A long-wavelength fluorescent substrate for continuous fluorometric determination of alpha-mannosidase activity: Resorufin alpha-D-mannopyranoside

作者：Coleman Daniel J; Kuntz Douglas A; Venkatesan Meenakshi; Cook Gabriele M; Williamson Staci P; Rose David R; Naleway John J^*

来源：Analytical Biochemistry, 2010, 399(1): 7-12.

DOI：10.1016/j.ab.2009.11.039

• Continuous fluorescent enzyme assay
• Kinetic assay
• GMII
• Golgi mannosidase II
• dGMII
• Drosophila melanogaster mannosidase II
• Fluorogenic substrate
• Res-Man
• Resorufin alpha-D-mannopyranoside

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 mu M 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.

• 出版日期2010-4-1