کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1175339 | 961797 | 2010 | 6 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: A long-wavelength fluorescent substrate for continuous fluorometric determination of α-mannosidase activity: Resorufin α-d-mannopyranoside A long-wavelength fluorescent substrate for continuous fluorometric determination of α-mannosidase activity: Resorufin α-d-mannopyranoside](/preview/png/1175339.png)
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 585 nm with excitation at 571 nm and has a pKa of 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 Km of 200 μM and Vmax of 11 nmol/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.
Journal: Analytical Biochemistry - Volume 399, Issue 1, 1 April 2010, Pages 7–12