Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells
Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells
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DOI：
10.1128/mcb.3.8.1343-1352.1983
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发表时间：
1983-08
影响因子：
5.3
通讯作者：
A. Frackelton;A. Ross;H. Eisen
中科院分区：
文献类型：
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作者：
A. Frackelton;A. Ross;H. Eisen
磷酸化蛋白质酪氨酸残基羟基的蛋白激酶与某些逆转录病毒的细胞转化以及某些多肽生长因子对正常细胞生长的调节有关。为了便于识别酪氨酸激酶底物，我们开发了偶氮苄基膦酸半抗原的单克隆抗体。这些抗体之一，MA-2G 8，被证明是特别有吸引力的，因为它结合了一个衍生物的氨基苯基磷酸盐，一个密切的磷酸酪氨酸类似物，具有更高的亲和力比它结合相应的衍生物的氨基苄基膦酸盐;然而，其亲和力磷酸丝氨酸是可以忽略不计的。在本文中，我们描述了使用这种抗体分离磷酸酪氨酸蛋白的最佳条件，特别强调，它与磷酸酪氨酸蛋白的相互作用是敏感的离子洗涤剂和抗体密度的免疫吸附剂基质。该抗体还结合ATP柠檬酸裂解酶;这种酶缺乏磷酸酪氨酸，但含有磷酸组氨酸，其结构与磷酸酪氨酸相似。通过将抗体以高密度附着在琼脂糖凝胶珠上，并从缓冲液中省去离子去污剂，可以通过微批量免疫吸附（随后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳）从Abelson鼠白血病病毒转化的细胞中分离120，000-道尔顿转化蛋白和几种其他磷酸酪氨酸蛋白。在相同条件下，还从用表皮生长因子刺激的人表皮癌细胞（A431）中分离出磷酸酪氨酰蛋白;这些蛋白中最突出的是表皮生长因子的170，000-道尔顿受体。
Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.