Anti‐peptide Antibody Identifies a 57 kDa Protein Tyrosine Kinase in the Sea Urchin Egg Cortex
Anti‐peptide Antibody Identifies a 57 kDa Protein Tyrosine Kinase in the Sea Urchin Egg Cortex
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抗肽抗体鉴定出海胆卵皮质中的 57 kDa 蛋白酪氨酸激酶
DOI：
10.1111/j.1440-169x.1993.00199.x
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发表时间：
1993
期刊：
影响因子：
4.6
通讯作者：
W. Kinsey
中科院分区：
文献类型：
--
作者：
G. Peaucellier;Kirsten L. Shartzer;W. Jiang;K. Maggio;W. Kinsey
卵细胞质膜和皮层结构高度富集蛋白酪氨酸激酶活性，其被认为在受精过程中起重要作用。为了鉴定卵皮质中的酪氨酸蛋白激酶，针对复制海胆c-abl基因产物的催化结构域的保守区域的肽产生定点多克隆抗体。选择作为抗原的区域与其他蛋白酪氨酸激酶具有高度同源性（57%）。发现该抗体以高度特异性结合S.紫色卵该抗体能够从溶解在NP-40中的纯化的卵皮质组分中免疫沉淀酶作为57 kDa磷蛋白。通过预先将抗体与用作抗原的合成肽温育，完全抑制免疫沉淀。抗体的结合完全抑制激酶活性。然而，免疫沉淀的激酶活性可以从琼脂糖偶联抗体洗脱，并且显示对含酪氨酸的肽底物具有催化活性。该酶在体外也发生酪氨酸自磷酸化。通过免疫电子显微镜对激酶的超微结构定位显示，该酶主要局限于卵质膜。
The egg plasma membrane and cortical structures are highly enriched in protein tyrosine kinase activity which is thought to play an important role in the fertilization process. In order to identify the tyrosine protein kinases in the egg cortex, a site directed polyclonal antibody was produced against a peptide duplicating a conserved region of the catalytic domain of the sea urchin c‐abl gene product. The region chosen as an antigen had a high degree of homology (57%) to other protein tyrosine kinases. The antibody was found to bind with a high degree of specificity to a 57 kDa protein tyrosine kinase in S. purpuratus eggs. The antibody was capable of immunoprecipitating the enzyme as a 57 kDa phosphoprotein from purified egg cortex fractions solubilized in NP‐40. Immunoprecipitation was completely inhibited by prior incubation of the antibody with the synthetic peptide used as an antigen. Binding of the antibody completely inhibited kinase activity. However, the immunoprecipitated kinase activity could be eluted from the Sepharose‐coupled antibody and was shown to have catalytic activity towards a tyrosine containing peptide substrate. The enzyme also underwent autophosphorylation on tyrosine in vitro. Ultrastructural localization of the kinase by immuno‐electron microscopy revealed that the enzyme was primarily restricted to the egg plasma membrane.