A long-wavelength fluorescent substrate for continuous fluorometric determination of alpha-mannosidase activity: resorufin alpha-D-mannopyranoside.
A long-wavelength fluorescent substrate for continuous fluorometric determination of alpha-mannosidase activity: resorufin alpha-D-mannopyranoside.
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用于连续荧光测定 α-甘露糖苷酶活性的长波长荧光底物：试卤灵 α-D-吡喃甘露糖苷。
DOI：
10.1016/j.ab.2009.11.039
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发表时间：
2010
影响因子：
2.9
通讯作者：
Naleway,JohnJ
中科院分区：
文献类型：
--
作者：
Coleman,DanielJ;Kuntz,DouglasA;Venkatesan,Meenakshi;Cook,GabrieleM;Williamson,StaciP;Rose,DavidR;Naleway,JohnJ
描述了一种简单可靠的连续测定α-甘露糖苷酶活性的方法，并证明了该方法可用于使用新底物试卤灵α-d-吡喃甘露糖苷（Res-Man）的两种重组人酶的分析。酶反应产物试卤灵在571 nm激发下在585 nm处发出荧光，pK_a为5.8，可在生理pH值或接近生理pH值时连续测定人溶酶体和果蝇高尔基体α-甘露糖苷酶的荧光转换。用重组果蝇高尔基体α-甘露糖苷酶（dGMII）进行的测定表明，每nmol dGMII的动力学参数Km为200μM，Vmax为11nmol/min。还提供了使用几种浓度的已知α-甘露糖苷酶抑制剂苦马豆素进行测定的方法，证明了将该测定用作高通量筛选可能用于癌症治疗的抑制剂的简单方法的潜力。
A simple and reliable continuous assay for measurement of α-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin α-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585nm with excitation at 571nm and has a pKaof 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi α-mannosidases. The assay performed using recombinant Drosophila Golgi α-mannosidase (dGMII) has been shown to give the kinetic parameters Kmof 200μM and Vmaxof 11nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known α-mannosidase inhibitor swainsonine are also presented, demonstrating a potential for use of the assay as a simple method for high-throughput screening of inhibitors potentially useful in cancer treatment.