Development of a continuous, fluorometric coupled enzyme assay for thyrotropin-releasing hormone-degrading ectoenzyme.
Development of a continuous, fluorometric coupled enzyme assay for thyrotropin-releasing hormone-degrading ectoenzyme.
复制标题
开发用于促甲状腺素释放激素降解胞外酶的连续荧光耦合酶测定法。
DOI：
10.1006/abio.1999.4276
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发表时间：
1999
影响因子：
2.9
通讯作者：
K. Bauer
中科院分区：
文献类型：
--
作者：
J. A. Kelly;G. R. Slator;K. Tipton;C. Williams;K. Bauer
促甲状腺激素释放激素降解酶（TRH-DE）（EC 3.4. 19.6），去除促甲状腺激素释放激素（TRH）的N-末端焦谷氨酰残基。不连续测定已被用来测量TRH-DE活性，然而，需要一个连续的测定，使可靠的测量初始速率，并促进动力学研究。提供了TRH-DE的连续偶联酶测定，其中TRH-DE水解底物焦谷氨酰-组氨酰-脯氨酰氨基-4-甲基香豆素（TRHMCA），得到His-ProMCA，然后用二肽基肽酶IV（EC 3.4.14.5）裂解，得到7-氨基-4-甲基香豆素（MCA）。通过测量MCA荧光的增加来连续监测反应进程。该检测方法对于快速筛选潜在的TRH-DE抑制剂特别有用。先前报道的不连续测定，其中在80 ℃下的非酶促环化用于从His-ProMCA中释放MCA，被发现低估了形成的产物的量。提出了一种避免这种情况的修改程序。TRH-DE水解TRHMCA的初始速率和动力学参数，使用这种改进的测定法确定与连续测定法确定的那些相对应。不连续和连续测定给出TRHMCA的K（m）值分别为3.4 +/- 0.7 μ M（n = 5）和3.8 +/- 0.5 μ M（n = 5）。通过TRH和TRH-OH的不连续测定法测定的K（i）值分别为35 +/- 4 microM（n = 3）和311 +/- 31 microM（n = 5）。
Thyrotropin-releasing hormone degrading-ectoenzyme (TRH-DE) (EC 3.4. 19.6), removes the N-terminal pyroglutamyl residue of thyrotropin-releasing hormone (TRH). Discontinuous assays have been used to measure TRH-DE activity; however, a continuous assay is needed to make reliable measurements of initial rates and facilitate kinetic studies. Presented is a continuous, coupled enzyme assay for TRH-DE in which TRH-DE hydrolyzed the substrate, pyroglutamyl-histidyl-prolylamido-4-methyl coumarin (TRHMCA), to give His-ProMCA, which was then cleaved by dipeptidyl peptidase IV (EC 3.4.14.5) to give 7-amino-4-methyl coumarin (MCA). Reaction progress was monitored continuously by measuring the increase in MCA fluorescence. This assay should be especially useful for rapid screening of potential TRH-DE inhibitors. A previously reported discontinuous assay, where nonenzymatic cyclization at 80 degrees C was used to liberate MCA from His-ProMCA, was found to underestimate the amount of product formed. A modified procedure that avoids this is presented. Initial rates and kinetic parameters for TRHMCA hydrolysis by TRH-DE determined using this modified assay correspond with those determined by the continuous assay. Discontinuous and continuous assays gave K(m) values for TRHMCA of 3.4 +/- 0.7 microM (n = 5) and 3.8 +/- 0.5 microM (n = 5), respectively. K(i) values determined by the discontinuous assay for TRH and TRH-OH were 35 +/- 4 microM (n = 3) and 311 +/- 31 microM (n = 5), respectively.