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Development of methods for detection of various protein kinases in sodium dodecyl sulfate-polyacrylamide gel

十二烷基硫酸钠-聚丙烯酰胺凝胶中多种蛋白激酶检测方法的建立

基本信息

批准号：
08557012
负责人：
FUJISAWA Hitoshi
金额：
$ 12.16万
依托单位：
Asahikawa Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

We had developed a sensitive method for detection of protein kinase activities in gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this method, the in situ renaturation of proteins in gels after SDS-PAGE was applied to the detection of protein kinase either autophosphorylation or by the phosphorylation of protein substrates included in the gel. However, extensive use of this method was hampered by the need for a relatively large amounts of substrate proteins, and therefore the use of synthetic oligopeptides in place of proteins as substrates were tried. The oligopeptides which were linked to amino acid polymers such as poly-L-lysine through their amino-terminal cysteinyl residue by a heterobifunctional reagent were efficiently retained in the gel matrix and served as substrates for protein kinases. Another advantage of the use of synthetic oligopeptides is that the sequence of the oligopeptides can be designed to be suited for studies of various protein kinases. Thus, various protein kinases in the crude tissue extracts were efficiently detected by various synthetic oligopeptides included in the gel.A method for detection of protein phosphatase activities toward phosphorylated oligopeptides in SDS-PAGE was also developed. With the use of a synthetic peptide corresponding to the autophosphorylation site of calmodulin-dependent protein kinase II (CaM-kinase II) which was conjugated to poly-L-lysine and then phosphorylated with [gamma-^<-32>P]ATP by the action of CaM-kinase II,as a substrate, several new protein phosphatase were detected in crude rat brain extracts. One of them was purified and characterized. The results suggest that the purifed eazyme is a specialized protein phosphatase for the regulation of CaM-kinase II.

建立了一种灵敏的检测十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）后凝胶中蛋白激酶活性的方法。在这种方法中，在原位复性的蛋白质在凝胶后，SDS-PAGE应用于检测蛋白激酶的自磷酸化或磷酸化的蛋白质底物包括在凝胶中。然而，由于需要相对大量的底物蛋白质，该方法的广泛使用受到阻碍，因此尝试使用合成寡肽代替蛋白质作为底物。通过异双功能试剂将寡肽通过其氨基末端半胱氨酰残基连接到氨基酸聚合物如聚-L-赖氨酸上，从而有效地保留在凝胶基质中，并作为蛋白激酶的底物。使用合成寡肽的另一个优点是寡肽的序列可以设计成适合于各种蛋白激酶的研究。因此，该凝胶可有效检测组织粗提液中的各种蛋白激酶，并建立了SDS-PAGE检测磷酸化寡肽蛋白磷酸酶活性的方法。利用与钙调素依赖性蛋白激酶II（CaM-激酶II）的自磷酸化位点相对应的合成肽作为底物，该合成肽与多聚-L-赖氨酸结合，然后<-32>通过CaM-激酶II的作用与[γ-^ P]ATP磷酸化，在粗鼠脑提取物中检测到几种新的蛋白磷酸酶。其中一个被纯化和表征。结果表明，纯化的酶是一个专门的蛋白磷酸酶的钙调蛋白激酶II的调节。