The synthetic peptide RPRAATF allows specific assay of akt activity in cell lysates
The synthetic peptide RPRAATF allows specific assay of akt activity in cell lysates
复制标题
DOI：
10.1006/abio.2002.5659
复制
发表时间：
2002-06-01
影响因子：
2.9
通讯作者：
Pearson, RB
中科院分区：
文献类型：
--
作者：
Bozinovski, S;Cristiano, BE;Pearson, RB
Akt蛋白激酶是一系列细胞过程中的关键信号分子。确定这种多效性激酶在任何特定过程中的作用的关键是定量其活性的能力。在这项研究中，我们表明，合成肽RPRAATF是一种特异性底物的激酶在粗细胞提取物，从而使快速，方便，灵敏的测定Akt的活性。肽激酶活性被限制在一个单一的高峰后，连续的离子交换色谱的Balb/c 3 T3成纤维细胞的全细胞提取物。这种活性刺激血小板衍生的生长因子和过钒酸盐，磷脂酰肌醇3-激酶依赖性，并抑制特异性免疫耗竭与抗Akt抗血清。此外，使用该肽对来自一系列细胞类型的粗提物进行的直接测定与使用特异性免疫沉淀测定获得的结果一致。(C)2002 Elsevier Science（美国）。
The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immuno-precipitation assays. (C) 2002 Elsevier Science (USA).