolabeled hormone, the [125I]TSH preparation was chromatographed on Sepharose 6B. Two peaks of radioactive material (Peaks I and II) were recovered, containing approx. 60% of the applied radioactivity. Upon elution with Mg2+, the remainder of the radiolabeled material was recovered as a single peak (Peak III). Characterization of these 3 peaks by radioinununoassay demonstrated that all 3 were immunocompetent, although Peaks I and III were 3–4-fold more immunoreactive than Peak II. Analysis by radioreceptor assay indicated that Peak III showed an increase in receptor-binding capacity (in comparison with the [125I]TSH preparation purified by Sephadex G100 alone), while both Peaks I and II exhibited significantly reduced binding activity. In contrast, human TSH (NIH) chromatographed mainly as a receptor inactive peak, although it was fully immunocompetent. Scatchard analysis of receptor binding to bovine [125I]TSH from Peak III yielded a curvilinear plot with affinities similar to those we have previously reported for [125I]TSH purified by Sephadex G100 chromatography. The total number of binding sites, however, increased proportionally with the active fraction of the [125I]TSH preparation. Since the mass of bound hormone is calculated from the percent bound of total radioactivity and only a fraction of the measured total participates in the binding, it is therefore necessary to correct for the inactive fraction when calculating the total receptor number.