Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations.
Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations.
复制标题
肝质膜制剂的胰岛素受体蛋白的光亲和标记。
DOI：
10.1021/bi00542a011
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发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
M. Moule
中科院分区：
文献类型：
--
作者：
C. Yip;C. Yeung;M. Moule
通过牛胰岛素与4-叠氮基苯甲酸的N-羟基琥珀酰亚胺酯反应合成光反应性胰岛素衍生物N epsilon B29-(叠氮基苯甲酰基)胰岛素(MAB-insulin)和N alpha A1, N epsilon B29-二(叠氮基苯甲酰基)胰岛素(DAB-insulin)。这些衍生物通过 SP-Sephadex 上的离子交换色谱纯化，并通过聚丙烯酰胺凝胶电泳、氨基酸分析和端基测定确定其身份。通过受体结合测定和脂肪细胞测定来测量它们的生物活性。这两种衍生物的光反应性通过光谱变化和暴露于光时形成高分子量共价聚合物来证明。放射性MAB-胰岛素和DAB-胰岛素通过用[125I]碘碘化制备。这些放射性衍生物的特征在于其光反应性、免疫反应性以及与肝质膜的受体结合。将大鼠、小鼠和豚鼠的肝质膜制剂与这些放射性胰岛素衍生物一起孵育并用光照射。这些质膜制剂在溶解和还原后的十二烷基硫酸钠凝胶电泳显示两种蛋白质被特异性标记。在所有三种动物中，两个放射性谱带的分子量估计约为 130 000 和 90 000。
The photoreactive insulin derivatives N epsilon B29-(azidobenzoyl)insulin (MAB-insulin) and N alpha A1, N epsilon B29-di(azidobenzoyl)insulin (DAB-insulin) were synthesized by reacting bovine insulin with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. These derivatives were purified by ion exchange chromatography on SP-Sephadex, and their identities were established by polyacrylamide gel electrophoresis, amino acid analysis, and end-group determination. Their biological activities were measured by receptor binding assay and fat cell assay. The photoreactivity of these two derivatives was demonstrated by spectral changes and by the formation of covalent polymers of high molecular weight when exposed to light. Radioactive MAB-insulin and DAB-insulin were prepared by iodination with [125I]iodine. These radioactive derivatives were characterized for their photoreactivity, immunoreactivity, and receptor binding to liver plasma membrane. Liver plasma membrane preparations of rat, mouse, and guinea pig were incubated with these radioactive insulin derivatives and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization and reduction showed that two proteins were specifically labeled. The molecular weights of the two radioactive bands were estimated to be about 130 000 and 90 000 in all three species of animals.