Protein tyrosine phosphatase assays.
Protein tyrosine phosphatase assays.
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DOI：
10.1002/0471142735.im1107s93
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发表时间：
2011-04
影响因子：
--
通讯作者：
Lorenz, Ulrike
中科院分区：
文献类型：
--
作者：
Lorenz, Ulrike
蛋白质的酪氨酸磷酸化和去磷酸化在免疫系统的许多过程中起关键作用，从早期发育到完全分化的效应子功能。由于蛋白质酪氨酸激酶（PTK）和蛋白质酪氨酸磷酸酶（PTPs）的相反作用决定了给定蛋白质上酪氨酸磷酸化的稳态水平，因此确定PTK和PTPs的比活性对于机制研究通常是重要的。PTP通过其催化磷酸酪氨酸残基去磷酸化的酶活性来定义。本单元着重于确定PTP酶活性的方法。虽然有多种PTP检测，但本单元的重点是免疫复合物PTP检测，它不需要精细的生化纯化，通常用于检测免疫系统中特定PTP的活性。尽管人们越来越认识到PTP显示底物特异性，但大多数体外PTP测定都是基于使用通用替代底物。在本单元提供的基本方案中，测量显色底物磷酸对硝基苯基酯（pNPP）的去磷酸化，以确定PTP的酶活性。在第一种替代方案中，使用孔雀石绿色测定来定量无机磷酸盐从磷酸肽的释放。第二种替代方案使用放射性标记的磷蛋白作为底物，释放的放射性的量用于评估PTP活性。
Tyrosine phosphorylation and dephosphorylation of proteins play a critical role during many processes of the immune system, from early development to fully differentiated effector function. Since the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) determine the steady state level of tyrosine phosphorylation on a given protein, it is often important for mechanistic studies to determine the specific activities of PTKs and PTPs. PTPs are defined by their enzymatic activity that catalyzes the dephosphorylation of phosphotyrosine residues. This unit focuses on methods to determine the enzymatic activity of PTPs. While there are many varieties of PTP assays, the focus in this unit is on immune complex PTP assays, which do not require elaborate biochemical purifications and are commonly used to test the activities of specific PTPs in the immune system. Although it has become increasingly recognized that PTPs show substrate specificities, most in vitro PTP assay are based on the use of generic surrogate substrates. In the basic protocol provided in this unit, dephosphorylation of the chromogenic substrate p-Nitrophenyl Phosphate (pNPP) is measured to determine the enzymatic activity of the PTP. In the first alternative protocol, the release of inorganic phosphate from a phosphopeptide is quantified using the Malachite Green Assay. The second alternative protocol uses a radioactively-labeled phosphoprotein as substrate, and the amount of released radioactivity is used to assess PTP activity.