Use of pseudosubstrate affinity to measure active protein kinase A.
Use of pseudosubstrate affinity to measure active protein kinase A.
复制标题
使用假底物亲和力来测量活性蛋白激酶 A。
DOI：
10.1016/j.ab.2006.06.002
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发表时间：
2006
影响因子：
2.9
通讯作者：
O'Connor,KathleenL
中科院分区：
文献类型：
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作者：
Paulucci-Holthauzen,AdrianaA;O'Connor,KathleenL
传统的基于底物磷酸化的cAMP依赖的蛋白激酶(又称蛋白激酶A[PKA])检测方法通常具有较高的背景活性，从而限制了敏感性，并使在细胞裂解产物中检测低水平的活性PKA变得困难。因此，一种更好的检测粗细胞裂解物中活性PKA的技术无疑是必要的。我们建立了一种有效而灵敏的方法来比较活性蛋白激酶A的活性水平，该方法是基于活性蛋白激酶A催化亚基与其假底物结构域抑制物结合，并结合谷胱甘肽S转移酶。这种假底物亲和分析可以检测在常见的PKA活性诱导剂存在的情况下，活性PKA水平的变化。该方法能在线性范围内检测活性PKA的浓度依赖曲线，对2.5 ng的活性酶也有较高的检测灵敏度。在PKA抑制剂N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide(H89)存在下观察到的PKA与PKI结合亲和力的变化表明，该方法可以成功地用于测量特定抑制剂对活性PKA的影响。我们的结论是，该方法是一种简单、廉价和无害的方法，可以在可以忽略的背景下，以高灵敏度和高特异度来比较活性PKA水平。
Traditional cAMP-dependent protein kinase (also known as protein kinase A [PKA]) assays, which are based on substrate phosphorylation, often have high background activity from other kinases, thereby limiting sensitivity and making it difficult to detect low levels of active PKA in cell lysates. Therefore, a better technique that measures active PKA in crude cell lysates undoubtedly is necessary. We developed an efficient and sensitive assay to compare active PKA levels based on binding of the active PKA catalytic subunit to its pseudosubstrate domain inhibitor (PKI) fused with glutathione S-transferase (GST–PKI). This pseudosubstrate affinity assay can detect variations in the active PKA levels in the presence of common inducers of PKA activity such as forskolin and prostaglandins. It has resolution to detect a concentration-dependent curve of active PKA in a linear range, and it also has sensitivity to detect up to 2.5ng of active enzyme. An observed change in the binding affinity between PKA and PKI in the presence of the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) shows that this assay can be successfully used to measure how active PKA is affected by specific inhibitors. We conclude that this method is a simple, inexpensive, and nonhazardous method to compare active PKA levels with high sensitivity and specificity with negligible background.