Marker Gene Technologies Inc., University of Oregon Riverfront Research Park, Eugene, OR 97403, USA.
Anal Biochem. 2010 Apr 1;399(1):7-12. doi: 10.1016/j.ab.2009.11.039. Epub 2009 Dec 21.
A simple and reliable continuous assay for measurement of alpha-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin alpha-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK(a) of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi alpha-mannosidases. The assay performed using recombinant Drosophila Golgi alpha-mannosidase (dGMII) has been shown to give the kinetic parameters K(m) of 200 microM and V(max) of 11 nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known alpha-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.
本文描述并验证了一种用于测量α-甘露糖苷酶活性的简单可靠的连续分析方法，该方法使用新的荧光底物——瑞氏素α-D-甘露吡喃糖苷（Res-Man），对两种重组人酶进行了分析。酶反应的产物——瑞氏素，在 571nm 激发下，发射 585nm 的荧光，pKa 值为 5.8，允许在接近生理 pH 值的条件下连续测量人溶酶体和果蝇高尔基体α-甘露糖苷酶的荧光周转率。使用重组果蝇高尔基体α-甘露糖苷酶（dGMII）进行的测定表明，该测定的动力学参数 K(m)为 200μM，V(max)为 11nmol/min per nmol dGMII。还介绍了使用几种已知的α-甘露糖苷酶抑制剂——苦马豆素进行测定的方法，证明该测定方法有望成为筛选潜在用于癌症治疗的抑制剂的高通量筛选方法。
