Enzyme assays by fluorescence polarization in the presence of polyarginine: study of kinase, phosphatase, and protease reactions.
Enzyme assays by fluorescence polarization in the presence of polyarginine: study of kinase, phosphatase, and protease reactions.
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在聚精氨酸存在下通过荧光偏振进行酶测定：激酶、磷酸酶和蛋白酶反应的研究。
DOI：
10.1006/abio.2002.5599
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发表时间：
2002
影响因子：
2.9
通讯作者：
T. Nikiforov
中科院分区：
文献类型：
--
作者：
A. Simeonov;Xiahui Bi;T. Nikiforov
我们之前报道过，通过测量聚精氨酸存在下的荧光偏振信号，可以方便地跟踪荧光标记的磷酸受体肽在激酶催化下转化为相应的磷酸肽。在目前的工作中，我们证明了该方法可以用于除激酶外的其他酶，如磷酸酶和蛋白酶。通过调整缓冲液的离子强度，可以在底物和酶产物都带高度负电荷的情况下使用该方法。通过在聚精氨酸存在的情况下进行反应并连续测量荧光偏振信号，可以实时跟踪所有这些酶促转化。研究发现，聚精氨酸对所研究的蛋白酶（组织蛋白酶G）的酶促转化速率没有影响，但它的存在降低了观察到的蛋白激酶A的磷酸化速率，可能是通过降低反应溶液中游离ATP的浓度来实现的。在聚精氨酸的存在下，白细胞抗原相关磷酸酶催化的去磷酸化反应更快。对于所有三种酶，发现存在聚精氨酸时的反应速率对已知酶抑制剂的存在敏感，但激酶抑制剂H-89和PKI的IC（50）值在存在时高于不存在聚精氨酸时。
We have previously reported that the kinase catalyzed conversion of fluorescently labeled phosphate acceptor peptides to the corresponding phosphopeptides can be conveniently followed by measuring the fluorescence polarization signal in the presence of polyarginine. In the present work, we demonstrate that the method can be used for other enzymes besides kinases, such as phosphatases and proteases. By adjustment of the ionic strength of the buffer it is possible to use this method in cases where both the substrate and the enzymatic product are highly negatively charged. All of these enzymatic transformations can be followed in real time, by performing the reactions in the presence of polyarginine and continuously measuring the fluorescence polarization signal. Polyarginine was found to have no effect on the rate of enzymatic conversion of the protease studied (cathepsin G), but its presence decreased the observed rate of phosphorylation by protein kinase A, presumably by decreasing the concentration of free ATP in the reaction solution. Leukocyte antigen related phosphatase catalyzed dephosphorylation reactions were faster in the presence of polyarginine. For all three enzymes, the reaction rates in the presence of polyarginine were found to be sensitive to the presence of known enzyme inhibitors, but the IC(50) values of the kinase inhibitors H-89 and PKI were higher in the presence than in the absence of polyarginine.
DOI：
10.1016/s0021-9258(19)34116-x
发表时间：
1990-03
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
T. Chijiwa;A. Mishima;M. Hagiwara;M. Sano;Kyozo Hayashi;Tsutomu Inoue;Naito Kenji;T. Toshioka
通讯作者：
T. Chijiwa;A. Mishima;M. Hagiwara;M. Sano;Kyozo Hayashi;Tsutomu Inoue;Naito Kenji;T. Toshioka